In recent years, naturalor synthetic TLR ligands have been used to boost DC driven Ag-specific immune responses in mice and humans (27
). A better understanding of the role of DCs during TLR-mediated B cell activation would provide important information about the use of TLR ligands as adjuvants during DC-based vaccine development. In this regard, we performed comprehensive in vitro
studies, including proliferation, cell cycle progression, differentiation, antibody production, and apoptosis of B cells in the presence or absence of iBMDCs, mBMDCs, BM-RDCs and Spl-RDCs. iBMDCs or BM-RDCs, but not mBMDCs or Spl-RDCs, strongly inhibited TLR (2, 3 & 4)-induced B cell proliferation.
Among various mitogens used in our study iBMDCs specifically inhibited TLR2, TLR3 and TLR4 as well as BCR-induced proliferation, but did not inhibit anti-CD40 or PMA-ionomycin induced B cell proliferation. These results suggest that iBMDC-mediated inhibition of B cells is signaling pathway specific and does not have global growth inhibitory effects. The cell cycle analysis data revealed that iBMDCs block B cell proliferation by inducing G1-S growth arrest. Further our study showed that the cell cycle arrest of B cells in the presence of iBMDCs is not due to their differentiation, as plasma cell formation and antibody production were also decreased in the presence of iBMDCs. Immature DCs have been shown to inhibit and tolerize T cells in vivo due to the absence of a second (co-stimulatory) signal, whereas maturation of DCs by TLR or CD40 overcomes this inhibitory effect on T cells (25
). Similar to the DC-mediated regulation of T cells, maturation of iBMDCs with TLR ligands overcame the inhibitory effect of DCs on B cells. Many different TLR ligands can mature DCs in either a MyD88 dependent or MyD88 independent manner (21
). Even though different TLR ligands are able to induce DC maturation, there were noticeable differences observed in terms of cytokine and chemokine secretion patterns depending on the TLR ligand (54
). We found that the maturation of iBMDCs via the MyD88 dependent pathway [LPS, Peptidoglycan (data not shown)] or the MyD88 independent pathway [Poly (I:C)] had similar effects in overcoming the inhibitory effect of iBMDCs on B cells. Co-stimulating B cells with anti-CD40 also overcame inhibitory effects of iBMDCs on B cells.
The iBMDC-mediated inhibition of B cells was dependent significantly on cell contact. This is in agreement with Santos et al. who showed that iBMDCs inhibited BCR mediated B cell responses in a contact dependent manner through CD22 mediated signaling on B cells (26
). We observed that CD22 expression is also critical for the iBMDC-mediated inhibition of TLR-induced B cell proliferation. The absence of CD22 on B cell surfaces completely abrogated iBMDC-mediated inhibitory effects during TLR3 or TLR4 induced B cell proliferation. Interestingly, maturation of BMDCs has been reported to decrease the expression of sialic acid ligands (55
), which is consistent with the role of CD22 in DC-B cell interaction. In parallel with finding of Santos et al. (26
), we found that expression of ST6Gal I, one of the enzymes that induces sialylation, was not required for DC-B cell interaction. Presently we cannot rule out the possibility that CD22 on B cells binds some other ligand on iBMDCs and inhibits their response to BCR and TLR mediated signaling. Despite the importance of CD22, present studies also cannot rule out a role for other sialic acid binding proteins like Siglec-G, which have been shown to be important for B-1 cell responses and for antigen induced tolerance in B cells (56
). Moreover, the inability of mature DCs to inhibit B cells could also be due to generation of rescue signals such as the gene expression pattern induced by CD40 ligation.
Our study with resident DCs from spleen and BM showed that inhibitory effect of DCs on B cells is a characteristic of BM-RDCs but not Spl-RDCs. To our knowledge, such a difference between BM-RDCs and Spl-RDCs in affecting B cell activation was never reported before. Spl-RDCs without stimulation were immature, characterized by low CD86 and Class II expression on the surface, compared to LPS matured Spl-RDCs. However, Spl-RDCs did not inhibit TLR induced B cell responses. In contrast, DCs isolated from the BM showed strong inhibition of TLR-induced proliferation of B cells from both BM and spleen. BM-RDCs resembled iBMDCs in enhancing CD40 mediated proliferation of B cells from both BM and spleen. This difference between BM-RDCs and Spl-RDCs is interesting in the context of the requirement for CD22, as there is no data about differences in the ubiquitously expressed ligands containing sialic acids on these two DC populations. Although iBMDCs normally are a heterogeneous population of DCs, flow cytometric sorting established that myeloid DCs in this population have potent inhibitory effects on B cells. It is of interest that Kilmon et al found that myeloid DCs and macrophages, but not plasmacytoid DCs inhibited auto-antibody production by self-reactive B cells but not antibody production by normal B cells (57
Finally, the inhibitory effects of iBMDCs in BM B cell growth responses led us to examine the role of iBMDCs in Ag-dependent selection of B cells. Negative selection of self-reactive B cells may occur via receptor editing, anergy or deletion. B cell tolerance involves anergy when soluble antigen is present but utilizes clonal deletion mechanisms when self-antigens are membrane bound. This was well established by the studies of Good now et al using soluble and membrane bound HEL and by Nemazee et al using membrane bound MHC class I molecules (51
). Therefore DCs may provide an opportunity to present self-antigens in a membrane bound form leading to deletion of self-reactive B cells. We found that HEL specific BM B cells showed increased apoptosis when HEL was presented by iBMDCs. Currently we don’t know the underlying mechanism. In the literature there is evidence for presentation of native antigen by DCs (59
). Hence one scenario for increased apoptosis with HEL pulsed BM B cells could be simultaneous binding of antigen to BCR and CD22 ligands on DCs to CD22 on B cells, although other explanations cannot be ruled out at this time. In thymus, DCs present self-antigen in the context of their MHC and play a decisive role in the negative selection of T cells (60
). Our study is first to suggest that DCs in the BM might play a similar role in B cell negative selection by presenting self-antigens to B cells in the context of CD22/SIGLEC mediated inhibitory signals. In this context, recently it has been shown that decoration of T-independent antigens with sialic acid epitopes made them tolerogenic through their ability to cross-link SIGLEC family proteins (56
). It was proposed that self-antigens that behave like T-independent antigens may utilize this pathway for self-tolerance. In support of such an idea, mice doubly deficient for CD22 and Siglec G developed autoantibodies and a moderate form of immune complex mediated glomerular nephritis (61
). Although the significance of inhibition of TLR responses of BM B cells is at present unclear, it must be noted that several endogenous TLR ligands have been identified and have been implicated in the breakdown of self-tolerance in several autoimmune models (62
). Some of the endogenous TLR ligands such as high-mobility group box 1 and heat shock proteins have their origin in cell death (66
), which is known to occur extensively during B cell development. Defects in clearance of dead cells have a critical role in development of autoimmune diseases like lupus (67
). In the periphery DCs in mucosal areas and epidermal Langerhans cells (sites of extensive cell turnover) have anti-inflammatory properties. Further in vivo
studies are warranted to address the potential role of DCs in the negative selection of B cells in the BM.
Overall, our study gives valuable insight on the role that immature DCs play in B cell function. As there is increasing use of BM derived DC vaccines and TLR ligands as adjuvants, a careful selection of DC subset and maturation state is warranted to generate B cell specific immune responses.