Nature Reviews Immunology 2011-1 (January 2011)

ReseaRch highlights in brief i N N AT E i M M U N i T Y Statins enhance formation of phagocyte extracellular traps Cho...

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ReseaRch highlights

in brief i N N AT E i M M U N i T Y

Statins enhance formation of phagocyte extracellular traps Chow, O. A. et al. Cell Host Microbe 8, 445–454 (2010)

Statins inhibit cholesterol synthesis and are prescribed to those at high risk of developing cardiovascular disease. The widespread use of these drugs has promoted interest in their other effects: this study shows that statins affect phagocyte functions. Pre-treatment with statins increased the capacity of human neutrophils and macrophages to kill various bacteria in vitro, but paradoxically, statins decreased the ability of neutrophils to phagocytose Staphylococcus aureus or induce the oxidative burst. Instead, statins promoted microbial killing by inducing phagocyte production of extracellular traps (mesh-like structures composed of nuclear DNA, histones and antimicrobial peptides). In a model of S. aureus-induced pneumonia, pre-treating mice with statins decreased bacterial loads and pathology in the lungs, and this was associated with increased formation of extracellular traps. Patients with pneumonia or sepsis have better survival rates if they are receiving statin therapy; this study may explain these findings. i N F L A M M AT i O N

A role for mitochondria in NLRP3 inflammasome activation Zhou, R. et al. Nature 1 Dec 2010 (doi:10.1038/nature09663)

The NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome is activated in response to pathogens or damaged cells and promotes the maturation of inflammatory cytokines, such as interleukin-1β (IL-1β). It is currently unclear how diverse danger signals activate the NLRP3 inflammasome, but one model proposes that the generation of reactive oxygen species (ROS) is involved. This study shows that stressed mitochondria are a rich source of ROS that trigger inflammasome activation. Inhibition of mitochondrial function led to ROS release and subsequent IL-1β induction in wild-type, but not NLRP3-deficient, macrophages. Healthy cells remove ROS-generating mitochondria through mitophagy, a specialized form of autophagy, and inhibiting mitophagy in macrophages led to inflammasome activation. These findings could explain the link between mitochondrial malfunction and chronic inflammatory diseases. MHC MOLECULES

Structure of a classical MHC class I molecule that binds “non-classical” ligands Hee, C. S. et al. PLoS Biol. 8, e1000557 (2010)

This paper reports the crystal structure of one variant of the polymorphic YF1 MHC class I molecule in chickens, showing that this unusual molecule represents a structural link between the peptide-presenting classical MHC class I molecules and the lipid-presenting non-classical MHC class I molecules (CD1 molecules) that are present in mammals. The YF1*7.1 heavy chain associates with β2-microglobulin to form the typical structure of a classical MHC class I molecule, with anti-parallel β-helices forming the binding groove. However, the binding groove of YF1*7.1 is narrower than that of classical MHC class I molecules and is lined by hydrophobic residues. Binding assays showed that YF1*7.1 can bind lipid antigens, and modelling studies suggested that the type of lipid that is bound might be affected by allelic differences in the binding groove. So, the presentation of a large repertoire of lipids, which is accomplished using multiple non-polymorphic CD1 genes in mammals, might be achieved by multiple alleles of a single YF1 gene in chickens (which have only two CD1 genes).

nature reviews | Immunology

volume 11 | january 2011 © 2011 Macmillan Publishers Limited. All rights reserved

ReseaRch highlights

vIRAL ImmuNIty

Bad memories Seasonal strains of influenza virus cause more severe disease in infants and the elderly, but historically, pan demic strains of influenza virus have induced the most debilitating disease in healthy young adults. A study in Nature Medicine now offers an explanation for this paradox. To explore the mechanisms by which pandemic strains of influenza virus promote disease, the authors characterized samples from patients who had been infected with either pandemic or seasonal strains of influenza virus. Pandemic strains of influenza virus have been pro posed to cause disease by inducing a ‘cytokine storm’; however, naso pharyngeal secretions from patients with pandemic or seasonal influenza contained similar levels of inflamma tory cytokines. Furthermore, mono cytes from healthy donors showed similar cytokine induction following

culture with haemagglutinin 1 (HA1) from pandemic or seasonal influenza virus. The authors next measured the levels of HA1specific antibodies in healthy individuals of different ages. IgG antibodies specific for viral HA1 were not detected in infants, but could be found in sera from young and elderly adults. However, although the HA1specific antibodies from elderly adults could neutralize and protect against a pandemic influenza virus strain from 2009, antibodies from young adults showed lower avidity and could not neutralize the virus. This was despite the fact that antibodies from the young adults showed higher avidity for a seasonal influenza virus. Young adults who became severely ill during the 2009 pandemic had higher serum levels of HA1specific IgG than those who developed mild disease, but the

nATure revIewS | Immunology

HA1specific antibodies from the severely ill individuals showed lower overall avidity for the 2009 influenza virus strain. Together, these data suggest that high levels of low avidity antibody, which was probably generated during previous seasonal influenza virus infections, promoted severe disease during the 2009 influ enza virus pandemic. Lowavidity antibody responses are associated with immune complex mediated disease, and in keeping with this, extensive deposition of the com plement component C4d was seen in lung sections from young adults who were fatally infected during the 2009 pandemic. In addition, although immune complexes were detected in secretions from patients with pandemic influenza, they were rarely found in secretions from patients with seasonal influenza. Finally, extensive deposition of C4d was observed in archived lung sections from adults who died during a 1957 influenza pandemic, indicating that immune complexmediated disease may also have contributed to fatal cases during this pandemic. These findings offer a plausible explanation for the unusual age dis tribution of severe cases that occurs during influenza virus pandemics. Healthy young adults are more likely to have preexisting antibodies to seasonal strains of influenza virus, and these antibodies crossreact with the pandemic strain but are non protective. Instead, the lowavidity antibodies promote the deposition of immune complexes in the lung, leading to severe, and often fatal, respiratory disease.

Yvonne Bordon

ORIGINAL RESEARCH PAPER Monsalvo, A. C. et al. Severe pandemic 2009 H1N1 influenza disease due to pathogenic immune complexes. Nature Med. 5 Dec 2010 (doi:10.1038/nm.2262)

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ReseaRch highlights

in brief i N N AT E i M M U N i T Y

Plexin‑A4–semaphorin 3A signaling is required for Toll‑like receptor‑ and sepsis‑induced cytokine storm Wen, H. et al. J. Exp. Med. 22 Nov 2010 (doi:10.1084/jem.20101138)

This study shows that plexin A4 signalling synergizes with Toll-like receptor (TLR) signalling to promote pro-inflammatory cytokine responses. In the absence of plexin A4, macrophages showed defective production of interleukin-6 and tumour necrosis factor in response to various TLR agonists and bacteria. Activation of TLR signalling components was also defective in plexin A4-deficient macrophages. Physiological relevance for plexin A4-enhanced TLR responses was confirmed by the findings that plexin A4-deficient mice showed a reduced cytokine storm after lipopolysaccharide (LPS) treatment compared with wild-type mice and were protected from lethal challenge with LPS. Plexin A4-deficient mice were also resistant to septic inflammation induced by caecal ligation and puncture. Finally, administration of the plexin A4 ligand semaphorin 3A enhanced LPS-induced cytokine production, suggesting that this pathway could be a target in the treatment of sepsis. i N F L A M M AT i O N

Gene from a psoriasis susceptibility locus primes the skin for inflammation Wolf, R. et al. Sci. Transl. Med. 2, 61ra90 (2010)

The chronic skin inflammation of psoriasis could result from an abnormality of epidermal keratinocytes or from a dysregulated immune response. A combination of these factors is probably responsible for the disease, and a new study supports this idea by showing that S100 proteins expressed by keratinocytes activate an inflammatory cascade through the receptor for advanced glycation end products (RAGE). Transgenic mice overexpressing keratinocyte-restricted S100AA — the single mouse orthologue of the human proteins S100A7 and S100A15, which are encoded in psoriasis susceptibility locus 4 (PSORS4) and are highly expressed by keratinocytes from psoriatic lesions — had an exaggerated inflammatory response to wounding of the skin associated with increased levels of T helper 1 (TH1) and TH17 cell-associated cytokines. In turn, these cytokines further upregulated S100AA expression, showing the therapeutic potential of targeting the S100A7/A15–RAGE axis in psoriasis. MUCOSAL iMMUNOLOGY

T helper type 1 memory cells disseminate postoperative ileus over the entire intestinal tract Engel, D. R. et al. Nature Med. 16, 1407–1413 (2010)

Localized intestinal surgery can disrupt the motility of the entire gastrointestinal tract (a condition termed postoperative ileus); this is thought to result from neuronal dysfunction. Using a mouse model, this study shows that it is not the nervous system but the immune system that drives postoperative ileus. Intestinal manipulation activated local dendritic cells to produce interleukin-12 and induce interferon-γ (IFNγ)-producing T helper 1 (TH1) cells, which had dual roles in postoperative ileus: they drove the inflammation that disrupted the local environment and promoted the spread of intestinal dysfunction by migrating to other areas of the intestine. TH1 cells that were induced in manipulated areas expressed the gut-homing receptor CC-chemokine receptor 9 (CCR9), which may have promoted their spread to other regions of the intestine. Interestingly, in patients undergoing abdominal surgery, the number of IFNγ-producing CCR9+ memory T cells in the blood was markedly increased shortly after the operation, but this population remained stable in patients undergoing non-abdominal surgery. nature reviews | Immunology


ReseaRch highlights

I N f L A m m At I O N

Directions from the matrix Emerging evidence reveals that components of the extracellular matrix can directly regulate inflammatory processes. Now, researchers have identified a role for the matrix component biglycan in the pathogenesis of lupus nephritis through its ability to induce expression of the B cell chemoattractant CXC-chemokine ligand 13 (CXCL13). Biglycan, which exists in the extracellular matrix and as a soluble molecule, has previously been shown to act as an endogenous danger signal by activating Toll-like receptor 2 (TLR2), TLR4 and the NLRP3 (NOD-, LRRand pyrin domain-containing 3) inflammasome. This activity led the authors to investigate whether biglycan is involved in renal inflammation associated with systemic lupus erythematosus (SLE). They first observed that biglycan levels are increased in the plasma and kidneys from patients with lupus nephritis, as well as from MRL–lpr mice and NZB/W F1 mice

(mouse models of SLE). Moreover, plasma biglycan levels in MRL–lpr mice increased progressively over time, coinciding with the initiation and progression of disease. In keeping with a role for biglycan in the pathogenesis of lupus nephritis, biglycan-deficient MRL–lpr mice did not develop the enlarged kidneys, high albuminuria and high serum immunoglobulin levels that occurred in wild-type MRL–lpr mice with disease. Furthermore, transient overexpression of soluble human biglycan increased albuminuria and worsened renal pathology in MRL–lpr mice owing to a large influx of mononuclear cells, including macrophages and T cells. This increase in immune cell infiltration was associated with higher renal and plasma levels of the macrophage and T cell chemoattractants CC-chemokine ligand 2 (CCL2), CCL5 and CCL3. Greater numbers of B cells, mainly B-1 cell populations, also infiltrated the

NaTuRE REvIEWS | Immunology

kidneys of mice that overexpressed biglycan compared with the numbers in control mice. Finally, biglycan was also found to promote the production of active caspase 1 and mature interleukin-1β (products of NLRP3 inflammasome activation) in diseased MRL–lpr mice. Of particular interest to the authors was the finding that CXCL13 levels were increased in diseased mice and reduced in biglycan-deficient MRL–lpr mice. CXCL13 recruits B cells that express CXC-chemokine receptor 5 and has previously been described as a marker of disease activity in SLE. Importantly, in vitro incubation of peritoneal macrophages and splenic dendritic cells from wildtype mice with biglycan triggered CXCL13 production, and this was shown to depend on their expression of TLR2 and TLR4 and not on activation of the inflammasome. Finally, in vivo experiments using mice deficient in TLR2, TLR4 or both TLRs confirmed that biglycan acts through these TLRs to induce the production of pro-inflammatory mediators and CXCL13, which drive the infiltration of immune cells into the kidneys. These data identify a new biglycanmediated mechanism of immune regulation, one that could be involved in other B cell-mediated renal diseases, as suggested by the observation that biglycan and CXCL13 levels are increased in plasma and renal biopsies from patients with acute renal allograft rejection.

