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The CD40-TRAF6 axis controls affinity maturation and the generation of long-lived plasma cells

Nature Immunology volume 3, pages 451456 (2002) | Download Citation



Affinity maturation of the immune response and the generation of long-lived bone marrow (BM) plasma cells are hallmarks of CD40-dependent, thymus-dependent (TD) humoral immunity. Through disruption of the tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)-binding site within the CD40 cytoplasmic domain, we selectively ablated affinity maturation and the generation of plasma cells after immunization. Mutagenesis of both the TRAF6 and TRAF2-TRAF3 sites was essential for arresting germinal center formation in response to immunization. CD40-induced B cell proliferation and early immunoglobulin production occurred even when all TRAF sites were ablated. These studies show that specific CD40-TRAF associations control well defined aspects of humoral immunity. In addition, they define the roles that TRAF-dependent and TRAF-independent pathways play in regulating antigen-driven B cell differentiation.

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  1. 1.

    et al. The CD40 antigen and its ligand. Annu. Rev. Immunol. 12, 881–922 (1994).

  2. 2.

    et al. Assembly and regulation of the CD40 receptor complex in human B cells. J. Exp. Med. 186, 337–342 (1997).

  3. 3.

    et al. CD40-tumor necrosis factor receptor-associated factor (TRAF) interactions: regulation of CD40 signaling through multiple TRAF binding sites and TRAF hetero-oligomerization. Biochemistry 37, 11836–11845 (1998).

  4. 4.

    , , & CD40 signaling through tumor necrosis factor receptor-associated factors (TRAFs). Binding site specificity and activation of downstream pathways by distinct TRAFs. J. Biol. Chem. 274, 14246–14254 (1999).

  5. 5.

    & TANK, a co-inducer with TRAF2 of TNF- and CD 40L-mediated NF-κB activation. Genes Dev. 10, 963–973 (1996).

  6. 6.

    et al. TRAF6 deficiency results in osteopetrosis and defective interleukin-1, CD40, and LPS signaling. Genes Dev. 13, 1015–1024 (1999).

  7. 7.

    et al. Early lethality, functional NF-κB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7, 715–725 (1997).

  8. 8.

    , & The TNF-receptor-associated factor family: scaffold molecules for cytokine receptors, kinases and their regulators. Cell Signal. 13, 389–400 (2001).

  9. 9.

    et al. Targeted disruption of Traf5 gene causes defects in CD40- and CD27- mediated lymphocyte activation. Proc. Natl. Acad. Sci. USA 96, 9803–9808 (1999).

  10. 10.

    , & Targeted disruption of TRAF3 leads to postnatal lethality and defective T-dependent immune responses. Immunity 5, 407–415 (1996).

  11. 11.

    , & Characterization of the roles of TNF receptor-associated factor 6 in CD40-mediated B lymphocyte effector functions. J. Immunol. 164, 623–630 (2000).

  12. 12.

    et al. Differential requirements for tumor necrosis factor receptor-associated factor family proteins in CD40-mediated induction of NF-κB and Jun N-terminal kinase activation. J. Biol. Chem. 274, 22414–22422 (1999).

  13. 13.

    , & Characterization of CD40 signaling determinants regulating nuclear factor-κB activation in B lymphocytes. J. Immunol. 159, 4898–4906 (1997).

  14. 14.

    , & CD40-mediated activation of IgCγ1 and IgCɛ germ-line promoters involves multiple TRAF family proteins. Eur. J. Immunol. 29, 3908–3913 (1999).

  15. 15.

    , , , & Cellular responses to murine CD40 in a mouse B cell line may be TRAF dependent or independent. Eur. J. Immunol. 32, 39–49 (2002).

  16. 16.

    , , , & A vector driving the expression of foreign cDNAs in the MHC class II- positive cells of transgenic mice. J. Immunol. Meth. 166, 287–291 (1993).

  17. 17.

    et al. Recombinant soluble trimeric CD40 ligand is biologically active. J. Biol. Chem. 270, 7025–7028 (1995).

  18. 18.

    et al. TRAF2 deficiency results in hyperactivity of certain TNFR1 signals and impairment of CD40-mediated responses. Immunity 11, 379–389 (1999).

  19. 19.

    , & An 11-amino acid sequence in the cytoplasmic domain of CD40 is sufficient for activation of c-Jun N-terminal kinase, activation of MAPKAP kinase-2, phosphorylation of IκBα, and protection of WEHI-231 cells from anti-IgM-induced growth arrest. J. Immunol. 162, 4720–4730 (1999).

  20. 20.

    et al. TRAF2 is essential for JNK but not NF-κB activation and regulates lymphocyte proliferation and survival. Immunity 7, 703–713 (1997).

  21. 21.

    et al. CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation. Proc. Natl. Acad. Sci. USA 91, 12135–12139 (1994).

  22. 22.

    et al. In vivo CD40-gp39 interactions are essential for thymus-dependent humoral immunity. II. Prolonged suppression of the humoral immune response by an antibody to the ligand for CD40, gp39. J. Exp. Med. 178, 1567–1575 (1993).

  23. 23.

    et al. Mice deficient for the CD40 ligand. Immunity 1, 423–431 (1994).

