CXCR5

CXCR5 Reinhold FoÈrster* Molecular Tumor Genetics and Immunogenetics, Max-Delbruck-Center for Molecular Medicine, Robert Rossle Strasse 10, Berlin, 13092, Germany * corresponding author tel: +49-30-9406-3330, fax: +49-30-9406-3884, e-mail: [email protected] DOI: 10.1006/rwcy.2000.21005.

SUMMARY The chemokine receptor CXCR5/BLR1 is expressed on mature B cells and a small subpopulation of T memory cells. Using gene targeting in mice, CXCR5 could be identified as the first chemokine receptor involved in homeostatic trafficking of lymphocytes to and within secondary lymphoid organs. CXCR5deficient mice lack B cell follicles in spleen and Peyer's patches. As the chemokine BLC/BCA-1, a ligand for CXCR5, is expressed on follicular stroma cells, both molecules represent a receptor-ligand pair that mediates the formation of follicles in lymphoid organs. In addition, CXCR5-deficient mice lack defined lymph nodes, suggesting that this chemokine receptor also plays a pivotal role in lymphoid organ development.

BACKGROUND

Discovery Chromosomal translocations causing deregulated expression of the proto-oncogene MYC had been delineated as an essential but not sufficient event during the development of a B cell neoplasia known as Burkitt's lymphoma. In order to identify additional factors involved in the pathogenesis of this disease, Dobner et al. (1992) compared the Burkitt's lymphoma cell line BL64 with the lymphoblastoid cell line IARC549 by means of subtractive hybridization. Using this approach, a novel G protein-coupled receptor was identified which was predicted to belong to the chemokine family as it contained the DRYLA motif at the end of the third transmembrane domain. Northern blot analysis revealed that this transcript

was exclusively expressed in mature B cells and Burkitt's lymphoma cells and thus it was termed Burkitt's lymphoma receptor 1 (BLR1).

Alternative names Burkitt's lymphoma receptor 1, BLR1, NLR, CXCR5.

Structure CXCR5 is a member of the G protein-coupled seven transmembrane chemokine receptor family.

Main activities and pathophysiological roles The restricted expression pattern of CXCR5 on mature B cells and a subpopulation of T memory cells suggested that this receptor might function as a regulator of B cell migration (FoÈrster et al., 1994). This view was recently confirmed by analyzing genetargeted mice lacking CXCR5. These mice lack inguinal lymph nodes, possess none or only few aberrantly developed Peyer's patches and show altered primary and secondary lymphoid follicles in the spleen, but have normal mesenteric and peripheral lymph nodes. Upon adoptive transfer in wild-type animals, B cells isolated from CXCR5-mutant mice failed to migrate from the outer periarteriolar lymphoid sheath (PALS) to the B cell follicles (FoÈrster et al., 1996). These experiments identified CXCR5 as the first chemokine receptor directing the homing of B cells to lymphoid organs and defined anatomic compartments within. The chemokine BLC/BCA-1 is the only physiological ligand for CXCR5 identified so far.

2020 Reinhold FoÈrster Figure 1 Nucleotide sequence of the translated cDNA encoding human CXCR5. The codons for the first and last amino acid are underlined. GCTGCCACCT TGGTGACTCA AACCTGGAGG GTGGAAAATC TTCGTGCCCG CTGGTGATCC CTGGCCGTGG GTGGGCTGGG TTCTACTGCA CACGCCGTCC ATCTGGCTGG GGCCATCACA CATGCCTGGT GTGATGGGCT CAGCGGCAGA TCACCCTACC ACCTGCAAGC GCCCACTGCT CTGTCGCGGC CCTAGCTGGC TAGGTCCCAG

CTCTAGAGGC CAGCCGGCAC ACCTGTTCTG ATCTCTGCCC TGGCCTACAG TGGAGCGGCA CCGACCTCCT TCCTGGGGAC GCAGCCTGCT ATGCCTACCG TGGGCTTCCT ACAACTCCCT TCACCTCCCG GGTGCTACGT AGGCAGTCAG ACATCGTCAT TGAATGGCTC GCCTCAACCC TCCTGACCAA GCAGGAGCAG TGTCCCCTTT