Lucy Bird

ORIGINAL RESEARCH PAPER Moreth, K. et al. The proteoglycan biglycan regulates expression of the B cell chemoattractant CXCL13 and aggravates murine lupus nephritis. J. Clin. Invest. 120, 4251–4272 (2010)

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ReseaRch highlights

In the news tb’s protective shield Most individuals infected with Mycobacterium tuberculosis remain asymptomatic and do not develop tuberculosis, despite the persistent presence of the bacteria. Researchers based in India have discovered a new mechanism that could explain how M. tuberculosis can persist in the face of potent immune responses. They say the bug’s trick is to recruit mesenchymal stem cells (MSCs) to the site of infection, where these cells suppress anti-mycobacterial T cell responses (Proc. Natl Acad. Sci. USA, 6 Dec 2010). Joanne Flynn, an immunologist at the University of Pittsburgh School of Medicine, Pennsylvania, USA, acknowledged that this was “a novel finding” and suggested that MSCs “may be an important cell subset for balancing inflammation” (The Scientist, 7 Dec 2010). Indeed, MSCs, which are bone marrow-derived pluripotent stem cells, are known to have immunosuppressive properties. Here, the scientists suggest that these stem cells form “a protective coating around granulomas and produce a range of immunosuppressant molecules, such as nitric oxide” (ABC Online, 7 Dec 2010). MSCs that accumulated at the periphery of granulomas containing live M. tuberculosis organisms secreted nitric oxide, which was shown to inhibit T cell proliferation. The MSCs also promoted the induction of regulatory T cells. Sam Behr of Harvard University, Cambridge, Massachusetts, USA, explained: “What they’re suggesting is that these stem cells are interposed between T cells and the infected macrophages … preventing access of the T cells to the macrophages” (The Scientist). The authors claim that the findings “identify these cells as unique targets for therapeutic intervention in tuberculosis” (Bloomberg, 7 Dec 2010), a disease that causes 2 million deaths each year. Gobardhan Das, senior author of the study, says that “If you can target these MSCs then you can destroy the protective layer and expose the bacteria to the macrophages” (ABC Online). Lucy Bird

nature reviews | Immunology


REVIEWS Innate immune mechanisms of colitis and colitis-associated colorectal cancer Maya Saleh* and Giorgio Trinchieri‡

Abstract | The innate immune system provides first-line defences in response to invading microorganisms and endogenous danger signals by triggering robust inflammatory and antimicrobial responses. However, innate immune sensing of commensal microorganisms in the intestinal tract does not lead to chronic intestinal inflammation in healthy individuals, reflecting the intricacy of the regulatory mechanisms that tame the inflammatory response in the gut. Recent findings suggest that innate immune responses to commensal microorganisms, although once considered to be harmful, are necessary for intestinal homeostasis and immune tolerance. This Review discusses recent findings that identify a crucial role for innate immune effector molecules in protection against colitis and colitis-associated colorectal cancer and the therapeutic implications that ensue. Helminth therapy Helminth therapy is a form of immunotherapy aimed at modulating the T helper 1 (TH1)/TH2 immune balance. It involves the deliberate inoculation of patients suffering from immune-mediated inflammatory diseases with helminth (parasitic intestinal nematodes) or helminth larvae. The currently studied regimens in humans use Trichuris suis, which does not cross the intestinal barrier or cause an invasive infection.

*Department of Medicine, McGill University, Montreal, Quebec, H3G 0B1 Canada. ‡ Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA. e-mails: maya.saleh@mcgill. ca; [email protected] doi:10.1038/nri2891 Published online 10 December 2010

Innate immunity is the body’s alarm system. It senses the presence of ‘foreign’ entities derived from either infections (non-self recognition) or tissue damage (alteredself recognition) and confers protection by actively inducing inflammatory, anti-microbial and anti-stress responses. It also serves to alert and prime the adaptive immune system in case the insult persists. However, dysregulation of these pathways can lead to severe inflammatory and immunological diseases. Inflammatory bowel diseases (IBDs) are inflammatory disorders of the intestinal tract that are most common in developed countries, affecting the quality of life of roughly 1.4 million individuals in the United States and 2.2 million in Europe, mainly of Caucasian descent1,2. The two main types of IBD are Crohn’s disease and ulcerative colitis, which share as a feature an overactive immune response to the intestinal microbiota but differ in the site and nature of the inflammatory pathology. Management of IBD has so far relied on nonspecific immunosuppressive therapies (such as steroids), antibiotics, and biologicals targeting mainly the proinflammatory tumour necrosis factor (TNF) pathway 3,4; however, these treatments are not effective in all patients. In addition, probiotics5 and ‘helminth therapy’6 have been used to modulate the abnormal inflammatory response and have shown promise in various clinical trials. One of the consequences of chronic inflammation is the promotion of tumorigenesis, and patients with IBD have a higher risk of developing colitis-associated colorectal cancer with an odds ratio of approximately three7–10. However, most colorectal cancers develop without any

obvious pre-existing inflammatory pathology. Colorectal cancer is the third most common malignancy in humans, with the highest frequencies observed in North America, Europe and Australia and the lowest in Africa, Asia and South America11. Interestingly, Asian populations that migrate to North America acquire the same risk of colorectal cancer as local populations within one generation12. This suggests that environmental differences and particularly alimentary customs, which probably affect the commensal microbiota, are responsible for the geographical variation in colorectal cancer incidence. As discussed in this Review, recent findings indicate that the sensing of commensal microorganisms by the innate immune system maintains intestinal homeostasis and induces healing responses following injury that confer protection against colitis and colorectal cancer. In addition to inducing antimicrobial responses, this crosstalk also triggers cell survival, autophagy and mucosal regeneration, which are all salutary for the host. Consistently, genetic mutations in the innate immune system that alter the host–microbiota equilibrium and affect epithelial cell regeneration result in enhanced susceptibility to experimental colitis and carcinogenesis. Paradoxically, mutations in negative regulators of innate immunity pathways also favour carcinogenesis by breaking mucosal tolerance to commensal microorganisms and thus amplifying the inflammatory response in the gut. Here, we discuss the mechanisms used by the innate immune system in the intestine in light of recent advances in the study of the microbiology, immunology and genetics of IBD and colon cancer.

NATURE REvIEwS | Immunology

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REVIEWS Autophagy A tightly regulated catabolic process that involves degradation of the cell’s own intracellular components through the lysosomal machinery. It is a normal part of cell growth, development and homeostasis, and recently it has emerged as an efficient innate immunity mechanism.

Villi Villi are projections into the intestinal lumen. Their outer epithelial layer consists mainly of mature, absorptive enterocytes but also contains mucus-secreting goblet cells.

Crypts Crypts are tubular invaginations of the intestinal epithelium. At the base of the crypts are Paneth cells, which produce bactericidal defensins, and stem cells, which continuously divide and are the source of all intestinal epithelial cells.

The pros and cons of the microbiota Commensal microorganisms contribute to host defence by limiting the growth of potentially harmful enteric pathogens, and can control intestinal inflammation and pathology by producing symbiosis factors. For example, Bacteroides fragilis protects animals from experimental colitis induced by Helicobacter hepaticus through the synthesis of polysaccharide A, which suppresses interleukin-17 (Il-17) production in the intestine and promotes the function of Il-10-producing CD4+ T cells13. Through crosstalk with the innate immune system, the microbiota also regulates intestinal homeostasis by providing instructive signals that drive intestinal epithelial cell (IEC) turnover and maturation (see below), lymphocyte development and the conditioning of the immune system both at mucosal sites14,15 and systemically 16. In addition to regulating the inflammatory environment in the intestine, the microbiota has a direct impact on intestinal homeostasis and pathology through the generation of growth factors and hormones or by affecting how natural mutagens and carcinogens are catabolized by the body 17. However, these latter effects will not be discussed further in this article. Despite this mutualism between the microbiota and the host (BOX 1), changes in the composition of the intestinal microbiota or colonization with specific pathogens may alter intestinal homeostasis and the nature of the immune response and may result in spontaneous colitis and/or tumorigenesis. The constant threat posed by commensal organisms is exemplified by the intestinal microbiota of T-bet−/−Rag2−/− ulcerative colitis (TRUC) mice, in which spontaneous colitis and subsequent colorectal cancer develop because of alterations in both innate and

Box 1 | The human–microbiota mutualism The human body, which consists of 1013 cells, is colonized at birth by approximately ten times as many microorganisms. The maternal flora is a major determinant of the eventual composition of that of the progeny120, and the colonization continues for several days after birth with the exposure to the external environment. The intestinal tract is the largest reservoir of human flora and is densely colonized by diverse species of microorganisms: predominantly bacteria, but also protozoa and fungi. Although it is estimated that the microbiota contains 500–1,000 different species of bacteria, 99% of these are predicted to originate from around 30–40 species. The composition of the flora varies widely among individuals, depending on kinship, age, diet, lifestyle changes and stress. There is also variation between different anatomical locations, with the number of bacteria increasing dramatically in the more distal portions of the gastrointestinal tract (the cecum and the colon). Collectively, the gut microbiome contains around 100 times as many genes as there are in the human genome. Recent efforts, through human microbiome projects (for example, the National Institutes of Health (NIH) Roadmap Human Microbiome Project and the European Commission MetaHIT project), are aiming at identifying microbiome common cores (common sets of microbial species) at different anatomical sites and characterizing microorganisms that are associated with health and disease. An intestinal microbiome core has been recently reported, and variations from this core have been associated with obesity121. The mammalian host and its commensal microorganisms share a mutually beneficial, symbiotic and dynamic relationship. The host provides a rich habitat that is suitable for microbial survival and replication and, in return, commensal microorganisms, through their metabolic activities, aid in host physiological functions such as fermentation, digestion and absorption of dietary polysaccharides, synthesis of vitamins, and regulation of host genes needed for deposition of lipids in adipocytes and fat storage122,123. However, this symbiosis extends beyond mere substrate exchange (see main text).

adaptive immune responses18. Recombination-activating gene 2 (RAG2) deficiency eliminates adaptive immune responses, whereas T-bet deficiency in the innate immune system results in excessive TNF production by dendritic cells (DCs). This imbalance in mucosal immunity affects the composition of the gut microbiota, leading to a population of commensal organisms that can transmit the disease to wild-type mice18,19. In particular, two bacterial species, Proteus mirabilis and Klebsiella pneumoniae, are associated with colitis in TRUC mice and, in conjunction with an endogenous microbial community, transmit colitis to wild-type mice20. A second example of a modulatory effect of the microbiota on the mucosal immune response is the association between segmented filamentous bacteria (SFB) colonization (found in mice from certain commercial animal production facilities) and a predominance of T helper 17 (TH17) cells21. Interestingly, colonization of the gut by SFB also exerts systemic effects, as it is sufficient to enhance TH17 cell responses and induce arthritis in a T cell receptor (TCR)-transgenic mouse model of this disease22. Indeed, disruption of host–microbiota homeostasis in the intestine can have consequences on distant organs, resulting in DNA damage, autoimmunity and cancer 23,24. Commensal microorganisms therefore present a constant threat after a mechanical, physical or immunological breach of the intestinal barrier, and this forms the basis of local, as well as some systemic, inflammatory-mediated immune diseases.

Intestinal homeostasis pathways The unique microbial environment of the intestine places the innate immune system at the centre of intestinal homeostasis. This system is not simply a host defence mechanism against invading pathogens, functioning solely in direct killing of microorganisms; it also modulates bacterial handling through autophagy (see below) and affects IEC proliferation, differentiation and survival. As such, the innate immune system is an important determinant in the onset and development of IBD and intestinal cancers. Several host innate immune mechanisms have evolved to deal with the challenge posed by the microbiota. Anatomically, a physical barrier formed by a single layer of columnar IECs, arranged into villi and crypts and covering a surface area of approximately 100 m2, shields the rest of the body from the commensal microorganisms that reside in the intestinal lumen. Multipotent stem cells located at the base of the crypts replenish IECs and regenerate the mucosa in response to tissue injury. Accumulating evidence indicates that IEC barrier integrity and underlying immune tolerance depend on the crosstalk between commensal microorganisms and the innate immune system. For instance, prostaglandin-endoperoxide synthase 2 (PTGS2; also known as COX2)-expressing mesenchymal cells in the crypts sense the microbiota through Toll-like receptors (TlRs), and this is required to maintain the stem cell niche25. Alterations in this crosstalk, as occur in mice deficient in the TlR and Il-1 receptor signalling adaptor molecule MyD88 (myeloid differentiation primary response protein 88), lead to susceptibility to experimental colitis26.

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REVIEWS

Tight junction A ring of proteins that seals apical epithelium; these proteins include the integral membrane proteins occludin and claudin, in association with cytoplasmic zonula occludins proteins.

Lamina propria The connective tissue that underlies the mucosal epithelium and contains various myeloid and lymphoid cells, including macrophages, dendritic cells, B cells and T cells.

