Handbook of ELISPOT: Methods and Protocols, 2nd Edition (Methods in Molecular Biology 792)

METHODS IN MOLECULAR BIOLOGY™ Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfi...

Author: Alexander E. Kalyuzhny

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METHODS

IN

MOLECULAR BIOLOGY™

Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

Handbook of ELISPOT Methods and Protocols Second Edition Edited by

Alexander E. Kalyuzhny R & D Systems, Inc., Minneapolis, MN, USA

Editor Alexander E. Kalyuzhny, Ph.D R & D Systems, Inc. Minneapolis, MN, USA [email protected]

ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-324-0 e-ISBN 978-1-61779-325-7 DOI 10.1007/978-1-61779-325-7 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011936016 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface Enzyme-linked immunospot assay (ELISPOT) has been known for almost three decades as a unique state-of-the-art technique for studying the cytokine-secreting activity of immune system cells. ELISPOT appears to be one of the fast growing applications in biomedical research and has become an indispensable tool in vaccine development, HIV research, transplantation studies, and cancer and allergy research. After publishing the 1st edition of the Handbook of ELISPOT in 2005 which received a strong positive feedback from novices, advanced users, and ELISPOT experts, a wealth of new experience with this assay has been accumulated, bringing about the 2nd edition. The very fact that almost twice as many ELISPOT papers were published in 2010 than when the first edition was written in 2004, suggests that ELISPOT is gaining popularity as a must-have research tool. In addition, ELISPOT appears to be a very dynamic technique that can be modified and adapted for a large variety of diverse research tasks. In spite of its apparent simplicity, ELISPOT is complicated and capricious, and setting up an assay and executing it requires a great deal of understanding of its chemical and biological aspects. Furthermore, even knowing the latter is not sufficient enough because it is also critically important to understand the principles of analyzing ELISPOT images, spot quantification, extracting the biological information from the images of spots, and performing a statistical analysis. The 2nd edition of the Handbook of ELISPOT is not just a reformatted 1st edition but rather an extension of the former. It is only the second book in the field which is entirely dedicated to ELISPOT assay, helping researchers not only to learn it but also to advance and become experts. In addition, this book is also intended to assist both novice and experienced researchers from other areas of biomedical science, including stem cells, neuroscience, and endocrinology who are looking for additional cell-based research tools. Part I of the Handbook of ELISPOT includes two chapters introducing the reader to the strengths (Chapter 1) and challenges (Chapter 2) of ELISPOT assay. Part II covers veterinary applications of the ELISPOT assay with equine (Chapter 3), feline (Chapter 4), and canine (Chapter 5) species. Advanced applications are grouped in Part III, covering multicolor fluorescent ELISPOT (Fluorospot, Chapter 6), as well as using ELISPOT for such novel applications as studying oxidative stress (Chapter 7) and secretory activity of microglial cells (Chapter 8), stem cell research (Chapter 9), and combining ELISPOT with ELISA to measure amounts of cytokine secreted by a single cell (Chapter 10). Principles of ELISPOT image analysis are presented in Part IV (Chapters 11–13) along with protocols on statistical data analysis (Chapters 14 and 15). Finally, Part V concludes this volume with chapters on using ELISPOT for vaccine development (Chapters 16 and 17), a diagnostic tool (Chapter 18), and it ends with an overview of membranes and membrane plates used for research and diagnostic ELISPOT applications (Chapter 19). As with the 1st edition, the ultimate goal of putting the current volume together was a compilation of a technical reference and a troubleshooting guide for researchers worldwide. The material presented in this book is written by the leading scientists in their fields who

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translated their hands-on experience into concise how-to protocols, walking the reader step-by-step through their merits and shortcomings. Even after dedicating 10 years to this field with more than 50 developed ELISPOT assays and considering myself an expert, I found contributed chapters as excellent educational materials with a lot of new tricks and hints to learn. I wish to thank contributing authors for sharing their knowledge and expertise with the rest of us, and spending a lot of time (often their personal) on writing and reviewing their chapters to make them both highly informative and easy to comprehend by researchers at different knowledge levels and training skills. I also hope that protocols presented in this volume will serve as food for thought for inquisitive minds in their attempts to develop the next generation of ELISPOT assays that can better meet the challenges presented by the biomedical science. Minneapolis, MN

Alexander E. Kalyuzhny

Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

SETTING AND PERFORMING ELISPOT ASSAY

1 Unique Strengths of ELISPOT for T Cell Diagnostics . . . . . . . . . . . . . . . . . . . . . . Paul V. Lehmann and Wenji Zhang 2 The Impact of Harmonization on ELISPOT Assay Performance . . . . . . . . . . . . . . . Sylvia Janetzki and Cedrik M. Britten

PART II

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ELISPOT FOR VETERINARY RESEARCH

3 Equine ELISPOT Assay to Study Secretion of IFNg and IL-4 from Peripheral Blood Mononuclear Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jodi Hagen, Chris Hartnett, Jeffrey P. Houchins, Steeve Giguère, and Alexander E. Kalyuzhny 4 Utilization of Feline ELISPOT for Mapping Vaccine Epitopes . . . . . . . . . . . . . . . . Jeffrey R. Abbott, Ruiyu Pu, James K. Coleman, and Janet K. Yamamoto 5 Analyzing Cellular Immunity to AAV in a Canine Model Using ELISPOT Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zejing Wang, Rainer Storb, Stephen J. Tapscott, and Stanley Riddell

PART III

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ADVANCED ELISPOT TECHNIQUES

6 Dual- and Triple-Color Fluorospot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Niklas Ahlborg and Bernt Axelsson 7 ELISPOT Assay as a Tool to Study Oxidative Stress in Lymphocytes. . . . . . . . . . . . Jodi Hagen, Jeffrey P. Houchins, and Alexander E. Kalyuzhny 8 ELISPOT Assay for Neuroscience Research: Studying TNFA Secretion from Microglial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jodi Hagen, Jeffrey P. Houchins, and Alexander E. Kalyuzhny 9 ELISPOT Assay as a Tool to Study the Effects of Stem Cells on Cytokine Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jun-Seop Shin and Chung-Gyu Park 10 Combining ELISPOT and ELISA to Measure Amounts of Cytokines Secreted by a Single Cell. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jodi Hagen, Jeffrey P. Houchins, and Alexander E. Kalyuzhny

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PART IV

IMAGE AND DATA ANALYSIS

11 How ELISPOT Morphology Reflects on the Productivity and Kinetics of Cells’ Secretory Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alexey Y. Karulin and Paul V. Lehmann 12 Mathematical Algorithms for Automatic Search, Recognition, and Detection of Spots in ELISPOT Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sergey S. Zadorozhny and Nikolai N. Martynov 13 Objective, User-Independent ELISPOT Data Analysis Based on Scientifically Validated Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wenji Zhang and Paul V. Lehmann 14 Statistical Analysis of ELISPOT Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marcus Dittrich and Paul V. Lehmann 15 Response Determination Criteria for ELISPOT: Toward a Standard that Can Be Applied Across Laboratories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Zoe Moodie, Leah Price, Sylvia Janetzki, and Cedrik M. Britten

PART V

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ELISPOT ASSAY FOR VACCINE DEVELOPMENT AND DIAGNOSTICS

16 Detection of Vaccinia Virus-Specific IFNG and IL-10 Secretion from Human PBMCs and CD8+ T Cells by ELISPOT . . . . . . . . . . . . . . . . . . . . . . 199 Benjamin J. Umlauf, Norman A. Pinsky, Inna G. Ovsyannikova, and Gregory A. Poland 17 ELISPOT Assays to Enumerate Bovine IFN-G-Secreting Cells for the Development of Novel Vaccines Against Bovine Tuberculosis . . . . . . . . . . . 219 Martin Vordermeier and Adam O. Whelan 18 IL-7 Addition Increases Spot Size and Number as Measured by T-SPOT.TB ® . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229 Marsha L. Feske, Miguel Medina, Edward A. Graviss, and Dorothy E. Lewis 19 Overview of Membranes and Membrane Plates Used in Research and Diagnostic ELISPOT Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 Alan J. Weiss Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Contributors JEFFREY R. ABBOTT s Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA NIKLAS AHLBORG s MABTECH AB, Nacka Strand, Sweden BERNT AXELSSON s MABTECH AB, Nacka Strand, Sweden CEDRIK M. BRITTEN s Department of Internal Medicine, University Medical Center, Johannes Gutenberg-University Mainz III, Mainz, Germany JAMES K. COLEMAN s Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA MARCUS DITTRICH s Department of Bioinformatics, Biocenter, University of Wuerzburg, Würzburg, Germany MARSHA L. FESKE s Center for Molecular and Translational Human Infectious Disease Research/Molecular Tuberculosis Laboratory, Methodist Hospital Research Institute, Houston, TX, USA STEEVE GIGUÈRE s Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA, USA EDWARD A. GRAVISS s Center for Molecular and Translational Human Infectious Disease Research/Molecular Tuberculosis Laboratory, Methodist Hospital Research Institute, Houston, TX, USA JODI HAGEN s R&D Systems, Inc., Minneapolis, MN, USA CHRIS HARTNETT s R&D Systems, Inc., Minneapolis, MN, USA JEFFREY P. HOUCHINS s R&D Systems, Inc., Minneapolis, MN, USA SYLVIA JANETZKI s ZellNet Consulting, Inc., Fort Lee, NJ, USA ALEXANDER E. KALYUZHNY s R&D Systems, Inc., Minneapolis, MN, USA ALEXEY Y. KARULIN s Cellular Technology Limited, Shaker Heights, OH, USA PAUL V. LEHMANN s Cellular Technology Limited, Shaker Heights, OH, USA DOROTHY E. LEWIS s Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX, USA NIKOLAI N. MARTYNOV s MZ Computers, Ltd, Moscow, Russia MIGUEL MEDINA s Department of Internal medicine, Health Division Infection Diseases, Houston, TX, USA ZOE MOODIE s Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, WA, USA INNA G. OVSYANNIKOVA s Mayo Vaccine Research Group, Mayo Clinic and Foundation, Rochester, MN, USA CHUNG-GYU PARK s Department of Microbiology and Immunology, Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea; Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, South Korea; Transplantation Research Institute, Seoul National University College of Medicine, Seoul, South Korea

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NORMAN A. PINSKY s Mayo Vaccine Research Group, Mayo Clinic and Foundation, Rochester, MN, USA GREGORY A. POLAND s Mayo Vaccine Research Group, Mayo Clinic and Foundation, Rochester, MN, USA; Program in Translational Immunovirology and Biodefense, Mayo Clinic and Foundation, Rochester, MN, USA LEAH PRICE s Division of Biostatistics, Department of Environmental Medicine, New York University School of Medicine, New York, NY, USA RUIYU PU s Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA STANLEY RIDDELL s Program in Immunology, Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA, USA JUN-SEOP SHIN s Department of Microbiology and Immunology, Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea; Xenotransplantation Research Center, Seoul National University College of Medicine, Seoul, South Korea; Transplantation Research Institute, Seoul National University College of Medicine, Seoul, South Korea RAINER STORB s Program in Transplantation Biology, Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA, USA STEPHEN J. TAPSCOTT s Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA BENJAMIN J. UMLAUF s Mayo Vaccine Research Group, Mayo Clinic and Foundation, Rochester, MN, USA MARTIN VORDERMEIER s Department of Bacteriology, TB Research Group, Veterinary Laboratories Agency, WeybridgeAddlestone, Surrey, UK ZEJING WANG s Program in Transplantation Biology, Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA, USA ALAN J. WEISS s Strategy and Business Development, EMD Millipore, A Division of Merck, Acton, MA, USA ADAM O. WHELAN s Department of Bacteriology, TB Research Group, Veterinary Laboratories Agency, WeybridgeAddlestone, Surrey, UK JANET K. YAMAMOTO s Department of Infectious Diseases and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA SERGEY S. ZADOROZHNY s MZ Computers, Ltd, Moscow, Russia WENJI ZHANG s Cellular Technology Limited, Shaker Heights, OH, USA

Part I Setting and Performing ELISPOT Assay

Chapter 1 Unique Strengths of ELISPOT for T Cell Diagnostics Paul V. Lehmann and Wenji Zhang Abstract The T cell system plays an essential role in infections, allergic reactions, tumor and transplant rejection, as well as autoimmune diseases. It does so by the selective engagement of different antigen-specific effector cell lineages that differentially secrete cytokines and other effector molecules. These T cell subsets may or may not have cytolytic activity, can preferentially migrate to different tissues, and display variable capabilities to expand clonally. The quest of T cell immune diagnostics is to understand which specific effector function and T cell lineage is associated with a given clinical outcome, be it positive or adverse. No single assay can measure all of the relevant parameters. In this chapter, we review the unique contributions that ELISPOT assays can make toward understanding T cell-mediated immunity. ELISPOT assays have an unsurpassed sensitivity in detecting low frequency antigen-specific T cells that secrete effector molecules, including granzyme and perforin. They provide robust, highly reproducible data – even by first time users. Because ELISPOT assays require roughly tenfold less cell material than flow cytometry, ELISPOT is ideally suited for all measurements requiring parallel testing under multiple conditions. These include defining (a) T cell reactivity to individual peptides of extensive libraries, thereby establishing the fine–specificity of the response, and determinant mapping; (b) reactivity to different concentrations of the antigen in serial dilutions to measure the avidity of the T cell response; or (c) different secretory products released by T cells which define their respective effector lineage/functions. Further, because T cells survive ELISPOT assays unaffected, they can be retested for the acquisition of additional information in follow-up assays. These strengths of ELISPOT assays the weaknesses of flow cytometrybased measurements. Thus, the two assays systems compliment each other in the quest to understand T cell-mediated immunity in vivo. Key words: ELISPOT, Flow cytometry, Intracellular cytokine staining, Tetramers, Pentamers, Multimers, Cytokine bead array, Luminex, ELISA, T cell-mediated immunity, Cellular immune response, Immune monitoring, T cell affinity, T cell avidity, Determinant mapping, Epitope mapping, High-throughput T cell testing, Multiplexing, Cytokines, Frequency measurements, Single cell analysis

1. Introduction The ultimate goal of T cell diagnostics is to reliably and reproducibly measure those T cells which are mediators of clinical correlates of interest; for example, the specific T cell type that mediates protection Alexander E. Kalyuzhny (ed.), Handbook of ELISPOT: Methods and Protocols, Methods in Molecular Biology, vol. 792, DOI 10.1007/978-1-61779-325-7_1, © Springer Science+Business Media, LLC 2012

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against a certain infection, or causes transplant rejection, autoimmune disease, allergy, etc. Until recently, efforts to identify such T cells had been misled by a dichotomous concept of T cell effector functions being either Th1 (type 1) or Th2 (type 2). Thus, it was assumed that the measurements of IFN-G producing T cells by IFN-G ELISPOT assays would detect all pro-inflammatory T cells, including CD8 T cells that mediate cytotoxicity. As such, IFN-G ELISPOT assays have been widely used to measure, e.g., the HIVspecific “cellular immune response.” The danger of equating CD8 T cell-mediated immunity with IFN-G measurements was recently brought to the spotlight by a high profile HIV vaccine trial in which induction of HIV-specific IFN-G producing T cells was detected without the induction of protective immunity (1). While a central role for T cells in controlling HIV infection has been abundantly documented, measurements of IFN-G or other cytokines (that also had been assessed in that trial) failed to identify the protective T cell class. While we now know that T cells can differentiate into a multitude of effector lineages, each exerting unique effector functions, we still do not know which of these functions are of particular relevance for a specific condition, such as the control of HIV or other viruses. For HIV, it is tempting to speculate that the cytolytic potential of CD8 T cells rather than their cytokine production capacity is critical for controlling the virus. Cytotoxic activity of CD8 cells, however, is not necessarily associated with IFN-G secretion. We have recently shown that immunizations with different adjuvants can induce CD8 T cells that produce IFN-G and other cytokines (TNF-A, IL-2, and IL-17) and mediate delayed type hypersensitivity (DTH) but are noncytolytic, while immunizations with other adjuvants can induce CD8 T cells that are highly cytolytic, but do not produce IFN-G or other cytokines (TNF-A, IL-2, and IL-17) and do not mediate DTH (2). The measurement of IFN-G production by antigen-specific T cells does not permit to conclude whether cytolytic T cells had been induced, that, if induced, might have mediated protective immunity against HIV, and it should not matter which assay platform is utilized for the measurement of IFN-y production by T cells. It would be utterly wrong to conclude. That the ELISPOT assay itself is unsuitable for detecting clinical correlates of HIV protection (3). The correct conclusion is that IFN-G measurement per se (irrespective of the method used for detection) is not sufficient to reveal the protective T cell class in HIV because apparently T cell functions other than IFN-G production are essential for controlling HIV. Measurement of cytolytic activity might have provided the sought after information which could have been done with granzyme B or perforin ELISPOT assays (4–6). Furthermore, TNF-related apoptosis-inducing ligand (TRAIL) ELISPOT assays could have revealed whether the HIV antigenspecific CD8 T cells are “helped,” functional effector cells (7).

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These measurements could be done only by ELISPOT assays measuring the actual secretion of these molecules. As we start to understand more and more about the complexity of the T cell system (and we are apparently still at the beginning of the learning curve (8)), we also learn to appreciate the importance to account for this complexity in T cell diagnostics. Isolated observations within any such complex system are likely to trigger fundamentally wrong conclusions. This generally applicable wisdom has been captured in the ancient Indian metaphor “The blind men and the elephant” (see Fig. 1). As long as we do not know what the critical effector functions are for a certain clinical condition, it should be wise to attempt to measure multiple facets of T cell immunity: their production of various cytokines, cytolytic, proliferative, and migrational properties, including their abilities to control virus (1, 9). Various assays are needed for the comprehensive measurement of different T cell functions. In flow cytometry-based measurements, cells need to be “poisoned” (Golgi inhibitors) and “killed” (permeabilized) for the detection of secretory products, as such, flow-based measurements tell us more about physical phenotypes

Fig. 1. The danger of relying on single parameter measurements. Inspired by the ancient Indian parable of “The blind men and the elephant.” Graphic artist: Gabor Pesthy.

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Fig. 2. Measuring T cell functions by flow cytometry vs. ELISPOT. Left panel: For the detection of secretory products by ICS, the cells need to be “poisoned” first with Golgi inhibitors to prevent secretion, then permeabilized and fixed/“mummified.” The subsequent standard flow cytometric analysis does not make the distinction whether the analyte is indeed bound for secretion and thus is biologically active, or is retained in/on the cell. Right panel: In contrast, ELISPOT measures the actual secretory activity of pharmacologically untreated, living cells. The cells survive ELISPOT assays unharmed, and can be retested, phenotyped, expanded, cloned, or cryopreserved. Graphic artist: Gabor Pesthy.

of cells than their biological function (see Fig. 2). ELISPOT does not allow examining of cell surface or introcytoplasmic markers, or sorting of cells based on physical characteristics, however, unlike flow cytometry, it enables single cell measurements of the actual secretion of bioactive molecules. Cell surface marker positive cell populations can be readily obtained and tested in ELISPOT, should it be important to define the cell surface phenotype of the analyte secreting T cell. Not only the choice of “what” to measure is critical, the “how” is equally important. Antigen-specific T cells normally occur in low frequencies (1/100,000–1,000,000) in the test material, typically peripheral blood, and detecting them can be a major challenge. Because of the low frequency of antigen-specific T cells, and because of the need to measure their function in complex assay systems, particular consideration needs to be given to the reliability and reproducibility of T cell measurements. Finally,

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feasibility issues are also critical when selecting an approach for T cell immune monitoring, such as the number of cells needed and labor and cost involved in the procedure of data analysis. Among the approaches available for T cell immune monitoring, this chapter focuses on the ELISPOT technique’s unique contributions to T cell diagnostics.

2. Materials 1. PBMC: Cryopreserved PBMC, high resolution HLA-typed, characterized for peptide and protein antigen reactivity. 2. CTL-CryoABC™ Kit: PBMC freezing medium for loss-free cryopreservation of PBMC without the component of serum. 3. CTL-AntiAggregate™ Wash 20×: PBMC thawing solution with anti-DNAse without the need of serum supplement. 4. CTL-Test™ Medium. ELISPOT assay medium, optimized for low background and high signal without the need to supplement with serum. 5. CEF – MHC Class I Control Peptide Pool “Plus”. 6. CMV – MHC Class I Control Peptide Pool. 7. EBV – MHC Class I Control Peptide Pool. 8. CEFT – MHC class II Control Peptide Pool “Plus”. 9. ImmunoSpot® Analyzer. 10. PBMC Reference Sample QC set. 11. Practical suggestions for standardized ELISPOT work can be found in Notes 1–17.

3. Methods 3.1. Unique Strengths of ELISPOT 3.1.1. ELISPOT Measures the Functionality of Single Cells via Their Secretory Activities

ELISPOT is the only technique that allows for the quantification of the actual secretory activity of individual cells. Intracellular cytokine staining (ICS) detects, as the name tells, intracellular analyte. The detection of actually secreted vs. intra cellular analyte can be critical for understanding functional properties of T cells. For example, a cytokine which is posttranslationally regulated will be detected upon de novo synthesis by ICS, or by measuring mRNA, but it will not exert biological effects unless it is actually secreted.

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Similarly, some highly relevant molecules are stored in granules of T cells – perforin and granzyme being prime examples. The specific release of these molecules upon antigen activation permits to selectively detect antigen-specific cytolytic CD8 effector cells by granzyme B or perforin ELISPOT assays (4–7). In contrast, by ICS all effector memory cells stain positive irrespective of their antigenspecificity, i.e., up to 20% of all CD8 T cells will be positive. Furthermore, several cell surface molecules important for T cell diagnostics become bioactive only after being cleaved and released from the cells – TNF family members, including TRAIL, fall in this category. ELISPOT detects only the functionally-relevant released molecules upon specific antigen activation. Flow cytometry measures the cell surface molecules, thus, leading to false positive results concerning functional information (7). Therefore, one needs to be thoughtful when interpreting what has been measured by flow cytometry: is it functionally relevant information, or is it a phenotype that possibly bears no functional significance. In all of the above situations, ELISPOT allows the investigator to detect the secreted, bioactive analytes. 3.1.2. ELISPOT Provides High Content Information on Analyte Secretion at Single Cell Resolution

With the advanced platform that recently have become available for ELISPOT data analysis, scientists now can gain information on the quantity and kinetics of analyte secretion as reflected by the size and density of the spots (see Chapter 11 and 13 on this topic). Such information can provide critical insights for T cell diagnostics beyond the frequency measurements. For example, T cells that have been activated recently in vivo, show increased per cell IFN-G productivity, i.e., produce larger and denser IFN-G spots (10). This observation made in the context of vaccinations might help to distinguish between long-term T cell memory and ongoing T cell activity. This distinction is especially important for the T cell diagnostic of autoimmune diseases, allergies, or chronic infections, including hepatitis and tuberculosis. Under conditions of immune suppression, T cells show a decreased per cell IFN-G productivity rate (11). High avidity T cells produce significantly more cytokine than low avidity T cells (12). Per cell productivity information cannot be obtained by supernatant-based measurements, including ELISAs or cytokine bead arrays (CBA/Luminex). The latter assays measure only the net amount of analyte produced, without revealing how many cells produced it, and at what rate.

3.1.3. ELISPOT Is the Most Sensitive Technique for Single Cell Functional Analysis

In systematic comparisons with ELISPOT, ICS was found to be less sensitive with a detection limit around 0.02% (13). In typical ELISPOT assays, 400,000 PBMC are tested per well, in which case the detection limit is 0.00025% (1 analyte producing cell in 400,000 bystander cells) (12). ELISPOT per se is inherently without a detection limit. In regular 96-well plates, the numbers of PBMC plated and spots detected are linear in the range from

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100,000 to 800,000 PBMC per well (14). Thus, by plating one million PBMC per well, the lower detection limit of ELISPOT assays can be readily extended to 0.0001%. ELISPOT assays can be performed in larger than 96-well plate format, like in 6-well plates with ten million cells per well, lowering the detection limit to 0.00001%. Practically, the number of cells available for testing is the only limiting factor when it comes to configuring ELISPOT assays for ultra low frequency measurements (but keep in mind, T cells survive ELISPOT assay intact and can be retested in a secondary ELISPOT or any other assays). Further, when compared to measurements of soluble analyte in supernatant, e.g., by ELISA, CBA or Luminex, ELISPOT has been shown to outperform the latter by far in sensitivity (12). There are two main reasons for this. First, in ELISPOT assays, the analyte is captured around the secreting cell before it is diluted into the supernatant, degraded or captured by receptors of bystander cells. Supernatant-based assays, in contrast, need to detect the analyte after dilution, absorption, and degradation has occurred. Second, unlike in supernatant-based assays that measure net analyte produced by all cells, in ELISPOT assays the secretory activity of individual cells is detected. Due to this quantitative nature of the ELISPOT measurements, even a moderate increase in the numbers of secreting cells becomes detectable, and can provide a statistically highly significant result identifying a T cell response (see Chapters 13–15). The ability to reliably detect rare antigen-specific T cells is at the very core of immune diagnostic. T cells each express a unique T cell receptor (TCR) which is specific for a single antigen. In order to be able to recognize the universe of antigens, the T cell system relies on an astronomical number (~1012) of various T cell specificities. Subsequently, the frequencies of T cells recognizing individual antigens are very low. While the frequency of antigenspecific effector T cells can transiently rise to as high as 1:100 after acute infections, it typically settles in the range of under 1:10,000 (0.01%) in chronic infections, or after the antigen is cleared (1, 2, 5–7, 10, 11, 14, 15). This frequency is at the lower detection limit of flow cytometry-based techniques, such as ICS, but is well within the linear detection range of standard ELISPOT measurements. 3.1.4. ELISPOT Is Most Economic in Sample Utilization

In ELISPOT assays, every single cell plated is being measured – no cells are lost, as for example, in the tubing of the flow cytometer. While for flow cytometry typically one million PBMC are stained per assay condition, for ELISPOT assays one tenth that number is required (100,000 PBMC per well). Furthermore, ELISPOT assays can be performed with even fewer cells. PVDF plates have become available in the 384-well format, permitting to downscale the cell numbers 1:4, thus 25,000 PBMC per well. Recently, we published a study in which ELISPOT assays were done with a

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single drop of blood obtained from the tail vein of mice: the cells obtained from each drop of blood were tested for medium background control and antigen-induced production of IFN-G and IL-17 in a dual color ELISPOT assay (15). Moreover, when antigen-presenting cells (APC) are provided as a monolayer, even single T cells can be studied in ELISPOT assays (12). Similarly, ELISPOT assays are well suited to run functional tests on the few T cells obtained by needle biopsy. The economic utilization of cells in ELISPOT compared with flow cytometry-based techniques is critical when either the numbers of cells available are limiting (which is the case with essentially any clinical trial, in particular for pediatric studies or with immune suppressed test subjects) or when several antigens or assay conditions need to be tested for determinant mapping, for measurements of functional affinity, or multiplexing (see below). PBMC can be efficiently frozen without loss of function when tested in ELISPOT assays (16). For valuable samples, it is wise to freeze them in aliquots so that data can be independently reproduced, or the range of measurements/analytes extended. Freezing away aliquots, however, cuts down on the cell material available for each test, which can make PBMC limiting even from healthy donors. Here again, the efficient cell utilization of ELISPOT assays is of major advantage. 3.1.5. T Cells Survive ELISPOT Assays, Intact, and Can Be Further Utilized

In ELISPOT assays, PBMC are cultured with antigen and remain otherwise untreated. While the cells are typically discarded after an initial incubation period (the optimal duration of which is different for different analytes, Fig. 3), they can be transferred to regular tissue culture plates for later testing. In one such example, we utilized only 11 million PBMC from subjects with type 1 diabetes to study their T cell reactivity to 70 individual peptides first ex vivo, and then again after 12 days of antigen-driven in vitro expansion while measuring IFN-G and IL-4 in a dual color assay at both time points (17). In Parallel, on day 12 ELISPOT testing was done with the cells transferred from the day 0 ELISPOT assay. We found that the results of the secondary ELISPOT testing were identical for such cells rescued from a primary ELISPOT testing, and PBMC that have been cultured in regular tissue culture plates in parallel (without initially performing an ELISPOT assay on them), further confirming that the T cells survived the primary ELISPOT assay unharmed for further utilization. While we retested them in ELISPOT, they could have been tested by flow cytometry, grown into T cell lines, or frozen down for further characterization at a later time. This “recycling” strategy can be very useful when one works with valuable clinical samples. It cannot be applied to assays in which the primary testing is done

1

Why ELISPOT?

