Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5

Nature Immunology volume 5, pages 943–952 (2004)Cite this article

This article has been updated

Abstract

Germinal center (GC) dark and light zones segregate cells undergoing somatic hypermutation and antigen-driven selection, respectively, yet the factors guiding this organization are unknown. We report here that GC organization was absent from mice deficient in the chemokine receptor CXCR4. Centroblasts had high expression of CXCR4 and GC B cells migrated toward the CXCR4 ligand SDF-1 (CXCL12), which was more abundant in the dark zone than in the light zone. CXCR4-deficient cells were excluded from the dark zone in the context of a wild-type GC. These findings establish that GC organization depends on sorting of centroblasts by CXCR4 into the dark zone. In contrast, CXCR5 helped direct cells to the light zone and deficiency in CXCL13 was associated with aberrant light zone localization.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: CXCR4 deficiency results in GC disorganization.
Figure 2: Treatment with a CXCR4 inhibitor results in GC disorganization.
Figure 3: Deficiency of CXCR4 in B cells disrupts FDC and CXCL13 polarity in the GC.
Figure 4: GC B cells upregulate CXCR4 and show enhanced chemotaxis to SDF-1.
Figure 5: Detection of SDF-1 protein and mRNA in the GC.
Figure 6: CXCR4 is upregulated on centroblasts and is required for dark zone localization.
Figure 7: CXCR5 and CXCL13 function in determining light zone position.

Similar content being viewed by others

Change history

  • 08 August 2004

    appended erratum PDF to back of AOP PDF; placed footnote in XML at every instance of Fig. 1; corrected online date will appear in issue PDF.

Notes

  1. *Note: In the version of this article originally published online, the labels in Figure 1b were positioned incorrectly. This error has been corrected for the HTML and print versions of this article.

References

  1. Röhlich, K. Beitrag zur Cytologie der Keimzentren der Lymphknoten. Z. Mikrosk. Anat. Forsch. 20, 287–297 (1930).

    Google Scholar 

  2. MacLennan, I.C. Germinal centers. Annu. Rev. Immunol. 12, 117–139 (1994).

    Article  CAS  PubMed  Google Scholar 

  3. Kelsoe, G. Life and death in germinal centers (redux). Immunity 4, 107–111 (1996).

    Article  CAS  PubMed  Google Scholar 

  4. McHeyzer-Williams, M.G. & Ahmed, R. B cell memory and the long-lived plasma cell. Curr. Opin. Immunol. 11, 172–179 (1999).

    Article  CAS  PubMed  Google Scholar 

  5. Berek, C., Berger, A. & Apel, M. Maturation of the immune response in germinal centers. Cell 67, 1121–1129 (1991).

    Article  CAS  PubMed  Google Scholar 

  6. Liu, Y.J., Zhang, J., Lane, P.J., Chan, E.Y. & MacLennan, I.C. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens. Eur. J. Immunol. 21, 2951–2962 (1991).

    Article  CAS  PubMed  Google Scholar 

  7. Koburg, E. in Germinal Centers in Immune Responses. (eds. Cottier, H., Odartchenko, N, Schindler, R. & Congdon, C.C.) 177–182 (Springer-Verlag, New York, University of Bern, Switzerland; 1966).

    Google Scholar 

  8. Koopman, G. et al. Adhesion of human B cells to follicular dendritic cells involves both the lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 and very late antigen 4/vascular cell adhesion molecule 1 pathways. J. Exp. Med. 173, 1297–1304 (1991).

    Article  CAS  PubMed  Google Scholar 

  9. Freedman, A.S. et al. Adhesion of human B cells to germinal centers in vitro involves VLA-4 and INCAM-110. Science 249, 1030–1033 (1990).

    Article  CAS  PubMed  Google Scholar 

  10. Voigt, I. et al. CXCR5-deficient mice develop functional germinal centers in the splenic T cell zone. Eur. J. Immunol. 30, 560–567 (2000).

    Article  CAS  PubMed  Google Scholar 

  11. Ansel, K.M. et al. A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 406, 309–314 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Forster, R. et al. A putative chemokine receptor, BLR1, directs B cell migration to defined lymphoid organs and specific anatomic compartments of the spleen. Cell 87, 1037–1047 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. Roy, M.P., Kim, C.H. & Butcher, E.C. Cytokine control of memory B cell homing machinery. J. Immunol. 169, 1676–1682 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Bowman, E.P. et al. Developmental switches in chemokine response profiles during B cell differentiation and maturation. J. Exp. Med. 191, 1303–1318 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bleul, C.C., Schultze, J.L. & Springer, T.A. B lymphocyte chemotaxis regulated in association with microanatomic localization, differentiation state, and B cell receptor engagement. J. Exp. Med. 187, 753–762 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Forster, R. et al. Intracellular and surface expression of the HIV-1 coreceptor CXCR4/fusin on various leukocyte subsets: rapid internalization and recycling upon activation. J. Immunol. 160, 1522–1531 (1998).