In addition to the presence of stem cells, there are four differentiated cell types within the intestinal epithelium that have fundamental roles in instating barrier function through specialized physical, biochemical and immunological mechanisms. The predominant IECs are the absorptive enterocytes. These cells exit the crypts and form the surface epithelium that restricts commensal microorganisms and other antigenic particles to the lumen through an apical brush border and intercellular tight junctions. Alterations in the composition and structure of tight junctions have been noted in inflamed intestinal segments of patients with Crohn’s disease27, and targeted disruption of IEC tight junctions contributes to the development of experimental colitis28. Goblet cells reinforce the enterocyte barrier by secreting mucins, which form a polysaccharide- and glycoproteincontaining glycocalyx bilayer, in which the firm inner layer is devoid of bacteria29. Deficiency in mucin 2, the most abundant gastrointestinal mucin, or missense mutations in its gene, lead to the development of spontaneous chronic colitis and colorectal cancer 30–32. Enteroendocrine cells and Paneth cells, found in the proximity of the stem cell zone, exert innate immune functions by secreting lysozymes and a broad range of antimicrobial peptides, including α-defensins, β-defensins, cathelicidins, calprotectins, lipocalins and the C-type lectin REG3γ (regenerating islet-derived protein 3γ)33. Paneth cells are not found in the colon but antimicrobial products are also produced by colonocytes and have a homeostatic role in the gut by regulating microbial ecology and the makeup of commensal bacteria34. A balance between cell death and survival is key for the maintenance of intestinal homeostasis. IECs are eliminated daily by apoptosis, yet resistance to apoptosis is essential to maintain barrier function and dysregulation of apoptosis has been linked to intestinal pathologies. Interestingly, inflammation and innate immunity are tightly intertwined with apoptosis, and the net outcome is likely to be a consequence of the thresholds set by endogenous inhibitors and counteracting mechanisms. For example, nuclear factor-κB (NF-κB), a master transcriptional regulator that is activated by various cytokine receptors and pattern-recognition receptors (PRRs), controls the expression of pro-inflammatory mediators such as TNF and enhances the survival of cells through the induction of anti-apoptotic genes35. However, TNF triggers both cell survival and cell death depending on the cellular context. Enterocyte-specific ablation of NF-κB signalling — through conditional deletion of NF-κB essential modulator (NEMO; also known as IKKγ) or both IκB kinase-α (IKKα) and IKKβ — leads to spontaneous enterocyte apoptosis and massive intestinal inflammation36. Similarly, mice with IECspecific deletion of the NF-κB component RElA exhibit increased susceptibility to chemically induced colitis37. In these mice, IEC apoptosis is completely driven by TNF, as inhibition of TNF receptor 1 (TNFR1) prevents the development of intestinal inflammation. Furthermore, the E3 ligase editing enzyme A20 (also known as TNFAIP3) terminates NF-κB signalling and inhibits TNF-induced apoptosis, and A20 mutations

have been associated with Crohn’s disease38. Enterocytespecific deficiency of A20 results in susceptibility to experimental colitis owing to a hyper susceptibility to TNF-induced apoptosis, further supporting a role for TNF-induced apoptosis in barrier disruption 39. Thus, we argue that induction of IEC apoptosis is one mechanism by which TNF sustains chronic inflammation in the intestine and that IEC resistance to TNFinduced apoptosis is crucial for protection against colitis. Consistently, TNF-targeting biologicals confer protection in IBD by differentially modulating apoptosis in the gut. Indeed, TNF-blocking antibodies have been proven to inhibit IEC apoptosis and restore barrier function in patients with Crohn’s disease40, whereas they induce apoptosis of lamina propria mononuclear cells41, which contributes to the resolution of the inflammatory response. However, although preventing IEC apoptosis is beneficial in colitis, impaired IEC death is linked to colitis-associated colorectal cancer. Indeed, enterocytespecific deletion of IKKβ leads to decreased tumour incidence owing to enhanced IEC apoptosis in the absence of NF-κB anti-apoptotic target gene expression42. Both environmental triggers and genetic predisposition affect IEC maintenance and survival in IBD and intestinal cancers, and innate immune mechanisms appear to orchestrate the fate of IECs and mucosal homeostasis. In the case of tissue injury, the innate immune system senses the damage and shifts the homeostatic balance towards IEC proliferation, restoration and production of cytoprotective and repair factors, which together trigger tissue repair. we propose that impairment of these responses results in the translocation of commensal microorganisms to the lamina propria, and this leads to excessive stimulation of resident immune cells, chronic inflammation, colitis and colitis-associated colorectal cancer. The innate immune system is thus generally protective in the context of tissue injury. However, colorectal cancer can also arise in the absence of tissue injury because of intrinsic mutations in oncogenes or tumour suppressor genes. In this instance, the innate immune system is deleterious, as it promotes tumorigenesis through the induction of inflammation in the intestine (see below).

Innate immunity, colitis and tissue repair TLRs and MYD88. Triggering of TlRs culminates in cellular responses aimed at killing microorganisms while preserving host cell integrity, and these responses include antimicrobial peptide production, inflammation, maturation of antigen-presenting cells and induction of tissue repair and cell survival pathways43. In the gut, multiple tolerance mechanisms ensure minimal TlR activation by commensal microorganisms. TlRs are expressed at low levels on IECs and are mostly distributed in endosomal vesicles or on the basolateral surface away from luminal content44. In addition, TlR signalling is blunted through distinct molecular mechanisms including: expression of inhibitory molecules such as Toll-interacting protein (TOllIP), single immunoglobulin Il-1-related receptor (SIGIRR; also known as TIR8), Il-1R-associated kinase 3 (IRAK3; also known as IRAK-M) and A20



REVIEWS (Ref. 44);

a paucity to respond to commensal products upon constant exposure33,45; and conditioning of resident leukocytes towards an anti-inflammatory phenotype46. Uncontrolled TlR activation, such as in mice deficient in SIGIRR (an inhibitory member of the TlR and Il-1R families), leads to defects in intestinal homoeostasis in

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Wnt pathway A signalling pathway that regulates cell fate determination, proliferation, adhesion, migration and polarity during development. In addition to the crucial role of this pathway in embryogenesis, Wnt ligands and their downstream signalling molecules have been implicated in tumorigenesis and have causative roles in human colon cancers.

Figure 1 | Innate immune effector molecules function in IECs to maintain intestinal homeostasis. Apical 0CVWTG4GXKGYU^+OOWPQNQI[ Toll-like receptor 9 (TLR9) stimulation leads to tolerance via the activation of the Wnt pathway and induction of type I interferon (IFN) production, whereas basolateral TLRs trigger inflammation and tissue repair partially through nuclear factor-κB (NF-κB). Apical TLR9 also leads to accumulation of NF-κB inhibitor α (IκBα), which blocks NF-κB activation. SIGIRR (single immunoglobulin IL-1-related receptor) inhibits TLR signalling. Activation of nucleotide-binding oligomerization domain (NOD) proteins expressed by colonocytes and ileal intestinal epithelial cells (IECs) triggers the release of antimicrobial peptides including defensins that maintain homeostasis by regulating the composition of the microbiota. NOD signalling also leads to the production of tissue repair factors including tumour necrosis factor (TNF), interleukin-6 (IL-6) and CXC-chemokine ligand 1 (CXCL1). NF-κB is one of the downstream effector transcription factors that induce the expression of pro-inflammatory and pro-survival factors. Stimulation of NOD-, LRR- and pyrin domain-containing 3 (NLRP3) by tissue damage and/or commensal-derived products leads to the recruitment of caspase 1 through apoptosis-associated speck-like protein containing a CARD (ASC), forming the inflammasome. This activates caspase 1, enabling it to process pro-IL-18 into its active cytokine form (IL-18), which is required for compensatory proliferation of IECs and for tissue repair. This process is inhibited by caspase 12, which antagonizes the NLRP3 inflammasome. MYD88, myeloid differentiation primary response protein 88.

the steady state. These defects depend on the presence of commensal microorganisms and include persistent IEC survival and enhanced expression of pro-inflammatory cytokines (fIG. 1). Notably, these defects render Sigirr–/– mice susceptible to experimental colitis induced with the IEC cytotoxic agent dextran sulphate sodium (DSS) and to colitis-associated colorectal cancer induced with the procarcinogen azoxymethane (AOM) in conjunction with chronic DSS treatment (BOX 2). Interestingly, tissue-specific transgenic expression of SIGIRR by IECs in Sigirr–/– mice restores immune tolerance and abrogates the susceptibility of Sigirr–/– mice to colitis and tumorigenesis47. Thus, exaggerated TlR signalling in IECs contributes to the development of intestinal pathologies. Nevertheless, although excessive TlR activation in the gut is pathogenic, sensing of the commensal flora by TlRs, particularly by TlR2 and TlR4, is required for intestinal homeostasis and control of tissue repair. MyD88-deficient mice, which cannot signal through Il-1 family receptors and most TlRs have significant defects in the mucosa with increased numbers of proliferating cells in the crypts48. This results in deficient repair of the intestinal barrier following radiation or chemical injury and enhanced susceptibility to colitis and colitisassociated colorectal cancer 48,49. Mice in which the commensal flora is decreased by antibiotic treatment have a similar phenotype to that of MyD88-deficient mice and have very low constitutive expression of factors (such as Il-6, TNF, CXC-chemokine ligand 1 (CXCl1) and heat shock proteins) that are important in preserving intestinal homeostasis50 (fIG. 1). The polarity of IECs also has a major role in regulating the response of TlR9 to bacterial DNA51. TlR9 is expressed both on the apical and basolateral membrane in polarized epithelial cells but only intracellularly in haematopoietic cells. Stimulation of basolateral TlR9 induces NF-κB activation and a pro-inflammatory response, whereas that of apical TlR9 leads to accumulation of NF-κB inhibitor α (IκBα), which blocks NF-κB activation. Apical TlR9 engagement triggers tolerance to various microbial stimuli and results in the production of ligands of the Wnt pathway that regulate the production of antibacterial factors and activate an alternative wnt pathway-dependent antimicrobial mechanism51 (fIG. 1). Thus, activation of apical TlR9 by commensal bacteria contributes to intestinal homeostasis by suppressing inflammation through a mechanism that is partly due to the induction of type I interferons (IFNs), which have been proposed to protect against colonic inflammation by preventing epithelial barrier dysfunction 52. In the presence of mucosal damage, however, microbial translocation takes place and leads to basolateral activation of TlR9, which results in a classical pro-inflammatory response51. NOD2. Mounting evidence points to a paramount role of NlRs (nucleotide-binding oligomerization domain (NOD)- and leucine-rich repeat (lRR)-containing proteins) in the intestine, and their dysregulation has been linked to IBD and colitis-associated colorectal cancer in experimental animal models. Notably, gene-linkage



REVIEWS Adenomatous polyposis coli (APC). A scaffold protein that sequesters β-catenin in the cytoplasm of resting cells. APC mutations, which are found in 90% of intestinal cancers, lead to constitutively active β-catenin.

Box 2 | Animal models of colorectal cancer Various rodent experimental models have been established that reproduce aspects of human colorectal cancer. These models are based on genetic alteration of colorectal cancer pathways (such as Wnt–β-catenin, mismatch repair and transforming growth factor-β (TGFβ) pathways), modulation of factors involved in the mucosal immune response (such as interleukin-2 (IL-2), IL-10, β2-microglobulin, T cell receptor α-chain and G protein-coupled receptors) or treatment with carcinogens, particularly azoxymethane (AOM) or its precursor 1,2-dimethylhydrazine (DMH)124,125. Both DMH and AOM are procarcinogens with organotropism for the colon, and they need to be further metabolized into methylazoxymethanol to induce methylation of the O6 position of guanine, the primary pro-mutagenic lesion produced by AOM treatment125. Different mouse strains are variably susceptible to AOM-mediated induction of colorectal cancer: A/J mice are the most susceptible strain whereas the widely used C57Bl/6 mouse strain has low but variable susceptibility depending on the sub-line (for example C57Bl/6N, C57Bl/6J and C57Bl/6Ha mice)125. AOM induction of colorectal cancer is linked to the ability of AOM to induce mutations in genes that regulate several signalling pathways, including the KRAS, SRC–PI3K–AKT, Wnt–β-catenin, TGFβ and p53 pathways126. Unlike human colorectal cancer, in which adenomatous polyposis coli (APC) mutations are frequent and β-catenin mutations are present in only a minor subset of patients, in the mouse AOM model mutations in β-catenin predominate127. AOM-induced tumorigenesis is significantly enhanced by chronic colitis. Indeed, the number of adenomas and adenocarcinomas is dramatically increased when AOM administration is coupled with repeated treatments with dextran sulphate sodium (DSS), mimicking the pathological process of colitis-associated cancer128. The ApcMin/+ mouse model is among the most widely used mouse genetic models of colorectal cancer. ApcMin/+ mice are heterozygous for a nonsense mutation at codon 850 of Apc, the murine homologue of the human APC gene129. This mutation is analogous to one seen in patients with familial adenomatous polyposis. ApcMin/+ mice develop numerous tumours in the small intestine, and under some conditions develop colorectal cancers. These mice are also more susceptible to mammary and alveolar neoplasia compared with wild-type mice129. AOM treatment of ApcMin/+ mice induces tumorigenesis in the colon, which is otherwise rare in untreated mice. The mutant mice also form larger polyps after AOM treatment than wild-type mice. Interestingly, unlike AOM-induced colorectal cancer in wild-type mice, tumours that form in the colon of ApcMin/+ mice following AOM treatment do not harbour β-catenin mutations130.