11

Fig. 3. The different time course of cytomegalovirus (CMV )-induced cytokine production. PBMC were plated with (or without, not show since negative) heat inactivated CMV into IL-2, -4, -5, -17, or IFN-G coated PVDF plates, and cultured in an incubator for the time period specified before the respective detection antibodies were added, and the analyte was visualized. Since the maximal numbers of spots differed for each cytokine, the maximal number was set as one. Note, the different cytokines each have very different secretion kinetics, which needs to be accounted for when measuring these cytokines.

by flow cytometry, because in such cases, the cells need to be killed (fixed, permealized) for analysis. Occasionally, the frequency of antigen-specific T cells is very low ex vivo, even below the detection limit of standard 96-well ELISPOT assays, where normally 100,000–500,000 PBMC are plated per well. This has been seen with some cancer vaccines or after immunizations with protein antigens. In such situations, scientists frequently rely on in vitro T cell expansion strategies: the PBMCs are first cultured with antigen plus T cell growth factors for a longer time period (typically 1–2 weeks) in the attempt to detect the antigen-reactive T cells following this expansion. However, frequencies measured after expansion do not necessarily match up with ex vivo frequencies, (17) because different T cell populations do not have uniform expansion potential. Thus, when tested after expansion, the ex vivo measurement is clouded by the proliferative capacity of the T cells. The expansion strategy is advisable only if no ex vivo signal can be obtained via an ex vivo ELISPOT assay. The two approaches can be elegantly combined, however. The fact that the T cells can be harvested without loss after an initial ex vivo ELISPOT assay makes it feasible to test a sample first ex vivo and then again, after expansion. Thus, the PBMC can be first tested in an ELISPOT assay in a 6-well membrane plate at ten million PBMC per well. After the 24-h incubation of an ex vivo ELISPOT assay, the cells can be transferred into 6-well tissue culture plates for further expansion, and after 14 days

12


of cell culture, can be retested in ELISPOT and/or other assays. In this way, the chances of obtaining direct ex vivo frequency measurements are maximized while still maintaining the option of learning about the frequencies after expansion via retesting. Moreover, by comparing the ex vivo frequencies with the frequencies after expansion, one can learn about the proliferative potential of the antigen-specific T cells, assessing an additional important parameter of T cell-mediated immunity, which one-time measurements by ELISPOT or flow cytometry cannot provide. 3.1.6. ELISPOT Is an Ideal Technique for High-Throughput Testing and Screening

A combination of qualities makes the ELISPOT assay the primary choice for high-throughput testing, e.g., for screening of PBMC for reactivity to a multitude of antigens/peptides (i.e., determinant mapping) or establishing antigen dose–response curves (i.e., T cell avidity measurements), or for testing a high number of donor samples (in CTL’s GLP lab, we test up to 300 PBMC samples per day), or for multiplexing by ELISPOT. One important quality that enables high-throughput testing by ELISPOT is the efficient cell utilization in this assay. An example was provided above (17) where only 11 million PBMC were used to test T cell reactivity to 70 individual peptide pools, measuring two cytokines, in that case even testing the cells repeatedly. Second, the simplicity of the assay favors high-throughput testing – the cells and reagents can all be handled in 96-well format, all being pipetted in batches. (The afore mentioned experiments were performed by one single student within a few days). Third, ELISPOT data analysis, including spot recognition and gating, can all be done in a fully automated and walk-away fashion (see Chapter 13 dedicated to this issue in this volume). For the above example of testing 70 peptides individually for two cytokines per test subject, the ImmunoSpot Analyzer requires less than 2 min. These 2 min include the fully automated process of acquiring the images from the wells, analyzing them for two colors, feeding the counts to a database while also saving raw and counted images for audit trails, and automatically preparing the publication-ready graph with the results. By flow cytometry, it would take many hours of intense manual work of highly experienced personal to accomplish the same. Finally, the low cost of ELISPOT assays relative to flow cytometric measurements has also contributed to it being the method of choice for high-throughput testing and screening.

3.1.7. ELISPOT Is the Ideal Technique for Determinant Mapping

T cells recognize peptide fragments of antigens presented on MHC molecules. MHC molecules are polymorphic (there are hundreds of alleles for each locus in the human population), whereby each allele has a unique antigen-peptide binding pattern. Moreover, MHC molecules are polygenic (T cells use several class I and class II gene products as restriction elements). As a consequence, antigenic peptide recognition by T cells in different individuals is highly individualized, being dictated by MHC polymorphism/polygenism,

1

Why ELISPOT?

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and other yet poorly understood rules of antigen processing and repertoire selection. This diversity is an insurmountable hurdle for comprehensive tetramer analysis. The peptides of an antigen that are recognized in the context of an MHC molecule are called determinants (or epitopes). Due to its high-throughput capability, ELISPOT is ideally suited for determinant (epitope) mapping, whereby extensive libraries of overlapping peptides are screened (18). The validity of the ELISPOT approach for determinant mapping was first validated on inbred mice using model antigens, such as hen egg-white lysozyme (HEL) or ovalbumin (OVA), whose determinant recognition in the context of different MHC haplotypes had been well established (19). Since then, screening large peptide libraries has become a standard method for testing the fine specificity of T cell responses and has been applied to many fields of T cell diagnostics. Here, we would like to give an illustration of the feasibility of high-throughput determinant mapping by ELISPOT – and why ELISPOT is the only technique currently available that can realistically accomplish this. The assumed task is the detection of T cell responses to an entire pathogen’s proteome using a library of overlapping peptides. For HIV, for example, a total of 410 peptides of 18 amino acid length, overlapping by 10 amino acids, are sufficient to cover the entire HIV proteome. Testing of these 410 peptides on, e.g., ten donors by ELISPOT requires a simple blood draw of about 40 ml from each individual (41 million PBMC if the PBMC are tested at 100,000 cells/well) or 10 ml of blood if the test is done in the corresponding 384-well format. The plating of the cells and developing the plates can be done by a single experienced scientist (assuming the peptides had been pre-aliquoted) – and it would not even fill his/her work day. The fully automated scanning, analysis and graphing time would be 10 min per test subject, thus less than 2 h for all ten subjects. The entire test could be easily done by a single investigator in 3 days, as a part time effort. If the mapping would be done by ICS, about 400 ml blood would be needed from each donor, and the analysis time alone would take days for the ten test subjects. Supernatant measurements by ELISAs or CBA/Luminex are high-throughput assays; however, these techniques are not sensitive enough to detect the peptide-induced production of cytokine by the low frequency T cells. 3.1.8. ELISPOT Is the Ideal Technique for Measurements of Functional T Cell Avidity

Typically, in functional T cell assays, antigens/peptides are tested at a single dose. This pragmatic approach misses important information about the T cell’s affinity/avidity for antigen. (Avidity is the appropriate term, since during T cell activation multiple TCRs bind to multiple MHC-peptide ligands on the APC, whereby the off-rate contributes more to T cell activation than the on-rate.) In practical terms, T cell avidity can be readily measured by titrating

14


Fig. 4. The different functional avidities of antigen-reactive T cells. PBMC of an HLA-A2 positive subject were plated with different concentrations of individual A-2 restricted CEF peptides, as specified by the different symbols. A standard 24 h IFN-G ELISPOT assay was performed. Note how far apart the maximum stimulatory concentrations of the different peptides are.

the peptide dose while measuring T cell activation (12). Figure 4 provides an example of the dose–response curves obtained when PBMC are tested for reactivity to different doses of peptides. Some peptides activate T cells only at relatively high concentration (in the 1–10 Mg/ml range), other peptide can cause full-blown T cell activation at concentrations as low as 1 pg/ml. High avidity T cells will be stimulated by trace amounts of antigen on APC in vivo, and are likely to exert effector functions. In contrast, the high peptide concentrations that can lead to the stimulation of low avidity T cells in vitro may not be reached in vivo – such T cells might be “ignorant” of the antigen in vivo. These considerations are of particular relevance for studies of autoimmunity and tumor immunity. We showed, using the example of myelin basic protein (MBP), that T cells in wild-type mice require 10,000-fold higher antigen doses to become activated, relative to T cells in MBP gene defective “shiverer” mice (20). In the wild-type mice, MBP is a “self-antigen” that causes negative selection of the high avidity MBP-specific T cell repertoire; whereas in the MBP deficient mice it is a foreign antigen encountering an unselected T cell repertoire. Due to negative selection, most tumor antigens (that are self-antigens) are recognized by low avidity T cells. Thus, when immunizing with such antigens, there is the danger of loading APC with a higher concentration of the antigen/ peptide than that which is present on the tumor cell. This would

1

Why ELISPOT?

15

result in the induction of low avidity antigen-specific T cells. Such T cells would be detected ex vivo when a high concentration of the antigen is used for their activation. The data would truthfully show the induction of a tumor antigen-specific T cell response, but, will not reveal whether those T cells could also recognize lower concentrations of the peptide on the tumor cells, i.e., whether they could function as effector cells. Measurements of T cell avidity by titrating the peptide in the recall assay will add an extra dimension to these tests providing important information toward the latter. T cell avidity measurements require functional assays that are highly efficient in cell utilization to permit testing of antigen in serial dilution while at the same time being sensitive enough to detect low frequency T cells. Among T cell assays, ELISPOT is the only technique that readily fulfills these requirements. 3.1.9. ELISPOT Is Readily Standardized and Validated for Immune Monitoring

Ever since T cell assays have been around, they have been surrounded by the stigma of being an art form that only few can successfully perform after a high level of specialization. Also there has been a perception that data from such assays are hard to reproduce. Indeed, the magnitude of this problem has been recently highlighted by a multicenter assay harmonization attempt (21). The same PBMC were tested in different laboratories for reactivity to the same antigen, yet the frequency measurements were more than 3,000% apart. It remains unclear to what extent this alarming variation resulted from the different level of expertise and training by the participants, the variations of protocols and reagents that were permitted to be used, subjective analysis of the data, or whether such variations are inherent to complex biological assays. Are T cell assays really so complex and their results so hard to reproduce? The authors of this chapter helped provide evidence that ELISPOT assays can produce very reproducible data among different laboratories, even in the hands of first time users, if all assay parameters are standardized and the data analysis is performed with scientifically validated principles (14). Expertise and GLP structure were found to be not critical, only the adherence to an optimized protocol that eliminates the variables in the ELISPOT assay, and importantly the utilization of an automated, scientifically validated algorithm for user-independent analysis of the test results. Note, the same PBMC tested in this study, along with reagents, are available from CTL to anyone who wishes to reproduce this claim. The finding in this study is also particularly encouraging for anyone who would like to get started with ELISPOT. Alerted by the high level of variation caused by the subjectivity of flow cytometry data analysis – which is still done manually – the iSBTc/SITC recently announced an “ICS Gating Panel” which invites scientists experienced in ICS to develop a gating harmonization strategy. While the field is struggling to come up with a software that is capable of automated, objective analysis of flow

16


cytometry data, this goal has been accomplished for ELISPOT with the ImmunoSpot platform. Scientifically validated and statistical-based analysis is used by ImmunoSpot® analyzers to define spot recognition parameters and to set gates automatically, making sure that the results are objective and user independent, hence ELISPOT data become reproducible between laboratories (see Chapter 13). Thus, as the first among T cell assays, ELISPOT has transited from an “art” form into an exact science – a technique that provides solid, reproducible measurements. 3.1.10. ELISPOT Is Well Suited for Multiplexing

Because T cells occur in many different effector classes, and because most of the time we do not know which of the effector functions are relevant, it is important to measure as many parameters as we can (see Fig. 1). Bead-based multiple analyte measurements in supernatants (CBA/Luminex) seem to be one of the ways to proceed in these efforts. However, being supernatantbased, they are most of the time not sensitive enough to reliably detect antigen-specific T cell activities that occur at low frequencies. Multiparameter flow cytometry is also an option for such measurements. However, anything more than four colors is presently an art form – even “high art” – such measurements can be reliably performed and reproduced by few researchers. Moreover, by the very nature of the measurements, flow cytometry excels in defining phenotypes of cells, not their functions. For many key functions however, such as antigen-specific killing, we have no reliable corresponding phenotypes. Dual color ELISPOT assay has been established since a decade (22). Cytokine combinations have been defined that, when measured in the double color format, provide the same spot count for each color as the corresponding analytes measured in parallel in single color ELISPOT assay (23). Also, cytokine coexpression can be studied by dual color ELISPOT, detecting coexpressing cells with the same frequency as measured by ICS (22). Double Color ELISPOT, therefore is well suited for detecting polyfunctional T cells that coexpress cytokines. Fully-automated double color analysis software largely facilitates such studies. Double color ELISPOT analysis can be done via the classical enzymatic approach using precipitating red and blue substrates, or by fluorescent detection (fluorospot). Both approaches provide equal sensitivity in the detection of two analytes simultaneously, and coproducers. Fluorospot becomes indispensable, however, when it comes to detecting more than two analytes. Fully-automated instrumentation and software for up to 8-color multiplexing via fluorospot analysis is already available from CTL. We believe that reliable, readily applicable and standardized 8-color fluorospot analysis will be sooner realized than 8 parameter flow cytometry with the ELISPOT-based approach having the additional advantage

1

Why ELISPOT?

17

of high sensitivity for the detection of low frequency cells, economy with cells, high-throughput capacity, and being a functional assay that measures biologically relevant secreted analyte. It also should be noted that “multiplexing” T cell measurements by ELISPOT, can be readily done by running multiple single- or double-color assays in parallel, or in succession. Since ELISPOT requires only 100,000 PBMC as a standard sample size, with one million PBMC, that a standard flow cytometry sample requires, one can obtain 10 single- or ten double color ELISPOT measurements, detecting 10 or 20 analytes, respectively – a target that is hard to match by multiparameter flow cytometry. One can further increase the number of analytes measured in ELISPOT assays by testing cells in succession. For example, granzyme and perforin are released within 4 h after antigen stimulation while the production of IL-4, IL-5, or IL-17 requires a longer activation period. Thus, the cells can be tested in a granzyme/perforin assay first, and then transferred into an IL-4/5 assay, doubling the number of analytes measured with one sample of 100,000 PBMC. One can also easily combine ELISPOT assays with proliferation assays. Because the cells can be retrieved from the ELISPOT assay unaffected, they can be transferred afterward into a proliferation assay or used for measuring other functions or for identifying phenotypes. Cells treated with Golgi inhibitors, permeabilized and fixed, in contrast, will no longer provide functional information. 3.2. Concluding Remarks

Clearly, reliable measurements of several key T cell functions will be required for a better understanding of these cells’ roles in diverse immune processes, and for mediating different clinical outcomes. These parameters include the type of cytokine, chemokine, and other mediators T cells produce, their cytolytic activity, migratory properties, proliferative potential, and their functional avidity. It will take the thoughtful utilization and combination of several different techniques to assess these functions. ELISPOT will continue to be the technique of choice for screening, measurements of effector functions mediated by secretory products, fine specificity, and avidity. Flow cytometry will continue to be indispensable for multiparameter phenotypic analysis. Neither of the two, however, will obviate the need for a new generation of killer assays, or migration assays. Each of these techniques excels in providing a specific type of information – and does not permit interpretations beyond what actually is being measured. Interpreting only one type of read-out inherently goes with the danger of being one of the “blind men studying the elephant.” The sum of the information gained, however, can help reveal the true nature of the “beast” studied. When used to its full potential, ELISPOT will continue to make major contributions to this quest.

18


4. Notes In the following, we provide some practical suggestions for ELISPOT work: 1. Blood draw: The use of heparin as anticoagulant is recommended. 2. Blood storage/shipping: Never chill blood or the PBMC! Keep at room temperature. Store in dark. If shipping in winter, add warm packs to keep at ambient temperature. Do not use cold media for Ficoll gradient separation or washing – it is better to prewarm media in a water bath to 37°C. If handled in this way, PBMC can be stored/shipped for 24 h without significant loss of CD4 or CD8 cell function (more than 90% of reactivity being retained after 24 h). 3. Media: Do not use untested serum for testing or even for washing or freezing – even brief exposure to a mitogenic or suppressive serum can ruin an assay (14). It is best to use special serum-free media that have been developed specifically for ELISPOT work for freezing, thawing, washing, and testing. Such media are available from CTL (14). Do not use PBS or similar minimal buffers for washing cells – it can ruin your assay! 4. Freezing and thawing: PBMC can be frozen without any loss in function, i.e., the frequencies of antigen-induced CD4 or CD8 cells producing IFN-G, IL-2, IL-4, IL-5, and IL-17 are identical in freshly isolated PBMC, and after freeze–thawing (16). To achieve this result, specific protocols that are available from CTL need to be followed. One of the key factors for success is that the freezing medium and the cells need to be at room temperature when mixed, and not chilled on ice, as commonly recommended (16). Also for thawing, the cells need to be warmed up to 37°C and warm washing media needs to be added, slowly, to avoid osmotic lysis of cells. Use pretested (ideally) serum-free media for processing the cells. Detailed protocols are available from CTL. 5. Final storage temperature: After rate-controlled freezing of cells in a −80°C freezer (e.g., using Mr. Frosty cryo-freezing container, Nalgene), or sealed, plastic wrapped Styrofoam racks, transfer them to liquid nitrogen within 48 h – do not store them at −80°C after freezing, or for short- or long-term storage – they will gradually lose functionality. Also for shipping, use dry ice only for overnight shipping – ship in vapor nitrogen containers. 6. Resting of PBMC: For work with freeze–thawed PBMC, the notion has been put forth that a resting period (keeping the cells in a tissue culture incubator overnight before recounting

1

Why ELISPOT?

19

and plating them for the assay) would increase the spot counts without increasing the medium background, thus resulting in a higher signal to noise ratio. We have tested this many times – different members of the lab on different PBMC samples – and we could not verify a real benefit of resting. We occasionally observed a 90%) are ideal (Reutlingen, Germany; or Sigma, St. Louis, MO). 2. Dissolve each peptide in 100% DMSO at a concentration of 20 mg/mL. Number the peptide stocks sequentially and store at −80°C. 3. Design a two-dimensional array for the individual peptides. For AAV6 VP1, there are 182 peptides in the panel, so a 14 × 13 array was designed (Table 1). 4. Generate a peptide pool by combining equal quantity of each peptide stock along an axis and a volume of DMSO sufficient to result in a final concentration of 2 mg/mL/peptide in each pool. For example, pool 1 contains all peptides in the first column, and pool 15 contains all the peptides in the top row. There are total 27 pools for AAV6 VP1 (Table 1). Store pools at −80°C until use.

16

30

44

58

72

86

100

114

128

142

156

170

15

29

43

57

71

85

99

113

127

141

155

169

16

17

18

19

20

21

22

23

24

25

26

27

87

73

59

45

31

17

3

171

157

143

129

115

101

3

88

74

60

46

32

18

4

172

158

144

130

116

102

4

89

75

61

47

33

19

5

173

159

145

131

117

103

5

90

76

62

48

34

20

6

174

160

146

132

118

104

6

91

77

63

49

35

21

7

175

161

147

133

119

105

7

92

78

64

50

36

22

8

176

162

148

134

120

106

8

93

79

65

51

37

23

9

177

163

149

135

121

107

9

178

164

150

136

122

108

94

80

66

52

38

24

10

10

179

165

151

137

123

109

95

81

67

53

39

25

11

11

180

166

152

138

124

110

96

82

68

54

40

26

12

12

181

167

153

139

125

111

97

83

69

55

41

27

13

13

182

168

154

140

126

112

98

84

70

56

42

28

14

14

A total of 182 peptides was synthesized each of which is 15 amino acids long and overlapping by 11 amino acids with adjacent peptides. Twenty-seven peptide pools are generated containing all peptides in their corresponding columns or rows

2

2

1

1

15

È

Pools Æ

Table 1 AAV6 VP1 peptide panel

70 Z. Wang et al.

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Analyzing Cellular Immunity to AAV in a Canine Model Using ELISPOT Assay

3.3.2. ELISPOT Assay

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1. Add 50 ML of 70% ethanol to each well. 2. Wash immediately 4× 200 ML of sterile PBS. 3. Calculate the total volume of capture antibody needed and dilute to the working concentration using PBS. 4. Add 100 ML diluted capture antibody to each well, cover, and incubate overnight at 4°C. 5. Aspirate capture antibody and wash four times with wash buffer (0.05% Tween 20 in PBS, 350 ML/well). After the final wash, remove any remaining liquid by inverting the plate and blotting it against a clean paper towel. 6. Block plates with 200 ML of blocking buffer (1% BSA, 5% sucrose in PBS) for 2 h at RT. 7. During incubation, thaw peptide pool stocks and dilute each pool in culture medium (Way-ISC) to 4 Mg/mL. The culture medium is used as negative control, and 5 Mg/mL PHA is used as positive control. 8. Aspirate blocking buffer, and wash the plates once with 350 ML of culture medium. 9. Aspirate medium, and fill with 100 ML/well of each peptide pools or controls. 10. Add 100 ML of PBMC suspensions (2 × 106 cells/mL) to each well (final concentration of 2 × 105 cells/well, see Notes 1 and 2) and incubate overnight at 37°C (see Note 3). 11. Aspirate and wash plates four times with 350 ML wash buffer. After the final wash, remove any remaining liquid by inverting the plate and blotting it against a clean paper towel. 12. Calculate the total volume of detection antibody needed and dilute to the working concentration using reagent diluent (1% BSA in PBS). 13. Add 100 ML of the diluted detection antibody per well. Cover the plate with the lid and incubate overnight at 4°C. 14. Aspirate and wash plates four times with 350 ML wash buffer. After the final wash, remove any remaining liquid by inverting the plate and blotting it against a clean paper towel.

3.3.3. Color Development

1. Calculate total volume of streptavidin–AP needed and dilute 1:60 in diluent reagent. 2. Add 100 ML to each well and incubate for 2 h at RT. 3. Wash the plates 4× 350 ML wash buffer, rinse again with deionized water; after the final wash, remove any remaining liquid by inverting the plate and blotting it against a clean paper towel. 4. Add 100 ML/well BCIP/NBT solution, cover, incubate for 30 min in dark at RT.

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5. Rinse six times with deionized water, invert plate, and tap to remove excess water. 6. Allow plates to dry at RT for at least 3 h. 7. Count spots using automated ELISPOT reader (see Notes 4 and 5).

4. Notes 1. When using frozen cells, treat cells with 50 U/mL DNase for 3 min at RT after thaw to prevent cell aggregates. Then, spin down at 1,000 × g for 5 min, wash two times with 10 ml of the culture medium, spin at 800 × g for 7 min, and resuspend cell pellet in appropriate volume that gives 2 × 106/mL. 2. Cell numbers per well should be determined empirically using 2 × 105 cells/well as a starting point. Too much background would require using reduced number of cells or increasing cell numbers to obtain sufficient positive spots. Or alternatively, expand antigen-specific T cells before subjecting to ELISPOT assay (see Note 5 below). 3. Incubation time can range from overnight to 3 days depending on the number or size of positive spots. 4. Once a positive pool is identified, each peptide within the pool should be subjected to a second round of ELISPOT assay as stimulant for identifying individual antigenic peptides in a positive pool. 5. To reduce background and increase the number of antigenspecific T cells, in vitro T cell expansion can be performed as follows: – Plate 1.5 × 106 cells/well in 500 ML medium in a 48-well plate. – Dilute peptide pools in culture medium and add 500 ML to the cells for final concentration of 4 Mg/mL. Incubate at 37°C for 48 h. – Take out 500 ML of the medium, and add IL-2 in 500 ML medium for final concentration of 2 U/mL. Incubate at 37°C for 5 days. – Spin down cells at 1,000 rpm for 5 min. – Resuspend in 350 ML of medium, and add 100 ML/well for ELISPOT assay.

