IgG-Immune Complexes Promote B Cell Memory by Inducing BAFF.

Memory B cell responses are vital for protection against infections but must also be regulated to prevent autoimmunity. Cognate T cell help, somatic hypermutation, and affinity maturation within germinal centers (GCs) are required for high-affinity memory B cell formation; however, the signals that commit GC B cells to the memory pool remain unclear. In this study, we identify a role for IgG-immune complexes (ICs), FcγRs, and BAFF during the formation of memory B cells in mice. We found that early secretion of IgG in response to immunization with a T-dependent Ag leads to IC-FcγR interactions that induce dendritic cells to secrete BAFF, which acts at or upstream of Bcl-6 in activated B cells. Loss of CD16, hematopoietic cell-derived BAFF, or blocking IC:FcγR regions in vivo diminished the expression of Bcl-6, the frequency of GC and memory B cells, and secondary Ab responses. BAFF also contributed to the maintenance and/or expansion of the follicular helper T cell population, although it was dispensable for their formation. Thus, early Ab responses contribute to the optimal formation of B cell memory through IgG-ICs and BAFF. Our work defines a new role for FcγRs in GC and memory B cell responses.

Receptor cross-talk spatially restricts p-ERK during TLR4 stimulation of autoreactive B cells.

To maintain tolerance, autoreactive B cells must regulate signal transduction from the BCR and TLRs. We recently identified that dendritic cells and macrophages regulate autoreactive cells during TLR4 activation by releasing IL-6 and soluble CD40 ligand (sCD40L). These cytokines selectively repress Ab secretion from autoreactive, but not antigenically naive, B cells. How IL-6 and sCD40L repress autoantibody production is unknown. In this work, we show that IL-6 and sCD40L are required for low-affinity/avidity autoreactive B cells to maintain tolerance through a mechanism involving receptor cross-talk between the BCR, TLR4, and the IL-6R or CD40. We show that acute signaling through IL-6R or CD40 integrates with chronic BCR-mediated ERK activation to restrict p-ERK from the nucleus and represses TLR4-induced Blimp-1 and XBP-1 expression. Tolerance is disrupted in 2-12H/MRL/lpr mice where IL-6 and sCD40L fail to spatially restrict p-ERK and fail to repress TLR4-induced Ig secretion. In the case of CD40, acute signaling in B cells from 2-12H/MRL/lpr mice is intact, but the chronic activation of p-ERK emanating from the BCR is attenuated. Re-establishing chronically active ERK through retroviral expression of constitutively active MEK1 restores tolerance upon sCD40L, but not IL-6, stimulation, indicating that regulation by IL-6 requires another signaling effector. These data define the molecular basis for the regulation of low-affinity autoreactive B cells during TLR4 stimulation; they explain how autoreactive but not naive B cells are repressed by IL-6 and sCD40L; and they identify B cell defects in lupus-prone mice that lead to TLR4-induced autoantibody production.

Autoreactive preplasma cells break tolerance in the absence of regulation by dendritic cells and macrophages.

The ability to induce Ab responses to pathogens while maintaining the quiescence of autoreactive cells is an important aspect of immune tolerance. During activation of TLR4, dendritic cells (DCs) and macrophages (MFs) repress autoantibody production through their secretion of IL-6 and soluble CD40L (sCD40L). These soluble mediators selectively repress B cells chronically exposed to Ag, but not naive cells, suggesting a means to maintain tolerance during TLR4 stimulation, yet allow immunity. In this study, we identify TNF-α as a third repressive factor, which together with IL-6 and CD40L account for nearly all the repression conferred by DCs and MFs. Similar to IL-6 and sCD40L, TNF-α did not alter B cell proliferation or survival. Instead, it reduced the number of Ab-secreting cells. To address whether the soluble mediators secreted by DCs and MFs functioned in vivo, we generated mice lacking IL-6, CD40L, and TNF-α. Compared to wild-type mice, these mice showed prolonged anti-nuclear Ab responses following TLR4 stimulation. Furthermore, adoptive transfer of autoreactive B cells into chimeric IL-6(-/-) × CD40L(-/-) × TNF-α(-/-) mice showed that preplasma cells secreted autoantibodies independent of germinal center formation or extrafollicular foci. These data indicate that in the absence of genetic predisposition to autoimmunity, loss of endogenous IL-6, CD40L, and TNF-α promotes autoantibody secretion during TLR4 stimulation.

Regulation of ENT1 expression and ENT1-dependent nucleoside transport by c-Jun N-terminal kinase.

Equilibrative nucleoside transporters (ENTs) are facilitative transporters broadly selective for pyrimidine and purine nucleosides and are essential for the modulation of nucleoside concentration and nucleoside analog availability. Resistance to nucleoside-derived drugs strongly correlates with a deficiency of ENT1 expression in several tumor cells. Thus, it is crucial to understand the mechanisms by which this transporter is modulated. Using a mouse myeloid leukemic cell line as a model, we investigated whether stress-activated kinases regulate ENT1 expression and function. JNK activation, but not p38 MAPK results in rapid loss of mENT1 function, mRNA expression and promoter activity. c-Jun but not the mutant c-Jun Ser63/73Ala, decreased mENT1 promoter activity. Moreover cJun bound to an AP-1 site identified at -1196 of the promoter, suggesting a specific role for this transcription factor in mENT1 regulation. We propose that activation of JNK-cJun pathway negatively regulates mENT1 and suggest that this mechanism might contribute to the development of nucleoside analog-derived drug resistance.

Imatinib-resistant CML cells have low ENT activity but maintain sensitivity to gemcitabine.

Philadelphia chromosome-positive chronic myelogenus leukemia (CML) is widely treated with imatinib mesylate (imatinib), a potent inhibitor of the Bcr-Abl tyrosine kinase. However, resistance to this compound remains a concern. Current treatment approaches include combinations of imatinib with nucleoside analogs such as gemcitabine, which requires equilibrative nucleoside transporters (ENTs) for uptake, to overcome this resistance. Here we report that imatinib treatment decreased ENT1-dependent activity and mRNA expression. Although, imatinib-resistant cells showed decreased levels of both ENT1 and ENT2 activity and expression, these cells remained sensitive to gemcitabine, suggesting that nucleoside analogs can be used as adjunctive therapy.