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T follicular helper (Tfh) cells are essential providers of help to B cells. The transcription factor B-cell CLL/lymphoma 6 (Bcl6) is a lineage-defining regulator of Tfh cells and germinal center B cells. In B cells, Bcl6 has the potential to recruit distinct transcriptional corepressors through its BTB domain or its poorly characterized middle domain (also known as RDII), but in Tfh cells the roles of the Bcl6 middle domain have yet to be clarified. Mimicked acetylation of the Bcl6 middle domain (K379Q) in CD4 T cells results in significant reductions in Tfh differentiation in vivo. Blimp1 (Prdm1) is a potent inhibitor of Tfh cell differentiation. Although Bcl6 K379Q still bound to the Prdm1 cis-regulatory elements in Tfh cells, Prdm1 expression was derepressed. This was a result of the failure of Bcl6 K379Q to recruit metastasis-associated protein 3 (MTA3). The loss of Bcl6 function in Bcl6 K379Q-expressing CD4 T cells could be partially rescued by abrogating Prdm1 expression. In addition to Prdm1, we found that Bcl6 recruits MTA3 to multiple genes involved in Tfh cell biology, including genes important for cell migration, cell survival, and alternative differentiation pathways. Thus, Bcl6 middle domain mediated repression is a major mechanism of action by which Bcl6 controls CD4 T-cell fate and function.

A member of the IFN regulatory factor (IRF) family of transcription factors, IRF-4 is expressed in lymphocytes and macrophage/dendritic cells. Studies using IRF-4-deficient mice have revealed the critical roles of IRF-4 in lymphocyte responses. However, the role of IRF-4 in innate immune responses is not clearly understood. Here, we demonstrate that IRF-4 negatively regulates the production of proinflammatory cytokines by macrophages in response to Toll-like receptor (TLR) stimulation. Mice lacking IRF-4 are sensitive to LPS-induced shock, and their macrophages produce high levels of proinflammatory cytokines, including TNF-alpha and IL-6, in response to TLR ligands. The inhibitory role of IRF-4 in response to TLR stimulation was confirmed by the down-regulation of IRF-4 expression in normal macrophages by using the small interfering RNA technique and by the overexpression of IRF-4 in macrophage line RAW264.7. Activation of the important signaling pathways for cytokine production, NF-kappaB and JNK (c-Jun N-terminal kinase), was enhanced after LPS stimulation in IRF-4(-/-) macrophages. These results imply that IRF-4 negatively regulates TLR signaling and is inhibitory to the production of proinflammatory cytokines in response to TLR stimulation.

Acquired immunity depends on proliferation and differentiation of antigen (Ag)-specific B cells in germinal centers (GCs) of lymphoid follicles in response to T cell-dependent Ags. Here, we studied the function of GC-associated nuclear protein that is selectively up-regulated in GC-B cells. B cell-specific ganp-deficient mice were compromised in affinity maturation of hapten-specific antibodies against T cell-dependent Ags with retarded development of GCs. B cell numbers and development, serum Ig levels, mitogen-induced B cell proliferation in vitro, and responses to T cell-independent Ag were nearly normal; however, the mutant B cells showed a decrease in anti-CD40-induced proliferation and an increased susceptibility to B cell apoptosis in vitro and in vivo. B cell-specific ganp-deficient mice showed a decreased frequency of variable-region somatic mutations, especially of the high-affinity type (W(33) --> L) in the V(H)186.2 region against nitrophenyl-chicken gamma globulin, whereas the class switching was normal. We conclude that GC-associated nuclear protein is necessary for generation or maintenance of B cells with high-affinity B cell Ag receptors during the maturation in GCs.

TRAF5 [tumor necrosis factor (TNF) receptor-associated factor 5] is implicated in NF-kappaB and c-Jun NH(2)-terminal kinase/stress-activated protein kinase activation by members of the TNF receptor superfamily, including CD27, CD30, CD40, and lymphotoxin-beta receptor. To investigate the functional role of TRAF5 in vivo, we generated TRAF5-deficient mice by gene targeting. Activation of either NF-kappaB or c-Jun NH(2)-terminal kinase/stress-activated protein kinase by tumor necrosis factor, CD27, and CD40 was not abrogated in traf5(-/-) mice. However, traf5(-/-) B cells showed defects in proliferation and up-regulation of various surface molecules, including CD23, CD54, CD80, CD86, and Fas in response to CD40 stimulation. Moreover, in vitro Ig production of traf5(-/-) B cells stimulated with anti-CD40 plus IL-4 was reduced substantially. CD27-mediated costimulatory signal also was impaired in traf5(-/-) T cells. Collectively, these results demonstrate that TRAF5 is involved in CD40- and CD27-mediated signaling.