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Acknowledgments We thank Drs. Jeffery Chamberlain and Dusty Miller for providing vectors used in the protocols and Dr. Kathy High for providing the cFIX plasmid. We thank Drs. Christine Halbert and Carolina Berger for technical advice, E. Zellmer and E. Finn for technical assistance, A. Joslyn, and the canine team, and M. Spector, DVM, and J Duncan, DVM, for their care of the dogs. We further thank S. Carbonneau, H. Crawford, B. Larson, K. Carbonneau, and D. Gayle for administrative assistance and manuscript preparation. This work was supported by NIH R01 AR056949-01A1, NIH CA15704, and by Career Development Award for Z. Wang from the Muscular Dystrophy Association (MDA 114979). References 1. Athanasopoulos, T., Fabb, S., and Dickson, G. (2000) Gene therapy vectors based on adenoassociated virus: characteristics and applications to acquired and inherited diseases (review). Int J Mol Med 6, 363–375. 2. Sun, B., Zhang, H., Franco, L. M., Young, S. P., Schneider, A., Bird, A., et al. (2005) Efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type II. Molecular Therapy 11, 57–65. 3. Warrington, K. H., Jr., and Herzog, R. W. (2006) Treatment of human disease by adenoassociated viral gene transfer. Hum Genet 119, 571–603. 4. Bostick, B., Yue, Y., Lai, Y., Long, C., Li, D., and Duan, D. (2008) Adeno-associated virus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic abnormalities in mdx mice. Hum Gene Ther 19, 851–856. 5. Yue, Y., Ghosh, A., Long, C., Bostick, B., Smith, B. F., Kornegay, J. N., et al. (2008) A single intravenous injection of adeno-associated virus serotype-9 leads to whole body skeletal muscle transduction in dogs. Molecular Therapy 16, 1944–1952. 6. Athanasopoulos, T., Graham, I. R., Foster, H., and Dickson, G. (2004) Recombinant adenoassociated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD) (Review). Gene Ther 11 Suppl 1, S109–S121. 7. Sabatino, D. E., Mingozzi, F., Hui, D. J., Chen, H., Colosi, P., Ertl, H. C., et al. (2005) Identification of mouse AAV capsid-specific CD8+ T cell epitopes. Molecular Therapy 12, 1023–1033. 8. Gao, G., Lu Y., Calcedo, R., Grant, R. L., Bell, P., Wang, L., et al. (2006) Biology of AAV

serotype vectors in liver-directed gene transfer to nonhuman primates. Molecular Therapy 13, 77–87. 9. Manno C. S., Pierce G. F., Arruda V. R., Glader B., Ragni M., Rasko J. J., et al. (2006) Successful transduction of liver in hemophilia by AAVFactor IX and limitations imposed by the host immune response (erratum appears in Nat Med. 2006 May;12(5):592). Nat Med 12, 342–347. 10. Wang, Z., Allen, J. M., Riddell, S. R., Gregorevic, P., Storb, R., Tapscott, S. J., et al. (2007) Immunity to adeno-associated virusmediated gene transfer in a random-bred canine model of Duchenne muscular dystrophy. Hum Gene Ther 18, 18–26. 11. Brantly, M. L., Chulay, J. D., Wang, L., Mueller, C., Humphries, M., Spencer, L. T., et al. (2009) Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1AAT gene therapy (Erratum appears in Proc Natl Acad Sci U S A. 2009 Oct 13;106(41):17606). Proc Natl Acad Sci USA 106, 16363–16368. 12. Halbert, C. L., Madtes, D. K., Vaughan, A. E., Wang, Z., Storb, R., Tapscott, S. J., et al. (2010) Expression of human A1-antitrypsin in mice and dogs following AAV6 vector-mediated gene transfer to the lungs. Molecular Therapy 18, 1165–1172. 13. Engers, H. D., Thomas, K., Cerottini, J. C., and Brunner, K. T. (1975) Generation of cytotoxic T lymphocytes in vitro. V. Response of normal and immune spleen cells to subcellular alloantigens. J Immunol 115, 356–360. 14. Altman, J. D., Moss, P. A., Goulder, P. J., Barouch, D. H., McHeyzer-Williams, M. G., Bell, J. I., et al. (1996) Phenotypic analysis of

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antigen-specific T lymphocytes Science 274, 94–96. 15. Appay, V., and Rowland-Jones, S. L. (2002) The assessment of antigen-specific CD8+ T cells through the combination of MHC class I tetramer and intracellular staining (Review). J Immunol Methods 268, 9–19. 16. Murali-Krishna, K., Altman, J. D., Suresh, M., Sourdive, D. J., Zajac, A. J., Miller, J. D., et al. (1998) Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8, 177–187. 17. Kern F., Faulhaber, N., Frommel, C., Khatamzas, E., Prosch, S., Schonemann, C., et al. (2000) Analysis of CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides. Eur J Immunol 30, 1676–1682. 18. Lalvani, A., Brookes, R., Hambleton, S., Britton, W. J., Hill, A. V., and McMichael, A. J. (1997) Rapid effector function in CD8+ memory T cells. J Exp Med 186, 859–865. 19. Tobery, T. W., Wang, S., Wang, X. M., Neeper, M. P., Jansen, K. U., McClements, W. L., et al. (2001) A simple and efficient method for the monitoring of antigen-specific T cell responses using peptide pool arrays in a modified ELISpot assay. J Immunol Methods 254, 59–66. 20. Schmittel, A., Keilholz, U., and Scheibenbogen, C. (1997) Evaluation of the interferon-gamma ELISPOT-assay for quantification of peptide specific T lymphocytes from peripheral blood. J Immunol Methods 210, 167–174. 21. Kumar, A., Weiss, W., Tine, J. A., Hoffman, S. L., and Rogers, W. O. (2001) ELISPOT assay for detection of peptide specific interferongamma secreting cells in rhesus macaques. J Immunol Methods 247, 49–60. 22. Tobery, T. W., and Caulfield, M. J. (2004) Identification of T-cell epitopes using ELISpot and peptide pool arrays. Methods in Molecular Medicine 94, 121–132.

23. Maecker, H. T., Moon, J., Bhatia, S., Ghanekar, S. A., Maino, V. C., Payne, J. K., et al. (2005) Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT. BMC Immunology 6, 17. 24. Smith, J. G., Joseph, H. R., Green, T., Field, J. A., Wooters, M., Kaufhold, R. M., et al. (2007) Establishing acceptance criteria for cell-mediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. Clinical and Vaccine Immunology 14, 527–537. 25. Arlen, P., Tsang, K. Y., Marshall, J. L., Chen, A., Steinberg, S. M., Poole, D., et al. (2000) The use of a rapid ELISPOT assay to analyze peptidespecific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines. Cancer Immunol Immunother 49, 517–529. 26. Kaufhold, R. M., Field, J. A., Caulfield, M. J., Wang, S., Joseph, H., Wooters M. A., et al. (2005) Memory T-cell response to rotavirus detected with a gamma interferon enzyme-linked immunospot assay. J Virol 79, 5684–5694. 27. Larsson, M., Jin, X., Ramratnam, B., Ogg, G. S., Engelmayer, J., Demoitie, M. A., et al. (1999) A recombinant vaccinia virus based ELISPOT assay detects high frequencies of Pol-specific CD8 T cells in HIV-1-positive individuals. AIDS 13, 767–777. 28. Wang, R., Richie, T. L., Baraceros, M. F., Rahardjo, N., Gay, T., Banania, J. G., et al. (2005) Boosting of DNA vaccine-elicited gamma interferon responses in humans by exposure to malaria parasites. Infection & Immunity 73, 2863–2872. 29. Firbas, C., Jilma, B., Tauber, E., Buerger, V., Jelovcan, S., Lingnau, K., et al. (2006) Immunogenicity and safety of a novel therapeutic hepatitis C virus (HCV) peptide vaccine: a randomized, placebo controlled trial for dose optimization in 128 healthy subjects. Vaccine 24, 4343–4353.

Part III Advanced ELISPOT Techniques

Chapter 6 Dual- and Triple-Color Fluorospot Niklas Ahlborg and Bernt Axelsson Abstract Cytokine ELISPOT has become a powerful routine tool for the analysis of disease- as well as vaccine-induced T-cell responses. The method is limited, however, in that only one cytokine at a time is assessed. Fluorospot is based on the principle of ELISPOT, but facilitates the analysis of single cells secreting several cytokines, e.g., polyfunctional T cells, suggested to be of protective importance in various infectious diseases. By detecting each cytokine with a specific fluorophore and analyzing differentially colored spots by fluorophore-specific filter systems, cells producing single or multiple cytokines are identified. Fluorospot maintains the simplicity and sensitivity of the ELISPOT while taking the analysis a step forward toward multiplex analysis. Key words: Fluorospot, ELISPOT, Cytokine, T cell, Immune response, Interferon-gamma, Interleukin-2

1. Introduction ELISPOT has, due to its high sensitivity and simplicity, proven valuable for assessing antigen-specific T-cell responses, both with regard to specificity and magnitude. One limitation of the regular ELISPOT method is, however, that only one cytokine at a time is measured. Still, in many settings, it is desirable to measure the production of multiple cytokines in a single well. For example, in studies of HIV, TB, and malaria infection or in the development of vaccines against these and other diseases, enumeration of antigen-specific T cells secreting, e.g., IFN-G, may not yield a complete picture of the quality of the immune response. Recent studies in the field have highlighted the importance of polyfunctional T cells that secrete multiple cytokines. The ability of CD4+ and CD8+ T cells to respond to antigen with a combination of, e.g., IFN-G, IL-2, and TNF-A, rather than only one of the cytokines, has been associated with enhanced protective immunity in viral, bacterial, as well as parasitic diseases (1–4).

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An ELISPOT-based assay analyzing multiple cytokines would not only be useful for defining polyfunctional T cells, it may as well be used for simultaneous measurement of functionally distinct cell populations of various types, e.g., T-cell populations, predominantly secreting single key cytokines representing, e.g., Th1, Th2, Th17, or Tregs. The possibility to measure multiple cytokines simultaneously in the same well also has other advantages, such as a need for less sample cells, valuable, e.g., in studies of mucosaderived cells or studies on newborns and children (5). Over the years, efforts to broaden the ELISPOT technique to include staining with substrates of two colors have been made, first for B cells secreting different Ig isotypes (6) and later for analysis of cytokines (7). Although successfully used in several studies (8, 9), the dual ELISPOT technique can be technically challenging to perform, suffers from several inherent analytical difficulties, and is limited to the analysis of two cytokines. Building on the principle of ELISPOT, but using detection based on fluorescence instead of substrates, the fluorospot assay was developed (10). At present, two-color fluorospot has been described in several publications and reagents/kits for various cytokine combinations and species are commercially available. The most common protocol for fluorospot includes the use of biotinylated detection antibodies for one cytokine and an FITC-labeled detection antibody for the other cytokine (Fig. 1). As a second step in the detection, streptavidin conjugated to a red fluorophore (Cy3) and anti-FITC antibodies labeled with a green fluorophore, respectively, are used (10, 11). The resulting spots are subsequently analyzed using an automated reader equipped with filters for FITC and Cy3. Fluorescent detection can be as sensitive as ELISPOT, or even more sensitive, and offers several advantages compared to dual ELISPOT. Most prominently, by using readers equipped with several narrow-band fluorophore filters, spots derived from cells secreting multiple cytokines are identified by the colocalization of single-colored spots in an overlay analysis of images from different filters (Fig. 2). Importantly, this enables the analysis of not only two, but also three and potentially even more cytokines simultaneously. Experimental systems for triple-color fluorospot have been described (12), but the method may need further development before commercial reagents become available. The major limitation is the availability of additional amplification systems compatible with, e.g., FITC/anti-FITC and biotin/avidin and, in particular, automated readers designed for the analysis of three-colored spots. One amplification strategy that has been evaluated is to use a third detection antibody from a unique species that can be detected by fluorophore-labeled species-specific anti-Ig antibodies, reactive only with the third detection antibody (12). However, in a wider perspective, this may be a limiting factor since most highly functional

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2# detection reagent Detection mAb

Cytokine secreted by cells

IL-10

IFN-G

Capture mAb

a

b

c

Fig. 1. Principle of dual and triple fluorospot. For dual fluorospot, a combination of two capture mAbs is used for coating. The cell incubation step is followed by addition of two detection mAbs and the corresponding secondary detection reagents (a, b). In the example above, IFN-G is detected by an FITC-labeled mAb followed by an anti-FITC mAb labeled with a green fluorophore emitting light at the same wavelength as FITC, whereas IL-2 is detected by a biotinylated mAb in combination with streptavidin (SA)-Cy3. To enable detection of a third cytokine, an additional capture antibody is included. The detection requires the use of a third detection mAb combined with yet another secondary detection reagent (c). For the third cytokine, here IL-10, the detection mAb is labeled with a tag recognized by an antitag mAb labeled with Cy5. Several different tags and antitag mAbs are presently being evaluated to optimize the triple fluorospot.

Fig. 2. Dual fluorospot for the detection of antigen-specific T cells secreting IFN-G and/or IL-2. Human PBMCs (250,000 cells/well) were stimulated with a class I-restricted EBV-derived 9-mer peptide. Images show IFN-G (green), IFN-G/IL-2 (yellow), and IL-2 (red ) spots after stimulation. The image in the middle, displaying IFN-G and/or IL-2 spots, is an overlay of the flanking IFN-G and IL-2 spot images. Note that the double-positive spots, visualized here in yellow, are identified by the position of red and green spots in a computerized overlay and not by their color. The bars below indicate the number of single positive IFN-G (green) and IL-2 (red ) spots. The yellow bar indicates double-positive spots. In wells with unstimulated cells, single green and red spots were 10. Each laboratory should determine an acceptable threshold for the variance ratio based on their data. Using 10 as the threshold for the variance filter, the experimental well responses (10, 10, 50) would fail as the variance-to-median ratio is 48. 3. An example of correctly formatted data is provided in Table 3. The “id” column is all numeric (no character or dashes), the “day” column is all numeric, “antigen” column contains consistently labeled antigen names, and the fourth through ninth columns contain the spot counts in the wells with missing data denoted as NA. Once the data have been saved as a .csv file, point the browser to http://www.scharp.org/zoe/runDFR/ and enter the relevant information: Number of antigens: 2. Number of experimental wells: 3. Number of control wells: 6. Name of negative control: negctl. Submit your .csv data file here (view an example): myfile.csv file created as described above. Click on Run Me icon and wait for output to print on screen. The results may also be downloaded as a .csv file. DFR(eq) positivity calls are listed in the “DFR(eq) response” column and DFR(2×) calls in the “DFR(2×) response” column. The corresponding p-values are listed in the respective adjusted p-value (adjp) columns.

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References 1. Czerkinsky, C., Andersson, G., Ekre, H.P., Nilsson, L.A., Klareskog, L., and Ouchterlony, O. (1988) Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. J Immunol Methods 110:29–36 2. Herr, W., Schneider, J., Lohse, A.W., Meyer zum Buschenfelde, K., and Wolfel, T. (1996) Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor a in response to HLA-A2.1-binding melanoma and viral peptide antigens. J Immunol Methods 191:131–142 3. Herr, W., Protzer, U., Lohse, A.W., Gerken, G., Meyer zum Buschenfelde, K.H., and Wolfel, T. (1998) Quantification of CD8+ T lymphocytes responsive to human immunodeficiency virus (HIV) peptide antigens in HIV-infected patients and seronegative persons at high risk for recent HIV exposure. J Infect Dis 178:260–265 4. Smith, S.G., Joosten. S.A., Verscheure, V., Pathan, A.A., McShane, H., Ottenhoff, T.H., et al. (2009) Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from Mycobacterium tuberculosis. PLoS ONE 4(11): e7972. doi:10.1371/journal.pone.0007972 5. Schloot, N.C., Meierhoff, G., Karlsson, F.M., Ott, P., Putnam, A.,and Lehmann, P., et al. (2003) Comparison of cytokine ELISpot assay formats for the detection of islet antigen autoreactive T cells. Report of the third immunology of diabetes society T-cell workshop. J Autoimmun 21:365–376 6. Asai, T., Storkus, W.J. and Whiteside, T.L. (2000) Evaluation of the modified ELISPOT assay for gamma interferon production in cancer patients receiving antitumor vaccines. Clin Diagn Lab Immunol 7:145–154 7. Cox, J.H., Ferrari, G., and Janetzki, S. (2006) Measurement of cytokine release at the single cell level using the ELISPOT assay. Methods 38:274–282 8. Speiser, D.E., Pittet, M.J., Guillaume, P., Lubenow, N., Hoffman, E., Cerottini, J.C., et al. (2004) Ex vivo analysis of human antigen-specific CD8+ T-cell responses: quality assessment of fluorescent HLA-A2 multimer and interferon-gamma ELISPOT assays for patient immune monitoring. J Immunother 27:298–308 9. Scheibenbogen, C., Romero, P., Rivoltini, L., Herr, W., Schmittel, A., Cerottini, J.C., et al. (2000) Quantitation of antigen-reactive T cells in peripheral blood by IFNgamma-ELISPOT

assay and chromium-release assay: a four-centre comparative trial. J Immunol Methods 20;244: 81–89 10. Janetzki, S., Panageas, K.S., Ben-Porat, L., Boyer, J., Britten, C.M., Clay, T.M., et al. (2008) Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI). Cancer Immunol Immunother 57:303–315 11. Britten, C.M., Gouttefangeas, C., Welters, M.J., Pawelec, G., Koch, S., Ottensmeier, C., et al. (2008) The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays. Cancer Immunol Immunother 57:289–302 12. Cox, J.H., Ferrari, G., Kalams, S.A., Lopaczynski, W., Oden, N., and D’Souza, M.P. (2005) Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency virus type 1 vaccine trials. AIDS Res Hum Retroviruses 21:68–81 13. Janetzki, S., Britten, C.M., Kalos, M., Levitsky, H.I., Maecker, H.T., Melief, C.J., et al. (2009) “MIATA”-minimal information about T cell assays. Immunity 31:527–528 14. Smith, J.G., Joseph, H.R., Green, T., Field, J.A., Wooters, M., Kaufhold, R.M., et al (2007) Establishing acceptance criteria for cellmediated-immunity assays using frozen peripheral blood mononuclear cells stored under optimal and suboptimal conditions. Clin Vaccine Immunol 14(5):527–37 15. Janetzki, S., Cox, J.H., Oden, N., Ferrari, G. (2005) Standardization and validation issues of the ELISPOT assay. Methods Mol Biol 302:51–86 16. Janetzki, S., Schaed, S., Blachere, N.E., BenPorat, L., Houghton, A.N., Panageas, K.S. (2004) Evaluation of Elispot assays: influence of method and operator on variability of results. J Immunol Methods 291(1–2): 175–83 17. Ryan, J.E., Ovsyannikova, I.G., Dhiman, N., Pinsky, N.A., Vierkant, R.A., Jacobson, R.M., et al. (2005) Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-gamma-secreting T cells. Scand J Clin Lab Invest 65(8):681–9 18. Cox, J.H., Ferrari, G., Kalams, S.A., Lopaczynski, W., Oden, N. and D’Souza, M.P. (2005) Results of an ELISPOT proficiency panel conducted in 11 laboratories participating in international human immunodeficiency

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virus type 1 vaccine trials. AIDS Res Hum Retroviruses 21(1):68–81 19. Moodie, Z., Price, L., Gouttefangeas, C., Mander, A., Janetzki, S., Lower, M., et al. (2010) Response definition criteria for ELISPOT assays revisited. Cancer Immunol Immunother 59(10):1489–501 20. Dubey, S., Clair, J., Fu, T.M., Guan, L., Long, R., Mogg, R., et al. (2007) Detection of HIV vaccine-induced cell-mediated immunity in HIV-seronegative clinical trial participants using an optimized and validated enzymelinked immunospot assay. J Acquir Immune Defic Syndr 45:20–27 21. Hudgens, M.G., Self, S.G., Chiu, Y.L., Russell, N.D., Horton, H. and McElrath, M.J. (2004) Statistical considerations for the design and

analysis of the ELISpot assay in HIV-1 vaccine trials. J Immunol Methods 288:19–34 22. Moodie, Z., Huang, Y., Gu, L., Hural, J. and Self, S.G. (2006) Statistical positivity criteria for the analysis of ELISpot assay data in HIV-1 vaccine trials. J Immunol Methods 315:121–132 23. Herr, W., Linn, B., Leister, N., Wandel, E., Meyer zum Buschenfelde, K., and Wolfel, T. (1997) The use of computer-assisted video image analysis for the quantification of CD8+ T lymphocytes producing tumor necrosis factor spots in response to peptide antigens. J Immunol Methods 203:141–152 24. Westfall, P.H. and Young, S.S. (1993) Resampling-based multiple testing: examples and methods for p-value adjustment. John Wiley and Sons, New York

Part V ELISPOT Assay for Vaccine Development and Diagnostics

Chapter 16 Detection of Vaccinia Virus-Specific IFNg and IL-10 Secretion from Human PBMCs and CD8+ T Cells by ELISPOT Benjamin J. Umlauf, Norman A. Pinsky, Inna G. Ovsyannikova, and Gregory A. Poland Abstract High-throughput in vitro assays, which rapidly and succinctly assess the immune status of large cohorts of individuals, are essential tools for conducting population-based studies, including vaccine research. The enzyme-linked immunospot (ELISPOT) assay has emerged as a sensitive, reliable high-throughput tool to measure functional recall immunity by assessing the frequency of antigen-specific cytokine-secreting lymphocytes present in peripheral blood mononuclear cells (PBMCs). For the past 10 years, ELISPOT method has been the dominant platform and a standard for the cell-mediated immune (CMI) assays. ELISPOT assays are used extensively as a measure of CMI response to vaccines, including smallpox (vaccinia), following primary or secondary vaccination. Here, we present detailed methodology for using ELISPOT assays to detect the frequency of cytokine secreting vaccinia-specific lymphocytes including optimized protocols for growing, titrating, and inactivating vaccinia virus; isolating, cryopreserving, and thawing human PBMCs; and finally, detecting vaccinia-specific IL-10 and IFNG secreting lymphocytes, as well as CD8+ IFNG T cells following in vitro stimulation of PBMCs with vaccinia virus. The methods presented below, although optimized for vaccinia virus, emphasize principles that can be generally applied to create ELISPOT assays capable of assessing the immune status as well as antiviral CD8+ T cell response of individuals following primary or secondary vaccination with other licensed or novel vaccines. Key words: Vaccinia virus, IFNG ELISPOT, IL-10 ELISPOT, CD8+ ELISPOT, Smallpox vaccine, Secreted cytokine

1. Introduction Vaccinia virus is the prototypic orthopoxvirus that has been widely used in the twentieth and twenty-first centuries to vaccinate individuals against smallpox (variola) as it induces cross-protective immunity against variola (1, 2). Although routine vaccination of individuals with vaccinia virus ceased during the mid-1970s in


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the USA, President George W. Bush initiated the US National Smallpox Vaccination Program in 2002 to vaccinate healthcare workers, first responders, and mission-critical forces with smallpox vaccine due to the possible threat that smallpox or another orthopoxvirus could be weaponized and used as a bioterorrist weapon (3). The resurgence of individuals receiving smallpox vaccination as a result of the US National Smallpox Vaccinization program has fueled the development and implementation of high-throughput assays that are designed to rapidly and succinctly assess the immune status of large populations postsmallpox vaccination. Successful vaccination against smallpox requires both humoral and cell-mediated immune (CMI) responses (1, 4). The vacciniaspecific humoral response can be measured using several methods; however, a neutralizing antibody (Ab) assay is considered the gold standard by the greater scientific community to quantify circulating vaccinia-specific Ab titers in sera (5). While no single gold standard assay exists to measure vaccinia-specific CMI response, an enzyme-linked immunospot (ELISPOT) assay is a high-throughput assay that assesses vaccinia-specific CMI response following smallpox vaccination (2, 6, 7). ELISPOT assays can be used to functionally assess recall immunity (8), to determine the frequency of vaccinia-specific cytokine-secreting lymphocytes at a single cell level (9), and to determine the Th-type immune response profile of a vaccinated individual (10). Mayo Clinic’s Vaccine Research Group has optimized the use of both IFNG and IL-10 ELISPOT assays to accurately assess both Th1-like and Th2-like cytokine profiles, respectively, for subjects previously vaccinated with vaccinia virus based on frequency of cytokine-secreting lymphocytes following in vitro stimulation with vaccinia virus. In addition, we also describe an ELISPOT assay to measure IFNG secretion of vaccinia-specific CD8+ T cells that does not require any expansion, presorting, or isolation of T cells. The antiviral CD8+ T cell-specific IFNG ELISPOT allows for a more precise measurement of vacciniaspecific IFNG response to vaccinia antigens by removing lymphocytes that are not specific to vaccinia, but still have the potential to secrete IFNG in response to vaccinia virus stimulation (e.g., NK cells). Moreover, a CD8+-specific IFNG ELISPOT allows for a measurement of vaccinia-specific cytotoxic T lymphocyte (CTL) response in individuals previously vaccinated with vaccinia virus. Most importantly, ELISPOT assays allow for a per cell measure of vaccinia-specific CMI response without in vitro expansion of the T-cell population or purification of T-cell subsets; thus, ELISPOT assays are a rapid, reliable in vitro method to monitor immune response postsmallpox vaccination. Here, we describe in detail vaccinia-specific IL-10 ELISPOT, IFNG ELISPOT, and IFNG CD8+ T-cell ELISPOT assays as well as techniques that are essential for measuring human vaccinia virus-specific CMI response using ELISPOT assays.

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2. Materials 2.1. Infecting HeLa Cells with Vaccinia Virus

1. HeLa S3 Cells [American Type Culture Collection (ATCC), Manassas, VA, Number CCL-2.2]. 2. Approximately 500 mL of DMEM with high glucose and L-glutamine supplemented with 10% fetal calf serum (FCS) and 100 U/mL penicillin–100 Mg/mL streptomycin. 3. Hank’s balanced salt solution (HBSS), prewarmed to 37°C. 4. Vaccinia virus, New York City Board of Health (NYCBOH) strain (ATCC). 5. 75-cm2 Sterile tissue culture flasks.

2.2. Harvesting Vaccinia Virus

1. Cell scrapers. 2. 16-mL 10 mM Tris–HCl, pH 9.0. 3. 4-mL 1 mM Tris–Cl, pH 9.0. 4. 50-mL Sterile polypropylene conical centrifuge tubes. 5. 17-mL 36% Sucrose. 6. 1.8-mL Cryogenic tubes.

2.3. Titrating Vaccinia Virus

1. Vero cells (ATCC, Number CCL-81). 2. MEM with L-glutamine and Earle’s salts supplemented with 10% FCS (Hyclone) and 100 U/mL penicillin–100 Mg/mL streptomycin. 3. 1× Sterile phosphate-buffered saline (PBS). 4. 1% Crystal violet in 70% methanol. 5. 12-Well sterile tissue culture plates. 6. 2.5% Trypsin in HBSS.