studies and genome-wide association (GwA) studies of patients with Crohn’s disease have consistently identified mutations and single-nucleotide polymorphisms (SNPs) in the NOD2-encoding gene CARD15 (Ref. 53). NOD2 contains ten carboxy-terminal lRRs that mediate its ability to sense bacterial peptidoglycans, particularly mycobacterial N-glycolyl muramyl dipeptide (MDP)54. Most of the Crohn’s disease-associated mutations in CARD15 fall within this lRR region, resulting in reduced affinity for MDP. It is generally accepted that alterations in NOD2 function affect susceptibility to IBD because of the key role of this receptor in linking innate signals to the induction of adaptive immune tolerance to the intestinal microbiota. However, the exact mechanism or mechanisms by which defects in NOD2 signalling lead to pathology in Crohn’s disease have been heavily debated. At least five non-mutually exclusive models have been proposed (fIG. 2). The first model conjectures that NOD2 is a negative regulator of TlR signalling and that deficiency in NOD2 function leads to dysregulated TlR signalling, inflammation and colitis55. Consistently, MDP-mediated activation of NOD2 protects animals from experimental acute colitis56. It is possible, however, that the effect of MDP in this study was due to the triggering of IEC compensatory proliferation and tissue repair rather than inhibition of TlR-induced inflammation. The second model suggests that NOD2 signalling leads to polarization of the adaptive immune response towards a TH2-type response and that defects in NOD2 activation lead to excessive TH1 and TH17 cell-mediated inflammation57. The third model proposes that NOD2 is essential for the production of α-defensins by Paneth cells and that alterations in this process change the composition

of the microbiota or result in an overgrowth of pathogenic bacteria, leading to the characteristic granulomatous inflammation of the ileum in Crohn’s disease. Consistent with this third model, it has recently been reported that Nod2–/– mice that are inoculated with the opportunistic commensal pathogen H. hepaticus develop ileal inflammation, which was suppressed by the transgenic expression of α-defensin 5 by Paneth cells of Nod2–/– mice58. However, the reduced α-defensin production in the ileum of patients with Crohn’s disease has been shown to be independent of the CARD15 genotype59. Similarly, it has been reported that patients with Crohn’s disease have a suppressed inflammatory response irrespective of whether or not they carry a mutation in CARD15 (Ref. 60). The fourth model proposes a mechanism that applies to the regulation of human but not rodent NOD2 signalling, emphasizing the importance of interrogating immunological mechanisms in humans. It has been shown that mutant human NOD2 actively inhibits the expression of the anti-inflammatory cytokine Il-10 by suppressing the activity of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1)61. In the fifth model, NOD2 is proposed to trigger autophagy in response to bacterial sensing 62. Autophagy is a catabolic process that is essential for maintaining cell homeostasis and is also required for bacterial clearance and antigen presentation by DCs62. Defective autophagy has been linked to increased susceptibility to infectious diseases, both in vitro and in vivo63. Recent findings from GwA studies of patients with Crohn’s disease have identified two autophagy loci, ATG16L1 (autophagyrelated 16-like 1) and IRGM (immunity-related GTPase family M), that are linked to Crohn’s disease susceptibility 64–66, suggesting that inefficient autophagy or handling



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Figure 2 | Proposed mechanisms of noD2 function in intestinal homeostasis. In addition to being expressed by ileal intestinal epithelial cells0CVWTG4GXKGYU^+OOWPQNQI[ (IECs) and colonocytes, nucleotide-binding oligomerization domain 2 (NOD2) is predominantly expressed by myeloid cells such as macrophages and dendritic cells. Five different models have been described to account for the role of NOD2 in suppressing the inflammatory response in the gut. The first proposes that NOD2 inhibits Toll-like receptor (TLR) signalling (a). The second describes a role of NOD2 in skewing the T helper (TH) cell response towards TH2 cells (b). The third implicates NOD2 in α-defensin production and subsequent limitation of commensal bacterial numbers and microbiome composition (c). The fourth argues that human NOD2 stimulates the production of the anti-inflammatory cytokine interleukin-10 (IL-10) by regulating heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) (d) and that mutant NOD2 inhibits this process. Finally, the fifth model conjectures that NOD2 stimulates autophagy by interacting with autophagy-related 16-like 1 (ATG16L1), which inhibits the inflammasome thereby suppressing the production of the pro-inflammatory cytokines IL-1β and IL-18 (e). NF-κB, nuclear factor-κB.

Inflammasome A large multiprotein complex formed by a NOD- and LRR-containing (NLR) protein, the adaptor protein apoptosisassociated speck-like protein containing a CARD (ASC; also known as PYCARD) and pro-caspase 1. The assembly of the inflammasome leads to the activation of caspase 1, which cleaves pro-interleukin-1β (pro-IL-1β) and pro-IL-18 to generate the active pro-inflammatory cytokines.

of enteric bacteria in genetically compromised individuals may contribute to disease pathogenesis. Interestingly, NOD2 was reported to interact with ATG16l1 to stimulate autophagy 67,68. DCs expressing Crohn’s disease risk variants of NOD2 or ATG16l1 (NOD2 l1007fsinsC (NOD2fs; a truncated protein resulting from a single nucleotide insertion and associated frameshift) or ATG16l1 T300A) display reduced induction of autophagy following stimulation of NOD2, and this results in reduced bacterial killing and defective antigen presentation67. It has been proposed that NOD2fs prevents ATG16l1 from localizing to sites of bacterial entry by retaining it in the cytoplasm68. Furthermore, ATG16l1-deficient mouse Paneth cells exhibit ultrastructural alterations and impaired secretion of antimicrobial peptides into the intestinal lumen in response to infection69. This phenotype seems to be triggered by infection with certain strains of norovirus (that are endemic in most animal colonies) and is dependent on the presence of commensal microorganisms70, suggesting that in the absence of autophagy, mucosal damage

induced by the virus alters the physiological interaction with the microbiota, leading to inflammation and colitis. whether this is fully dependent on NOD2 is not clear, but it is consistent with the dampened production of antimicrobial peptides observed in Crohn’s disease59. Moreover, ATG16l1-deficient macrophages exhibit enhanced responsiveness to TlR stimulation and exaggerated activation of the inflammasome71, which is similarly linked to increased susceptibility to experimental colitis71 (fIG. 2). Together these results indicate that aberrant bacterial handling could act as a trigger for inflammation in Crohn’s disease. The inflammasomes. Unlike NOD2, which triggers inflammation by activating NF-κB and mitogen-activated protein kinase (MAPK) pathways72, most NlRs recruit and activate inflammatory caspases in macromolecular complexes termed inflammasomes 73. These NlRs include NlRP (NlR family, pyrin domain-containing) proteins, NlRC4 (NlR family, CARD-containing protein 4; also known as IPAF) and NAIP (NlR family, apoptosis inhibitory protein; also known as NlRB1). Caspase 1 (encoded by CASP1) is the main effector inflammatory caspase; it directly processes pro-Il-1β and pro-Il-18 into their mature biologically active forms and induces an inflammatory form of cell death termed pyroptosis74,75. This process is distinct from apoptosis, which is immunologically silent. By contrast, caspase 12 is a repressor of the inflammasome and a molecular ‘brake’ on caspase 1 activity 76–78 (fIG. 1). A recent report has identified SNPs in a regulatory region downstream of the human NLRP3 gene, and these SNPs were found to be associated with Crohn’s disease susceptibility in individuals of European descent 79. SNPs in this region result in decreased NlRP3 expression and dampened Il-1 family cytokine production79. Of these Il-1 family cytokines, Il-18 is probably the most relevant for Crohn’s disease80. Il-18 is generally considered as a pro-inflammatory cytokine. It was originally described as an inducer of IFNγ, functioning mainly by amplifying the effect of other IFNγ inducers such as Il-12, and it was later shown to also enhance the production of other cytokines including TH2 cell-associated cytokines. The functions of Il-18 are complex and their possible contribution to the maintenance of chronic inflammation in the intestine is unclear. Il-18 receptor accessory protein (IL18RAP) polymorphisms have been associated with IBD81, whereas genetic data on polymorphisms of the IL18 gene promoter have been controversial82. Several studies have suggested that Il-18 could be an effector cytokine in IBD as circulating or local Il-18 levels have been associated with disease severity 83. For instance, it has been shown that Il-18 is required for DSS-induced colitis83,84, and this is consistent with its pro-inflammatory role. However, more recent investigations have shown that Il-18- or Il-18R-deficient mice are more susceptible rather than resistant to DSSinduced colitis and colitis-associated colorectal cancer 49,85,86. The mechanism by which Il-18 confers its protective effect on the colonic mucosa is reminiscent of its role in wound healing and repair in the skin87, but



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Pyroptosis A form of cell death that is distinct from immunologically silent apoptosis. It is triggered concomitantly with the activation of the inflammasome and requires caspase 1 activity. During pyroptotic cell death, an inflammasome complex forms to activate caspase 1.

whether it acts directly or indirectly on IECs remains to be investigated. However, excessive production of Il-18 is pathogenic: hyperactivation of the inflammasome and subsequent elevated production of Il-1β and Il-18 by macrophages results in enhanced susceptibility to DSSinduced colitis71. Thus, it appears that Il-18 exerts a dual role in intestinal homeostasis and colitis. Early in the mucosal immune response, its expression by IECs mediates a protective effect88, but under chronic inflammation its excessive production by IECs and lamina propria mononuclear cells results in deleterious effects85,88. we and others have recently demonstrated a role for caspase 1 activation by the inflammasome in epithelial cell regeneration and tissue repair following injury in mice89–91 (fIG. 3). Casp1–/– mice are susceptible to DSS-induced injury with early mortality compared with wild-type animals. This phenotype is primarily ascribed to a lack of Il-18 production by Casp1–/– mice, as it is completely reversed by exogenous administration of this cytokine89,90. Regulation of the function of caspase 1 by caspase 12 is necessary for immune tolerance in the gut. Casp12–/– mice, in which the inflammasome is derepressed, are resistant to acute and chronic colitis89. However, as detailed below, the excessive repair response in these mice, together with an enhanced inflammatory response, renders them significantly more susceptible to colitis-associated tumorigenesis. Therefore, a physiological level of inflammasome activation, triggered by the commensal microbiota in the presence of mucosal injury, is necessary for epithelial cell regeneration and is protective from colitis and colitis-associated colorectal cancer (fIG. 3).

Innate immunity and colorectal cancer Animal models of colorectal cancer (BOX 2) have provided much information on the role of inflammatory mediators in the development of colitis-associated colorectal cancer. In particular, these studies have focused on the roles of innate immune cells, cytokines (such as TNF, Il-1, Il-6, Il-10, Il-11, Il-17, Il-18, Il-22 and Il-23), and the signal transducer and activator of transcription 3 (STAT3) and NF-κB axes92. Notably, the activation of STAT3 downstream of Il-6 and Il-11 signalling has a central role in intestinal mucosal regeneration after injury and in the development of colitis-associated colorectal cancer 93–95 (fIG. 4). The link between inflammation and gastrointestinal cancers is widely established; the canonical example is that of Helicobacter pylori and promotion of gastric cancer. Similarly, enterotoxigenic B. fragilis, which can activate the survival and inflammatory pathways mediated by wnt and NF-κB96, and attaching and effacing Escherichia coli strains, which induce severe colitis and downregulate genes encoding DNA mismatch repair proteins97, have been linked to increased risk of colorectal cancer. The important role of the commensal flora in the development of colon cancer is further underlined by the above-mentioned studies showing that alterations in the gut microbiota that induce colitis in TRUC mice also induce spontaneous progression to colonic dysplasia and rectal adenocarcinoma18. lack of tumour formation in germ-free mice was also observed in several genetic models of colorectal cancer (Il10–/– mice, Gpx1–/–Gpx2–/– mice and Tcrb–/–Trp53–/– mice)



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Figure 4 | A dual role for innate immunity in colitis‑associated colorectal cancer and intestinal malignancy. a | Signalling by the inflammasome, caspase 1, interleukin-18 (IL-18)–IL-18 receptor (IL-18R) and myeloid differentiation primary response protein 88 (MYD88) regulates the homeostasis of the intestinal epithelium and stimulates tissue repair after injury. Loss of these innate immune effector molecules impairs tissue repair, leading to microbial translocation to the lamina propria and chronic stimulation of mononuclear cells. This triggers excessive inflammation and tumour necrosis factor (TNF)-dependent intestinal epithelial cell (IEC) death, which exacerbates the vicious cycle of colitis. Ultimately, the inflammatory environment induced by damage and bacterial translocation promotes the development of colitis-associated colorectal cancer. However, the oncogenic mechanisms involved in the IEC transformation are not clear. b | Although innate immune responses are protective in the case of tissue injury, these responses need to be tightly controlled, as exaggerated caspase 1 activation in the absence of the inflammasome antagonist caspase 12 or exaggerated IL-18R or Toll-like receptor (TLR) activation in the absence of the antagonist single immunoglobulin IL-1-related receptor (SIGIRR) leads to increased incidence of colitis-associated colorectal cancer. This is also observed in the absence of the signal transducer and activator of transcription (STAT) inhibitor suppressor of cytokine signalling 1 (SOCS1; not shown), owing to increased STAT3 function in IECs. STAT3 is a critical effector of IEC proliferation during tissue repair and tumorigenesis and is activated downstream of the IL-6R or IL-11R signalling pathways. In these instances, excessive tissue repair and inflammatory responses drive the tumorigenic phenotype. c | By contrast, tumorigenesis initiated by intrinsic defects in pathways regulating cell proliferation, primarily in the Wnt–APC–β-catenin pathway as observed in ApcMin/+ mice or in mice treated with genotoxic compounds, is driven by inflammation and innate immune signalling pathways. Agonists from the commensal flora and alarmins produced in the tumour microenvironment trigger innate immune pathways to promote tumorigenesis. For instance, signalling through the adaptor MYD88 has been shown to mediate tumorigenesis in ApcMin/+ mice through extracellular signal-regulated kinase (ERK)-dependent MYC phosphorylation and stabilization. In the absence of MYC or MYD88, tumorigenesis in ApcMin/+ mice is markedly reduced. Thus defective innate immune signalling, as in MYD88-deficient mice, leads to decreased tumour burden owing to dampened activation of oncogenic and survival mechanisms. APC, adenomatous polyposis coli; DAMP, damage-associated molecular pattern; NF-κB, nuclear factor-κB; NLRP3, NOD-, LRR- and pyrin domain-containing 3; PRR, pattern recognition receptor; TNFR1, TNF receptor 1.