    CAS  PubMed  Google Scholar 

  17. Casamayor-Palleja, M., Mondiere, P., Verschelde, C., Bella, C. & Defrance, T. BCR ligation reprograms B cells for migration to the T zone and B-cell follicle sequentially. Blood 99, 1913–1921 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Estes, J.D. et al. Follicular dendritic cell-mediated up-regulation of CXCR4 expression on CD4 T cells and HIV pathogenesis. J. Immunol. 169, 2313–2322 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Corcione, A. et al. Stromal cell-derived factor-1 as a chemoattractant for follicular center lymphoma B cells. J. Natl. Cancer Inst. 92, 628–635 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Shinall, S.M., Gonzalez-Fernandez, M., Noelle, R.J. & Waldschmidt, T.J. Identification of murine germinal center B cell subsets defined by the expression of surface isotypes and differentiation antigens. J. Immunol. 164, 5729–5738 (2000).

    Article  CAS  PubMed  Google Scholar 

  21. Maeda, K. et al. Murine follicular dendritic cells and low affinity Fc receptors for IgE (FcεRII). J. Immunol. 148, 2340–2347 (1992).

    CAS  PubMed  Google Scholar 

  22. Cyster, J.G. et al. Follicular stromal cells and lymphocyte homing to follicles. Immunol. Rev. 176, 181–193 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Vonderheide, R.H. & Hunt, S.V. Does the availability of either B cells or CD4+ cells limit germinal centre formation? Immunology 69, 487–489 (1990).

    CAS  PubMed Central  PubMed  Google Scholar 

  24. Tamamura, H. et al. Enhancement of the T140-based pharmacophores leads to the development of more potent and bio-stable CXCR4 antagonists. Org. Biomol. Chem. 1, 3663–3669 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Strasser, A. et al. Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and elicits autoimmune disease. Proc. Natl. Acad. Sci. USA 88, 8661–8665 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Secord, E.A., Edington, J.M. & Thorbecke, G.J. The Eμ-bcl-2 transgene enhances antigen-induced germinal center formation in both BALB/c and SJL mice but causes age-dependent germinal center hyperplasia only in the lymphoma-prone SJL strain. Am. J. Pathol. 147, 422–433 (1995).

    CAS  PubMed Central  PubMed  Google Scholar 

  27. Smith, K.G. et al. Bcl-2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells. J. Exp. Med. 191, 475–484 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hargreaves, D.C. et al. A coordinated change in chemokine responsiveness guides plasma cell movements. J. Exp. Med. 194, 45–56 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Okada, T. et al. Chemokine requirements for B cell entry to lymph nodes and Peyer's patches. J. Exp. Med. 196, 65–75 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Krug, A. et al. IFN-producing cells respond to CXCR3 ligands in the presence of CXCL12 and secrete inflammatory chemokines upon activation. J. Immunol. 169, 6079–6083 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Casamayor-Palleja, M. et al. Expression of macrophage inflammatory protein-3alpha, stromal cell-derived factor-1, and B-cell-attracting chemokine-1 identifies the tonsil crypt as an attractive site for B cells. Blood 97, 3992–3994 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Amara, A. et al. Stromal cell-derived factor-1alpha associates with heparan sulfates through the first β-strand of the chemokine. J. Biol. Chem. 274, 23916–23925 (1999).

    Article  CAS  PubMed  Google Scholar 

  33. Nieuwenhuis, P. & Opstelten, D. Functional anatomy of germinal centers. Am. J. Anat. 170, 421–435 (1984).

    Article  CAS  PubMed  Google Scholar 

  34. Wehrli, N. et al. Changing responsiveness to chemokines allows medullary plasmablasts to leave lymph nodes. Eur. J. Immunol. 31, 609–616 (2001).

    Article  CAS  PubMed  Google Scholar 

  35. Guinamard, R. et al. B cell antigen receptor engagement inhibits stromal cell-derived factor (SDF)-1α chemotaxis and promotes protein kinase C (PKC)-induced internalization of CXCR4. J. Exp. Med. 189, 1461–1466 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Marchese, A. & Benovic, J.L. Agonist-promoted ubiquitination of the G protein-coupled receptor CXCR4 mediates lysosomal sorting. J. Biol. Chem. 276, 45509–45512 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Balogh, P., Aydar, Y., Tew, J.G. & Szakal, A.K. Appearance and phenotype of murine follicular dendritic cells expressing VCAM-1. Anat. Rec. 268, 160–168 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Egawa, T. et al. The earliest stages of B cell development require a chemokine stromal cell-derived factor/pre-B cell growth-stimulating factor. Immunity 15, 323–334 (2001).