2.4. Inactivation of Vaccinia Virus

1. 1 mg/mL Psoralen diluted in sterile H2O. 2. 0.1% Bovine serum albumin in HBSS. 3. 35 mm Petri dishes. 4. DNA Cross-linker with 365-nm long-wave UV bulb.

2.5. Collecting and Isolating Peripheral Blood Mononuclear Cells

1. HISTOPAQUE-1077. 2. Accuspin™ tube (Sigma). 3. 1× Sterile PBS. 4. ACK lysis buffer (Invitrogen – Life Technologies, Carlsbad, CA). 5. Cell strainer. 6. Trypan blue. 7. Hemacytometer.

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8. RPMI freezing medium: RPMI 1640 with L-glutamine supplemented with 20% FCS (Hyclone) and 10% DMSO. 9. 1.8-mL Cryogenic freezing tubes. 2.6. Thawing Cryopreserved PBMCs

1. 15-mL Sterile conical centrifuge tubes. 2. RPMI culture medium supplemented with DNase [RPMI 1640 with L-glutamine supplemented with 10% FCS, 100 U/mL penicillin–100 Mg/mL streptomycin, 1 mM sodium pyruvate, and 10 Mg/mL DNase (Sigma)]. 3. RPMI culture medium [RPMI 1640 with L-glutamine supplemented with 5% FCS (Hyclone), 100 U/mL penicillin–100 Mg/ mL streptomycin, and 1 mM sodium pyruvate]. 4. Cell strainers. 5. 50-mL Sterile conical centrifuge tubes. 6. Trypan blue. 7. 1× Sterile PBS.

2.7. Resting PBMCs in the Presence of IL-2

1. 24-Well sterile tissue culture plate. 2. Recombinant IL-2 cytokine (Chiron). 3. RPMI culture medium: RPMI 1640 with L-glutamine supplemented with 5% FCS (Hyclone), 100 U/mL penicillin– 100 Mg/mL streptomycin, and 1 mM sodium pyruvate. 4. 0.25% Trypsin–EDTA (Gibco). 5. Trypan blue. 6. 1u Sterile PBS.

2.8. Infecting PBMCs with Vaccinia Virus for IL-10 Secretion

1. Human IL-10 ELISPOT kit (BD Pharmingen cat. no. 551018). 2. RPMI culture medium: RPMI 1640 with L-glutamine (Gibco) supplemented with 5% FCS (Hyclone), 100 U/mL penicillin–100 Mg/mL streptomycin, and 1 mM sodium pyruvate. 3. Inactivated vaccinia virus multiplicity of infection (MOI) 0.05. 4. Phytohemagglutinin-P (PHA-P) (Sigma). 5. Aluminum foil.

2.9. Infecting PBMCs with Vaccinia Virus for IFNg Secretion

1. Human IFNG ELISPOT kit (either total PBMCs or CD8+ T cells) (R&D cat. no. PEL285 and PEL3094, respectively). 2. RPMI culture medium: RPMI 1640 with L-glutamine (Gibco) supplemented with 5% FCS (Hyclone), 100 U/mL penicillin–100 Mg/mL streptomycin (Gibco), and 1 mM sodium pyruvate. 3. Inactivated vaccinia virus MOI = 5.0. 4. PHA-P (Sigma). 5. Aluminum foil.

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1. Human IL-10 ELISPOT kit (BD Pharmingen cat. no. 551018). 2. 3,3’, 5,5’-Tetramethylbenzidine (TMB) substrate (Moss Inc. cat. no. TMBH-500). 3. Stereomicroscope or automated ELISPOT reader.

2.11. Detection of IFNg Secreting Cells 2.12. Detection of IFNg CD8+ Secreting Cells

1. Human IFNG ELISPOT kit (R&D cat. no. PEL285). 2. Stereomicroscope or automated ELISPOT reader. 1. Human CD8+ IFNG ELISPOT kit (R&D cat. no. PEL3094). 2. Stereomicroscope or automated ELISPOT reader.

3. Methods The following methods describe the steps required to detect human IFNG and IL-10 secreting lymphocytes (1) infecting HeLa cells with vaccinia virus, (2) harvesting vaccinia virus, (3) titrating vaccinia virus, (4) inactivating vaccina virus, (5) collecting and isolating PBMCs, (6) thawing cryopreserved PBMCs, (7) resting PBMCs in the presence of IL-2, (8) infecting PBMCs with vaccinia virus for IL-10 secretion, (9) infecting PBMCs with vaccinia virus for IFNG secretion, (10) detecting IL-10 secreting cells, (11) detecting IFNG secreting cells, and (12) detecting CD8+ IFNG secreting T cells. 3.1. Infecting HeLa Cells with Vaccinia Virus

1. Seed 1 × 106 to 5 × 106 HeLa cells per T75 culture flask. 2. Incubate flasks at 37°C in a 5% CO2 humidified incubator until cells are 80–90% confluent (approximately 2–3 days). 3. Dilute virus stock to MOI = 0.05–0.1 in 2 mL of medium per tissue culture flask. 4. Aspirate all medium from tissue culture flask using a sterile Pasteur pipette connected to a vacuum flask. 5. Wash flask once with HBSS and aspirate. 6. Infect each sterile T75 flask containing HeLa cells with 2 mL of virus suspension. 7. Swirl flask gently to ensure that virus suspension covers all cells. 8. Incubate for 2 h at 37°C in a 5% CO2 humidified incubator swirling flask every 15 min. 9. After 2 h, add approximately 20 mL of complete medium to the flask. 10. Incubate cells for 2–3 days checking for the formation of plaques [i.e., cytopathic effect (CPE)] daily until >90% CPE is observed.

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3.2. Harvesting Vaccinia Virus

The method below is a modified protocol for culturing and purifying vaccinia virus (11). HeLa cells should be harvested only after >90% CPE is observed. 1. Remove all medium from flask and pipette into a 50-mL sterile polypropylene conical centrifuge tube. 2. Centrifuge medium at 500 u g for 10 min at room temperature, and aspirate supernate without disturbing the cell pellet. Save the cell pellet (see Note 1). 3. Using cell lifters, scrape the remaining cells from the flasks into 2 mL of 10 mM Tris–Cl, pH 9.0. If harvesting multiple flasks, scrape cells into 2 mL of 10 mM Tris–Cl, pH 9.0 per flask. 4. Pool cells harvested from each flask with the cell pellet collected in step 2 into a 50-mL sterile polypropylene conical centrifuge tube, and centrifuge the conical tube at 500 u g for 10 min at room temperature. 5. Aspirate supernate then resuspend the cell pellet in 14 mL of 10 mM Tris–Cl, pH 9.0. 6. Transfer cell/virus suspension to a 15-mL U bottom tube and place on ice. 7. Sonicate cell/virus suspension for 30 s at full power (wear proper protective equipment during sonication including a laboratory coat and eye protection). 8. Allow suspension to cool down for 1–2 min on ice, then resonicate cell/virus suspension for 30 s. 9. Layer sonicated cell/virus suspension onto 17 mL of 36% sucrose solution in a sterile ultra centrifuge tube. Place cell/ virus suspension on ice without disturbing the virus suspension-sucrose interface. 10. Centrifuge suspension at 15,800 rpm, 4°C for 80 min. Place ultra centrifuge tube on ice. 11. Aspirate supernatant and resuspend pellet in 4 mL of 1 mM Tris–Cl, pH 9.0, in a U bottom tube. 12. Sonicate suspension for 1 min as described in steps 7 and 8. 13. Store viral suspension at 4°C until the virus is titrated (see Note 2).

3.3. Titrating Vaccinia Virus

The method presented below is slightly modified from established protocols for determining the titer of vaccinia virus in pfu/mL (12, 13). Two separate aliquots of virus should be titrated in parallel to ensure that the observed titer is reflective of the complete viral stock. The titer of each aliquot should have a pfu/mL within 0.5 log of each other. If both titers are within 0.5 log, average the pfu/mL to determine titer of vaccinia viral stock. If the observed

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titers are greater than 0.5 log apart, repeat titration with a third aliquot. 1. Seed 12-well tissue culture (TC) plates with 3 × 105 Vero cells per well 1–3 days before titrating. Cells should be >90% confluent before infection with virus (see Note 3). One 12-well TC plate is sufficient for testing one dilution of the virus in quadruplicate along with the negative controls and standard reference virus. 2. Dilute 2.5% trypsin (10× trypsin) to 1× concentration (0.25%) with calcium and magnesium free 1× PBS. 3. Mix equal volumes (1:1) of 1× trypsin and virus stock in a microcentrifuge tube. Incubate mixture in a 37°C water bath for 30 min. 4. After incubation, perform a tenfold series of dilutions of the virus using medium. Generally, 10−3 to 10−11 dilutions are used for determining virus titers. 5. Dilute reference standard virus, obtained from ATCC or wellcharacterized vaccinia virus with known pfu, to 500 pfu/mL. 6. Remove medium from the wells of one TC plate leaving 50–100 ML of medium in each well. 7. Add 100 ML of diluted viral test samples to the top four wells of the 12-well TC plate. 8. Add 100 ML of standard reference virus to the middle four wells of the 12-well TC plate. 9. Add 100 ML of medium to the bottom four wells of the 12-well TC plate for negative controls (see Note 4). 10. Rock plate vigorously to ensure the inoculum covers the entire surface of each well. 11. Incubate plates at 37°C in a 5% CO2 humidified incubator for 1 h. Rock TC plates every 15 min during the incubation period. 12. After 1 h, add 1 mL of medium to each well. 13. Incubate plates at 37°C in a 5% CO2 humidified incubator for 72 h. 14. After 72 h of incubation, remove medium from the plate (see Note 5). 15. Gently add 1–2 mL of 1× PBS to each well of the TC plate. 16. Gently swirl PBS and then aspirate. 17. Add approximately 1 mL of 1% crystal violet in 70% methanol to each well. Allow the plate to incubate at room temperature for at least 20 min to fix and stain the remaining Vero cells as well as inactivate the virus.

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18. Dump crystal violet solution into the sink. Wash wells in a gentle stream of tap water until the water runs clear. 19. Allow plates to dry. 20. Count plaques per well in the wells with the highest number of countable plaques (usually no more than 120 plaques) per well to calculate titer. 21. Titer in pfu/mL average plaques/well × dilution × 10 (100 ML added per well) × 2 (virus is diluted 1:1 with trypsin). 22. Count plaques in the next lower dilution and average the titers to get the overall titer in pfu/mL (see Note 6). 3.4. Inactivation of Vaccinia Virus

Inactivated virus protects laboratory workers from live virus exposure while not damaging the surface antigens that stimulate a host response. Importantly, inactivated virus has been shown to stimulate cells in assays measuring recall immunity comparable to live virus (10, 14). 1. Dilute virus stock to proper pfu/mL (1 × 108) with 0.1% bovine serum albumin (BSA) in HBSS. 2. Add 5.0 Mg/mL psoralen to the virus stock. 3. Place virus suspension in a 35-mm Petri dish. 4. Incubate at room temperature for 10 min. 5. After 10 min, place 35-mm Petri dish under cross-linker and UV irradiate for 60 s at 365 nm. 6. Remove virus suspension from cross-linker and aliquot 0.3 mL of inactivated virus suspension into prelabeled cryogenic freezing tubes. 7. Repeat titration assay (Subheading 3.4) to determine pfu/mL of inactivated virus. Inactivation should result in a 7–8 log reduction in titer. 8. Store aliquots at −80°C.

3.5. Collecting and Isolating PBMCs

The method below is used to separate PBMCs from whole blood that is collected in tubes treated with heparin or EDTA to prevent coagulation. This procedure is based on the manufacturer’s protocol for separating PBMCs in Accuspin™ tubes. Using cryopreserved PBMCs rather than fresh PBMCs to perform ELISPOT assays has several benefits. Freezing isolated PBMCs allows ELISPOT assays to be performed at a later date postblood draw. This minimizes assay drift because subjects can be tested in consecutive tests and saves money because the entire microplate can be filled with samples. Furthermore, using cryopreserved PBMCs allows for the use of multiple recruitment sites to collect the subject’s specimens, but have ELISPOT assays performed at a single site thus minimizing variability. Importantly,

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cryopreserving human PBMCs does not affect the ability of lymphocytes to produce cytokines (15, 16). 1. Warm HISTOPAQUE-1077 to room temperature using a 37°C water bath. Keep HISTOPAQUE-1077 out of direct light. 2. Pipette 15 mL of HISTOPAQUE-1077 into the upper chamber of each Accuspin™ tube. 3. Centrifuge Accuspin tubes at 800 × g for 30 s to move HISTOPAQUE-1077 into the lower chamber of the Accuspin™ tube. 4. Gently pipette the whole blood from a tube treated with anticoagulant (Heparin or EDTA) into the upper chamber of the Accuspin™ tube (see Note 7). 5. Add sterile 1× PBS into the Accuspin™ tube up to the 45-mL mark. 6. Gently mix blood and PBS; do not force any blood below the frit. 7. Centrifuge Accuspin™ tubes at 1,000 × g for 15 min at 25°C with the brake OFF. 8. After centrifugation carefully remove approximately half of the plasma layer using a sterile Pasteur pipette. Do not disturb the white layer (buffy coat) of PBMCs directly above the frit. 9. Using a sterile Pasteur pipette, carefully remove the layer of PBMCs (white hued layer directly above the frit) and transfer it to a 15-mL sterile conical centrifuge tube. 10. Add 1× sterile PBS to PBMCs bringing the volume of liquid in the 15-mL conical centrifuge tube up to the 10-mL mark to wash cells. 11. Resuspend PBMCs by inverting the tube several times. 12. Centrifuge at 500 × g for 10 min at 25°C with brake ON. 13. Remove supernatant without disturbing the cell pellet. 14. Add 5 mL of ACK lysis buffer to the cell pellet. Resuspend cells by pipetting cell suspension up and down. 15. Allow cells to incubate at room temperature for 5 min in the ACK lysis buffer. 16. Add 1× sterile PBS to the cells + ACK lysis buffer, to bring the volume of liquid in the 15-mL conical centrifuge tube up to the 10-mL mark. 17. Centrifuge at 500 × g for 10 min at 25°C with brake ON. 18. Remove supernatant without disturbing the cell pellet then resuspend pellet in 5 mL of 1× sterile PBS. 19. Place a cell strainer on top of a 50-mL conical centrifuge tube. Transfer the cell suspension from the 15-mL conical centrifuge

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tube to the 50-mL conical centrifuge through the cell strainer (see Note 8). 20. To count the number of live and dead cells, place 200 ML of 1u PBS, 37.5 ML of Trypan blue, and 12.5 ML of cell suspension into a 5-mL falcon tube; mix well then fill a hemacytometer with 10 ML of sample. Count and record the number of unstained (live) cells in the outer four quadrants of the hemocytometer. 21. Total number of cells Number of live cells/4 × 10,000 × 20 (dilution factor [250/12.5]) × total volume of cells (5 mL or pooled total). 22. Centrifuge cell suspension at 500 × g for 10 min at 25°C with brake ON. 23. Adjust cell concentration to 1 u 107 cells/mL with 4°C RPMI freezing medium (see Note 9). 24. Aliquot 1 mL of cell suspension into prelabeled cryogenic freezing tubes. 25. Place cryogenic freezing tubes into a −80°C freezer in a controlled-rate freezing container overnight. 26. Transfer cells to a liquid nitrogen storage tank for long-term storage (see Note 10). 3.6. Thawing Cryopreserved PBMCs

1. Warm RPMI culture medium supplemented with DNase in a 37°C water bath for a minimum of 15 min. 2. Add 100 ML of RPMI culture medium supplemented with DNase into a 15-mL conical centrifuge for each sample being thawed. 3. Remove one vial of PBMCs (cell concentration 1 × 107) for each sample from liquid nitrogen storage tank. 4. Rapidly thaw PBMCs stored in cryogenic freezing tubes using a 37°C water bath by swirling the vial in the water bath until a small amount of ice remains. 5. Quickly wipe the vial with 70% ethanol and place in a sterile tissue culture hood. 6. Pipette each sample from the cryogenic freezing tube into a 15-mL conical centrifuge tube containing 100 ML of RPMI culture medium supplemented with DNase (prepared in step 2). Do not pipette cells up and down. 7. Mix the cells and medium by gently shaking the 15-mL conical centrifuge tube. 8. Slowly add 500 ML of RPMI culture medium supplemented with DNase while swirling the tube gently to mix the cells and medium together.

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9. In 1 min add double the amount (1 mL) of RPMI culture medium supplemented with DNase to the cell suspension in the15-mL conical centrifuge tube. 10. Continue adding double the amount of RPMI culture medium supplemented with DNase every minute until the cell suspension reaches a final volume of 10 mL. 11. Cap each conical tube and invert it five times to mix the cells; do not vortex cell suspension. 12. Centrifuge at 300 u g for 7 min at 25°C with brake ON. 13. Remove supernatant then resuspend cells in 10 mL of RPMI culture medium supplemented with DNase. 14. Cap each conical tube and invert it five times to mix the cells; do not vortex cell suspension. 15. Incubate cells at 37°C for 20 min by placing the 15-mL conical centrifuge tubes in a 37°C water bath. Invert tubes once 10 min into the incubation period. 16. After 20-min incubation, place cells on ice for 7 min. 17. Centrifuge cells at 300 u g for 7 min at 4°C with brake ON. 18. Carefully remove all supernatant and resuspend cells in 1 mL of RPMI culture medium supplemented with 5% FCS. 19. Place a cell strainer on top of a 50-mL conical centrifuge tube. Transfer the cell suspension from the 15-mL conical centrifuge tube to the 50-mL conical centrifuge through the cell strainer (see Note 11). 20. To count the number of live and dead cells, place 200 ML of 1u PBS, 37.5 ML of Trypan blue, and 12.5 ML of cell suspension into a falcon tube. Mix well and fill a hemacytometer with 10 ML of sample. Count and record the number of unstained (live) cells in the outer four quadrants of the hemocytometer. 21. Total number of cells = Number of live cells/4 × 10,000 × 20 (dilution factor [250/12.5]) u total volume of cells (1 mL or pooled total). 22. Adjust the cell concentration to 2 u 106 cells/mL by adding RPMI culture medium supplemented with 5% FCS. 3.7. Resting PBMCs in the Presence of IL-2

This step is only required if PBMCs were damaged or stressed before or during the isolation procedure, for example, if blood has to be shipped overnight before isolation of PBMCs. IL-2 has been shown to increase T-cell survival and proliferation as well as prevent apoptosis (17–19). By resting PBMCs overnight in 50 IU/mL, then recounting and plating for ELISPOT assay, T-cell viability is maintained, thus increasing the precision and reproducibility of the assay. Resting PBMCs in IL-2 is cost and labor intensive and only needs to be performed if the background (negative controls) is

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above the acceptable thresholds or if PBMCs have low-viability postthawing procedure. If PBMCs were isolated and frozen from a blood sample within 12 h, it is unnecessary to perform an IL-2 resting phase (20). 1. Add 2-mL of cell suspension from Subheading 3.7, step 22, into each well of a 24-well sterile tissue culture plate (final concentration 4 u 106 cells/well) (see Note 12). 2. Add 1 ML of IL-2 (concentration 1 × 105 IU/mL) per well such that the final concentration of IL-2 in each well is 50 IU/mL. 3. Incubate plate at 37°C in a 5% CO2 humidified incubator for 18 h. 4. After 18 h, remove medium from wells and pool all medium from one subject into a single 15-mL conical centrifuge tube. 5. Add 0.5 mL of prewarmed (see Note 13) 0.25% Trypsin– EDTA to each well. 6. Place plate back in a 37°C in a 5% CO2 humidified incubator until cells detach (approximately 10 min). Confirm detachment with a microscope. 7. Remove cells/trypsin suspension from each well and add to the corresponding 15-mL conical centrifuge tube which contains medium harvested from the same wells in step 4. 8. Add another 0.5 mL of prewarmed 0.25% Trypsin–EDTA to each well (see Note 14), and incubate plate for 10 min in a 37°C in a 5% CO2 humidified incubator. 9. Add 0.5 mL of RPMI culture medium supplemented with 5% FCS to each well and mix by pipetting up and down. Pool suspension from each well with the corresponding 15-mL conical centrifuge tube which contains cells harvested from that well in steps 4 and 7. 10. Bring the volume of each 15-mL conical centrifuge tube up to 10 mL by adding RPMI culture medium supplemented with 5% FCS. 11. Centrifuge cells at 300 × g for 7 min at 4°C with brake ON. 12. Remove supernatant without disturbing the cell pellet then resuspend cell pellet in 0.5 mL of RPMI culture medium supplemented with 5% FCS. Keep cells on ice once resuspended. 13. Count the number of live and dead cells by placing 200 ML of 1× PBS, 37.5 ML of Trypan blue, and 12.5 ML of cell suspension into a falcon tube. Mix well and fill a hemacytometer with 10 ML of sample. Count and record the number of unstained (live) cells in the outer four quadrants of the hemocytometer. 14. Total number of cells Number of live cells/4 u 10,000 u 20 (dilution factor [250/12.5]) × total volume of cells (0.5 mL).

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15. Adjust the cells to the desired concentration by adding RPMI culture medium supplemented with 5% FCS (see Note 15). 16. Keep cells on ice until they are ready to be plated (see Note 16). 3.8. Infecting PBMCs with Vaccinia Virus for IL-10 Secretion

The method below is used for a kit that does not come with precoated microplates. These microplates must be coated with capture antibody (purified antihuman IL-10) before use. The protocols below do not use HeLa cell lysate as a negative control; rather, culture medium was used as a negative control for ELISPOT assays. Using HeLa cell lysate as a negative control could be beneficial because only one variable is altered (absence of vaccinia virus in the culture). However, the presence of HeLa cell lysate could alter the cytokine secretion patterns of isolated PBMCs and as such it is not a true negative control. In addition, manufacturing and storing HeLa cell lysate costs personnel time, money, and freezer space; thus, using culture medium rather than HeLa cell lysate as a negative control is a scientifically as well as economically attractive option (see Note 17). 1. One day prior to plating PBMCs, coat the PVDF (polyvinylidene difluoride)-backed microplate with capture antibody (purified antihuman IL-10) by adding 100 ML of dilute capture antibody (1:200 in 1× sterile PBS) per well in a sterile culture hood (see Note 18). 2. Cover microplate with the lid and allow it to incubate overnight at 4°C. 3. Two hours before plating cells in the microplate, remove microplate from 4°C refrigerator and discard the contents of the microplate wells inside a sterile culture hood by flicking. Invert plate and blot dry on paper toweling. 4. Wash wells once with RPMI culture medium supplemented with 10% FCS. Discard the contents of the microplate wells inside a sterile culture hood by flicking then invert microplate and blot dry on paper toweling. 5. Block microplate by adding 200 ML/well of RPMI culture medium supplemented with 10% FCS to each well and incubate at room temperature for 2 h. 6. Remove and discard all medium from the microplate inside a sterile culture hood by flicking; invert microplate and blot dry on a paper toweling. 7. Remove vaccinia virus aliquots from −80°C freezer and thaw under a cold stream of water. 8. Dilute vaccinia virus stock aliquots to MOI = 0.05 with 4°C RPMI culture medium supplemented with 5% FCS. 9. Add 100 ML of cell suspension (adjusted to 1 × 106 cells/mL) to each well (final concentration 1 u 105 cells/well).

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10. Add 100 ML of RPMI culture medium supplemented with 5% FCS to columns 1–3 of each row (see Note 19). 11. Add 100 ML of vaccinia virus MOI 0.05 to columns 4–6 of each row. 12. Add 100 ML of PHA-P (concentration 5 Mg/mL) to column 7 of each row. 13. Wrap microplate with aluminum foil and incubate for exactly 24 h at 37°C in a 5% CO2 humidified incubator (see Note 20). 3.9. Infecting PBMCs with Vaccinia Virus for IFNg Secretion

The method below describes the detection and visualization of IFNG secreting lymphocytes (and specific lymphocyte subsets) using PVDF microplates that are precoated with human anti-IFNG capture Ab from the manufacturer. 1. Thirty minutes prior to plating, remove a microplate precoated with human anti-IFNG Ab from 4°C storage. 2. Block microplate by adding 200 ML of RPMI culture medium supplemented with 5% FCS per well and incubate at room temperature for 20 min. 3. Remove and discard medium from all wells inside a sterile culture hood by flicking; invert microplate and blot dry on paper toweling. 4. Remove vaccinia virus aliquots from −80°C freezer and thaw under a cold stream of water. 5. Dilute vaccinia virus stock aliquots to MOI = 5.0 with 4°C RPMI culture medium supplemented with 5% FCS. 6. Add 50 ML of cell suspension (for CD8+ IFNG [cell] 1 u 107, for total IFNG [cell] 4 u 106) to each well (final concentration CD8+ 5 u 105 cells/well; total IFNG = 2 u 105 cells/well). 7. Add 50 ML of RPMI culture medium supplemented with 5% FCS to columns 1–3 of each row (see Note 21). 8. Add 50 ML of vaccinia virus (MOI = 5.0) to columns 4–6 of each row. 9. Add 50 ML of PHA-P (concentration 5 Mg/mL) to column 7 of each row. 10. Cover microplate with aluminum foil and incubate for exactly 24 h for total IFNG or 6 h for CD8+ IFNG at 37°C in a 5% CO2 humidified incubator.

3.10. Detection of IL-10 Secreting Cells

1. After PVDF microplate has incubated 24 h, discard medium from the microplate into a sink by flicking. Invert microplate and blot dry on paper towels. 2. Wash the microplate two times with 200 ML/well of deionized (DI) water. Allow wells to soak for 3–5 min each wash.