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ApcMin/+ mice A mouse strain that carries a point mutation in one adenomatous polyposis coli (Apc) allele and spontaneously develops intestinal adenomas. It is used as a model for human familial adenomatous polyposis and for human sporadic colorectal cancer.

and promoted by bile injection in a subcutaneous cecal hernia was observed in conventionally reared rats but not in those raised under germ-free conditions99. These results indicate that tumour formation occurred only when the inflammatory response was initiated by stimuli derived from the commensal flora100. Importantly, intestinal infection of ApcMin/+ mice (a mouse model of sporadic colorectal cancer) with B. fragilis induces colorectal cancer 96 but, surprisingly, infection with H. hepaticus also induces mammary adenocarcinoma, providing evidence that the inflammatory status of the gut may have systemic effects101. Similarly, it has been shown that oral DSS treatment of wild-type mice induces DNA damage not only in intestinal cells but also in circulating T cells102. This damage seems to result in an altered response to the commensal flora and subsequently in an inflammatory environment that is responsible not only for colitis and colitis-associated colorectal cancer, but also for systemic damage in distant organs. Pro-inflammatory effector molecules released into the circulation and/or microbial translocation leading to systemic inflammation could mediate this systemic response. It has been suggested that a major mechanism by which MyD88 stimulates colorectal cancer in ApcMin/+ mice is through activation of a MAPK, extracellular signal-regulated kinase (ERK), which stabilizes the oncoprotein MyC by preventing its ubiquitylation and proteosomal degradation103. Indeed, in the ApcMin/+ mouse model, MyD88 signalling contributes to adenoma growth and progression104 (fIG. 4c). Myd88–/–ApcMin/+ mice display lower levels of phosphorylated ERK in the intestinal mucosa compared with wild-type mice and have fewer and smaller intestinal tumours. Interestingly, activation of ERK by epidermal growth factor (EGF), which is MyD88 independent, restores tumorigenicity in this model103. MyD88 signalling has also been shown to be required for colon carcinogenesis in two other mouse models: AOM-initiated colon carcinogenesis in Il10–/– mice98 and colon carcinogenesis induced by repeated treatment with AOM104. MyD88 is also necessary for skin chemical carcinogenesis105 and diethylnitrosamine (DEN)-induced liver cancer 106, two models in which host–microbiota interaction may have a role in initiating inflammation and promoting tumour initiation and progression. Thus, in different tissues, signalling through MyD88 is required for cell transformation and carcinogenesis. By contrast, MyD88 signalling is protective in the AOM plus DSS (injury)-induced colitis-associated colorectal cancer model49 (fIG. 4a). This observation suggests that the inability of Myd88–/– mice to heal ulcers generated by injury with DSS creates an altered inflammatory environment that exacerbates the mutation rate in mucosal epithelial cells and results in augmented adenoma formation and cancer progression. Recently, it was found that mice that are deficient in TlR2 reproduce, in part, the phenotype of Myd88–/– mice in the AOM plus DSS model, and display greater tumour incidence and an increased number and size of tumours compared with wild-type control mice107. However, Tlr4–/– mice, which largely reproduce the phenotype of

Myd88–/– mice in terms of their inability to efficiently repair the colonic mucosa following acute DSS-induced injury, are resistant to colitis-associated colorectal cancer 108. Although the contribution of other TlRs remains to be fully analysed, the susceptibility to colitisassociated colorectal cancer of Myd88–/– mice is not due to their inability to signal through TlR4. Instead, mice that lack expression of inflammasome components (caspase 1, apoptosis-associated speck-like protein containing a CARD (ASC; also known as PyCARD) or NlRP3), as well as mice that lack Il-18 or Il-18R expression, phenocopy Myd88–/– mice and display increased susceptibility to AOM plus DSS-induced colitis-associated colorectal cancer 49,91. This suggests that the susceptibility of Myd88–/– mice to colitis and colitis-associated colorectal cancer is in part due to their inability to signal through Il-18R49,91. Both tumour initiation and promotion are enhanced in mice that lack a component of the inflammasome–Il-18–Il-18R–MyD88 axis compared with wild-type animals, as illustrated by the enhanced number and size of polyps49. Interestingly, although the intestinal tissue repair response and IEC proliferation in response to damage are impaired in these mutant mice, their enterocytes express phosphorylated nuclear STAT3 and present evidence of induced activation of wnt–β-catenin, EGF receptor and MET proto-oncogene (HGF receptor) signalling pathways, as well as alterations in Smads expression. These features are suggestive of decreased signalling by the anti-proliferative factor transforming growth factor-β (TGFβ)49. Therefore, these cells are in a proliferation-prone state with activation of many of the cell cycle-inducing pathways, but an as yet unknown mechanism limits their ability to progress through the cell cycle in the repair response. Genetic alterations, possibly facilitated by a genomic instability that is secondary to the downregulation of mismatch repair genes in the absence of Il-18R–MyD88 signalling 49, may overcome such a block in proliferation, leading to colitis-associated colorectal cancer development. During DSS-induced colitis, Il-18 is probably produced prevalently by IECs89. Thus, studies implicating myeloid cells in DSS-induced colitis48 suggest that Il-18R–MyD88 signalling in these cells may be responsible for activating the still unknown mechanisms leading to efficient mucosal repair and tumour suppression. Unlike the ApcMin/+ mouse model of colorectal cancer, in which MyD88 is crucial for the activation of the Il-6–STAT3 pathway 104, in the AOM plus DSS model the damage-induced expression of Il-6 family cytokines and STAT3-dependent genes is independent of MyD88 (Ref. 49). It is plausible that the damage signal is transduced through TlR adaptor molecules other than MyD88, or through NlRs or other damage-associated molecular pattern (DAMP) receptors such as receptor for advanced glycation end-products (RAGE)109. Consistently, the DAMP high-mobility group box 1 (HMGB1) has been shown to induce Il-6 expression and mediate wound healing through RAGE110,111. An alternative hypothesis is that a pro-inflammatory response is intrinsically triggered by DSS-induced DNA damage



REVIEWS Tumour immunosurveillance Tumour immunosurveillance or attack refers to the identification and elimination of cancerous or pre-cancerous cells by the immune system. However, as tumours do still develop despite a functioning immune system, the concept of ‘immune editing’ has taken over.

1.

2. 3. 4. 5. 6. 7.

without involving classical PRRs112. The expression of the cytokine IFNγ, which is important for tumour immunosurveillance, and IFNγ-dependent genes is reduced in Myd88–/– mice compared with wild-type animals49, which is consistent with the role of Il-18 in the production of IFNγ. Thus, the AOM plus DSS-enhanced tumorigenic phenotype in MyD88- or inflammasome-deficient mice, which express reduced IFNγ levels, might be partially due to impaired tumour immunosurveillance113. Together, these data suggest that mouse colon carcinogenesis models that depend on a genotoxic agent (AOM) along with a mucosal damaging stimulus (DSS) are fuelled by the extensive and durable mucosal erosion that occurs in inflammasome-, MyD88- or Il-18-deficient mouse strains. Thus, even in the absence of signalling through these major pro-inflammatory pathways, pro-carcinogenic genes (such as IL6, IL11 and COX2) are induced through alternative PRRs, indirectly through TNFR1 (Ref. 114), or alternatively through other molecular mechanisms of cell-intrinsic inflammation. Notably, it was observed that despite the lack of mucosal repair in this context, many cell cycle-activating genes were induced in the damaged epithelium. Therefore, it is conceivable that mutations induced by the initial AOM genotoxic treatment or successively by the DSS-triggered inflamed environment would render the few remaining epithelial cells more fit to hijack the growth and proliferation factors from the microenvironment and expand into clonal neoplastic lesions. Interestingly, the direct interaction of certain bacterial species with IECs can induce the downregulation of DNA repair genes97,115, similarly to what was observed in the colons of DSS-treated MyD88- or Il-18-deficient mice49. Thus, altered host–microbiota interaction could favour genetic instability and successive mutations that would further enhance tumorigenesis. Consistently, chronic treatment with DSS alone induces polyp formation in MyD88-deficient mice even in the absence of a genotoxic compound49. In contrast to tumorigenesis that is induced as a consequence of tissue damage, carcinogenesis that occurs in the presence of an intact, or readily repaired, epithelial barrier depends on MyD88 signalling for tumour development (fIG. 4b). Thus, genetic mutations that strengthen

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the inflammatory response, in particular downstream of PRRs, the inflammasome or MyD88, enhance tumour formation by creating an inflammatory environment that favours excessive tissue repair and tumorigenesis. Indeed, Socs1–/–, Sigirr–/– and Casp12–/– mice that exhibit enhanced inflammatory responses with inflammasome hyperactivation and/or increased signalling through TlRs, Il-1 family receptors and IFNγ receptors are highly susceptible to AOM plus DSS-induced tumorigenesis47,89,116,117. Excessive activation of these inflammatory pathways also enhances spontaneous tumorigenesis; accordingly, ApcMin/+Sigirr–/– mice have increased colonic polyposis compared with ApcMin/+ mice118. Therefore, we propose that the mucosal damage induced by DSS in MyD88- or inflammasome-deficient mice, together with associated microbial mucosal translocation89,90,119, are responsible for a dramatically different state of inflammation compared with that elicited during tumorigenesis in the context of an intact mucosal barrier, such as in mice with increased repair responses or in ApcMin/+ mice.

Future directions we are beginning to appreciate the intricacies of innate immune regulation and function in the intestine and it is becoming clear that the ablation of innate immune signalling in the gut as a therapeutic approach for intestinal pathologies needs to be revisited. The net outcome of the interaction between the commensal microbiota and the innate immune system is complex. During physiological conditions, the innate immune system is important for maintaining the homeostasis of the intestinal mucosa, but when altered it becomes the direct cause of the pathways underlying chronic inflammatory and neoplastic diseases. An emerging picture from recent investigations distinguishes between mucosal injury-mediated colitis and colitis-associated colorectal cancer versus pathologies arising in the presence of an intact mucosal barrier because of dysregulated inflammatory responses. The characterization of the innate immune mechanisms implicated in these two scenarios is underway and will certainly set the stage for the development of new and targeted therapies for IBD and intestinal cancer.

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Acknowledgements

M.S. thanks the students and colleagues in her laboratory for reading and commenting on this manuscript. Work in M.S.’s laboratory is supported by the Canadian Institutes for Health Research (MOP-79410, MOP-82801, MOP-86546, CTP-87520) and the Burroughs Wellcome Fund. Work in G.T.’s laboratory is supported by the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Center for Cancer Research, USA.

Competing interests statement

The authors declare no competing financial interests.