    Article  CAS  PubMed  Google Scholar 

  39. Ma, Q. et al. Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc. Natl. Acad. Sci. USA 95, 9448–9453 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Zou, Y.R., Kottmann, A.H., Kuroda, M., Taniuchi, I. & Littman, D.R. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393, 595–599 (1998).

    Article  CAS  PubMed  Google Scholar 

  41. Ara, T. et al. A role of CXC chemokine ligand 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its receptor CXCR4 in fetal and adult T cell development in vivo. J. Immunol. 170, 4649–4655 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Plotkin, J., Prockop, S.E., Lepique, A. & Petrie, H.T. Critical role for CXCR4 signaling in progenitor localization and T cell differentiation in the postnatal thymus. J. Immunol. 171, 4521–4527 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Martin, C. et al. Chemokines acting via CXCR2 and CXCR4 control the release of neutrophils from the bone marrow and their return following senescence. Immunity 19, 583–593 (2003).

    Article  CAS  PubMed  Google Scholar 

  44. Bleul, C.C., Fuhlbrigge, R.C., Casasnovas, J.M., Aiuti, A. & Springer, T.A. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J. Exp. Med. 184, 1101–1109 (1996).

    Article  CAS  PubMed  Google Scholar 

  45. Hernandez, P.A. et al. Mutations in the chemokine receptor gene CXCR4 are associated with WHIM syndrome, a combined immunodeficiency disease. Nat. Genet. 34, 70–74 (2003).

    Article  CAS  PubMed  Google Scholar 

  46. Luther, S.A., Gulbranson-Judge, A., Acha-Orbea, H. & MacLennan, I.C. Viral superantigen drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production. J. Exp. Med. 185, 551–562 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Ngo, V.N., Tang, H.L. & Cyster, J.G. Epstein-Barr virus-induced molecule 1 ligand chemokine is expressed by dendritic cells in lymphoid tissues and strongly attracts naive T cells and activated B cells. J. Exp. Med. 188, 181–191 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Cinamon, G. et al. Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone. Nat. Immunol. 5, 713–720 (2004).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank D. Hargreaves, Y. Xu and M. Lesneski for technical assistance; C. Miller for training in laser-capture microdissection; J. Dietrich for surgical expertise; S. Jiang for cell sorting; D. Littman for Cxcr4+/− mice; M. Lipp for Cxcr5−/− mice; T. Roach for some Eμ-Bcl2-22 mice; F. Arenzana-Seisdedos for K15C antibody; J. Lin and other members of the Weiss Lab for Jurkat cells; the Werb lab for the use of equipment and supplies; the University of California San Francisco Diabetes Center for use of the ABI Prism 7900HT; S. Luther, T. Okada and G. Cinamon for advice and comments on the manuscript; and M. Matloubian, C. Lo and J. Cholfin for discussions. Work supported by Howard Hughes Medical Institute and grants AI40098 and AI45073 from the National Institutes of Health, and by predoctoral grants from Howard Hughes Medical Institute (C.D.C.A. and K.M.A.).

Author information

Author notes

  1. K Mark Ansel

    Present address: Center for Blood Research, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts, 02115, USA

Authors and Affiliations

  1. Howard Hughes Medical Institute and Department of Microbiology and Immunology, University of California San Francisco, San Francisco, 94143-0414, California, USA

    Christopher D C Allen, K Mark Ansel, Caroline Low, Robin Lesley & Jason G Cyster

  2. Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, 606-8501, Kyoto, Japan

    Hirokazu Tamamura & Nobutaka Fujii

Authors
  1. Christopher D C Allen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K Mark Ansel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Caroline Low
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robin Lesley
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hirokazu Tamamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Nobutaka Fujii
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jason G Cyster
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jason G Cyster.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

GCs in Eμ-bcl-2-22 transgenic mice have normal dark and light zone polarity. (PDF 74 kb)

Supplementary Table 1

Antibodies used in flow cytometry. (PDF 18 kb)

Supplementary Table 2

Primary antibodies used in mouse immunohistochemistry. (PDF 19 kb)

Supplementary Table 3

Secondary antibodies used in mouse immunohistochemistry. (PDF 17 kb)

Supplementary Table 4

Primer and probe sequences for quantitative real-time PCR. (PDF 18 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Allen, C., Ansel, K., Low, C. et al. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat Immunol 5, 943–952 (2004). https://doi.org/10.1038/ni1100

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni1100

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing