Intracellular signals generated by interactions between a number of TNF/TNFR family members have been shown to be critical for the function of B lymphocytes at multiple steps of humoral immune responses (1
). Stimulation of CD40 on B cells is essential for Ig isotype class switching of T-dependent antibody responses and germinal center formation (38
). Interactions between B lymphocyte stimulator (BLyS) and its receptors have been shown to influence multiple aspects of B cell biology, including development, proliferation, homeostasis, and humoral responses, especially to T-independent type II antigens (5
). Absence of DR6 was previously shown to enhance CD4+
T cell proliferation along with Th2 differentiation and cytokine production (25
). In the course of in vivo responses, however, CD4+
T cells undergo multiple interactions with other cell types to establish protective immunity. During T cell–dependent B cell responses, interactions between antigen–BCR, MHC class II–peptide-TCR, CD28–CD86, and CD40–CD40L generate intracellular signals in both T and B cells that are necessary for the progression of a humoral response (40
). Surface expression of DR6 was detected on resting B220+
cells and like CD4+
T cells (25
), this expression was down-regulated after stimulation (). These data suggested that DR6 may also have functions intrinsic to B cells that occur early in responses.
We investigated the impact of DR6 deficiency on modulating B cell responses both in vitro and in vivo. DR6−/− mice exhibited no gross defects in B cell development or in peripheral populations of conventional B2, B1, or marginal zone B cells (). The in vitro proliferation of DR6−/− B cells was markedly increased after stimulation with either anti-IgM, anti-CD40, or LPS (). These responses were due, at least in part, to increased cell mitosis () and reduced apoptosis () after activation. Together, these in vitro results indicate that lack of DR6 in B cells can influence their activation, proliferation, and survival.
NF-κB/Rel family members are among the many transcription factors implicated in controlling gene expression in B lymphocytes and also can serve to protect cells from apoptotic signals (22
). C-Rel is expressed at high levels in lymphoid cells (19
). C-Rel–deficient B lymphocytes have impaired proliferative responses to anti-IgM, anti-CD40, or LPS and exhibit defects in their ability to receive survival signals through anti-IgM or LPS stimulation (21
). In our study, increased nuclear levels of c-Rel capable of forming c-Rel–DNA complexes were observed in activated DR6−/−
B cells, suggesting that DR6 may regulate B cell proliferative responses, at least in part, through a c-Rel–mediated pathway. Our data is consistent with previous studies (21
) indicating that c-Rel is a critical transcription factor for both cell division and apoptosis, and DR6 appears to be involved in regulating nuclear c-Rel activity. The increase of nuclear c-Rel in DR6−/−
B cells could be caused by accelerating the nuclear translocation by increased NF-κB pathway activation. However, we did not observe any differences in NF-κB activation between WT and DR6−/−
B cells after stimulation (unpublished data), suggesting the increase of nuclear c-Rel is not due to increased NF-κB activation and accelerated c-Rel nuclear translocation.
Bcl-2 family proteins have been shown to play an important role for the survival of various stages of B lymphocytes and Bcl-xL
is crucial for the survival and maturation of germinal center B cells (42
). The expression of apoptosis inhibitor Bcl-xL
is known to be transcriptionally regulated by c-Rel (33
). Enhanced expression of Bcl-xL
was observed in activated DR6−/−
B cells ( B), suggesting that increased nuclear c-Rel levels and Bcl-xL
expression may contribute to both increased proliferation and decreased apoptosis of activated DR6−/−
The CD28–CD86 interaction has been demonstrated to be important for T cell activation (43
). Increased expression of CD86 upon stimulation of B cells through BCR or CD40 was found to contribute to enhanced production of IgG1
and IgE (44
). Additionally, it was recently reported that signals induced by cross-linking of CD86, but not CD80, on activated B cells enhanced proliferation and production of IgG1 along with augmenting levels of Bcl-xL
). Upon activation, a greater percentage of cultured DR6−/−
B cells exhibited high CD86 surface expression compared with WT B cells ( A). In addition, our data demonstrated increased allogeneic T cell proliferation in response to previously activated DR6−/−
B cells as APC ( C). The increased CD86 expression of DR6−/−
B cell populations may contribute, via costimulatory signals, to increased T cell responses and/or may also provide signals directly to the B cell itself.
T cells can influence antibody isotype secretion to T-dependent antigens. Th2 cells contribute to IgG1
and IgE isotype generation whereas Th1 cells influence IgG2a
). Previous studies showed that upon KLH challenge, production of Th2 cytokines was markedly higher in the activated DR6−/−
T cells (25
). Consistent with these previous studies, DR6−/−
mice immunized with NP-KLH exhibited increased IgM and IgG1
isotypes at day 7, however, no significant difference was observed in IgG2a
levels compared with WT controls.
In addition, we observed an increase in the size of germinal centers in DR6−/−
spleens after T-dependent antigen challenge (). Previous studies have shown that CD28 is a crucial factor for germinal center formation (48
) and CD28 expression is up-regulated in DR6−/−
T cells compared with WT mice (25
). This, along with higher B cell CD86 expression, suggests that DR6−/−
B cells received stronger stimulating signals in the T cell–rich regions of the white pulp to promote the formation of germinal centers. Although the absence of DR6 affects T-dependent humoral responses, it is unclear whether this is mediated through intrinsic effects on B cells, the preferential Th2 differentiation of CD4+
T cells, or a combination of both.
T-independent antigens are also critical for host defense and involve the stimulation of B cells by antigen-presenting dendritic cells without the initial involvement of CD4+
T cells (49
). In addition to T-dependent humoral responses, DR6−/−
mice also displayed increased Ig levels in response to T-independent type I (NP-LPS) and type II (NP-Ficoll) antigens. These data further indicate that DR6 serves a regulatory role that is intrinsic to B cells, although it does not exclude the possibility that DR6 influences cellular responses in both the innate and adaptive arms of host defense.
Our current studies provide evidence that DR6 has a fundamental role in activation-induced B cell expansion, survival, and humoral responses. Therefore, modulation of the DR6 signaling pathway might be able to control the extent of B cell responses in addition to CD4+ T cell responses and potentially provide therapeutic benefits to treat certain immune system disorders, such as asthma, allergy, and various autoimmune diseases.