FURTHER INFORMATION Maya Saleh’s homepage: http://www.mcgill.ca/hostres/investigators/saleh Giorgio Trinchieri’s homepage: http://ccr.nci.nih.gov/staff/staff.asp?profileid=11574 European Commission MetaHIT: http://www.metahit.eu National Institutes of Health Roadmap Human Microbiome Project: http://nihroadmap.nih.gov/hmp All lInks ARE ACTIvE In ThE onlInE PDf


REVIEWS

Imaging techniques for assaying lymphocyte activation in action Lakshmi Balagopalan*, Eilon Sherman*, Valarie A. Barr and Lawrence E. Samelson

Abstract | Imaging techniques have greatly improved our understanding of lymphocyte activation. Technical advances in spatial and temporal resolution and new labelling tools have enabled researchers to directly observe the activation process. Consequently, research using imaging approaches to study lymphocyte activation has expanded, providing an unprecedented level of cellular and molecular detail in the field. As a result, certain models of lymphocyte activation have been verified, others have been revised and yet others have been replaced with new concepts. In this article, we review the current imaging techniques that are used to assess lymphocyte activation in different contexts, from whole animals to single molecules, and discuss the advantages and potential limitations of these methods. Diffraction limit of light This refers to the physical impossibility of focusing light that is emitted from a point source into a single point owing to diffraction, which limits optical resolution to a distance of about half of the light wavelength (~200 nm for green light).

Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. Correspondence to L.E.S. e‑mail: [email protected] *These authors contributed equally to this work. doi:10.1038/nri2903

Lymphocytes are a central component of immune defence mechanisms and have a pivotal role in our battle against pathogens. During adaptive immune responses, lymphocytes bearing antigen receptors identify and respond to rare pathogen-derived antigens without responding to self antigens. These cells continuously patrol the body, each in search of its cognate antigen. In many cases, such as T cell activation, physical contact between an antigen-presenting cell (APC) and a lymphocyte is required for the antigenspecific receptor to recognize and bind antigen. This initial binding event must be translated into a productive signal in the lymphocyte to generate a successful immune response. The consequences of inappropriate activation in this system are significant. Autoimmunity could result from inappropriate recognition of self, whereas a compromised immune response could lead to infection and death. Information on the events that are triggered by the binding of an antigen receptor to its ligand was initially obtained by biochemical studies, which successfully identified a large number of signalling molecules (including receptors, enzymes, adaptors and second messengers) that are required for lymphocyte activation1–3. Genetic manipulations have confirmed the role of many of these proteins and have aided in understanding the functional hierarchy of molecules in these signalling cascades4. These techniques provide very limited temporal and spatial information at the level of a single cell or molecule. Imaging approaches are unique in providing the ability to monitor individual events and to follow these events in time, thus allowing the investigator to

determine heterogeneity in the immune response and to understand the dynamics of lymphocyte signalling. Consequently, imaging studies have led to unexpected observations of the diversity and dynamics of lymphocyte–APC contacts, the spatial organization of the contact zone between the two cells and the intracellular molecular events. Although imaging of the immune system began more than 100 years ago with Elie Metchnikoff ’s early work on phagocytosis5, in the past three decades rapid advances in light microscopy have revolutionized our understanding of immune processes. Electron and advanced light microscopy techniques have been used to produce high-resolution images of lymphocytes in vitro. The advent of two-photon microscopy in the past decade has also made available data from in vivo settings. Most recently, high-resolution methods have broken the diffraction limit of light to probe subcellular features as small as single molecules. Thus, advances in imaging techniques have enabled the visualization of signalling events in lymphocytes with progressively greater spatial and temporal precision. In this Review, we provide an overview of the imaging toolbox that is used for visualizing lymphocytes during activation. We start out at the whole animal or tissue level, then zoom in to the cellular and subcellular levels and finally discuss techniques for imaging cells at molecular resolution (FIG. 1). Whole body imaging methods such as positron emission tomography (PET), magnetic resonance imaging (MRI) and bioluminescence are not covered and in vivo imaging is discussed only briefly. Instead, we focus on microscopy techniques

nATuRE REvIEWs | Immunology

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100 infection endpoints feasible. Second, the candidate vaccine needs to induce the immune response of interest at a sufficient frequency to support a correlates analysis (30–70%). Third, there has to be clinical efficacy. Identifying and maintaining highrisk cohorts and the clinical infrastructure to conduct HIV vaccine efficacy trials is expensive and dynamic. Many factors other than the vaccine intervention tend to reduce HIV incidence in relevant study populations66–68. Education, counselling, self-esteem and access to medical care for trial participants all contribute to reduce

NATURE REVIEwS | Immunology

Box 2 | T cell immune correlates Several characteristics of T cells have been investigated as potential correlates of immune protection in HIV.

T cell phenotype • CD4+ or CD8+ T cells • Central memory, effector memory or effector T cells • Expression of homing markers • Expression of exhaustion markers T cell function • Expression of individual cytokines and chemokines • Production of single versus several cytokines and chemokines • Killing capacity or perforin expression • Inhibition of virus in vitro • Proliferation T cell antigen specificity • Responses to Gag, Env, Pol, Nef and accessory proteins • Number of epitopes targeted • Sequence conservation of epitopes • Ease of sequence escape within epitopes • Effect of sequence escape on viral fitness HlA restriction • Frequency in human population • Association with lack of disease progression T cell receptor • Diverse or restricted • Ability to cover several clades • Ability to cover potential escape variants • Public T cell receptors (dominant in multiple individuals) or private T cell receptors (rarely present in multiple individuals)

incidence of infection, and over time new options for prevention, such as circumcision, pre-exposure prophylaxis or microbicides, will change the constituency of study populations that are appropriate for HIV vaccine clinical trials. This will add to the expense and complexity of performing the trials that are needed to establish a correlate of protection. There are several candidate vaccine regimens in development, but few immunological hypotheses for how vaccine-induced immunity might protect. Candidate vaccine regimens with immunological endpoints that have distinct specificity or functional properties should be considered for analysis in test-of-concept efficacy trials to define a correlate of protection. Currently, it is typical in Phase I and Phase II clinical trials of candidate HIV vaccines to exclude individuals who are at high risk of HIV infection, VOLUME 11 | jANUARy 2011 | 67

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Figure 1 | Conventional and adaptive trial design. Conventional clinical trials explicitly follow a protocol that is designed to provide an unbiased answer to a discrete question stated as the primary 0CVWTG4GXKGYU^+OOWPQNQI[ objective. Achieving the predetermined number of study endpoints should then provide a statistically robust conclusion. Adaptive trial designs may be useful when there are multiple objectives or multiple treatment arms to test, or both. Adaptive designs provide an unbiased approach to shift from one study objective to another or shift the emphasis from many treatment groups to a few. For example, if a vaccine study using a 1/1 allocation of vaccine to placebo was discovered to have efficacy during a proscribed interim analysis, the allocation could be changed to a 2/1 ratio to shift the primary focus towards defining an immune correlate. Alternatively, if an immune correlate was known, it could be used as a surrogate endpoint in a study that tests multiple vaccine concepts. When a particular vaccine achieved the predetermined immunological endpoint, it would trigger an altered randomization scheme to assure more subjects would be enrolled in study arms that achieve the immunogenicity objective. This would allow the emphasis to shift from immunogenicity to efficacy evaluation.

so there are relatively few exposures and breakthrough infection cases to evaluate. The inclusion of subjects who are at risk of HIV infection in early Phase clinical trials would be one way of improving our knowledge of vaccine-induced immunity and would provide another parameter of safety to assess before commencing larger test-ofconcept efficacy trials. Importantly, robust and frequent sample collection is crucial. Extensive sample collection may not be necessary for a candidate vaccine being developed for licensure, but is essential for defining a correlate of protection. Frequent sampling allows the assessment of immunity closer to the time of infection and helps to define the timing of infection more precisely. The measurement of peak immunogenicity time points is useful for product validation but is much less likely to provide a correlate with efficacy than estimates of immunity near the time of HIV exposure that may be distant from the time of immunization. The use of similar collection time points and uniform assays across a series of clinical efficacy trials will improve the likelihood of identifying a correlate of

protection and would ultimately help to generalize findings by providing data for subsequent meta-analysis69. From trials that show partial protection? It is crucial for funding bodies and decisionmakers to appreciate that an immune correlate of protection can be defined from trials of vaccines that show partial efficacy. If properly designed with enough clinical endpoints and sufficient sample collection, efficacy as low as 10–15% may allow identification of an immune correlate of protection. This would then allow the subsequent evaluation of several vaccine delivery platforms and antigen concepts, and product development can proceed in a more logical and systematic way. The concept of using ‘adaptive’ trial designs for HIV vaccine evaluation has recently been a topic of considerable discussion. Adaptive trial design implies that flexibility to accommodate changes in study design in response to accumulating data is incorporated into the protocol (FIG. 1). Adaptive trial designs have gained some momentum in treatment-intervention

68 | jANUARy 2011 | VOLUME 11

studies in which a clinical endpoint can be detected soon after treatment initiation. The theoretical advantage of an adaptive approach is that it provides a mechanism to evaluate many approaches in a shorter amount of time. However, the practical advantage is lost if events that trigger adaptation are infrequent and distant from the time of subject randomization. There may be ways in which adaptive trial designs facilitate the identification of correlates of vaccine-induced immunity. Although vaccine efficacy is determined by comparing the frequency of study endpoints in recipients of the vaccine and the placebo, to detect immune correlates a case-controlled comparison is performed between vaccine recipients who become infected and those who do not. Therefore, enrolling additional vaccine recipients in trials that show early evidence of efficacy would improve the chances of defining immune correlates. However, this would not provide a notable time advantage. To attain the time efficiency promised by adaptive trial designs for defining efficacy, adjustments to group allocation would need to be done in real-time based on an immunological ‘surrogate’ endpoint (BOX 1). For example, if an antibody response with a particular specificity and function or a T cell response with certain phenotypic characteristics was found to be a correlate of protection, then parallel trials with several product concepts could be initiated, and every time an immunized subject achieved the ‘surrogate’ endpoint or the immune correlate of protection, that group would gain an advantage in future subject allocations. In that way, enrolment could proceed for many concepts, but only the ones achieving the correlate at a high frequency would accrue enough subjects to determine clinical efficacy. Defining a correlate of protection to use as a surrogate endpoint is the crucial step that would allow the best use of adaptive trial designs and improve the likelihood of eventually achieving a significant level of clinical efficacy. Adaptive vaccine clinical trials that use a clinical endpoint of infection or disease progression require too much time and clinical trial capacity to remain relevant. Performing efficacy trials that are designed to detect high levels of clinical efficacy (>50%) but are of insufficient size or intensity to define a correlate of protection may result in a fortuitous breakthrough that could support the further development of a selected product. However, they are much less likely to support incremental scientific advances that would lead to a highly effective HIV vaccine. As we have witnessed in the two www.nature.com/reviews/immunol

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PersPectives Box 3 | Antibody immune correlates Characteristics of antibodies that will be investigated as potential correlates of immune protection in the RV144 vaccine trial.

Antibody titre • Binding by enzyme-linked immunosorbent assay • Duration Antibody function • Neutralization (compared with several panels of isolates) • Antibody-dependent cell-mediated cytotoxicity • Antibody-dependent cell-mediated virus inhibition • Fc binding • Effect on viral mobility in mucous • Affinity and avidity Antibody specificity • Clade specificity of antibody functions • Cross-competition with known neutralizing antibodies • Linear epitope mapping Antibody phenotype • Immunoglobulin class and subclass • Fc modifications (sialylation and glycosylation) Antibody location • Serum • Mucosal samples

most recent efficacy trials, it is hard to guess based on established scientific paradigms what the outcome of a vaccine trial will be. The STEP trial focused on inducing Gagspecific CD8+ T cell responses and was considered promising by the scientific community but resulted in vaccine-enhanced infection rates, whereas the vaccine tested in the RV144 trial, which induced relatively weak CD8+ T cell responses and nonneutralizing antibodies and was highly criticized by the scientific community, showed partial efficacy. Unfortunately, neither trial was specifically designed to define an immune correlate of protection, and the retrospective analysis of available samples is not likely to reach a definitive answer. The current HVTN 505 efficacy trial has relatively extensive sample collection, but is not large enough to accumulate a sufficient number of subjects in the vaccine group to be sure of defining a correlate of protection if partial efficacy is achieved. It should also be noted that in the absence of a strong T cell response in the RV144 trial, an antibody correlate is most likely to derive from the correlates

analysis. This does not, however, diminish the complexity of the investigation. Similarly to the T cell response, there are many aspects of the antibody response that can and will be evaluated in the search for an antibody correlate of protection in the RV144 vaccine trial (BOX 3). Until there is sufficient investment in the process to define a correlate of protection to allow the establishment of surrogate immunological endpoints for efficacy trials, development of a vaccine for HIV will remain a distant hope, a point made recently by the Global HIV Vaccine Enterprise70. Conclusion The challenge presented in developing an HIV vaccine is both new and unique. Infected individuals do not clear the virus, are not immune to subsequent reinfection and do not typically survive in the absence of antiretroviral therapy. This separates the quest for an HIV vaccine from other vaccine efforts in which correlates of protection may arise from an empirical approach, instead of being prerequisites for the rational design of the vaccine. The search for correlates of protection in cohorts such as long-term non-progressors seemed to be a reasonable approach but may ultimately have led us astray. Although polyfunctional Gagspecific cytotoxic T cells were heralded as the goal to be achieved in a successful vaccine, in fact they may be only a correlate of lower viral load in chronically infected people. Observations in highly exposed uninfected people are equally consistent with innate rather than adaptive host immune factors as underlying mechanisms for protection, and it is unclear how these mechanisms may be relevant to a vaccine. Furthermore, although there has been much progress in the elicitation of Env-specific antibodies by vaccines, studies in infected individuals certainly did not reveal them as correlates of protection and, in fact, suggested that stimulating T cell responses to Env would be harmful. Thus the focus of our studies should shift to the establishment of correlates of protection in uninfected people in large vaccine trials, rather than protection from virus replication or disease progression in chronically infected people. when we embark on such trials we must bear in mind that should a trial fail, certain response thresholds may be found to be inadequate, but assays themselves cannot be formally negated. Only in a trial that has partial efficacy can a correlate be disregarded if it fails to distinguish protected from unprotected individuals. As transmission incidence is generally low, defining

NATURE REVIEwS | Immunology

correlates is inherently difficult, and therefore we should be guided in this endeavour by the necessity for the recruitment of large numbers of volunteers who should be sampled frequently. The path to a successful vaccine for HIV is likely to be an iterative one, driven forward by a process of successive approximation or, as it is more colloquially termed, trial and error. Richard A. Koup, Barney S. Graham and Daniel C. Douek are at the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892‑3017, USA. Correspondence to D.C.D. e‑mail: [email protected] doi:10.1038/nri2890 Published online 17 December 2010 1.