ACCTGGCGGG AGCCATGAAC GGAACTGGAC TGCCACAGAG CCTCATCTTC CCGGCAGACA GCTGGTCTTC CTTCCTCTGC CCTGGCCTGC CCACCGCCGC CCTTGCCTTG GCCACGTTGC ATTCCTCTAC GGGGGTAGTG GGTGGCCATC CTTCCTGGAC TCTCCCCGTG CATGCTCTAC GCTGGGCTGT TCTCTCTGAG TATTGCTGCT

GAGCCTCTCA TACCCGCTAA AGATTGGACA GGTCCCCTCA CTCCTGGGCG CGCAGTTCCA ATCTTGCCCT AAAACTGTGA ATCGCCGTGG CTCCTCTCCA CCAGAGATTC ACCTTCTCCC CATGTGGCGG CACAGGTTGC CTGGTGACAA ACCCTGGCGA GCCATCACCA ACTTTCGCCG ACCGGCCCTG TCAGAGAATG TTTCCTTGGG

GENE

Accession numbers EMBL: Human CXCR5 cDNA: X68149, S48709, S48717 Murine CXCR5 cDNA: X71788 Rat CXCR5 cDNA: X71463, S59748 Human CXCR5 promoter: X83755 Murine CXCR5 promoter: X83756

Sequence See Figure 1. The cDNA sequence of human CXCR5 contains an open reading frame of 1116 bp coding for a 372 amino acid protein. Genomic analysis of the human CXCR5 gene shows that it consists of two exons separated by a 9 kb intron (Dobner et al., 1992). The first exon encodes for the first 17 amino acids. A similar situation has been found in the mouse. Murine CXCR5 consists of 374 amino acids. The first 19 amino acids are encoded about 12 kb upstream of the second exon (Kaiser et al., 1993). In addition to the full-length transcript an alternative form of CXCR5, termed MDR15, has been reported. The two forms differ in the 50 region, where the open reading frame of MDR15 is shorter by 45 codons. This cDNA would encode a 327 amino acid protein lacking almost the entire N-terminus of CXCR5 (Barella et al., 1995). A single specific start side of the human CXCR5 transcript could be mapped 267 bp upstream of the

ACATAAGACA CGCTGGAAAT ACTATAACGA TGGCCTCCTT TGATCGGCAA CGGAGACCTT TTGCCGTGGC TTGCCCTGCA ACCGCTACCT TCCACATCAC TCTTCGCCAA AAGAGAACCA GATTCCTGCT GCCAGGCCCA GCATCTTCTT GGCTGAAGGC TGTGTGAGTT GCGTGAAGTT CCTCCCTGTG CCACCTCTCT GCAGGCAGTG

GTGACCAGTC GGACCTCGAG CACCTCCCTG CAAGGCCGTG CGTCCTGGTG CCTGTTCCAC CGAGGGCTCT CAAAGTCAAC GGCCATTGTC CTGTGGGACC AGTCAGCCAA AGCAGAAACG GCCCATGCTG GCGGCGCCCT CCTCTGCTGG CGTGGACAAT CCTGGGCCTG CCGCAGTGAC CCAGCTCTTC CACCACGTTC ATGCTGGATG

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Figure 2 Schematic representation of putative binding sites of the human CXCR5 promoter. Those sites which were identified by mutational analysis to confirm cell- and differentiation-specific expression of CXCR5 are depicted in red. (Full colour figure can be viewed online.)

ATG start codon (Wolf et al., 1998). Alignment of the nucleotide sequence of the human and murine CXCR5 promoter identified highly conserved regions for potential regulatory elements. They include an AP-1, a LEF-1, a purine-rich sequence (Pu box), a non-canonical octamer side (Oct), an E-box as well as three NFB-binding motifs. Neither the human nor the murine promoter show CCAAT or TATA boxes or initiator-like sequences. Mutational analysis revealed that three essential elements confer cell type and differentiation-specific expression to B cells: the octamer motif (‡157 with respect to the transcription start site), the NFBIII side (‡44), and a functional promoter region (ÿ36). Figure 2 depicts the putative binding sites of the human CXCR5 promoter and functionally important binding sites. The importance of the NFB- and Oct-binding sites was confirmed by in vivo studies using gene-targeted mice deficient either in both NFB subunits p50 and p52, in Oct-2 or Bob1. The latter factor is known to act as a B cellspecific coactivator of octamer-binding factors. In all these mutated animals, expression of CXCR5 on

CXCR5 2021 B cells was reduced or even totally absent in the case of p50/p52 double-mutant mice (Wolf et al., 1998).

Relevant homologies and species differences

PROTEIN

CXCR5 shows considerable homology to other members of the chemokine receptor family including CXCR2 (37% amino acid identity), CXCR1 (35%), CCR7 (33%), and CXCR4 (30%).

Accession numbers SwissProt: Human CXCR5: P32302 Murine CXCR5: Q04683 Rat CXCR5: P34997

Sequence See Figure 3.

Description of protein CXCR5 shows all the characteristic features of chemokine receptors including seven hydrophobic transmembrane-spanning domains and the DRYLAIVHA motif at the end of transmembrane domain III. Murine and rat CXCR5 consist of 374 amino acids. The human homolog lacks two residues at positions 13 and 14 of the extracellular N-terminus and thus consists of 372 amino acids. Human and murine CXCR5 show an overall identity of 83%. The extracellular N-terminal domain of the receptor is less conserved between the two species, where an identity of 47% can be observed (Kaiser et al., 1993). Murine and rat CXCR5 show an overall 92% amino acid identity (Kouba et al., 1993). Human CXCR5 contains two potential N-linked glycosylation motifs at position Asn28 (N-terminus) and Asn196 (second extracellular domain) and three putative C-terminal phosphorylation sites at Ser354, Ser359, and Ser361 (see Figure 3) (Dobner et al., 1992).

Affinity for ligand(s) The chemokine BLC/BCA-1 has been recently identified as a ligand for CXCR5 (Gunn et al., 1998; Legler et al., 1998). Binding affinities have not yet been determined.

Cell types and tissues expressing the receptor Using monoclonal antibodies directed against the N-terminus, CXCR5 was identified as the first lymphoid-specific member of the chemokine receptor family. In both human and murine peripheral blood cells, expression of CXCR5 is restricted to B cells (Figure 4a) and to minor subpopulations of CD4+ (approx. 15%; Figure 4b) and CD8‡ (approx. 2±8%, Figure 4c) T cells. However, in contrast to most other chemokine receptors, CXCR5 is not expressed on monocytes or neutrophils (Figure 4d) (FoÈrster et al., 1994, 1996). Immunohistology on secondary lymphoid organs such as human tonsils and spleen demonstrated that all B cells found in the marginal and mantle zone express high levels of CXCR5, whereas most of the germinal center cells do not express this chemokine receptor (FoÈrster et al., 1994). After the murine and the rat homolog of CXCR5 had been identified, it was reported that in addition to the lymphoid system CXCR5 is also expressed in defined areas of the cerebrum and the cerebellum

Figure 3 Amino acid sequence of human CXCR5: Putative transmembrane domains are shaded and depicted by Roman numbers. The boundaries of the two exons (blue), putative sites for disulfide bonds (yellow), phosphorylation (green), and glycosylation (red) are shown. (Full colour figure can be viewed online.) MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSL I I I IFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTF III IV LCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLL V EXON 1 EXON 2 ALPEILFAKVSOGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVG VI VVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSL VII PVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCOLFPSWRRSSL SESENATSLTIF