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3. After DI wash, wash wells three times with 200 ML/well with wash buffer (1× PBS supplemented with 0.05% Tween 20), discarding wash buffer in the sink after each wash. 4. Dilute detection Ab (biotinylated antihuman IL-10) 1:250 (2 Mg/mL) in 1× PBS supplemented with 10% FCS (see Note 22). 5. Add 100 ML of detection Ab suspension per well and incubate with lid covering the microplate for 2 h at room temperature. 6. After 2-h incubation, flick Ab suspension into a sink, invert microplate, and blot dry on paper towels. 7. Wash wells three times with 200 ML/well with wash buffer (1u PBS supplemented with 0.05% Tween 20). Allow wells to soak 1–2 min each wash. 8. Add 100 ML per well of dilute enzyme conjugate [(StreptavidinHRP) 1:100 in 1× PBS containing 10% FCS] (see Note 23). 9. Cover microplate and incubate it for 1 h at room temperature. 10. After 1 h of incubation, discard dilute enzyme conjugate into the sink by flicking. 11. Wash wells four times with 200 ML/well of wash buffer (1u PBS supplemented with 0.05% Tween 20). Allow wells to soak 1–2 min each wash. 12. Wash wells two times with 200 ML/well of 1u PBS. After final wash invert microplate and blot dry on paper toweling. 13. Add 100 ML of prewarmed to room temperature 3,3c,5,5c-tetramethylbenzidine (TMB) to each well and incubate microplates in the dark for 30 min (see Note 24). 14. Discard TMB into the sink by flicking. 15. Remove plastic backing from the microplate and discard. Stop substrate reaction by rinsing both the back and front of the microplate in DI water three times. 16. Invert microplate and blot dry on paper toweling; wipe the bottom of the microplate dry with paper toweling. 17. Allow microplate to air-dry overnight. 18. Once the microplate is completely dry, the spots per well can be counted using an automated ELISPOT reader such as an ImmunoSpot® reader from Cellular Technology Ltd. (CTL, Shaker Heights, OH) or manually using a stereomicroscope. 3.11. Detection of IFNg Secreting Cells

1. After PVDF microplate has incubated 24 h, discard medium from the microplate into a sink by flicking. Invert microplate and blot dry on paper towels. 2. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot until dry. To make wash buffer, add 50 mL of wash buffer concentrate (provided in the kit) to 450 mL of H2O.

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3. Immediately prior to use, prepare detection antibody (biotinylated antihuman IFNG) by mixing 100 ML of detection Ab concentrate with Dilution Buffer 1. Mix thoroughly, then add 100 ML of Ab suspension per well. 4. Incubate microplate overnight at 4°C. 5. The following day, discard detection Ab suspension from the microplate into a sink by flicking; invert microplate and blot dry. 6. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot dry. 7. Directly prior to use, prepare Streptavidin-AP by adding 100 ML of Streptavidin-AP concentrate A to bottle of Dilution Buffer 2. Mix thoroughly, then add 100 ML of Streptavidin-AP suspension per well. 8. Incubate microplate for 2 h at room temperature. 9. Prewarm BCIP/NBT Chromogen substrate to room temperature using a 37°C water bath. 10. After the microplate has incubated for 2 h, discard Streptavidin-AP solution from wells into a sink by flicking. 11. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot dry. 12. Add 100 ML of BCIP/NBT Chromogen substrate per well. 13. Incubate microplate in the dark for 30 min at room temperature. 14. After the microplate has incubated for 30 min in the dark, discard BCIP/NBT Chromogen substrate from wells into a sink by flicking; invert microplate and blot dry. 15. Remove plastic backing from the microplate and discard. Stop substrate reaction by rinsing both the back and front of the microplate in DI water three times. 16. Invert microplate and blot dry on paper toweling; wipe the bottom of the microplate dry with paper toweling. 17. Allow microplate to air-dry overnight. 18. Once the microplate is completely dried, the spots per well can be counted using an automated ELISPOT reader such as an ImmunoSpot® reader from CTL or manually using a stereomicroscope. 3.12. Detection of IFNg CD8+ Secreting T Cells

1. After the microplate has incubated for 6 h in 37°C in a 5% CO2 humidified incubator, discard the contents of the microplate wells into a container containing bleach within a sterile environment by flicking. Invert microplate and blot dry on paper toweling. 2. Remove any unbound cells by washing microplate with 250 ML of 1× sterile PBS three times. After each wash, invert microplate and blot dry.

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3. After three washes, add 100 ML of RPMI culture medium supplemented with 5% FCS to each well. 4. Wrap microplate in aluminum foil and incubate microplate for 18 additional hours (24 h total) in a 5% CO2 humidified incubator. 5. After the microplate has incubated for 18 h, discard medium from wells into a sink by flicking. 6. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot dry. To make wash buffer, add 50 mL of wash buffer concentrate (provided in the kit) to 450 mL of H2O. 7. Immediately prior to use, prepare detection antibody (biotinylated antihuman IFNG) by mixing 100 ML of detection Ab concentrate with Dilution Buffer 1. Mix thoroughly, then add 100 ML of Ab suspension per well. 8. Incubate microplate overnight at 4°C. 9. The following day, discard the content of the microplate wells into a sink by flicking; invert microplate and blot dry. 10. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot dry. 11. Directly prior to use, prepare Streptavidin-AP by adding 100 ML of Streptavidin-AP concentrate A to bottle of Dilution Buffer 2. Mix thoroughly, then add 100 ML of Streptavidin-AP suspension per well. 12. Incubate microplate for 2 h at room temperature. 13. Prewarm BCIP/NBT Chromogen substrate to room temperature using a 37°C water bath. 14. After the microplate has incubated for 2 h, discard Streptavidin-AP solution from wells into a sink by flicking. 15. Wash microplate four times using wash buffer provided with the kit. After each wash, invert microplate and blot dry. 16. Add 100 ML of BCIP/NBT Chromogen substrate per well. 17. Incubate microplate in the dark for 30 min at room temperature. 18. After the microplate has incubated for 30 min in the dark, discard BCIP/NBT Chromogen substrate from wells into a sink by flicking; invert microplate and blot dry. 19. Remove plastic backing from the microplate and discard. Stop substrate reaction by rinsing both the back and front of the microplate in DI water three times. 20. Invert microplate and blot dry on paper toweling; wipe the bottom of the microplate dry with paper toweling. 21. Allow microplate to air-dry overnight.

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22. Once the microplate is completely dry, the spots per well can be counted using an automated ELISPOT reader such as an ImmunoSpot® reader from CTL or manually using a stereomicroscope.

4. Notes 1. The tissue culture medium contains 103–10 4 pfu/mL of vaccinia virus and should be disposed properly in a hazardous waste container. 2. Vaccinia virus is stable at 4°C for several months. 3. Vero cells used for this assay should not be passaged more than 14 times before performing this assay. 4. Normally, one whole plate can be inoculated without the risk of cells drying out. Minimize cell drying by using the plate lid to cover the plate whenever possible and occasionally shake the plate vigorously to spread the residual medium equally across the wells. 5. Dispose medium as infectious waste. 6. Generally, acceptable vaccinia virus stock has a titer of >108 pfu/mL. 7. For optimal separation, do not add more than 20 mL of whole blood into the Accuspin™ tube. 8. At this point if you have multiple tubes for one subject, then they should be pooled into one 50-mL conical centrifuge tube before cell counting. 9. Required volume (mL) freezing medium 1 u 107/total number of cells. 10. When transferring cells keep on dry ice to prevent thawing. 11. At this point if you have multiple tubes for one subject, then they should be pooled into one 50-mL conical centrifuge tube before cell counting. 12. If any cells are remain after the 2 mL/well addition, then add remaining cells to a new well and bring the volume of the well up to 2 mL with RPMI culture medium supplemented with 5% FCS. 13. Warm 0.25% Trypsin–EDTA in a 37°C water bath for a minimum of 15 min prior to use. 14. Trypsinize wells twice to maximize recovery of PBMCs. 15. The desired concentration of PBMCs is dependent on the type of ELISPOT assay being performed. The required PBMC concentration by ELISPOT assays are as follows: IL-10

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ELISPOT = 1 × 106 cells/mL, IFNG ELISPOT = 4 × 106 cells/mL, and CD8+ IFNG ELISPOT = 1 × 107 cells/mL. 16. To minimize cell death, cells should be plated within 30 min after they have been counted. 17. HeLa cell lysate is manufactured by identical methods of infecting HeLa cells and harvesting HeLa cells (Subheadings 3.1 and 3.2, respectively) except cell cultures are not inoculated with vaccinia virus. 18. For one microplate, using BD Pharmingen IL-10 ELISPOT kit cat. no. 551018, add 50 ML of purified antihuman IL-10 Ab to 9.95 mL of sterile 1× PBS. 19. Using the microplate vertically will allow for 12 subjects to be tested on one microplate in triplicate as well as a positive control. 20. Wrapping microplate in aluminum foil during incubation reduces well-to-well variability (21). 21. See Note 19. 22. For one microplate, add 40 ML of detection Ab stock to 9.96 mL of 1× PBS supplemented with 10% FCS. 23. For one microplate, add 100 ML of Streptavidin-HRP to 9.9 mL of PBS containing 10% FCS. 24. If the background hue interferes with detecting spots, then decrease the development time.

Acknowledgments We would like to thank the entire Mayo Clinic Vaccine Research Group for their invaluable technical assistance and discussion during the development and execution of these assays. This work was supported by NIH contract AI40065. References 1. Kennedy, R.B., Ovsyannikova, I.G., Jacobson, R.M., and Poland, G.A. (2009)The immunology of smallpox vaccines. Curr Opin Immunol 21, 314–320. 2. Weltzin, R,. Liu, J., Pugachev, K.V., Myers, G.A., Coughlin, B., Blum, P.S., et al. (2003) Clonal vaccinia virus grown in cell culture as a new smallpox vaccine. Nat Med 9, 1125–1130. 3. Poland, G.A., Grabenstein, J.D., Neff, J.M. (2005) The US smallpox vaccination program: a review of a large modern era smallpox

vaccination implementation program. Vaccine 23, 2078–2081. 4. Damon, I.K., Davidson, W.B., Hughes, C.M., Olson, V.A., Smith, S.K., Holman, R.C., et al. (2009) Evaluation of smallpox vaccines using variola neutralization. J Gen Virol 90, 1962–1966. 5. Kennedy, R.B., Pankratz, V.S., Swanson, E., Watson, D., Golding, H., and Poland, G.A. (2009) Statistical approach to estimate vaccinia- specific neutralizing antibody titers using a high throughput assay. Clin Vaccine Immunol 16, 1105–1112.

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6. Frey, S.E., Newman, F.K., Cruz, J., et al. (2002) Dose-related effects of smallpox vaccine. N Engl J Med 346, 1275–80. 7. Kim, S.H., Yeo, S.G., Cho, J.H., et al. (2006) Cell-mediated immune responses to smallpox vaccination. Clin Vaccine Immunol 13, 1172–1174. 8. Helms, T., Boehm, B.O., Asaad, R.J., Trezza, R.P., Lehmann, P.V., and Tary-Lehmann, M. (2000) Direct visualization of cytokineproducing recall antigen-specific CD4 memory T cells in healthy individuals and HIV patients. J Immunol 164, 3723–3732. 9. Czerkinsky, C., Andersson, G., Ekre, H.P., Nilsson, L.A., Klareskog, L., and Ouchterlony, O. (1988) Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. J Immunol Methods 110, 29–36. 10. Ryan, J.E., Dhiman, N., Ovsyannikova, I.G., Vierkant, R.A., Pankratz, V.S., and Poland, G.A. (2009) Response surface methodology to determine optimal cytokine responses in human peripheral blood mononuclear cells after smallpox vaccination. J Immunol Methods 341, 97–105. 11. Earl, P.L., Moss, B., Wyatt, L.S., and Carroll, M.W. (2001) Generation of recombinant vaccinia viruses. Curr Protoc Mol Biol Chapter 16, Unit 16. 12. Earl, P.L., Cooper, N., Wyatt, L.S., Moss, B., and Carroll, M.W. (2001) Preparation of cell cultures and vaccinia virus stocks. Curr Protoc Mol Biol Chapter 16, Unit 16. 13. Newman, F.K., Frey, S.E., Blevins, T.P., Mandava, M., Bonifacio, A. Jr., Yan, L., et al. (2003) Improved assay to detect neutralizing antibody following vaccination with diluted or undiluted vaccinia (Dryvax) vaccine. J Clin Microbiol 41, 3154–3157. 14. Tsung, K., Yim, J.H., Marti, W., Buller, R.M., and Norton, J.A. (1996) Gene expression and

15.

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cytopathic effect of vaccinia virus inactivated by psoralen and long-wave UV light. J Virol 70, 165–171. Kreher, C.R., Dittrich, M.T., Guerkov, R., Boehm, B.O., and Tary-Lehmann, M. (2003) CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays. J Immunol Methods 2003 278, 79–93. Smith, J.G., Liu, X., Kaufhold, R.M., Clair, J., and Caulfield, M.J. (2001) Development and validation of a gamma interferon ELISPOT assay for quantitation of cellular immune responses to varicella-zoster virus. Clin Diagn Lab Immunol 8, 871–879. Salomoni, P., Perrotti, D., Martinez, R., Franceschi, C., and Calabretta, B. (1997) Resistance to apoptosis in CTLL-2 cells constitutively expressing c-Myb is associated with induction of BCL-2 expression and Mybdependent regulation of bcl-2 promoter activity. Proc Natl Acad Sci U S A 94, 3296–3301. Stern, J.B., and Smith, K.A. (1986) Interleukin-2 induction of T-cell G1 progression and c-myb expression. Science 233, 203–206. Letourneau, S., Krieg, C., Pantaleo, G., and Boyman, O. (2009) IL-2- and CD25dependent immunoregulatory mechanisms in the homeostasis of T-cell subsets. J Allergy Clin Immunol 123, 758–762. Kierstead, L.S., Dubey, S., Meyer, B., Tobery, T.W., Mogg, R., Fernandez, V.R., et al. (2007) Enhanced rates and magnitude of immune responses detected against an HIV vaccine: effect of using an optimized process for isolating PBMC. AIDS Res Hum Retroviruses 23, 86–92. Kalyuzhny, A., and Stark, S. (2001) A simple method to reduce the background and improve well-to-well reproducibility of staining in ELISPOT assays. J Immunol Methods 257, 93–97.

Chapter 17 ELISPOT Assays to Enumerate Bovine IFN-g-Secreting Cells for the Development of Novel Vaccines Against Bovine Tuberculosis Martin Vordermeier and Adam O. Whelan Abstract Enumeration of antigen-specific cells after vaccination is one of the prime immunological parameters determined when developing vaccines. Due to their exquisite sensitivity (limits of detection can be below 1/100,000 cells), ELISPOT assays are therefore an important tool in vaccine development programs. This is particularly the case for vaccines against diseases that require protective cell-mediated immunity, such as tuberculosis. This chapter describes ELISPOT assays detecting bovine IFN-G. Key words: ELISPOT, Cytokines, IFN-G, Effector and memory T-cell responses, Cultured ELISPOT

1. Introduction We are engaged in developing novel vaccines against bovine tuberculosis which is caused by infection with Mycobacterium bovis. In this disease, Th1 responses, and in particular IFN-G production, are considered to be major contributors to protective immunity (1). Therefore, this chapter describes ELISPOT assays detecting bovine IFN-G (2–4). An example of ELISPOT results observed in cattle after subunit vaccination with a mycobacterial antigen, delivered as DNA vaccine or as pool of synthetic peptides, is shown in Fig. 1 (4). The same assay principles apply to the detection of other cytokines or chemokines. However, due to the lack of bovinespecific reagents, important cytokines assessed in human systems, like interleukin-2 (IL-2), cannot be probed by ELISPOT in cattle at present. Cytokine ELISPOT assays in their basic form detect


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Fig. 1. Example of ex vivo ELISPOT assays used to assess vaccine-induced cellular immunity: in vitro cellular immune responses after vaccination. PBMCs were stimulated in vitro peptide pool containing the complete set of 14 overlapping peptides covering the Rv3019c sequence. (a) IFN responses measured by ELISPOT and expressed as mean SFC/106 PBMC ± S.E.M. per group (n = 8 calves/group). (b) Proliferative responses expressed as mean stimulation index (SI) (cpm with peptides/cpm of medium control) ± S.E.M per group (n = 8 calves/group). Symbols: circles, peptide vaccination; diamonds, DNA/peptide vaccination; triangles, DNA vaccination; squares, saline control group. *p < 0.05; **p < 0.003. Arrows indicate time of vaccinations. From ref. 4.

Fig. 2. Flow chart of ex vivo and cultured ELISPOT assay.

effector T-cell responses since assays are initiated directly using ex vivo peripheral blood mononuclear cells (PBMCs), see Fig. 1. However, assessment of memory responses is also a vital parameter to be assessed following vaccination. This can also be addressed using a modification of the ex vivo ELISPOT, the so-called cultured ELISPOT system (Fig. 2). The principle of the cultured ELISPOT is that the IFN-G ELISPOT is not performed on ex vivo PBMC, but on cells that have been expanded and differentiated in vivo for around 2 weeks by initial stimulation with antigen and then regular feeds of IL-2. The majority of effector cells that are initially stimulated by this approach die of IFN-G-induced cell

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Fig. 3. Example of cultured ELISPOT application: Correlation of memory responses with protection. Memory cell responses were determined by cultured IFN-G ELISPOT assay. Results of ELISPOT analysis are expressed as mean spot-forming cells (SFCs)/million cells. Cultured ELISPOT assays were performed before Mycobacterium bovis infection 14 weeks post-BCG vaccination and compared to outcome of infection with M. bovis at week 28 post-vaccination (animals were challenged with M. bovis at week 14 postvaccination). Protection has been determined by bacterial load (log CFU/g tissue). Shown is the correlation of mean cultured ELISPOT responses and mean bacterial loads using data from unvaccinated cattle as well as cattle vaccinated with three different vaccines that induced various degrees of protection (from ref. 2).

apoptosis during this culture and expansion step, whereas memory cells differentiate into secondary effector cells that can be enumerated at the end of this culture step by IFN-G ELISPOT. This assay was first applied in the human system and applied to diseases, such as malaria (5, 6). A study by Godkin et al. (7) has demonstrated that the CD4+ T-cell memory population probed with this assays consisted mainly of central memory cells expressing the chemokine receptor CCR7. We have adapted this system for use in cattle (8– 11), and its protocol is also described in this chapter. Interestingly, in our hands, cultured ELISPOT responses were a direct predictor of vaccine efficacy against M. bovis challenge in cattle (10, 11), as illustrated in Fig. 3.

2. Materials 2.1. Cell Culture

1. Nonessential amino acids (Sigma–Aldrich, Poole, UK). 2. Penicillin–streptomycin solution (penicillin 10,000 U/mL, streptomycin 10 Mg/mL). 3. Tissue culture medium (TCM): Add to 500 mL RPMI1640 (with Glutamax-1 and 25 mM HEPES buffer): 50 mL heat-activated fetal bovine serum (FBS), 5 mL penicillin– streptomycin solution, 5 mL nonessential amino acids, 0.5 mL 2-mercaptoethanol (from 50 mM stock solution).

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4. Human IL-2 (Sigma–Aldrich) (see Note 1). 5. Antigens as required for system: Antigens can be recombinant proteins or synthetic peptides used in our hands generally at 2–5 Mg/mL diluted in TCM (recombinant proteins) or 5–10 Mg/mL (synthetic peptides) (see Note 2). 6. Positive controls: Staphylococcal enterotoxin B (SEB) diluted to 2 Mg/mL in TCM (see Note 3). 7. Hank’s balanced salt solution (HBSS). 8. 24-well tissue culture plates. 2.2. ELISPOT

1. Coating buffer: 0.05 M carbonate/bicarbonate buffer, pH 9.6, to be prepared using buffer tablets. Stored at −20°C, filter sterilize before use through 0.2-Mm filter. 2. MABTECH Bovine/Ovine/Equine IFN-G ELISPOT kit (ALP) which includes the anti-bovine IFN-G-coating monoclonal antibodies (mAbs) bIFNG-I, biotinylated PAN IFN-G detection mAb (PAN-biotin), and Streptavidin–Horseradish Peroxidase (MABTECH, Stockholm, Sweden, www.mabtech. com: Product Code: 3115-2H) (see Note 4). 3. PBS-Tween (PBST); 0.5 mL Tween 20 to 1 L of PBS. 4. PBST/bovine serum albumin (BSA); 100 mg BSA to 1 L PBST, filter sterilize through 0.2-Mm filter. 5. 3-Amino-9-ethylcarbazole (AEC) staining kit (Sigma–Aldrich) (see Note 5).

2.3. Equipment

1. ELISPOT plates, Millipore MAIPS4510 (Millipore, Watford, UK) (see Note 6). 2. Humidified CO2 incubator. 3. Vortex mixer. 4. Orbital plate shaker. 5. Cell centrifuge. 6. ELISPOT plate reader (e.g. from AID GmbH, Strassberg, Germany; http://www.aid-diagnostika.com).

3. Method 3.1. ELISPOT Plate Preparation

1. Coat ELISPOT plates with 100 Ml/well capture antibody (bIFNG-1, at 7.5 Mg/mL) diluted in coating buffer. Wrap plates in cling film and store in refrigerator; plates can be kept up to 3 days in refrigerator until use (see Note 7).

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Note: Steps 1–4 need to be performed in class 2 biosafety cabinet. 1. Discard coating antibody solution and wash plates twice with 200 Ml/well RPMI-1640 using a multi-channel pipette to add wash fluid. Flick off fluid between and after washes. Then, block wells for 45–60 min with 200 Ml/well of TCM at 37°C. 2. Flick off blocking solution and add 100 Ml/well antigens in duplicate or triplicate diluted in TCM according to requirements of experiment (see Note 2). 3. Add 100 Ml/well of PBMC suspended in TCM at 1–4 × 106/mL. Incubate for 20–24 h at 37°C and 5% CO2. Ensure that plates are level so that cells are evenly distributed (see Note 8). 4. After this incubation period, shake plates on plate shaker (400– 500 rpm, 5–10 s) and flick off cells into container in a biosafety class 2 cabinet. Wash plates twice with H2Odd and then three times with PBST by adding 200 Ml/well wash fluids using multi-channel pipette. Place on shaker each time for 10 s before flicking off wash fluid. Remove as much wash fluid as possible by vigorously tapping plate on paper towels. 5. Add 100 Ml/well detection mAb (PAN-biotin, 0.25 Mg/mL, diluted in PBST–BSA) and incubate for 2 h at room temperature (above 20°C). 6. Wash three to four times with PBST as in step 4. Remove as much wash fluid as possible by tapping plate on paper towels. 7. Add 100 Ml/well streptavidin–peroxidase conjugate (diluted 1:100 in PBST/BSA) and incubate for 1 h at room temperature. 8. Wash plates six times with PBST and then add 100 Ml/well AEC substrate prepared as per kit instructions (see Note 5). 9. Once spots have developed (ca. 10 min, but this should be closely monitored to avoid over-development), flick off substrate and wash with copious amounts of tap water (under tap will do), remove plastic cover, and wash back of wells. Allow plates to dry either in air or using a drying oven set at 30°C. 10. Keep plates in dark before counting using an automated ELISPOT reader. Plates can also be read manually using a dissecting microscope. 11. Result presentation: Conventionally, responses are expressed as mean spot-forming cell (SFC)/106 cells of duplicate or triplicate wells.

3.3. Cultured ELISPOT

1. PBMCs are stimulated in 24-well plates (2 × 106 PBMC/mL, 1-mL aliquot in TCM) with antigens (e.g. recombinant proteins at 2–10 Mg/mL, see Note 2). PBMC should preferentially be

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freshly prepared, but can also be prepared from cryo-preserved stocks. Incubate in 37°C humidified CO2 incubator. Set up at least two wells per cell line (see Note 9). 2. PBMC cultures to be fed on days 3 and 7 with recombinant human IL-2 to a final concentration of 10 U/mL in the following way; on day 3, add 0.5 mL of IL-2 (30 U/mL in TCM) directly to each 1 mL culture; on day 7, carefully remove and discard 0.5 mL of culture supernatant without disturbing cell layer at bottom of wells and replace with 0.5 mL of IL-2 (30 U/mL in TCM) (see Note 10). 3. On day 10, carefully remove half of the supernatant and replace with equal volume of TCM without IL-2. Do not disturb cell layer. 4. On day 12, remove 1–1.25 mL medium from each well, and replace with same volume of fresh TCM without IL-2. 5. The day before the assay (day 12), prepare ELISPOT plates by coating them overnight at 4°C with a bovine IFN-G-specific mAb ELISPOT as described above in Subheading 3.1. 6. On the day of assay (day 13), prepare fresh PBMC to be used as antigen-presenting cells (APCs) and re-suspend PBMC at 2–4 × 106/mL in TCM. 7. To the pre-blocked ELISPOT plates (see step 1 in Subheading 3.2 above), add APC (2 × 105 PBMC/well) to required wells (see Note 11). 8. Incubate for 90–120 min at 37°C. 9. In the meantime, warm up TCM aliquot to 37°C. 10. After the 90–120-min incubation step (step 8), shake up plates vigorously (plate shaker) and flick off cells into container in cabinet. 11. Wash plates with warm TCM by addition of 200 Ml TCM/ well, shake plates on orbital plate shaker (100–200 rpm, 10 s), and discard medium in container in cabinet. Repeat this step twice more. 12. After last wash, add antigen solutions in TCM in 100 Ml/well in duplicate or triplicate and return plates to 37°C humidified CO2 incubator. Add 100 Ml TCM to “no antigen” control wells and add mitogen solution to positive control wells. Antigens can be recombinant proteins (assay concentrations 2–5 Mg/mL) or synthetic peptides (assay concentrations 2–10 Mg/mL) (see Note 2); positive control can be SEB (1–2 Mg/mL) or other mitogens (see Note 3). 13. Pool cells from 24-well T-cell line culture plates and wash cultured cells four times with HBSS solution by centrifugation (300 × g, room temperature), count viable cells, and re-suspend at required cell titres (see next step).

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14. Add between 5 × 103 and 2 × 104 cells to wells of ELISPOT plates containing the antigen solutions and APCs (see Note 11). 15. After 24 h of incubation in the presence of cells, develop spots as described for ex vivo ELISPOT (Subheading 3.2, steps 4–10). 16. Tabulation of results and interpretation criteria: There are a number of ways to tabulate the results. We advise to normalize results obtained (SFC/well) to the number of input cells (from step 1) by using the following formula: SFC/106 input cells = [S × (N/E)]/IR (see Note 12). Input cell ratio (IR): Number of input cells (step 1) divided by 106; N: output cell number (total viable cells counted at step 13); E: number of cells/well during ELISPOT (step 14); S: SFC/well (step 15).