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56. Hudgens, M. G. et al. Power to detect the effects of HIV vaccination in repeated low-dose challenge experiments. J. Infect. Dis. 200, 609–613 (2009). 57. Morgan, C. et al. The use of nonhuman primate models in HIV vaccine development. PLoS Med. 5, e173 (2008). 58. Nishimura, Y. et al. Highly pathogenic SHIVs and SIVs target different CD4+ T cell subsets in rhesus monkeys, explaining their divergent clinical courses. Proc. Natl Acad. Sci. USA 101, 12324–12329 (2004). 59. Shedlock, D. J., Silvestri, G. & Weiner, D. B. Monkeying around with HIV vaccines: using rhesus macaques to define ‘gatekeepers’ for clinical trials. Nature Rev. Immunol. 9, 717–728 (2009). 60. McElrath, M. J. et al. HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort analysis. Lancet 372, 1894–1905 (2008). 61. Shiver, J. W. et al. Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature 415, 331–335 (2002). 62. Hansen, S. G. et al. Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge. Nature Med. 15, 293–299 (2009). 63. D’Souza, M. P. & Altfeld, M. Measuring HIV-1-specific T cell immunity: how valid are current assays? J. Infect. Dis. 197, 337–339 (2008). 64. Streeck, H., Frahm, N. & Walker, B. D. The role of IFN-γ Elispot assay in HIV vaccine research. Nature Protoc. 4, 461–469 (2009). 65. Janetzki, S., Cox, J. H., Oden, N. & Ferrari, G. Standardization and validation issues of the ELISPOT assay. Methods Mol. Biol. 302, 51–86 (2005). 66. Baeten, J. M. et al. Trends in HIV-1 incidence in a cohort of prostitutes in Kenya: implications for HIV-1 vaccine efficacy trials. J. Acquir. Immune Defic. Syndr. 24, 458–464 (2000). 67. Kaul, R. et al. Reduced HIV risk-taking and low HIV incidence after enrollment and risk-reduction counseling in a sexually transmitted disease prevention trial in Nairobi, Kenya. J. Acquir. Immune Defic. Syndr. 30, 69–72 (2002). 68. van Loggerenberg, F. et al. Establishing a cohort at high risk of HIV infection in South Africa: challenges and experiences of the CAPRISA 002 acute infection study. PLoS ONE 3, e1954 (2008). 69. Qin, L., Gilbert, P. B., Corey, L., McElrath, M. J. & Self, S. G. A framework for assessing immunological correlates of protection in vaccine trials. J. Infect. Dis. 196, 1304–1312 (2007). 70. The Council of the Global HIV Vaccine Enterprise et al. The 2010 scientific strategic plan of the Global HIV Vaccine Enterprise. Nature Med. 16, 981–989 (2010).

Acknowledgements

We thank the many members of the Vaccine Research Center whose helpful discussions over the years have helped to frame the opinions expressed here.

Competing interests statement

The authors declare no competing financial interests.

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ReseaRch highlights Nature Reviews Immunology | AOP, published online 3 December 2010; doi:10.1038/nri2905

R E G u L At O Ry t C E L L S

Kings of the delta blues Regulatory T (TReg) cells prevent excessive inflammatory responses and are crucial for maintaining intestinal homeostasis. Several distinct leukocyte populations are known to be targets of TReg cell-mediated suppression; Sankar Ghosh and colleagues now report that TReg cells can also prevent uncontrolled γδ T cell-mediated attacks against the intestinal microbiota. Previous work described an essential role for 3-phosphoinositidedependent protein kinase 1 (PDK1) in transducing activatory signals from the co-receptor CD28 during CD4+ T cell activation. Surprisingly, the authors found that transgenic mice with a CD4+ T cell-specific deletion of PDK1 developed spontaneous colitis, despite showing defective CD4+ T cell activation. By characterizing colonic leukocyte populations, they found that γδ T cells (but not αβ T cell or B cell populations) were markedly increased in the colonic epithelium of mice with PDK1-deficient CD4+ T cells. In particular, an increased proportion of interleukin-17 (IL-17)producing γδ T cells was found in the colons of the transgenic mice compared with wild-type mice, suggesting

that these γδ T cells contributed to disease development. To explore this further, the authors crossed mice with PDK1-deficient CD4+ T cells with animals lacking γδ T cells and found that these double-deficient mice no longer developed colitis. Treatment with antibiotics also prevented disease in mice with PDK1-deficient CD4+ T cells. These findings confirmed that γδ T cells are required for colitis development in this model and suggested that γδ T cells are activated in response to the commensal microbiota. As the transgenic mice also lacked PDK1 expression in CD4+ TReg cells, the authors next explored whether colitis in these animals resulted from a TReg cell defect. In mice with PDK1-deficient CD4+ T cells, forkhead box P3 (FOXP3)+ TReg cells developed normally and were only slightly reduced in number in the periphery. However, in contrast to wild-type TReg cells, PDK1-deficient TReg cells failed to upregulate various antiinflammatory cytokines, including IL-10, following activation with CD3- and CD28-specific antibodies, and were only weakly suppressive

nATuRe RevIewS | Immunology

in vitro. Furthermore, in the classic T cell transfer model of colitis, PDK1-deficient TReg cells could not prevent disease development. In a series of transfer experiments, the authors showed that wild-type, but not PDK1-deficient, TReg cells can inhibit proliferation and IL-17 production by γδ T cells in vivo. TReg cell-mediated suppression of γδ T cells was not contactdependent but instead depended on TReg cell production of IL-10; this was an interesting finding, as the ability of TReg cells to inhibit CD4+ αβ T cell responses is independent of TReg cell-derived IL-10. Finally, the authors found that colitis did not develop in transgenic mice with PDK1-deficient CD4+ T cells following the transfer of wild-type TReg cells, indicating that disease in these mice is caused by defective TReg cellmediated suppression of γδ T cells, rather than due to an intrinsic defect in γδ T cell populations.

Yvonne Bordon

ORIGINAL RESEARCH PAPER Park, S. et al. T regulatory cells maintain intestinal homeostasis by suppressing γδ T cells. Immunity 33, 791–803 (2010)

vOLume 11 | jAnuARy 2011 © 2010 Macmillan Publishers Limited. All rights reserved

ReseaRch highlights Nature Reviews Immunology | AoP, published online 3 December 2010; doi:10.1038/nri2906

B CELLS

Illuminating the dark zone In a recent paper published in Cell, Dustin, Nussenzweig and colleagues illuminate the dynamics of germinal centre B cell migration and show that T cell help is the limiting factor for the selection of high-affinity B cells. Although much research has focused on defining the mechanisms of B cell selection in germinal centres and the importance of migration between germinal centre dark and light zones, a clear picture of the dynamics of B cell selection in these structures is lacking. To address this issue, the authors generated transgenic mice that expressed photoactivatable green fluorescent protein (PA-GFP) in their haematopoietic cells. These cells can be photoactivated with great precision

B cells expressing photoactivatable green fluorescent protein can be photoactivated with high spatial precision in germinal centres. Follicular dendritic cells in the germinal centre light zone are labelled with an antigen-conjugated red fluorescent protein and collagen is in blue. Image courtesy of G. D. Victora and M. Nussenzweig, The Rockefeller University, New York, USA.

(close to one cell diameter) in these mice and can be analysed by multiphoton laser scanning microscopy and flow cytometry. This method allows for the precise labelling, tracking and molecular analysis of cells within living tissues. The authors then generated antigen-specific germinal centres in which transferred antigenspecific PA-GFP-expressing B cells could be specifically photoactivated in either the dark zone or the light zone. The light zone was identified by injecting an antigen-conjugated red fluorescent protein. In antigenimmunized mice this forms immune complexes that bind follicular dendritic cells (FDCs), which are confined to the light zone. The authors found that dark zone B cells were CXCR4hiCD83lowCD86low and expressed genes associated with cell division and somatic hypermutation at high levels, whereas light zone B cells were CXCR4lowCD83hiCD86hi and expressed activation markers (associated with antigen encounter and T cell help) and apoptosis regulators. Analysis of B cell migration in germinal centres in the popliteal lymph nodes of living mice showed that photoactivated dark zone B cells migrated rapidly to the light zone, with up to 50% of cells migrating to the light zone in 4 hours. By contrast, migration from the light zone to the dark zone was slow, with only 15% of cells making the transition in 6 hours. These observations are consistent with the model in which the dark zone acts as a source of proliferating B cells with mutated B cell receptors (BCRs) that migrate to and undergo

NATuRe RevIewS | Immunology

selection in the light zone. B cells in the light zone must compete for a limiting factor that promotes their return to the dark zone for further rounds of proliferation. But what is the limiting factor? It has been proposed that affinity-based selection of B cells may be driven by BCR cross-linking by antigen that is deposited in immune complexes on FDCs and/or by help from follicular helper T (TFH) cells following antigen uptake and presentation by high-affinity B cells. The authors devised a model to address the exact mechanism of B cell selection, and found that targeting TFH cell help to a subpopulation of B cells resulted in migration of these B cells from the light zone to the dark zone. They also showed that TFH cell help was required for clonal expansion and affinity maturation of these B cells. BCR cross-linking was necessary but not sufficient for affinity maturation, indicating that TFH cell help is the limiting factor for B cell selection in germinal centres. So, using a unique method that combines PA-GFP expression in B cells with multiphoton microscopy and flow cytometry, this study defines specific mechanisms that govern B cell selection in germinal centres.

Olive Leavy

ORIGINAL RESEARCH PAPER Victora, G. D. et al. Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143, 592–605 (2010) fuRtHER REAdING Cyster, J. G. Shining a light on germinal center B cells. Cell 143, 503–505 (2010)

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I N N At E I m m u N I t y

Calling on the neighbours The enteric bacterium Shigella flexneri is known to produce multiple effector proteins that interfere with host cell inflammatory responses. But despite this, infection with this bacterium induces a dramatic inflammatory response, including the secretion of large amounts of CXC-chemokine ligand 8 (CXCL8; also known as IL-8). A recent study published in Immunity provides an explanation for this, by showing that infected intestinal epithelial cells communicate with uninfected neighbouring epithelial cells to promote inflammatory chemokine secretion that is unrestrained by bacterial effector proteins. A related, independent study in PLoS Pathogens describes another mechanism of intercellular communication that allows amplification of innate immune responses to Listeria monocytogenes. In the first study, Kasper et al. noted, to their surprise, that when epithelial cells were exposed to low doses of S. flexneri, not only infected cells but also uninfected cells showed activation of the pro-inflammatory transcription factor nuclear factor-κB (NF-κB). The uninfected cells with active NF-κB seemed to neighbour infected cells, suggesting the occurrence of bystander activation. Such activation was not a result of bacterial spread to neighbouring cells, as it was not reduced following infection with a non-motile mutant of S. flexneri.