  24. 24.

    et al. TRAF1 is a negative regulator of TNF signaling: Enhanced TNF signaling in TRAF1-deficient mice. Immunity 15, 647–657 (2001).

  25. 25.

    et al. Identification of the intracytoplasmic region essential for signal transduction through a B cell activation molecule, CD40. Eur. J. Immunol. 20, 1747–1753 (1990).

  26. 26.

    , , & A novel RING finger protein interacts with the cytoplasmic domain of CD40. J. Biol. Chem. 269, 30069–30072 (1994).

  27. 27.

    , , & A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78, 681–692 (1994).

  28. 28.

    , , & Different CD40-mediated signaling events require distinct CD40 structural features. J. Immunol. 157, 1047–1053 (1996).

  29. 29.

    , , , & Two differently regulated nuclear factor κB activation pathways triggered by the cytoplasmic tail of CD40. Proc. Natl. Acad. Sci. USA 96, 1234–1239 (1999).

  30. 30.

    , , , & Rewiring of CD40 is necessary for delivery of rescue signals to B cells in germinal centres and subsequent entry into the memory pool. Immunology 102, 263–272 (2001).

  31. 31.

    & Jak3 is associated with CD40 and is critical for CD40 induction of gene expression in B cells. Immunity 6, 379–87 (1997).

  32. 32.

    et al. Dissection of B cell differentiation during primary immune responses in mice with altered CD40 signals. Int. Immunol 14, 319–329 (2002).

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Confocal microscopy and flow cytometry was done at the Herbert C. Englert Cell Analysis Laboratory, which was established by equipment grants from the Fannie E. Rippel Foundation, the NIH Shared Instrument Program and Dartmouth Medical School and is supported, in part, by Core Grant CA 23108 from the National Cancer Institute to the Norris Cotton Cancer Center. Supported by NIH grant AI26296 (to R. J. N.) and the American Cancer Society (L. D. E.).

Author information

Author notes

    • Cory L. Ahonen
    •  & Eric M. Manning

    These authors contributed equally to this work.

    • Marilyn R. Kehry

    Present address, IDEC Pharmaceuticals Corporation, 3010 Science Park Rd, P.O. Box 919080, San Diego, CA 92191-9080, USA.


  1. Department of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756, USA.

    • Cory L. Ahonen
    • , Eric M. Manning
    • , Loren D. Erickson
    • , Brian P. O'Connor
    • , Evan F. Lind
    •  & Randolph J. Noelle
  2. Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., P.O. Box 368, Ridgefield, CT 06877-0368, USA.

    • Steven S. Pullen
    •  & Marilyn R. Kehry


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The authors declare no competing financial interests.

Corresponding author

Correspondence to Randolph J. Noelle.

Supplementary information

Image files

  1. 1.

    Web Figure 1.

    Transgenic CD40 expression in X-CD40 mice. T-depleted splenocytes from Flt3L-treated mice (10 days with 10 µg/mouse/day) were stained with anti-MHC class II (M5) or CD11c and anti–human CD40. Histograms depict anti–human CD40 staining on both class II and CD11c–positive cell populations in each X-CD40 strain. Open histograms depict staining on CD40-/- populations.

  2. 2.

    Web Figure 2.

    Assessment of signaling data in X-CD40 transgenic mice on a murine CD40+ genetic background. Where denoted by the letter M, murine CD40 was engaged with the monoclonal anti-murine CD40 agonistic antibody 1c10. Control experiments were performed at time points of maximal stimulation: 5 min for phospho-IκBα analysis and 10 min for Jnk and p38 MAPK phosphorylation analyses. The expression of endogenous CD40 does not affect the function of the transgenic receptor. (a) Primary cultures of splenic B cells from unimmunized X-CD40 transgenic mice were stimulated in vitro with 1000 ng/ml of shCD154 for the times indicated in minutes. Cells were lysed, and lysates were immunoblotted with antibodies against the phosphorylated TpY motif of Jnk2. Both isoforms of phosphorylated Jnk, p54 and p46, are recognized by the primary antibody. (b) Analysis of p38 MAPK activation in primary splenic B cells treated with shCD154. Cells were stimulated for indicated times, lysed and analyzed by immunoblot with an antibody against the phosphorylated TpY motif of p38 MAPK. (c) Analysis of NF-kB activation in primary splenic B cells. Cells were treated with shCD154 for the times indicated, lysed and analyzed by immunoblot using an antibody against phospho–Ser32-IkBa. For all the signaling experiments, the data presented are representative of 3 or more independent experiments.

  3. 3.

    Web Figure 3.

    Testing doses of shCD154 for induced B cell proliferation. Primary splenic B cells were cultured for 60 h with indicated doses of shCD154 plus IL-4 and IL-5. Cultures were supplemented with 1µCi of [3H]thymidine, and continued for an additional 12 h. After 72 h total culture time, cells were harvested, and genomic DNA was tested for incorporation of [3H]thymidine by scintillation counting. Proliferation was indistinguishable in all mice at doses above 200 ng/ml. In all mice, proliferation was diminished at doses below 100 ng/ml.

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