4. Notes 1. Human IL-2 works effectivly to expand bovine T cells and is easier to source than bovine IL-2. 2. The protein and peptide concentrations given should be viewed as guidelines based on our experiences with the mycobacterial antigens. However, antigen concentrations should always be optimized with the antigens to be investigated and in the system they are applied to. Please also note that the concentrations given are final assay concentrations and that the actual antigen solutions to be plated out should be of twice the final assay concentrations. 3. Other positive control stimuli can also be used, such as the mitogens Pokeweed mitogen (PWM) or phytohaemagglutinin (PHA). Both mitogens can be used at 5 Mg/mL final assay concentration. 4. Alternative bovine IFN-G mAb sources can also be used (e.g. see: AbD Serotec, http://www.abdserotec.com). 5. Tetramethylbenzidine (TMB, Sigma–Alrdrich) is an alternative substrate and can also be used with horseradish peroxidasebased conjugates. Alternatively, it is possible to substitute horseradish peroxidase conjugates with alkaline phosphatase-based reagents to be used in combination with substrates, such as Fast Red or 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT, Sigma–Aldrich). We have had excellent results using BCIP/NBT which appears to provide not only higher sensitivity, but also increased background staining. 6. Alternative sources of plates suitable for ELISPOT applications are available, although their use would require careful

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optimization. Our experience has found that polyvinylidene fluoride (PVDF)-based membranes provided improved spot formation compared with nitrocellulose or nylon membranes and that Millipore MAIPS4510 plates were found to be optimal for our application. 7. Pre-wetting of plates: Some sources recommend pre-wetting of membranes with 35–70% ethanol prior to mAb coating (http://www.mabtech.com). In our hands, this is not necessary. However, should a pre-wetting step be included, it is imperative to remove the ethanol by repeated washes of membranes with coating buffer or distilled water prior to the coating step. The volume of ethanol used and the incubation period need to be optimized depending on the source of ELISPOT plates used. 8. These cell titres are for guidance only and have to be optimized for respective application and experiment. Instead of PBMC, more defined T-cell populations can also be probed, although this requires the addition of APCs, such as magnetically sorted CD14+ monocytes, dendritic cells, or adherent cell populations, as described for the cultured ELISPOT assay. APCs also need to be titrated to optimize their concentration for the antigens and experimental systems under investigation. To ensure equal temperature distribution during the incubation step, and hence improved uniformity of spot quality, ELISPOT plates can be wrapped in tin foil. They should also not be stacked up in the incubator. Further, it is best avoided to place them in an incubator that is subject to vibrations, such as being sited on the same bench as a large centrifuge, since the cells’ agitation and movement could result in enlarged and/or diffuse spots. 9. In addition to PBMC, sorted cell populations can also be used, such as CD4+ T cells or different memory cell populations. These cultures need to be supplemented with APC, such as CD14+ monocytes. 10. Alternatively, lines can be fed with IL-2 at days 5 and 8. 11. When peptides are used as antigens, it is also possible to perform these assays without addition of APC. This not only reduces the number of spot visualized in the medium control wells, but also reduces the overall number of peptide-specific SFCs. When not using APCs, we recommend that at least 2 × 104 cultured cells/well are used during the ELISPOT. 12. Worked example: (a) Input cells: 4 × 106 PBMC (step 1); therefore, IR = 4 (b) Recovered cells at the end of culture step (step 13): N = 106 viable cells

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(c) Cell concentration used in ELISPOT (step 14): E = 104 cells/well (d) ELISPOT (step 15): S = 50 SFC/well SFC/106 input cells = (50 × (106/104))/4 = 1,250.

Acknowledgements The authors’ work was supported by the Department for Environment, Food and Rural Affairs, the UK. References 1. Vordermeier, H.M., Chambers, M.A., Buddle, B.M., Pollock, J.M. and Hewinson, R.G. (2006) Progress in the development of vaccines and diagnostic reagents to control tuberculosis in cattle. Vet J 171, 229–244. 2. Vordermeier, H.M., Chambers, M.A., Cockle, P.J., Whelan, A.O., Simmons, J., and Hewinson, R.G. (2002) Correlation of ESAT6-specific gamma interferon production with pathology in cattle following Mycobacterium bovis BCG vaccination against experimental bovine tuberculosis. Infect Immun 70, 3026–3032. 3. Vordermeier, H.M., Rhodes, S.G., Dean, G., Goonetilleke, N., Huygen, K., Hill, A.V., et al. (2004) Cellular immune responses induced in cattle by heterologous prime-boost vaccination using recombinant viruses and bacille CalmetteGuerin. Immunology 112, 461–470. 4. Vordermeier H.M., Pontarollo R., Karvonen B., Cockle P., Hecker R., Singh, M., et al. (2005) Synthetic peptide vaccination in cattle: induction of strong cellular immune responses against peptides derived from the Mycobacterium bovis antigen Rv3019c. Vaccine 23, 4375–4384. 5. Keating S.M., Bejon P., Berthoud T., Vuola J.M., Todryk S., Webster, D.P., et al. (2005) Durable human memory T cells quantifiable by cultured enzyme-linked immunospot assays are induced by heterologous prime boost immunization and correlate with protection against malaria. J Immunol 175, 5675–5680.

6. Todryk S.M., Bejon P., Mwangi T., Plebanski M., Urban B., Marsh, K., et al. (2008) Correlation of memory T cell responses against TRAP with protection from clinical malaria, and CD4 CD25 high T cells with susceptibility in Kenyans. PLoS ONE 3, e2027. 7. Godkin, A.J., Thomas, H.C. and Openshaw, P.J. (2002) Evolution of epitope-specific memory CD4(+) T cells after clearance of hepatitis C virus. J Immunol 169, 2210–2214. 8. Vordermeier H.M., Dean G.S., Rosenkrands I., Agger E.M., Andersen P., Kaveh, D.A., et al. (2009) Adjuvants induce distinct immunological phenotypes in a bovine tuberculosis vaccine model. Clin Vaccine Immunol 16, 1443–1448. 9. Vordermeier, H.M., Huygen, K., Singh, M., Hewinson, R.G. and Xing, Z. (2006) Immune responses induced in cattle by vaccination with a recombinant adenovirus expressing Mycobacterial antigen 85A and Mycobacterium bovis BCG. Infect Immun 74, 1416–1418. 10. Vordermeier H.M., Villarreal-Ramos B., Cockle P.J., McAulay M., Rhodes S.G., Thacker, T., et al. (2009) Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis. Infect Immun 77, 3364–3373. 11. Waters W.R., Palmer M.V., Nonnecke B.J., Thacker T.C., Scherer C.F., Estes, D.M., et al. (2009) Efficacy and immunogenicity of Mycobacterium bovis DeltaRD1 against aerosol M. bovis infection in neonatal calves. Vaccine 27, 1201–1209.

Chapter 18 IL-7 Addition Increases Spot Size and Number as Measured by T-SPOT.TB ® Marsha L. Feske, Miguel Medina, Edward A. Graviss, and Dorothy E. Lewis Abstract The interferon-gamma (IFN-J) release assay (IGRA) is an in vitro extension of the century-old in vivo tuberculin skin test, better known as the TST. Shortcomings to the TST are multifactorial and include limitations in sensitivity and specificity. IGRAs improve diagnostic specificity by using antigens not found in the Bacille Calmette-Guérin, a vaccine given in most countries. IGRAs capture the IFN-J produced by T cells in response to antigen stimulation. The ELISPOT immediately captures IFN-J produced directly from each cell, resulting in the generation of a cellular “footprint.” The dimensions and intensity of the generated footprint indicate the avidity of the secreting cell. We show a further improvement in IGRAs by addition of interleukin-7 (IL-7). IL-7 reduces T-cell apoptosis and stabilizes IFN-J message. In addition to increasing the number of spots in the ELISPOT T-SPOT.TB platform, IL-7 increased IFN-J production per cell as measured by an increase in spot size with no change in spot distribution. Key words: Interferon-gamma, Tuberculosis, Interleukin-7, T-SPOT.TB, IGRA, ELISPOT, TB, IFN-J, IL-7

1. Introduction The interferon-gamma (IFN-J) release assay (IGRA) is an in vitro extension of the century-old in vivo tuberculin skin test, better known as the TST. The TST was one of the first diagnostics (1) and is still used today. The need for a diagnostic replacement for the TST had been long recognized, but awaited scientific advancement. Shortcomings include limitations in specificity (2, 3) and sensitivity (4–6). Reduced specificity correlates with the use of the Bacille Calmette-Guérin (BCG) (7), a vaccine given in most countries.


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TST sensitivity is reduced in persons with immunosuppression, a characteristic that has become more pronounced with the worldwide HIV epidemic (8, 9). IGRAs employ scientific achievements in assay development and cytokine detection using either the ELISA or ELISPOT platform to capture IFN-J produced in response to antigen stimulation within 16–24 h. The first IGRA prototypes, like the TST, relied on cell-mediated responses to purified protein derivative (PPD), a purified fraction of Mycobacterium tuberculosis (MTB) proteins (5). Sequencing of the MTB genome revealed a region of difference (RD-1) that was not present in BCG (10). This finding led to the replacement of the IGRA PPD peptide pool with two specific RD-1 MTB peptides (ESAT-6 and CFP10) and recently TB 7.7 (11). The use of RD-1 peptides increased predictive values of IGRAs and further increased their viability as diagnostics (4, 12, 13). IGRAs also offer improved diagnostic feasibility compared to the TST because there is no requirement for a follow-up visit reducing the cost for the provider, and barriers to care, such as transportation, scheduling, and cost for the subject. The ELISPOT platform requires isolation of peripheral blood mononuclear cells (PBMCs) from whole blood, a step that requires more time and training. The isolation of PBMCs, however, is essential to both the specificity and sensitivity of the assay and serves a dual purpose. First, it ensures that the same number of PBMCs are stimulated, allowing the assay to be less dependent on the subjects’ T-cell number or underlying T-cell repertoire. Secondly, isolation of PBMCs allows spot visualization of individual effector cells. Spot size and density can be used to define the measured kinetics of the secreting cell. This function may prove to be valuable as the specificity of spot readers improves or as scientific knowledge concerning TB immunology advances (14). Most effector memory T cells secrete their products within 4–6 h of antigen presentation (15). In the ELISA assay, the secreted IFN-J may be diluted, degraded, or utilized in proportion to the timing of the antigen-presenting cell (APC) presentation and the stop point in the assay. The ELISPOT immediately captures IFN-J produced directly from each cell, resulting in the generation of a cellular “footprint” and the reduction of dilution. For a T cell, this footprint has a characteristic dark center with a halo effect that is the visual result of the degradation of the initial IFN-J produced upon activation (16). The dimensions and intensity of the generated footprint are indicators of the avidity of the secreting cell (17). ELISA-based QuantiFERON-TB Gold® (QFT-G) and QuantiFERON-TB Gold in Tube® (QFT-GIT) (Cellestis, Victoria Australia) and the ELISPOT-based T-SPOT.TB® (Oxford Immunotec, Oxford, England) have gained endorsements from medical entities, such as the American Thoracic Society (ATS) and Center for Disease Control and Prevention (CDC) (18), and

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approval as a latent TB infection (LTBI) diagnostic from regulatory agencies in many countries, including the US FDA, European CE Mark, and Chinese FDA. Since approval of the first TB IGRA in 2001, and the first M. tuberculosis-specific IGRA in 2004, extensive research has focused on the positive and negative predictive values of each platform (4, 7, 19–22). This type of scrutiny is not common for a diagnostic, but is expected because of the global burden of LTBI and the lack of a gold-standard for comparison. Without a gold-standard, those interested in reporting assay performance must explain the meaning of discordance with the TST and the discordance of serial assays. Overall, the use of IFN-J production as a diagnostic indicator has proven to be useful; but mechanisms that cause variability are imperative for understanding IGRA results in the context of disease progression and coinfection. Comparatively little research has focused on the cellular mechanisms involved in IGRA variability. The measurement of IFN-J responses to TB-specific antigens is dependent not only on the platform, but more importantly on the frequency of IFN-Jproducing memory cells (23), the time since exposure (14), interleukin-2 (IL-2) (24) and IL-12 availability (25), effective antigen presentation, which can vary depending on the APC type in vivo (26), and whether the T cell has been recently activated (17). We reasoned that since IL-7 is a cytokine known to cause immune activation (27, 28), affect dendritic cell maturation (29), and enhance antigen-specific responses (30) and T-cell survival (24), it might improve memory T-cell responses in vitro. We stimulated isolated PBMCs in both IGRA platforms, and measured antigen-specific responses with and without IL-7 in IGRA-positive subjects (ESTAT-6 and CFP10 peptides). We also measured responses in both control and IGRA-positive subjects to tetanus, viral peptide pools from influenza virus, cytomegalovirus, Epstein-Barr virus (CEF) and cytomegalovirus (IE-1), negativecontrol peptides, and a positive-control mitogen, phytohemagglutinin (PHA). In both diagnostic platforms, IL-7 augments memory-specific responses. In vitro, IL-7 increases the number of cells producing IFN-J as measured by the TSPOT.TB ® assay (31). In ELISA, the cell-to-volume ratio of 250,000 cells in 100 Pl increased the difference between IL-7-treated and -untreated samples (31). In addition to more spots, some IL-7-augmented responses have an increased production of IFN-J per cell or “spotforming unit” as measured by spot size comparisons to antigen stimulation without IL-7 (Figs. 1 and 2). The spots produced by IFN-J-producing cells vary in size (32). Clonal T-cell pools activated by a single type of APC demonstrate that although the spot sizes of a T cell vary, they follow a lognormal distribution (33). Software on the automated CTL ELISPOT reader (S4) (Cellular Technology Limited Ohio, USA) allows the comparison of histograms of the footprints in each well

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Number of Spots

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1.00 Category boundaries (log sqmm)

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Fig. 1. Spot sizes (log mm2) resulting from viral antigen stimulation of control PBMCs (n = 5). In this example, PBMCs were stimulated with viral peptides CEF, IE-1, or tetanus antigen for 20 h, the TSPOT.TB ® performed, and the plate counted by CTL plate reader using S4 software to get a distribution of the counted spot sizes. This clearly shows that IL-7 addition to viral antigen-stimulated PBMCs from healthy controls causes a right shift (increase size) in spot size distribution. The mean difference in spot size with IL-7 was significant (p < 0.05) at −2.20 log mm2 and −1.80 log mm2 (* = p < 0.05).

6.00

* 5.00

Number of Spot

TB peptides TB peptides + IL-7

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Average size gating boundary for NK cells

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1.00 0.00

3.00 2.80 2.60 2.40 2.20 2.00 1.80 1.60 1.40 1.20 1.00 0.80 0.60 Category Boundaries (log sq mm)

Fig. 2. Spot sizes (log mm2) resulting from RD-1 peptide stimulation of IGRA-positive PBMCs (n = 3). In this example, PBMCs were stimulated with RD-1 peptide pools A and B for 20 h, the TSPOT.TB ® performed, and the plate counted by CTL plate reader using S4 software to determine the distribution of the counted spot sizes. This clearly shows that IL-7 addition to RD-1 peptide-stimulated PBMCs from IGRA-positive subjects causes a right shift (increase size) in spot size distribution. The spot size boundary for NK cells is well below the mean spot size of −2.00 log mm2 for which a significant change was seen (p < 0.05) in PBMCs when IL-7 was added to RD-1 stimulation (* = p < 0.05).

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or in groups of wells. The kinetics of the IFN-J-secreting cell detected by the ELISPOT membrane is measured by the rate at which IFN-J is released (spot density) and the length of time that the cell secretes (spot size). Histogram comparisons of the category boundaries (the log of the spot size in square millimeters) measured by the spot reader show that the addition of IL-7 increased the length of time that the cell secreted (Figs. 1 and 2), indicating that APC contact time was increased (34). There are two likely cellular sources of IFN-J production in the IGRA assays: NK cells and T cells. Because IL-7 increased the number of spots, it was important to test whether the spots resulted from secreting NK cells. We, therefore, depleted PBMC samples (Miltinyi Biotech Bergisch Gladback, Germany) so that the only remaining cell type was NK cells. We also depleted PBMC samples to study isolated T-cell subsets. We found that the footprints made by NK cells were much smaller than those made by T cells (Figs. 1–3). The use of a plate reader (such as CTL) allows for spot gating based on size which can exclude NK cells when measuring T cell-specific responses. Fig. 3 shows the results of stimulating 250,000 PBMCs or NK cells with the mitogen control. The graphs in Figs. 1–3 show

Fig. 3. Examples of spot sizes (log mm2) of PBMC or NK cells stimulated with PHA mitogen or RD-1 antigen. In this example, PBMCs or PBMCs depleted of all cells, but NK cells, were stimulated with PHA or RD-1 antigens (T-SPOT.TB ® panel A and B) for 20 h, T-SPOT.TB ® performed, and the spot size determined. This data shows that NK cells leave spot “footprints” when stimulated with PHA and antigen. This data emphasizes the importance of using plate reader gating to ensure that the mitogen-stimulated positive control is indeed a non-NK cell response. This is achievable because the NK cells’ spot size (−3.0 log mm2) is at the tail of the normal distribution of secreting T cells and can be used as a gating parameter during spot counting.

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that the difference seen with the addition of IL-7 is greatest in spots with size ranges between −2.6 log mm2 and −1.0 log mm2 which is above the size (−3.0 log mm2) measured for NK cells (Fig. 3).

2. Materials 1. T-SPOT.TB kit® (Oxford Immunotec, Oxford, England). 2. Lithium heparin blood collection tubes. 3. Ficoll-Paque Plus/Histopaque-1077 or alternative density gradient. 4. Pasteur pipette for underlying of density gradient. 5. Conical tubes for blood centrifugation. 6. BSA supplemented Aim-V® media (Invitrogen Carlsbad, CA). 7. 1× tissue culture phosphate-buffered saline (PBS). 8. Distilled or deionized water. 9. Centrifuge and buckets for 15 or 50-mL conical tubes. 10. Pipette (0–2, 0–20, 20–200 Pl) and sterile pipette tips. 11. Reservoirs for multichannel pipette. 12. Trypan blue, hemocytometer, and microscope or hematology analyzer for cell counts. 13. 5% CO2 humidified incubator. 14. Plastic squirt bottle or plate washer. 15. Inverted microscope or an ELISPOT reader. 16. Recombinant human (rh) IL-7. 17. Antigens of interest (whole proteins or peptide pools).

3. Methods 1. Prior to starting, read the entire protocol. Familiarize yourself with technique (Fig. 4, Table 1) and the science behind the assay (see Note 1). 2. Obtain 8 mL or more of human peripheral blood using lithium heparin vacutainers. 3. Within 8 h of blood collection, place blood into appropriatesize conical tube (see Note 2). 4. Add an equal amount of PBS to dilute blood (e.g., to 8 mL of blood, add 8 mL of PBS). 5. Underlay blood/PBS mixture with a density gradient measuring one-fourth amount of total volume of blood/PBS mixture

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Fig. 4. Diagram of process.

Table 1 Template of assay set-up 1

2

Nil Control

100 ªl of cell sample

B

Panel A

100 ªl of cell sample + antigen 1

C

Panel B

100 ªl of cell sample + antigen 2

D

Positive Control 100 ªl of cell sample + positive control

E

IL-7 Control

A

100 ªl of cell sample + 1ul of IL-7 [1ng/ªl]

(e.g., 20 mL of blood + 20 mL of PBS + 10 mL of density gradient). 6. Immediately centrifuge for 30 min at 25°C and 1,000 × g with low acceleration and the brake off. 7. Carefully remove sample and collect white cloudy layer of PBMCs (Fig. 5). 8. As a washing step, add AIM-V® to the collected sample to equal the amount of the starting blood volume. 9. Centrifuge sample again at 1,000 × g and 25°C for 10 min (Fig. 6).

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Fig. 5. Characteristic PBMC layer after density gradient separation.

Fig. 6. Visible cell pellet after washing.

10. Remove the supernatant and resuspend the cells in a volume of AIM-V equal to one-fifth the original blood volume (e.g., if original blood volume is 20 mL, resuspend in 4 mL AIM-V®). 11. Perform cell count and bring PBMC concentration to 2.5 × 106 per mL (2.5 × 105 per 100 Pl) using supplemented AIM-V® media (see Notes 3 and 4). 12. After calculating the total numbers of PBMCs, decide if the test can be run in duplicate or triplicate based on PBMC volume (see Note 5).

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Fig. 7. Example of T-SPOT.TB ® wells.

13. Take the T-SPOT.TB® kit from storage (2–8°C) and remove the appropriate number of T-SPOT.TB® precoated eight-well strips from the packaging (Fig. 7). Clip strips into plate holder and allow product equilibration to room temperature (see Note 6). 14. Mix cells thoroughly and place 100 Pl of cell sample into each well (see Notes 7 and 8). 15. Add reagents (antigens and mitogens of choice) immediately to wells. If using the T-SPOT.TB® antigen panels A, B, and positive control, 50 Pl of each stimulating product is added to each well. If other whole proteins or peptide pools are used as stimulating antigen(s), the respective concentrations should be titrated to find the effective dose used as described by the manufacturer or as the literature recommends. In our experience with tetanus toxoid (TT) and keyhole limpet hemocyanin (KLH) protein, we found 1 Pg/mL to be an ideal concentration (see Note 9). 16. Add 1 ng of rh IL-7 per well to IL-7 costimulated test wells (in plate above, wells E, F, G, and H). 17. Cover plate with plate lid. Immediately and carefully transfer plate to 37°C incubator and incubate for 20 h (see Note 10). 18. After a 20-h incubation at 37°C, discard cells from plate by swiftly shaking the plate over sink. 19. Wash the plate by filling each well with 200 Pl PBS per well. Shake PBS from plate, and repeat wash three times. When finished, shake the plate and tap on paper towel to remove excess PBS (see Note 11).

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20. Reconstitute the conjugate (mouse monoclonal antibody to alkaline phosphatase-conjugated IFN-J) according to manufacturer’s protocol. Set out substrate solution (BCIP/NBTplus) to allow equilibration to room temperature. 21. Using a multichannel pipette and reservoir, add 50 Pl of PBSdiluted conjugate to each well and incubate in the dark at 2–8°C for 1 h. 22. Discard conjugate from the plate by shaking. Follow by four washes with PBS. 23. Add 50 Pl of substrate to each well using a multichannel pipette and incubate at room temperature for 7 min. 24. Stop the reaction step by filling the plate with deionized/distilled water. Allow to dry overnight (see Note 12). 25. Read spots with an automated reader and verify with blinded stereoscope counts. 26. Once an ELISPOT assay has been standardized (as is the case with using the T-SPOT.TB® platform), the same counting parameters can be used to count all assays for objective, standardized comparisons of results across a diverse population of individuals (see Note 13).

4. Notes 1. We recommend using the standardized/optimized T-SPOT.TB® kit for measurement of IFN-J production. This platform minimizes problems with coating antibody concentration and nonspecific absorption of unrelated proteins: two of the five known variables in ELISPOT platform. We found that the use of T-SPOT.TB® plates was an ideal strategy to ensure the consistency of our results regardless of the antigen used to stimulate IFN-J from PBMCs and various cell subsets. In addition, we were able to forgo the laborious tasks of finding the ideal combination of coating and detection antibodies and the verification of each antibody lot. 2. Ensure that isolated PBMCs are stimulated within 8 h of the blood draw time and within 2–3 h of PBMC isolation. When longer time periods are used between PBMC isolation and stimulation, no spots or less-distinct spots result (unpublished observation). 3. The concentration and number of cells are important and have a direct impact on the amount of cytokine detected (31).

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4. The cell counting step is extremely important as it is one of the steps, where interassay variability can be reduced. All counts are based on the sample from which they were taken. Additionally, counting via the hemocytometer or cell counter is a sampling of the sample. To improve statistical validity, we suggest that a minimum of three samples be counted and the average used to determine the concentration. 5. We recommend, at minimum, one negative-control well of cells in media alone, one positive-control well of mitogenstimulated cells, and all test wells in duplicate (though triplicate is preferred). 6. If using T-SPOT.TB®, plan experiment and plate layout through the use of a template to minimize waste. 7. Because well-to-well results are compared based on the antigens and substrates added, it is essential to mix the sample well prior to plating to ensure homogeneity of each sample. 8. When adding reagents and samples, be careful not to damage ELISPOT membranes with pipette tips. 9. If performing test wells in duplicate or triplicate, we suggest that cell stimulations are done first in the total volume of cells to be tested for that parameter. For instance, if stimulating with tetanus toxoid in triplicate: (a) Pipette 650 Pl of cell sample into a 1-mL Eppendorf tube and add 0.65 Pg of TT. (b) Mix well and pipette 100 Pl each into the three respective antigen-stimulated wells. (c) Add 3.5 ng of IL-7 to mixture in Eppendorf tube. (d) Mix well and pipette 100 Pl each into the three respective antigen + IL-7-stimulated wells. 10. Placing a paper towel dampened with filter-purified water under the plate reduces background (unpublished observation). 11. Be careful not to tear the plate membrane when removing excess PBS from plate. 12. Although spot counts can be assessed any time after the completion of the assay, spots are the darkest and most distinct after the plate is completely dry. 13. If using a validated assay, such as the T-SPOT.TB®, and a plate counter, the same parameters can be used to assess all plate counts (32). For our experiments, we used a sensitivity of 100, background of 100, the maximum and minimum cell size parameters, and a cell separation value of 3 as programmed on the CTL Immunospot S4 software®.