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2022 Reinhold FoÈrster Figure 4 Conserved expression pattern of CXCR5 on human and murine white blood cells (WBC). Leukocytes were isolated from peripheral blood and stained with biotinylated monoclonal antibodies specific for human or murine CXCR5. Cells were stained with streptavidin-cychrome and FITC- or PE-labeled antibodies as indicated and analyzed by flow cytometry.

monoclonal antibodies and IL-4 leads to complete downregulation of surface CXCR5. The same has been observed on T cells after stimulation with antiCD3 monoclonal antibody (FoÈrster et al., 1994). In contrast, activating naõÈ ve T cells with OX40 ligand induces expression of CXCR5 on these cells (Flynn et al., 1998).

SIGNAL TRANSDUCTION As shown for most members of the chemokine receptor family, pertussis toxin can inhibit B cell migration and Ca2‡-flux in vitro, suggesting that CXCR5 couples to Gi proteins.

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors The chemokine BLC/BCA-1 is the only natural ligand for CXCR5 identified so far. Stimulating leukocytes in vitro with this chemokine induces B cell migration with high efficacy but also induces a weak chemotactic response in T cells and macrophages (Gunn et al., 1998; Legler et al., 1998).

(Kaiser et al., 1993; Kouba et al., 1993). In contrast to the situation observed in the immune system, there are, at present, no data available concerning a possible role of CXCR5 in the central nervous system. Organs, tissues, and cells expressing CXCR5 include spleen, lymph nodes, tonsil, brain, bone marrow, B cells, T memory cells, cerebrum, cerebellum, hippocampus, pituitary. CXCR5-positive cell lines: BL64, BL21, JBL2, JI, LY66, LY91, Daudi, BL40, BL70, BL72, ESIII, Raji, BL60, BL90, BL99, BL106, LY67, WEHI 231, WEHI 279, 2PK3, A20.2J, M12-13, BFO.3.

Regulation of receptor expression CXCR5 is primarily expressed on resting cells. Activation of B cells in vitro with anti-CD40

Phenotypes of receptor knockouts and receptor overexpression mice Data derived from CXCR5-gene targeted mice support the idea that CXCR5 is a major regulator of B cell migration (FoÈrster et al., 1996). CXCR5deficiency prevents the formation of inguinal lymph nodes and severely affects the development of Peyer's patches (PPs). In 55% of all CXCR5ÿ/ÿ mice no PPs could be identified. In about 30% of the mice, small rudimentary structures could be identified resembling PPs, whereas in about 15% of the mice few regularly shaped PPs were found. Although outwardly normal, these PPs revealed severe histological alterations as no differentiation in B cell- and T cell-rich areas could be detected. Further morphological and functional alterations in CXCR5-mutant mice can be found in the spleen. Compared with the situation in wild-type animals,

CXCR5 2023 Figure 5 Transferred B cells from CXCR5-deficient mice fail to enter splenic B cell follicles. B cells (green) isolated from wild-type (left) or CXCR5-mutant (right) mice were intravenously transferred into wild-type recipients. After 5 hours wild-type B cells (left) had populated the T cell-rich zone (blue) and the B cell follicle (red). In contrast, B cells derived from CXCR5-deficient mice accumulated at the outer edge of the T cell zone but failed to migrate into the B cell follicle (right). (Full colour figure may be viewed online.)

primary splenic follicles of CXCR5-deficient mice show three major differences: (1) the T cell zone is surrounded by a small rim of IgD+IgMÿ cells, whereas very few cells can be found expressing both, IgM and IgD, (2) the marginal zone is enlarged, and (3) the T cell zone is situated centrally but not polarized in the follicle. After immunization with T cell-dependent antigens, the development of germinal centers is also impaired. Peanut agglutinin (PNA)positive cells are not found in the B cell follicle but are scattered through the red pulp and within the T cell-rich zone around the central artery. Interestingly, mesenteric and peripheral lymph nodes do not show obvious differences in CXCR5-deficient mice when compared with wild-type animals. The aberrant follicular structure of spleen and PPs is caused by impaired B cell migration. Upon adoptive transfer, B cells isolated from CXCR5-mutant animals are not able to migrate to the B cell follicles in spleen (Figure 5) and PPs of wild-type recipients but accumulate at the outer edge of the T cell zone (FoÈrster et al., 1996). These data support the multistep model for lymphocyte migration and identify CXCR5 as the first chemokine receptor regulating microenvironmental homing.

Human abnormalities Clinical studies demonstrate that CXCR5 is aberrantly expressed during the progression of the acquired immune deficiency syndrome (AIDS). As deregulated expression of CXCR5 on peripheral blood B and T cells can be observed even at the clinically latent stage, it has been suggested that this observation might reflect activating and remodeling processes leading to the destruction of lymphoid tissues by the human immunodeficiency virus, HIV (FoÈrster et al., 1997).

References Barella, L., Loetscher, M., Tobler, A., Baggiolini, M., and Moser, B. (1995). Sequence variation of a novel heptahelical leucocyte receptor through alternative transcript formation. Biochem. J. 309, 773±779. Dobner, T., Wolf, I., Emrich, T., and Lipp, M. (1992). Differentiation-specific expression of a novel G protein-coupled receptor from Burkitt's lymphoma. Eur. J. Immunol. 22, 2795± 2799. Flynn, S., Toellner, K. M., Raykundalia, C., Goodall, M., and Lane, P. (1998). CD4 T cell cytokine differentiation: the B cell activation molecule, OX40 ligand, instructs CD4 T cells to

2024 Reinhold FoÈrster express interleukin 4 and upregulates expression of the chemokine receptor, Blr-1. J. Exp. Med. 188, 297±304. FoÈrster, R., Emrich, T., Kremmer, E., and Lipp, M. (1994). Expression of the G-protein-coupled receptor BLR1 defines mature recirculating B cells and a subset of T memory helper cells. Blood 84, 830±840. FoÈrster, R., Mattis, E. A., Kremmer, E., Wolf, E., Brem, G., and Lipp, M. (1996). A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen. Cell 87, 1037±1047. FoÈrster, R., Schweigard, G., Johann, S., Emrich, T., Kremmer, E., Nerl, C., and Lipp, M. (1997). Abnormal expression of the B-cell homing chemokine receptor BLR1 during the progression of acquired immunodeficiency syndrome. Blood 90, 520±525. Gunn, M. D., Ngo, V. N., Ansel, K. M., Ekland, E. H., Cyster, J. G., and Williams, L. T. (1998). A B-cell homing chemokine made in lymphoid follicles activates Burkitt's lymphoma receptor-1. Nature 391, 799±803. Kaiser, E., FoÈrster, R., Wolf, I., Ebensperger, C., Kuehl, W. M., and Lipp, M. (1993). The G protein-coupled receptor BLR1 is involved in murine B cell differentiation and is also expressed in neuronal tissues. Eur. J. Immunol. 23, 2532±2539.

Kouba, M., Vanetti, M., Wang, X., SchaÈfer, M., and HoÈllt, V. (1993). Cloning of a novel putative G-protein-coupled receptor (NLR) which is expressed in neuronal and lymphatic tissue. FEBS Lett. 321, 173±178. Legler, D. F., Loetscher, M., Roos, R. S., Clark-Lewis, I., Baggiolini, M., and Moser, B. (1998). B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CXCR5. J. Exp. Med. 187, 655±660. Wolf, I., Pevzner, V., Kaiser, E., Bernhardt, G., Claudio, E., Siebenlist, U., FoÈrster, R., and Lipp, M. (1998). Downstream activation of a TATA-less promoter by Oct-2, Bob1, and NFB directs expression of the homing receptor BLR1 to mature B cells. J. Biol. Chem. 273, 28831±28836.

LICENSED PRODUCTS Monoclonal antibodies against human CXCR5 are distributed by R&D Systems.