Further analysis revealed that the uninfected bystander cells also showed activation of JUN N-terminal kinase (JNK), extracellular signalregulated kinase (ERK) and the mitogen-activated protein kinase p38, all of which are involved in inflammatory responses to S. flexneri. In particular, p38 activation was much higher in these uninfected cells than in the infected cells, and this was shown to be a result of dephosphorylation of p38 in the infected cells by the bacterial effector protein OspF; accordingly, p38 activation was increased in cells that were infected with an OspF-deficient S. flexneri mutant. In keeping with the observed activation of pro-inflammatory signalling, uninfected epithelial cells produced much more CXCL8 than infected cells, as assessed on a singlecell level using immunofluorescence microscopy or mRNA hybridization. So, how is the infection communicated to bystander cells? Treatment of the epithelial cells with brefeldin A, which blocks protein secretion, had no effect on bystander activation during S. flexneri infection, indicating that activation was not mediated by paracrine signalling involving secreted proteins. By contrast, pharmacological blockade of gap junctions, which allow the passage of small molecules between adjacent epithelial cells, did reduce CXCL8 secretion by bystander cells. Moreover, S. flexneri infection


of an epithelial cell line that lacks expression of the gap junction protein connexin 43 (also known as GJA1) failed to induce activation of NF-κB, JNK, ERK and p38 in bystander cells. Finally, the finding that connexin 43 had to be expressed by both the infected and the uninfected bystander cells confirmed that epithelial cell inflammatory responses are propagated during bacterial infection through gap junction communication. In the study by Dolowschiak et al., communication between intestinal epithelial cells did not seem to depend on gap junctions but instead was shown to be mediated by reactive oxygen intermediates that were produced by NADPH oxidase in cells that were infected with the cytosolic bacterium L. monocytogenes. However, despite the differing mechanisms of intercellular communication that are described, these papers identify an important new way in which innate immune responses can be amplified at the early stages of bacterial infection.

Lucy Bird

ORIGINAL RESEARCH PAPERS Kasper, C. A. et al. Cell-cell propagation of NF-κB transcription factor and MAP kinase activation amplifies innate immunity against bacterial infection. Immunity 33, 804–816 (2010) | Dolowschiak, T. et al. Potentiation of epithelial innate host responses by intercellular communication. PLoS Pathog. 6, e1001194 (2010)

vOLUME 11 | JANUARy 2011 © 2010 Macmillan Publishers Limited. All rights reserved

ReseaRch highlights Nature Reviews Immunology | AoP, published online 17 December 2010; doi:10.1038/nri2908

T cells

Shaping Il4 gene expression Transcription of the T helper 2 (TH2)-associated cytokine genes — interleukin-4 (IL4), IL5 and IL13 — is controlled by the TH2 cell master regulator GATA-binding protein 3 (GATA3). However, the molecular basis of GATA3-mediated gene regulation during TH2 cell development is unclear and controversial. Tanaka et al. now show that binding of GATA3 to DNase I hypersensitive site 2 (HS2) in the second intron of the Il4 locus is specifically required for chromosomal modifications at this locus that allow transcription of Il4. Numerous regulatory elements in the TH2 cytokine locus have been identified, but whether TH2-associated cytokine expression is controlled by a single element or by the coordinated activity of multiple elements is not known. To address this issue, the authors generated a series of mutant mice that lack each hypersensitive element in the Il4–Il13 locus and assessed the effect of each deletion on cytokine production. TH2 cells from mice that lack HS2 produced the lowest levels of IL-4 following activation, whereas the expression of other TH2-type cytokines by these cells was similar to wild-type TH2 cells. These data suggest a specific role for HS2 in IL-4 expression. Deletion of other regulatory elements also impaired IL-4 expression, but to a lesser extent, suggesting that multiple elements are required for complete lineage-specific expression of IL-4. By contrast, naive T cells that lack the conserved GATA3-response element (GCRE) in the Il13 locus gave rise to wild-type

numbers of IL-4-producing T cells but few IL-13-producing T cells in TH2 cell-inducing conditions, indicating that this element regulates Il13 transcription. Next, the authors assessed whether GATA3 is linked to the function of the HS2 enhancer. Unlike in wild-type TH1 cells, overexpression of GATA3 in HS2-deficient TH1 cells


did not result in IL-4 expression. Furthermore, GATA3 directly binds to HS2 during TH2 cell differentiation, as determined by chromatin immunoprecipitation analysis. GATA3 functions mainly as an epigenetic modifier, so it is possible that binding of GATA3 to HS2 is required for transcription-permissive epigenetic changes at the Il4 locus. Indeed, acetylation of histone H3 at Lys9 and Lys14, and trimethylation of histone H3 at Lys4 (all of which are permissive modifications) were impaired in HS2-deficient TH2 cells, but only at the Il4 locus. By contrast, deletion of GCRE resulted in impaired methylation of histone H3 at Lys4 at the Il13 locus but not the Il4 locus. Finally, antigen-specific IgG1 and IgE levels, eosinophilia and airway hyperresponsiveness to acetylcholine were reduced in HS2-deficient mice compared with wild-type mice in models of allergic lung inflammation, confirming that the TH2 cell response was impaired in HS2-deficient mice. So, HS2 is a crucial GATA3binding site in the Il4 locus and is required for the GATA3-mediated epigenetic modifications that are necessary for lineage-specific IL-4 expression.

Olive Leavy

ORIGINAl ReseARcH PAPeR Tanaka, S. et al. The enhancer HS2 critically regulates GATA‑3‑mediated Il4 transcription in TH2 cells. Nature Immunol. 5 Dec 2010 (doi:10.1038/ni.1966) FURTHeR ReADING Wilson, C. B., Rowell, E. & Sekimata, M. Epigenetic control of T‑helper‑cell differentiation. Nature Rev. Immunol. 9, 91–105 (2009)

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ReseaRCh highlights Nature Reviews Immunology | AoP, published online 17 December 2010; doi:10.1038/nri2909

dENdRItIC CELLS

pDCs play off scratch Plasmacytoid dendritic cells (pDCs) express Toll-like receptor 7 (TLR7) and TLR9 and produce large amounts of type I interferons (IFNs) in response to viral nucleic acids. However, the contribution of this rare circulating cell population to host immunity remains unclear. Two recent studies in the Journal of Experimental Medicine now show that, although they are absent from normal skin, pDCs are rapidly recruited to sites of cutaneous inflammation. Here, they serve as an early source of type I IFNs and contribute to wound healing in normal mice, but can promote an autoimmune skin reaction in lupus-prone animals. Using a mechanical tape-stripping model to induce acute skin inflammation in mice, Gregorio et al. found that pDCs infiltrated and transiently accumulated in the injured skin around 24 hours after injury. Infiltration of pDCs to injured skin was associated with the production of type I IFNs, and antibody-mediated depletion of pDCs abrogated the IFN response, suggesting that pDCs were the chief source of the IFNs. Mice that were treated with IRS954 (a selective inhibitor of TLR7 and TLR9) prior to tape stripping failed to upregulate type I IFNs, indicating that activation of pDCs through these TLRs is necessary for IFN production. The sterile nature of the model suggested that pDCs were activated by host-derived nucleic acids, possibly those released by damaged keratinocytes. Depletion of pDCs or blockade of IFN-mediated signalling prior to tape stripping decreased the production of cytokines that are involved in wound

repair, such as interleukin-6 (IL-6), IL-17 and IL-22, and led to delayed regrowth of the epidermis. In healthy human volunteers, mechanical or chemical-induced skin injury also led to pDC recruitment and upregulation of type I IFNs early in the immune response, suggesting that the transient recruitment of IFN-producing pDCs may contribute to wound healing in humans as well as in mice. Similarly, Guiducci et al. found that tape stripping led to early recruitment of IFNα-producing pDCs to the skin, and that treating mice with IRS954 reduced the expression of IFNs and other inflammatory cytokines in injured skin. In addition, these authors found that in a lupus-prone mouse strain, tape stripping led to the development of chronic skin lesions resembling those seen in patients with cutaneous lupus. Treating lupus-prone mice with IRS954 or depleting pDCs before tape stripping prevented the development of these skin lesions, suggesting that pDCs and signalling through TLR7 and TLR9 are necessary for this pathological response. Furthermore, IRS954 promoted healing in lupus-prone mice with already established skin lesions, indicating that continued signalling through TLR7 and TLR9 was necessary for the chronic inflammatory response; however, the exact role of pDCs was not identified. The ability to explore the functions of pDCs in chronic skin lesions may have been hampered by a lack of suitable reagents to deplete pDCs. Antibodies against bone marrow stromal antigen 2 (BST2) are commonly used to deplete

NATURe RevIewS | Immunology

pDCs in mice, but BST2 is upregulated by other cell types during inflammation. A new transgenic mouse, in which pDCs can be conditionally depleted, has been described in a recent Immunity article and could clarify this matter. Using this mouse to explore pDC functions during viral infection, Swiecki et al. found that pDCs are only essential for type I IFN production during the early stages of infection with murine cytomegalovirus or vesicular stomatitis virus; however, this early pDC response can, depending on the viral load, be crucial for containing these viruses. Together, these studies suggest that the physiological role of pDCs is to serve as an early, transient source of type I IFNs following activation by foreign or endogenous damage-associated nucleic acids. As such, pDCs seem to be important for containing early viral infections and promoting tissue repair following acute injury. However, in genetically susceptible individuals, pDCs may become chronically activated and contribute to the breakdown of tolerance and the development of autoimmunity in the skin.

Yvonne Bordon

ORIGINAL RESEARCH PAPERS Gregorio, J. et al. Plasmacytoid dendritic cells sense skin injury and promote wound healing through type I interferons. J. Exp. Med. 29 Nov 2010 (doi:10.1084/jem.20101102) | Guiducci, C. et al. Autoimmune skin inflammation is dependent on plasmacytoid dendritic cell activation by nucleic acids via TLR7 and TLR9. J. Exp. Med. 29 Nov 2010 (doi:10.1084/jem.20101048) | Swiecki, M. et al. Plasmacytoid dendritic cell ablation impacts early interferon responses and antiviral NK and CD8+ T cell accrual. Immunity. 2 Dec 2010 (doi:10.1016/j.immuni.2010.11.020)

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tumOuR ImmuNOLOGy

CD4+ T cells sponsor oncogene addicts Naturally occurring tumours have multiple and complex genetic abnormalities, but their growth and survival can often be inhibited by the inactivation of a single oncogene. This phenomenon — known as ‘oncogene addiction’ — can explain the effects of some of our most successful cancer therapeutics, such as the tyrosine kinase inhibitor imatinib mesylate (Gleevec; Novartis). Oncogene inactivation was thought to have cell-autonomous effects on tumour cell apoptosis, proliferation, differentiation and senescence, but new research published in Cancer Cell shows that an intact immune system is necessary for ‘addicted’ tumour cells to respond to oncogene withdrawal. The authors used a transgenic mouse model of Myc-induced haematopoietic tumorigenesis, in which Myc can be inactivated by administering doxycycline,

to investigate the role of the immune system in oncogene addiction. Following transplantation of tumour cells from these mice into various immunocompromised mouse strains and subsequent inactivation of Myc, the kinetics of tumour regression were found to be significantly delayed compared with wild-type hosts. The immunodeficient hosts also showed a significant increase in minimal residual disease and in tumour recurrence. Deficiency of CD4+ T cells, but not CD8+ T cells, in the transplantation hosts was sufficient to impede tumour regression after Myc inactivation. There were no differences between wild-type and immunodeficient hosts in terms of the increased tumour cell apoptosis or decreased proliferation that occur after Myc inactivation, showing that these effects do not depend on the immune system. However, whereas tumour cells that were transplanted into wild-type hosts had increased expression of senescence-associated markers after Myc inactivation, this did not occur in immunodeficient Cd4–/– mice. Similarly, in Cd4–/– mice, Myc inactivation failed to inhibit angiogenesis, and the higher mean vascular density in Cd4–/– mice compared with wild-type mice after Myc inactivation was associated with decreased production of the antiangiogenic protein thrombospondin 1 (TSP1; encoded by Thbs1). So, the absence of CD4+ T cells impairs the induction of cellular senescence and the inhibition of angiogenesis following oncogene inactivation.

NATuRe ReviewS | Immunology

The role of CD4+ T cells in the effects of oncogene inactivation was confirmed by showing that the reconstitution of immunodeficient hosts with CD4+ T cells, but not CD8+ T cells, completely eliminated minimal residual disease and prolonged tumour-free survival after Myc inactivation. Also, CD4+ T cells rapidly localized to the tumour site after Myc inactivation and were associated with the increased production of ‘antitumour’ cytokines such as TSP1 and the decreased production of ‘pro-tumour’ cytokines such as vascular endothelial growth factor. Reconstitution of immunodeficient mice with Thbs1–/–Thbs2–/– splenocytes failed to prevent tumour relapse after Myc inactivation, showing the importance of thrombospondins for tumour regression. These data indicate that oncogene addiction is not entirely cell autonomous and that changes in the cytokine milieu elicited by CD4+ T cells are required for cellular senescence and the shutdown of angiogenesis, which might be involved in constraining minimal residual disease. This highlights the importance of testing targeted oncogene therapies in immunocompetent models and the potential for combining such therapies with immunotherapeutic agents that boost the CD4+ T cell response. Kirsty Minton

ORIGINAL RESEARCH PAPER Rakhra, K. et al. CD4+ T cells contribute to the remodeling of the microenvironment required for sustained tumor regression upon oncogene inactivation. Cancer Cell 18, 485–498 (2010)

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