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References 1. Lebedeva, Z. A. (1977) [Early Diagnosis of Tuberculosis (on the Centenary of the Birth of Charles Mantoux)]. Med Sestra 36, 51–52. 2. Lalvani, A., and Pareek, M. (2010) A 100 Year Update on Diagnosis of Tuberculosis Infection. Br Med Bull 93, 69–84. 3. Okada, K., Mao, T. E., Mori, T., Miura, T., Sugiyama, T., Yoshiyama, T., et al., (2008) Performance of an Interferon-Gamma Release Assay for Diagnosing Latent Tuberculosis Infection in Children. Epidemiol Infect 136, 1179–1187. 4. Diel, R., Loddenkemper, R., and Nienhaus, A. (2010) Evidence-Based Comparison of Commercial Interferon-J Release Assays for Detecting Active TB. Chest 137, 952–968. 5. Lodha, R., and Kabra, S. K. (2004) Newer Diagnostic Modalities for Tuberculosis. Indian J Pediatr 71, 221–227. 6. Pai, M., Zwerling, A., and Menzies, D. (2008) Systematic Review: T-Cell--Based Assays for the Diagnosis of Latent Tuberculosis Infection: An Update. Ann Intern Med 149, 177–184. 7. Dheda, K., van, Z. S., Badri, M., and Pai, M. (2009) T-Cell Interferon-Gamma Release Assays for the Rapid Immunodiagnosis of Tuberculosis: Clinical Utility in High-Burden Vs. Low-Burden Settings. Curr Opin Pulm Med 15, 188–200. 8. Cattamanchi, A., Ssewenyana, I., Davis, J. L., Huang, L., Worodria, W., den Boon, S., et al., (2010) Role of Interferon-Gamma Release Assays in the Diagnosis of Pulmonary Tuberculosis in Patients with Advanced HIV Infection. BMC Infect Dis 10, 75–75. 9. Mazurek, G. H., Jereb, J., Lobue, P., Iademarco, M. F., Metchock, B., and Vernon, A. (2005) Guidelines for using the QuantiFERON-TB Gold Test for Detecting Mycobacterium Tuberculosis Infection, United States. MMWR Recomm Rep 54, 49–55. 10. Prabha, C., Karthic, S., Das, S. D., Swaminathan, S., Subramaniam, S., and Sukumar, B. (2005) Impact of Tuberculosis on Serum Leptin Levels in Patients with HIV Infection. Horm Res 63, 228–233. 11. Cellestis. QuantiFERON-TB Gold Package Insert. Cellestis. 12. Keeler, E., Perkins, M. D., Small, P., Hanson, C., Reed, S., Cunningham, J., et al., (2006) Reducing the Global Burden of Tuberculosis: The Contribution of Improved Diagnostics. Nature 444 Suppl 1, 49–57. 13. Pai, M., Minion, J., Sohn, H., Zwerling, A., and Perkins, M. D. (2009) Novel and Improved

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Technologies for Tuberculosis Diagnosis: Progress and Challenges. Clin Chest Med 30, 701. Ewer, K., Millington, K. A., Deeks, J. J., Alvarez, L., Bryant, G., and Lalvani, A. (2006) Dynamic Antigen-Specific T-Cell Responses After Point-Source Exposure to Mycobacterium Tuberculosis. Am J Respir Crit Care Med 174, 831–839. Klinman, D. (2008) ELISPOT Assay to Detect Cytokine-Secreting Murine and Human Cells. Curr Protoc Immunol Chapter 6, 6.19-6.19. Kalyuzhny, A. E. (2005) Handbook of ELISPOT: Methods and Protocols, in Methods in molecular biology, 302 Totowa, N.J.: Humana Press, c2005. Schlingmann, T. R., Shive, C. L., Targoni, O. S., Tary-Lehmann, M., and Lehmann, P. V. (2009) Increased Per Cell IFN-J Productivity Indicates Recent in Vivo Activation of T Cells. Cell Immunol 258, 131–137. CDC. (2000) Targeted Tuberculin Testing and Treatment of Latent Tuberculosis Treatment. CDC MMWR. Pai, M., Riley, L. W., and Colford, John M, Jr. (2004) Interferon-Gamma Assays in the Immunodiagnosis of Tuberculosis: A Systematic Review. Lancet Infect Dis 4, 761–776. Orlando, G., Merli, S., Cordier, L., Mazza, F., Casazza, G., Villa, A. M., et al., (2010) Interferon-Gamma Releasing Assay Versus Tuberculin Skin Testing for Latent Tuberculosis Infection in Targeted Screening Programs for High Risk Immigrants. Infection 38, 195–204. Leyten, E. M. S., Arend, S. M., Prins, C., Cobelens, F. G. J., Ottenhoff, T. H. M., and van Dissel, J. T. (2007) Discrepancy between Mycobacterium Tuberculosis-Specific Gamma Interferon Release Assays using Short and Prolonged in Vitro Incubation. Clin Vaccine Immunol 14, 880–885. Grimes, C. Z., Hwang, L., Williams, M. L., Austin, C. M., and Graviss, E. A. (2007) Tuberculosis Infection in Drug Users: Interferon-Gamma Release Assay Performance. Int J Tuberc Lung Dis 11, 1183–1189. Helms, T., Boehm, B. O., Asaad, R. J., Trezza, R. P., Lehmann, P. V., and Tary-Lehmann, M. (2000) Direct Visualization of CytokineProducing Recall Antigen-Specific CD4 Memory T Cells in Healthy Individuals and HIV Patients. J Immunol 164, 3723–3732. Marrack, P., and Kappler, J. (2004) Control of T Cell Viability. Annu Rev Immunol 22, 765–787.

18 25. Flynn, J. L., and Chan, J. (2001) Immunology of Tuberculosis. Annu Rev Immunol 19, 93. 26. Ott, P. A., Tary-Lehmann, M., and Lehmann, P. V. (2007) The Secretory IFN-J Response of Single CD4 Memory Cells After Activation on Different Antigen Presenting Cell Types. Clinical Immunology 124, 267–276. 27. Rosenberg, S. A., Sportès, C., Ahmadzadeh, M., Fry, T. J., Ngo, L. T., Schwarz, S. L., et al., (2006) IL-7 Administration to Humans Leads to Expansion of CD8+ and CD4+ Cells but a Relative Decrease of CD4+ T-Regulatory Cells. J Immunother 29, 313–319. 28. Fry, T. J., and Mackall, C. L. (2005) The Many Faces of IL-7: From Lymphopoiesis to Peripheral T Cell Maintenance. J Immunol 174, 6571–6576. 29. Li, Masucci, Levitsky, and Levitsky, V. (2000) Effect of Interleukin-7 on the in Vitro Development and Maturation of Monocyte Derived Human Dendritic Cells. Scand. J Immunol 51, 361–371.


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30. Carreno, B. M., Becker-Hapak, M., and Linette, G. P. (2009) CD40 Regulates Human Dendritic Cell-Derived IL-7 Production that, in Turn, Contributes to CD8(+) T-Cell Antigen-Specific Expansion. Immunol Cell Biol 87, 167–177. 31. Feske, M., Nudelman, R. J., Medina, M., Lew, J., Singh, M., Couturier, J., et al., (2008) Enhancement of Human Antigen-Specific Memory T-Cell Responses by Interleukin-7 may Improve Accuracy in Diagnosing Tuberculosis. Clin Vaccine Immunol 15, 1616–1622. 32. Lehmann, P. V. (2005) Image Analysis and Data Management of ELISPOT Assay Results. Methods Mol Biol 302, 117–132. 33. Hesse, M. D., Karulin, A. Y., Boehm, B. O., Lehmann, P. V., and Tary-Lehmann, M. (2001) A T Cell Clone’s Avidity is a Function of its Activation State. J Immunol 167, 1353–1361. 34. Slifka, M. K., and Rodriguez, F. (1999) Rapid on/off Cycling of Cytokine Production by Virus-Specific CD8+ T Cells. Nature 401, 76.

Chapter 19 Overview of Membranes and Membrane Plates Used in Research and Diagnostic ELISPOT Assays Alan J. Weiss Abstract Polyvinylidene fluoride (PVDF) membrane-bottomed, 96-well plates and 8-well strips constitute the formats in which the overwhelming majority of ELISPOT assays used in research and diagnostic applications are performed. PVDF is well suited for ELISPOT because it has a high antibody-binding capacity and because its white color provides an excellent backdrop for ELISPOT enumeration. Nitrocellulose (NC) and PVDF membranes and 96-well plates containing those membranes used in ELISPOT assays were initially commercialized for filtration applications and later optimized for a range of different protein analytical applications. An overview of the development and biotechnology applications of PVDF membrane is provided. Characteristics and attributes of the membrane that are relevant to ELISPOT are summarized. Enhancements in PVDF membrane performance and optimization of devices for automation compatible and diagnostic ELISPOT applications are presented. Key words: ELISPOT, Polyvinylidene fluoride membrane, Filter plate, Diagnostics

1. A Chronology of Relevant Developments

There are two different types of membranes (in 96-well plate formats) that are used in ELISPOT applications: Nitrocellulose (NC) and polyvinylidene fluoride (PVDF) with PVDF becoming increasingly dominant over the past 10 years (although some features and guidance on how the use of NC plates will be provided, the emphasis in this chapter will be on PVDF and the use of PVDFbased devices in ELISPOT). The reason that NC and PVDF are the principal membrane types used in ELISPOT is based largely on their fortuitous suitability in a range of non-ELISPOT applications rather than on the development of optimized assay substrates to address the specific needs of the ELISPOT assay. The chronology of relevant membrane and application developments is outlined in Fig. 1.


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A.J. Weiss 1954: NC membranes become commercially available. Principal use is in removing bacteria from aqueous samples (sterile filtration) 1975: 0.45μm NC membrane used in DNA hybridization (Southern Blotting) assay (1) 1975: PVDF membranes become commercially available. Initially, principal use is in removing bacteria from aqueous samples. PVDF is more durable and more solvent resistant than NC and as such is more suited to large-scale filtration applications. 1979: 0.45μm NC membrane used in immunodetection of electro-blotted proteins (Western Blotting assay; see reference 2 ). 1983: ELISPOT assay developed on plastic, 96-well plates (3,4 ). 1985: 96-well plate with NC membrane becomes commercially available. Intended application is dot blotting of nucleic acids using vacuum transfer (instead of capillary transfer). 1986: 0.45μm PVDF membrane used in Western Blotting Assay (8,9 ). 1988: 96-well plate with NC membrane used in ELISPOT (5 ). 1992: 96-well plate with PVDF membrane becomes commercially available. Intended application is dot blotting of proteins using vacuum transfer (instead of electro-blotting). Millipore Corporation (Bedford, MA) reports in its 1994/5 Catalog that this plate can be used in ELISPOT 1995: Articles citing improved ELISPOT results using PVDF plates are published (6 ). 2002: Automation compatible 96-well plates become commercially available making it possible to use robotic liquid handlers to perform many of the reagent additions and plate washing steps associated with high throughput screening (HTS) 2003: Automation compatible, 96-well plates designed specifically for ELISPOT becomes commercially available 2003: Membrane-bottomed, 8-well strip plates become commercially available for diagnostic and research ELISPOT applications 2005: PVDF membranes are made with low background fluorescence making it much more possible to perform fluorescent ELISPOT assays 2008: Patented (7 ) T-Spot ELISPOT assay receives FDA PMA approval as a clinical diagnostic test for Tuberculosis

Fig. 1. Timeline of membrane, applications, and ELISPOT developments.

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As can be seen in Fig. 1, NC and then PVDF membranes were first developed to serve the needs of sterile filtration applications. In ways that were never anticipated by membrane manufacturers, the porosity and binding properties of these membranes enabled them to be used in two extremely important and burgeoning research applications; nucleic acid hybridization assays and Western blotting. Eventually, to serve the specific requirements of molecular biology and protein chemistry applications, NC and PVDF membrane-bottomed 96-well plates were developed and made commercially available. Independently and separately, ELISPOT assays were developed on 96-well plastic plates and took advantage of enzyme-linked immunosorbent assay (ELISA) techniques that had been perfected in that format. Since the immunodetection component of ELISPOT assays and Western blotting is essentially identical, it was only a matter of time until the overwhelming majority of ELISPOT assays were performed on membrane-bottomed, 96-well plates.

2. PVDF Fluoride Membrane Development and Applications

PVDF membranes were developed in part to overcome major limitations of NC, including poor chemical compatibility, shedding of particulates, and brittleness. Many of the same companies that were able to produce NC also developed the ability to make PVDF membranes over a range of different pore sizes. The PVDF polymer itself is highly resistant to chemical degradation – except in the presence of strong alkali (pH greater than 12) – and membranes made from PVDF are sufficiently elastic to withstand a broad range of fabrication conditions (including sonic welding and pleating) and high-pressure filtration applications. Millipore, an early provider of PVDF membranes, created the trademark, “Durapore®” to call attention to these attributes. PVDF, like NC, is intrinsically hydrophobic. Whereas NC is only marginally hydrophobic and can be made water wettable by adding a surfactant (e.g., glycerin) or detergent (e.g., Triton®-X 100) to the membrane, PVDF is extremely hydrophobic and requires significant surface modification to make it compatible with aqueous solutions (see Note 1). A considerable amount of chemistry and patented technology was subsequently developed to make PVDF membranes hydrophilic. The important point to note is that even though the polymer itself is very hydrophobic, membranes made from PVDF can be either hydrophilic or hydrophobic depending on whether the manufacturer has modified or covered over the polymer surface with a secondary chemical treatment that is water compatible. The discussion of PVDF is relevant to ELISPOT in that the hydrophobic version of the 0.45 Mm (8) membrane was found to

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Fig. 2. A scanning electron micrograph of a 0.45 Mm PVDF membrane.

H

F

C

C

H

F

n

Fig. 3. Chemical structure of polyvinylidene fluoride.

be an excellent Western blotting substrate. A scanning electron micrograph of a 0.45 Mm PVDF membrane appears in Fig. 2. PVDF binds proteins by hydrophobic interactions (van der Waal’s forces). This applies, of course, only to hydrophobic PVDF membranes. Most types of hydrophilic PVDF will not bind proteins to any appreciable degree. Interestingly, hydrophobic PVDF will bind single-stranded DNA and RNA, but will not bind double-stranded DNA. The chemical structure of PVDF is illustrated in Fig. 3. The use of PVDF membranes in Western blotting type applications grew rapidly. Unlike NC membranes, PVDF membranes could stand up to automated protein sequencing chemistries and were better able to retain low molecular weight proteins and peptides. PVDF was also better suited to a wider range of detection techniques, including fluorescence and chemiluminescence. One significant drawback of PVDF is the need to pre-wet the membrane

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in an alcohol solution prior to using it in Western blotting and most other applications that include immunodetection. The requirement to pre-wet with alcohol – which is completely necessary in Western blotting applications – is not universally applicable in ELISPOT. Differences in ELISPOT protocols with regard to the pre-wetting step are significant and are likely to have an impact on the performance of the assay. This topic is discussed in greater detail later in this chapter (see Note 2).

3. 96-Well Filter Plate Development and Applications The rationale behind the development and commercialization of NC and PVDF is clear from the perspective of filtration applications. As has been pointed out already, the use of each of these membrane types in molecular biology and protein chemistry applications was based on a fortuitous combination of membrane properties; high permeability (due to high porosity), and high DNA/ RNA and protein binding. The development of 96-well PVDF and NC bottomed plates was primarily driven by the secondary (i.e., biochemistry) applications. One of the attributes of both Southern and Western blotting is that (DNA) hybridization and antibody binding are diffusion limited reactions. In other words, the reactant in solution (complimentary DNA or antibody) must diffuse to the surface of the membrane before it can couple with the immobilized reactant (DNA or protein). In these types of solid phase reactions, the times required to reach equilibrium binding are much longer as compared to reactions in which both reactants are in solution. Typically, DNA hybridization (Southern blotting) and immunodetection (Western blotting) require from 2 to 24 h. Membrane-bottomed, 96-well plates, an example of which is seen in Fig. 4, made it possible to reduce the time requirements associated with solid phase binding. (Earlier, simpler versions of these plates, called “Dot Blot” or “Slot Blot” apparatuses provided the first opportunity to exploit the benefits of filtration in these applications). Filtration of the reactant in solution through the membrane brings it into intimate contact with the reactant immobilized on the membrane surface. So long as the filtration rate is kept low enough for hybridization or binding to take place, the time required to achieve efficient capture can be dramatically reduced. Additionally, all wash steps in between various reactions can be accomplished using filtration. The development of membrane-bottomed plates in conjunction with compatible vacuum manifolds made it possible to carry out from 1 to 96 different hybridization or immunodetection assays using less reagents and requiring less time as compared to standard methodologies.

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Fig. 4. 96-well filter-bottomed plate used for ELISPOT and HTS applications.

In addition to the membranes and other features that were useful for these applications, the plates were also designed to allow for discrete liquid transfer from the top (membrane-containing) plate to a (standard, plastic, 96-well) receiver plate. The filterplate components that allow for this to occur have the potential to interfere with ELISPOT applications in at least three different ways. 1. If alcohol is used to pre-wet membrane (this applies only to the use of PVDF membrane plates), and becomes trapped under the membrane, the alcohol can suppress or completely inhibit cytokine release (see Note 2). 2. If biotinylated antibody or avidin-enzyme conjugate becomes trapped under the membrane, high background will likely result (see Note 2). 3. If the membrane cannot be removed because of these components, it may be difficult to analyze and essentially impossible to archive ELISPOT results. Preventing these types of problems during the ELISPOT assay and optimizing the different parts of the protocol are reviewed elsewhere. There are other plate attributes that may have an impact on ELISPOT assay performance or analysis. The membrane inside each well needs to be planar within a fraction of a millimeter

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(e.g., ±0.1 mm) in order to provide the best assay results. Lack of membrane flatness may contribute to difficulties in imaging depending on the type of microscopy being used. Additionally, if the membrane is bowed, cells may tend to settle unevenly or roll to the relative low points (often either the center or the periphery) during incubation. Consequently, spots may become very unevenly distributed and difficult to enumerate accurately especially if there is any spot confluence. The plate itself should also be flat (within a tolerance of perhaps 1 mm corner to corner) to assure compatibility with plate washers and most of the imaging software that supports automated image acquisition and analysis. There is one other feature of 96-well plates that has the potential to introduce variability into the ELISPOT assay. The 96-well plate is arrayed as 8 rows of 12 wells. Wells at the periphery (columns 1 and 12, rows A and F) of the plate are fundamentally different from “interior” wells insofar as they are in the most direct contact with the plate surroundings. Depending on incubation conditions and other protocol steps, this physical distinction may have some impact on one or more parts of the ELISPOT assay (see Note 3).

4. 96-Well Filter Plates in ELISPOT As evidenced by the chronology laid out in Fig. 1, ELISPOT assays were first developed on plastic, 96-well plates. Shortly after NC-bottomed filter plates became available, the majority of ELISPOT assays were carried out in those plates. When PVDF filter plates were introduced, some investigators chose to use PVDF plates and some continued to use NC. The reasons for choosing one plate (membrane) over the other are highly varied and will not be addressed in detail here although over the past 10 years, the percentage of ELISPOT assays performed on PVDF plates has steadily increased. The fact that some laboratories and individual researchers feel strongly that one membrane is superior to the other runs contrary to the large body of Western blotting experience: Despite some clear-cut differences in how each of the membranes can be used, there is essentially no reported difference in terms of detection sensitivity or signal to noise on NC versus PVDF in the Western blotting application. This having been said, it is clear that the two membranes and their properties are quite different. Subheading 5 will be devoted to reviewing these properties which are summarized in Table 1, and highlighting the differences – especially as they might pertain to ELISPOT applications.

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Table 1 Comparison of PVDF and NC membranes

a

Attribute or characteristic

NC (used in ELISPOT) [nominal or average values]

PVDF (used in ELISPOT) [nominal or average values]

Pore sizea

0.45 Mm

0.45 Mm

Porosityb

70–75%

65–70%

Thickness

150 Mm

135 Mm

2

BET surface area (9)

6.5 m /g

6 m2/g

Surface area ratioc

250

350

Saturation binding capacity (IgG)

250 Mg/cm2

350 Mg/cm2

Binding capacity of top 1 Mm (IgG)

2 Mg

3 Mg

Wettability

Not wettable without prior addition of surfactants or detergents

Not directly wettable in water. Must be pre-wet with alcohol and then exchanged with water

Additives

Glycerin

None

Solvent compatibility

Not compatible with methanol or ethanol

Broadly compatible with a wide range of aqueous and organic solvents. Avoid prolonged exposure to strong alkali (e.g., pH > 12)

Mechanism of binding

Electrostatic

Hydrophobic

Things which will interfere with or destabilize binding of anti-cytokine antibodies

Chaotropes (e.g., Tween-20, Triton-X 100, etc.). Water (if never dried), Proteins, especially larger molecular weight proteins

Detergents (e.g., SDS), low polarity solvents (e.g., dimethyl formamide, etc.)

Compatibility with different detection modes

Ú Colorimetric Ù Fluorescence Ú Chemiluminescence

Ú Colorimetric Ú Fluorescence (marginal) Ú Chemiluminescence

Pore size is nominal and corresponds to the diameter of the largest particle that can pass through the membrane structure. A 0.45 Mm pore size membrane is expected to retain 100% of particles whose diameter exceeds 0.45 Mm b Porosity is the portion of the membrane volume that is occupied by air (not occupied by polymer). 1 cm2 of membrane whose thickness is 140 Mm (i.e., 0.014 cm) will have a volume of 0.014 cm3 (14 ML). If the membrane is 70% porous, it will contain approximately 10 ML of void space c Surface area ratio is the ratio of internal to frontal surface area. A surface area ratio of 250 means that in a 1 cm2 piece of membrane, the polymer surface area is 250 cm2

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5. Properties of PVDF Membrane that Affect Its Performance in ELISPOT


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Like NC, and for the same reasons, the PVDF membrane in plates used for ELISPOT has a nominal pore size of 0.45 Mm. PVDF is nominally 135 Mm thick and about 65–70% porous. The BET surface area is about the same as NCs and its surface area ratio is somewhat higher – around 350 (see Note 4). Consequently, PVDF can bind upward of 350 Mg/cm2 of IgG or in excess of 100 Mg per well of a 96-well plate. As with NC, blocking of PVDF should occur within a few hours (or less) of antibody coating. Failure to do so may result in a rapid and significant loss of antibody activity. Once antibody has been coated and the membranes have been blocked (and washed using deionized water or very low molarity buffer), plates can be stored (desiccated and at room temperature) for weeks or even months (see Note 5). PVDF that has been coated with protein (e.g., as a consequence of antibody coating and blocking) will rewet spontaneously upon the addition of aqueous media. The major difference between NC and PVDF in ELISPOT applications is related to their mechanisms of binding and associated differences in handling or pretreatment. PVDF is very hydrophobic (its surface energy is approximately 21 dyn/cm) and will not wet out in water. In Western blotting applications, PVDF is always pre-wet in alcohol (typically 50–100% methanol), then normally exchanged in water, and ultimately equilibrated in a (transfer) buffer solution before applying the membrane to the polyacrylamide gel for electrophoresis. The fact that hydrophobic PVDF membranes will not wet out spontaneously in water – unless coated by (blotted) proteins – is even exploited in a Western blotting application called “Transillumination” (10). The overwhelming majority of literature in Western blotting references the pre-wetting step, so it is not a surprise that many ELISPOT protocols also include an alcohol pre-wet step. What is surprising is that some ELISPOT protocols do not include a pre-wet step. At Millipore (Danvers, MA), experiments were performed to determine the relative performance PVDF 96-well plates (catalogue number: MSIPS4510) that were either pre-wet with 15 MmL of 70% v/v methanol in water and rinsed, or not pretreated at all prior to antibody coating. Briefly, following the alcohol pretreatment (or no pretreatment), plates were coated with 1 Mg of anti-human interferon-gamma (Mabtech, Stockholm, Sweden), and blocked for 2 h in tissue culture media (RPMI, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen). 50,000 peripheral blood mononuclear cells (see Note 6) were added per well to 16 wells per plate, stimulated with 0.5 Mg phytohemagglutinin

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Table 2 The effect of alcohol pretreatment of PVDF on ELISPOT assay performance

a

Experiment number

Pre-wet spot number (Mean ± SDa)

Non pre-wet spot number (Mean ± SDa)

Non pre-wet as a percent of pre-wet

1

606 ± 46

413 ± 37

68%

2

577 ± 37

416 ± 34

72%

3

604 ± 35

440 ± 42

73%

4

609 ± 40

391 ± 28

64%

n = 12–16

(PHA-L, Sigma, St. Louis MO) and the plates were incubated overnight in a humidified, 37°C, 5% CO2 tissue culture incubator. ELISPOTs were visualized using biotinylated anti-human interferon-gamma (Mabtech), conjugated avidin-alkaline phosphatase (Mabtech) and BCIP/NBT Plus (Moss, Inc., Pasadena, CA) and then enumerated using an automated microscope (KS Elispot, Zeiss, Thornwood, New York) and its associated software. The results of four different experiments are summarized in Table 2. In these experiments, the cells in the untreated (nonpre-wet) plates produced approximately 30% fewer detectible spots. However, the consistency well to well and plate to plate was equivalent. Spot quality (intensity, uniformity, and size) and overall assay background were comparable in both plate types. Considering that half these results were obtained without pre-wetting, the comparable, side-by-side performance is quite remarkable. It would appear that the determination to pre-wet with alcohol or not can be made by individual laboratories based on their reagent selections and particular assay requirements. PVDF, like NC, is fully compatible with ELISA detection involving precipitating, color-forming substrates and chemiluminescent substrates. Although the fluorescence background of PVDF is also high, due principally again to light scattering, ELISPOT assays have been developed on PVDF that are based on the use of fluorescently labeled antibodies (11). The ability to develop fluorescent immunoassays on PVDF membranes – particularly useful for applications in which more than one antigen is being simultaneously detected – was significantly enhanced with the commercialization of a specialized PVDF membrane (Immobilon-FL™; Millipore, Billerica, MA, USA in 2003) that was specifically developed for fluorescence detection.

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6. 96-Well Plates and 8-Well Strip Plates Specifically Developed for ELISPOT and Diagnostic ELISPOT Applications


253

As was stated earlier in the chapter, 96-well plates were initially designed to enable sample filtration and discrete transfer of fluids. These design elements, which are not useful for ELISPOT, can actually interfere with the ELISPOT assay if the user is not careful. Consequently, in 2003, a 96-well plate with PVDF was commercialized that was designed specifically for ELISPOT applications (Fig. 5). There is no underdrain to facilitate discrete fluid transfer in these ELISPOT-specific plates. Consequently, problems associated with alcohol pre-wetting and membrane removal after ELISPOT development are virtually eliminated. One of the issues with 96-well plates of all types is that many ELISPOT assays do not end up requiring the use of all 96-wells and unused wells essentially get wasted. This is especially true in diagnostic applications in which only one or two patients may be tested at a time. For these types of low-throughput applications, there are now 8-well strip plates available (Fig. 6). Individual (8-well) strips can be antibody coated, blocked, and used in an assay without any impact on the remaining strips. This particular format was first used in a diagnostic

Fig. 5. 96-well filter-bottomed plate used specifically for ELISPOT.

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Fig. 6. Membrane-bottomed 8-well strip plate used specifically for ELISPOT (especially diagnostic applications).

test approved by the FDA in 2008 for tuberculosis infection and is now also commercially available for research and development applications

7. Final Remarks

Although with proper optimization, it is likely that comparable ELISPOT performance can be achieved using either type of membrane, it is unlikely that the same protocol will work equally well in both cases. Neither PVDF nor NC is a drop-in for the other (see Note 7). Fundamental differences in the two membranes, especially with regard to their mechanisms of binding and their ability to wet directly in water will affect their behavior in ELISPOT. Modifications that have been made in the design of 96-well plates to make them compatible with automation – including stricter dimensional specifications and rigid sidewalls (to allow handling by laboratory robotics and provide space for bar codes) have also benefited ELISPOT applications. These plates are now fully compatible with standard plate washers as well as with imaging equipment and image analysis software. It is reasonable to believe, based on the importance of ELISPOT and the impressive growth in the number of assays being performed, that membranes and membrane-based plates may someday be optimized specifically for this application.

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8. Notes 1. PVDF in 96-well plates (for example, Millipore catalogue number MAIPS4510) used for ELISPOT is extremely hydrophobic. It will not wet out spontaneously upon the addition of water. The behavior of PVDF in this regard is significantly different from that of NC. 96-well plates containing hydrophilic PVDF (for example, Millipore catalogue number MAHVS4510) will not work at all in the ELISPOT application. 2. As required or desired, the hydrophobic PVDF membrane should be pre-wet by adding 15 ML of 70% methanol to each well. Within 1 or 2 min (or less) of adding the methanol solution, the membrane should be rinsed by adding 100 ML of water or coating buffer to the well and aspirating or decanting immediately thereafter. The rinse step may be repeated once more. The antibody solution should be added immediately after rinsing the membrane. Adding larger (e.g., 50 ML) or more concentrated (e.g., 100%) volumes of methanol creates the risk of liquid collecting under the membrane. This liquid cannot be washed out effectively and may create serious problems later on in the ELISPOT assay. 3. 96-well plates containing NC (for example, Millipore catalogue number MAHAS4510) may contain some wells, especially at the periphery of the plate, that will not wet out immediately upon addition of aqueous solution (e.g., antibody coating buffer). This may be the result of storage and handling conditions. In any event, wells that do not wet out immediately may produce spurious results and should not be used in the ELISPOT assay. 4. The binding capacity of both NC and PVDF (q75 Mg/well) far exceeds the amount of specific antibody (typically a 5 Mg/well) that is used to coat the membrane. Whereas this is beneficial insofar as it results in the localization of the antibody at or very near the membrane’s top surface, it makes it absolutely necessary to block the membrane to prevent high levels of background due to nonspecific binding. Membrane should be blocked within a few hours of antibody coating to prevent the loss of antibody activity. 5. Once membranes have been coated and blocked, they may be rinsed in water or low molarity buffer (e.g., 10 mM phosphate, pH 7.4) and stored desiccated for weeks or months. 6. The membrane frontal surface area in a typical 96-well plate is approximately 0.3 cm2. If the responding T-cell is assumed to be round and estimated to have a nominal diameter of 10–15 Mm, approximately 150,000 cells would constitute a

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monolayer. Adding more than approximately 100,000 cells creates the risk of some of these cells not being in intimate contact with the membrane. If the cell secreting cytokine is not in direct contact with the antibody-coated membrane, it is possible that the shape and intensity of its corresponding ELISPOT may be so irregular as to disqualify it from being enumerated. In instances when the response rate to antigen is anticipated to be so low that it is advisable to stimulate 500,000 or 1,000,000 cells to get a significant response above background, it might be best to add 100,000 cells per well to 5 or 10 wells and determine the aggregate response £10 wells , not the average response. 7. It is nearly certain that substituting a PVDF plate into a protocol that has been optimized using an NC plate will produce satisfactory results. The opposite is also true. The ELISPOT assay, especially the antibody coating, blocking, and color development steps should be optimized for whichever plate is going to be used. References 1. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517 2. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350–4354 3. Czerkinsky, C.C., Nilsson, L.A., Nygren, H., Ouchterlony, O., and Tarkowski, A. (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods 65, 109–121 4. Sedgwick, J.D. and Holt, P.G. (1983) A solidphase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J Immunol. Methods 57, 301–309 5. Czerkinsky, C., Andersson, G., Ekre, H.-P., Nilsson, L.-A., Klareskog, L., and Ouchterlony, O. (1988) Reverse ELISPOT assay for clonal analysis of cytokine production. I. Enumeration of gamma-interferon-secreting cells. J Immunol Methods 110, 29–36 6. Schielen, P., van Rodijnen, W., Tekstra, J., Albers, R., and Seinen, W. (1995) Quantification of natural antibody producing B cells in rats by

an improved ELISPOT technique using the polyvinylidene difluoride membrane as the solid support. J Immunol Methods 188, 33–41 7. Lalvani; Ajit, and Brookes; Roger Hamilton. “Assay method for peptide specific T-cells.” US Patent 7,575,870. August 18, 2009 8. Pluskal, M.F., Przekop, M.B., Kavonian, M.R., Vecoli, C. and Hicks, D.A. (1986) Immobilon PVDF transfer membrane: a new membrane substrate for Western blotting of proteins. BioTechniques 4, 272–282 [Japanese reference to the use of 0.2 Mm membrane (GVHP) in protein blotting] 9. Brunauer, S., Emmett, P.H., and Teller, E. (1938) Adsorption of Gases in Multimolecular Layers. J. Am. Chem. Soc. 60, 309–319 10. Reig, J.A. and Klein, D.C. (1988) Submicron quantities of unstained proteins are visualized on polyvinylidene fluoride membranes by transillumination. Applied and Theoretical Electrophoresis 1, 59–60. 11. Gazagne, A., Claret, E., Wijdenes, J., Yssel, H., Bousquet, F., Levy, E., Vielh, P., Scotte, F., Goupil, T.L., Fridman, W.H., Tartour, E. (2003) A Fluorospot assay to detect single T lymphocytes simultaneously producing multiple cytokines. J Immunol Methods. 283, 91–98.

INDEX A Adeno-associated virus (AAV) ....................................65–72 Anesthesia reagents acepromazine ...............................................................67 atropine........................................................................67 glycopyrrolate ..............................................................67 lidocaine hydrochloride ...............................................67 propofol .......................................................................67 Antigens FIVBang ................................................................... 51, 57 FIV IWV ...............................................................51, 57 FIVPet ........................................................................... 57 FIV p24 recombinant protein ................................51, 57 HIV–1HXB2 .............................................................51, 57 HIV–1LAV p24..............................................................57 HIV–1 recombinant protein ..................................51, 57 Association for Cancer Immunotherapy (CIMT) .............30

B BALB/c ................................................................... 108–113 Bioterorrist weapon .........................................................200 Blood draw ............................................................... 13, 18, 238 shipping .......................................................................18 storage ........................................................... 18, 93, 120

C Calcium ionomycin (CaI) .................40–42, 91, 92, 116, 118 Cancer Immunotherapy Consortium of the Cancer Research Institute (CIC/CRI) ...................29–31 Cat specific-pathogen-free ...........................................50, 55 C57BL/6 ................................................................. 108–113 CD cells isolation Dynabeads® ................................................................. 50 Ficoll............................................................................ 50 MyOne™ ....................................................................50 Untouched™ ...............................................................50 CEF.................................................... 7, 14, 27, 29, 231, 232 Cells antigen-presenting cells (APCs) ................10, 13, 14, 20, 65, 157, 161, 167, 174, 224–226, 230, 231, 233

BV2 ..................................................................... 98–101 CD14................................................................. 112, 226 CD31......................................................................... 112 CD45......................................................................... 112 CD106....................................................................... 112 CD11c ....................................................................... 112 dendritic cells (DC) .....................................20, 105, 157, 159, 226, 231 fibroblast .............................................106, 108–110, 112 HeLa .......................................... 201, 203, 204, 211, 217 mesenchymal stem cells (MSC) ........................ 105–113 microglial ............................................................. 97–102 productivity.........................................125–142, 157, 161 splenocytes ................................................. 106, 108–113 T cell line ..................................10, 21, 27, 179–182, 224 Cellular immune response ....................4, 65, 66, 68, 69, 220 CMV-canine factor IX (cFIX) ..........................................66 Concanavalin A (Con A)......................40–42, 51, 53, 57, 58 Crystal violet ................................................... 201, 205, 206 CTL-AntiAggregate™........................................................7 CTL-CryoABC™...............................................................7 CTL-Test™ .........................................................................7 Cytokine bead array (CBA) ............................... 8, 9, 13, 157 Cytokines granzyme B.......................................... 4, 8, 26, 126, 161 IFN-G ......................... 4, 8, 10, 11, 14, 18, 26, 27, 30, 50, 77, 79–84, 88, 89, 91–93, 113, 116, 117, 126, 137, 140, 156–159, 161, 174, 179, 180, 185, 219–227, 229–231, 233, 238 IL–2......................... 4, 11, 18, 21, 22, 47–52, 54, 72, 77, 79–84, 88, 89, 91–93, 113, 126, 140, 161, 169, 202, 203, 209–210, 219, 220, 222, 224–226, 231 IL–3........................................................................... 161 IL–4................................... 10, 17, 18, 39–44, 84, 88–93, 113, 126, 140, 161 IL–5................................ 17, 18, 88, 89, 91–93, 140, 161 IL–6............................................................159, 161, 169 IL–7................................................................... 229–239 IL–8............................................................88, 89, 91–93 IL–10 ...... 79, 80, 106, 107, 109, 110, 112, 113, 126, 159, 161, 169, 199–217 IL–12 ................................................................... 49, 231 IL–17 ........................................ 4, 10, 17, 18, 21, 22, 140

Alexander E. Kalyuzhny (ed.), Handbook of ELISPOT: Methods and Protocols, Methods in Molecular Biology, vol. 792, DOI 10.1007/978-1-61779-325-7, © Springer Science+Business Media, LLC 2012

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HANDBOOK OF ELISPOT 258 Index Cytokines (Continued) perforin .............................................4, 8, 17, 26, 47, 161 TNF-A ............. 4, 8, 47, 77, 88, 89, 91–93, 97–102, 129 TRAIL .............................................................. 4, 8, 161 Cytomegalovirus (CMV) ................... 7, 11, 27, 66, 186, 231

D Determinant mapping ...........................................10, 12–13 Dulbecco’s phosphate-buffered saline................................67

E ELISA Assay Diluent RD1–51 .............................................119 Calibrator Diluent RD6–21 ......................................117 microplate reader ...............................................117, 119 Quantikine.................................................................117 ELISPOT antibodies affinity ..........................126, 133–136, 138, 142, 158 anti-CD3 ...............................................................84 anti-CD28 .......................................................81, 83 anti-IFN-G-FITC ............................................81, 84 anti-IL–2-biotin ..............................................81, 84 association rate............................. 128, 134–136, 142 avidity ........8, 12–15, 17, 21, 126, 134, 157, 169, 230 7-B6–1 ................................................................... 80 binding....83, 126, 128–130, 133–136, 138, 142, 250, 255 coating ..................26, 80, 81, 83, 137, 138, 223, 238, 251, 255, 256 9D7........................................................................ 80 dissociation rate ................................... 128, 134–136 1-D1K ...................................................................80 12G8......................................................................80 JES5–2A5 .................................................... 106, 107 JES5–16E3 ..................................................106, 107 asynchronous analyte production .......................139–140 background .....................7, 10, 19–21, 26, 34, 44, 53, 61, 72, 94, 101, 102, 121, 142, 148, 153, 158–159, 168, 169, 173, 175–178, 182, 186–190, 192, 193, 209, 225, 244, 252, 255, 256 BCIP ..................................40, 42, 51, 60, 71, 91, 93, 98, 100, 106, 109, 116, 118, 158, 214, 215, 225, 238, 252 bovine ..................................... 50, 81, 201, 206, 219–227 ex vivo ............................... 10–12, 15, 125, 220, 223, 225 fluorescent Cy3 ............................................................ 78–82, 84 Cy5 ..................................................................79–82 DAPI .....................................................................80 enhancer ..........................................................80, 82 FITC .........................................................78–82, 84 fluorospot............................16, 77–84, 126, 140–142

harmonization ..................15, 25–34, 156, 162, 177, 189 horse .............................................................. 40, 42, 106 image analysis algorithm ....................................15, 49, 61, 145–153 AutoGate™ ................................................. 164, 165 automatic construction ................................ 150–151 brightness .............................146, 147, 150, 151, 153 cluster analysis algorithms ...........................149, 153 Code for Federal Regulations (CFR) Part 11 guidelines .......................................................155 color classification........................................148–150 data analysis ....................8, 12, 15, 19, 155–170, 182 data documentation .............................................166 data management.........................................167, 168 differential equation .............................................129 Good Laboratory Practice (GLP) ..... 12, 15, 21, 166 illumination ................40, 91, 98, 116, 145, 148, 151, 153, 161, 162 image form construction ......................................146 ImmunoSpot® ......................7, 16, 21, 127, 137–139, 156–160, 162, 163, 165–169, 213, 214, 216 iSpot spectrum .......................................................80 local minimums ...................................................148 logarithmic function ............................ 130, 131, 141 morphological approach ..............................147–148 numerical solution ............................... 127, 129–131 pixels ............................................................148, 161 QuantiHub ............................................ 41, 150–152 SmartCount™ .............................................158, 163 SpotMap™ ..................................................162, 167 spot morphology ......................... 125, 126, 132–139, 142, 156–159, 163, 164, 169 spot recognition ...............12, 16, 145–153, 156–160, 163, 164, 179 international Society of Biological Treatment of cancer (iSBTc) ....................................................15, 156 negative control ......... 57, 60, 69, 71, 175–177, 186–190, 192–194, 205, 209, 211, 239 neuroscience research...........................................97–102 nitro blue tetrazolium (NBT) ..............40, 42, 51, 60, 71, 91, 93, 98, 100, 106, 109, 116, 118, 158, 214, 215, 225, 238, 252 optimization ................................................... 31–33, 83, 226, 254 pre-validation.........................................................31, 32 proficiency panel .................................... 27–34, 187, 191 spots automatic search ..........................................145–153 classifying ............................................................146 concentric profile .................................................146 density .................. 130–134, 137, 138, 141, 142, 233 density profile ..............................................130, 134 detection ...................................... 130, 142, 145–153 diffusion ...............................................................158

HANDBOOK OF ELISPOT 259 Index distribution kinetics .....................................127–129 formation .................. 26, 84, 113, 132, 135, 142, 226 fuzzy .........................44, 94, 102, 121, 142, 157, 158, 169, 174, 175 kinetic model ....................................... 127–129, 141 mathematical model ....................................126, 132 morphology .........................125, 126, 132–139, 142, 156–159, 163, 164, 169 numerical solution ............................... 127, 129–131 profile .................................................. 130–132, 135 recognition .......................12, 16, 145–153, 156–160, 163, 164, 179 shape .............................121, 130, 145, 148, 149, 157 size ......................... 21, 126–128, 130–135, 139, 141, 157–159, 161, 164, 166, 167, 170, 173–175, 229–239 spot forming cells (SFC)......................... 42, 43, 101, 186, 188, 190, 220, 221, 223, 225–227 spot forming units (SFU) .............. 53, 54, 56, 59–61 spot size distribution ....................127, 130, 141, 161, 164, 173, 175, 232 too numerous to count (TNTC)..........................169 well-to-well spot consistency ................... 44, 94, 121 standardization Cancer Immunotherapy Immunoguiding Program (CIP) ............................................... 30, 186, 194 empirical approach............................... 177, 187–188 false positive rates ........................ 177, 187, 189–193 HLA-A*0201 restricted epitopes of CMV ..........186 Influenza ..............................................................186 mean responses ....................................................187 negative control ...................57, 60, 69, 71, 175–177, 186–190, 192–194, 205, 209, 211, 239 Q2R1 guideline ...................................................190 signal-to-noise ratio .....................................176, 190 true negative rates ................................................187 true positive rates .................................................187 statistical analysis analysis of variance (ANOVA).....................180, 182 Anderson-Darling test .........................................179 distribution-free resampling (DFR).............188–194 GraphPad Prism® .................................................179 Kolmogorov-Smirnov test ...................................179 MATLAB ...........................................................179 negative binomial distribution .....................178, 181 Poisson distribution .....................................179–183 SAS .....................................................................179 Shapiro-Wilks test ...............................................179 software package R ..............................................179 spot-size gating parameters ................. 159–161, 233 SPSS ....................................................................179 Statistica .............................................................. 179 T-test ...................... 21, 179, 180, 182, 188, 191, 192 type I errors ......................................................... 177

Westfall-Young Stepdown max T adjustment......188 Wilcoxon Rank Sum Test ............................181, 182 streptavidin-AP (Streptavidin conjugated to alkaline phosphatase) .........................51, 59, 71, 214, 215 substrate build-up .............................................. 139, 140 validation .............................................13, 15–16, 26, 27, 31–33, 155–170 ELISPOT suppliers AID Diagnostika .........................................................80 CTL ......................... 7, 12, 15, 16, 18–21, 48, 49, 52, 55, 57, 66, 68, 127, 137, 138, 162, 179, 200, 213, 214, 216, 231–233, 239 Mabtech ................................................ 51, 80, 222, 252 Millipore ...........................50, 68, 80, 106, 108, 116, 222, 226, 244, 245, 251, 252, 255 MVS Pacific ...................................91, 99, 117, 146, 151 R&D Systems....................... 40, 50, 51, 91, 98, 116, 117 Epitope mapping ................................................... 13, 47–61 Epstein Barr virus (EBV) ................................ 7, 27, 79, 231

F FACS......................................................................... 52, 112 Feline immunodeficiency virus (FIV)......................... 47–49, 51–55, 57–59 Feline immunodeficiency virus (FIV) vaccine dual-subtype .................................................... 49, 52, 53 FD–1 adjuvant .............................................................49 Fel-O-Vax®FIV ................................................ 49, 53, 55 FIVPet ..................................................................... 49, 57 FIVShi ........................................................................... 49 IWV .....................................................49, 51–55, 57–59 FIV vaccine. See Feline immunodeficiency virus (FIV) vaccine Frequency measurements ................................8, 9, 12, 15, 20

H Hepatocyte growth factor (HGF) ...................................106 High throughput T cell testing.............................. 12, 13, 66 HiTrap heparin column .....................................................67 HIV vaccine ........................................ 4, 29, 48, 49, 51, 52, 55

I Immune monitoring ..................... 6, 7, 15–16, 26, 28, 33, 34 Immunosuppression ................................ 105–107, 112, 230 Indoleamine 2,3-dioxygenase (IDO)............................... 106

K Keyhole limpet hemocyanin (KLH) ................................237

L Lipopolysaccharide (LPS) ......................57, 98, 99, 101, 159 Luminex .......................................................8, 9, 13, 16, 157

HANDBOOK OF ELISPOT 260 Index M Major histocompatibility complex (MHC) .............7, 12, 13, 48, 49, 55, 65, 66 Media AIM-V® ........................................... 50, 51, 59, 234–236 amphotericin................................................................98 beta-mercaptoethanol .................................... 40, 88, 116 bovine serum albumin (BSA) ................50, 81, 201, 206, 222, 234 conditioned ........................................................106–113 DMEM ............................................... 98, 106, 108, 201 DMSO .......................................................... 67, 69, 202 Eagle’s ........................................................................ 106 fetal bovine serum (FBS) ........................... 106, 221, 251 fetal calf serum (FCS)..........................40, 43, 80, 81, 83, 88, 93, 98, 116, 120, 201, 202, 209–213, 215–217 gentamicin ......................................40, 88, 106, 108, 116 Hanks’ balanced salt solution (HBSS) .................. 50, 91, 201, 222 heat inactivated dog serum ..........................................68 HEPES ....................................40, 80, 88, 106, 116, 221 HIV-seronegative human sera .....................................50 horse serum................................................................106 ISCOVE................................................................67, 69 MEM ........................................................................ 201 penicillin ...................................68, 69, 80, 201, 202, 221 RPMI .................................................. 40, 41, 43, 80, 88, 91–93, 116–118, 120, 202, 208–212, 215, 216, 221, 223, 251 serum free ............................. 7, 18, 20, 30, 169, 176, 179 specific-pathogen-free (SPF) .................................50, 55 Waymouth .............................................................67, 69 Membrane-removal device ............................ 41, 91, 99, 117 MHC. See Major histocompatibility complex (MHC) MIATA ............................................................................. 31 Mixed cellulose ester membranes .................................... 137 Mixed lymphocyte reaction (MLR) .........106, 107, 110–113 Multiplexing .....................................10, 12, 16–17, 137, 140 Myelin basic protein (MBP)..............................................14

N Nitric oxide (NO) ............................................................ 106

P PBST................................................107, 109–111, 222, 223 Peptide library ....................................................... 13, 66, 69 Peripheral blood mononuclear cell (PBMC) apoptotic ...................................................... 19, 176–177 canine .......................................................................... 39 cat ................................................................ 7, 48, 52–57 CD3....................................................................... 60, 84 CD4............................................ 18, 48, 50, 55, 126, 226 CD8.........................4, 18, 48, 50, 55, 126, 186, 199–217

counting ............................ 18, 20, 94, 120, 176, 179, 209 cytotoxic T lymphocyte (CTL) ............7, 15, 48, 66, 200 dog ......................................................................... 66, 68 effector and memory T cell responses ................ 220, 230 equine .................................................................... 40–42 feline ................................................................ 39, 50, 59 freezing ..........................7, 10, 18, 19, 176, 179, 206, 208 HLA-typed ............................................................. 7, 21 horse ............................................................................ 42 infecting with vaccinia virus ...............199–203, 211–212 NK cells ..................................................... 159, 232–234 oxidative stress ............................................89, 90, 92, 93 reference samples ........................................7, 21, 27, 179 resting ..................................... 18–19, 202, 203, 209–210 T cells .................................10, 11, 14, 20, 27, 48, 50, 52, 157, 174, 176, 199–217 Th1 ........................................................................ 48, 93 Th2 ...........................................................78, 88, 93, 200 Th17 ............................................................................ 78 thawing ........7, 18, 19, 174, 176, 179, 202, 203, 208–210 Tregs ............................................................................ 78 trypan blue ........................ 40, 91, 92, 116, 117, 201, 202 Phorbol 12-myristate 13-acetate (PMA) ............. 40–42, 91, 92, 116, 118, 244 Phosphate buffered saline (PBS) .........18, 40, 50, 57, 59, 60, 67, 68, 71, 80–83, 88, 92, 98–100, 102, 107–109, 111, 113, 116, 117, 201, 202, 205, 207–211, 213, 214, 217, 222, 234, 235, 237–239 Plates aluminum foil .......................... 44, 81, 94, 101, 121, 202, 212, 215, 217 autofluorescence.....................................................80, 83 ethanol ..............................................57, 81, 83, 226, 250 IPFL ............................................................................ 80 membranes alcohol pretreatment .................................... 251, 252 blocking ................................................. 83, 116, 251 brittleness.............................................................245 characteristic ........................................................ 250 chemical compatibility .........................................245 chemiluminescence .............................. 246, 250, 252 compatibility with different detection modes ......250 Dot Blot ...................................................... 244, 247 Durapore® ............................................................ 245 flatness ................................................................. 249 fluorescence ......................................................... 250 fluorescence background .............................. 244, 252 Immobilon-FLTM ..............................................252 MAHAS4510......................................................255 MAIPS4510 .........................................222, 226, 255 mechanism of binding ..........................250, 251, 254 methanol ............................... 201, 205, 250, 251, 255 nitrocellulose (NC) ..............226, 243–247, 249–252, 254–256

HANDBOOK OF ELISPOT 261 Index particulates ...........................................................245 polyvinylidene fluoride (PVDF) ............ 50, 226, 243 pore size ............................................... 245, 250, 251 pre-wetting ...................................226, 247, 251–253 protein sequencing ............................................... 246 saturation binding capacity .................................. 250 shedding .............................................................. 245 Slot Blot .............................................................. 247 solvent compatibility ............................................250 surface area ratio ..........................................250, 251 thickness ..............................................................250 wettability ............................................................250 Nunc-Immuno™ washer ......................... 41, 91, 98, 117 washing ................................................ 84, 113, 168, 244 Pokeweed mitogen (PWM).............................................225 Prostaglandin E2 (PGE2) ...............................................106

R Red blood cells (RBC) lysing solution.......................92, 116

S Single cell analysis secretion ......... 3, 6–8, 115–122, 156, 200 Smallpox vaccine .............................................................200 Standardized operating procedures (SOPs) .......... 26, 27, 29, 30, 32, 34 Staphylococcal enterotoxin B (SEB) .......................222, 224

T T cell affinity .............................................................13, 176 T cell avidity ......................... 8, 12–15, 17, 21, 126, 157, 169

T cell mediated immunity ................................. 12, 107, 141 T cell receptor (TCR).................................. 9, 13, 49, 65, 66 Tetanus toxoid (TT) ................................................237, 239 Transforming growth factor-B1 (TGF-B1) ..... 106, 107, 113 Tris ...................................................................106, 201, 204 TrypLE™.......................................................................... 106 Tuberculosis Bacille Calmette-Guérin (BCG) vaccine..................221, 229, 230 bovine ................................................................ 219–227 CFP10 peptides ................................................. 230, 231 ESAT–6..................................................................... 230 human.........................................................219, 221, 234 interferon-gamma (IFN-G) release assay (IGRA) ............ 229–233 Mycobacterium tuberculosis (MTB) ............................. 230 QuantiFERON-TB Gold® (QFT-G)........................ 230 QuantiFERON-TB Gold in Tube® (QFT-GIT) ...... 230 region of difference (RD–1) .......................230, 232, 233 TB 7.7 ....................................................................... 230 T-SPOT.TB® ..................................................... 229–239 tuberculin skin test (TST) ................................. 229–231 Tween 20 ...................................... 68, 71, 107, 213, 222, 250

U Unibrain Fire-i ........................................................ 146, 151 US National Smallpox Vaccinization Program ................ 200

V Vaccinia ................................................................... 199–217

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