SUMMARY Immunoglobulin M (IgM) memory cells undergo differentiation in germinal centers following antigen challenge, but the full effector cell potential of these cells is unknown. We monitored the differentiation of enhanced yellow fluorescent protein (eYFP)- labeled CD11c+ and CD11cneg T-bet+ IgM memory cells after their transfer into naive recipient mice. Following challenge infection, many memory cells differentiated into IgM-producing plasmablasts. Other donor B cells entered germinal centers, down- regulated CD11c, underwent class switch recombination, and became switched memory cells. Yet other donor cells were maintained as IgM memory cells, and these IgM memory cells retained their multi-lineage potential following serial transfer. These findings were corroborated at the molecular level using immune repertoire analyses. Thus, IgM memory cells can differentiate into all effector B cell lineages and undergo self-renewal, properties that are characteristic of stem cells. We propose that these memory cells exist to provide long-term multi-functional immunity and act primarily to maintain the production of protective antibodies.

CD11c + T-bet + B cells generated during ehrlichial infection require CD4 + T cell help and IL-21 signaling for their development, but the exact T cell subset required had not been known. In this study, we show in a mouse model of Ehrlichia muris that type 1 T follicular helper (T FH1 ) cells provide help to CD11c + T-bet + B cells via the dual secretion of IL-21 and IFN-g in a CD40/CD40Ldependent manner. T FH1 cell help was delivered in two phases: IFN-g signals were provided early in infection, whereas CD40/ CD40L help was provided late in infection. In contrast to T-bet + T cells, T-bet + B cells did not develop in the absence of B cellintrinsic Bcl-6 but were generated in the absence of T-bet. T-bet-deficient memory B cells were largely indistinguishable from their wild-type counterparts, although they no longer underwent switching to IgG2c. These data suggest that a primary function of T-bet in B cells during ehrlichial infection is to promote appropriate class switching, not lineage specification. Thus, CD11c + memory B cells develop normally without T-bet but require Bcl-6 and specialized help from dual cytokine-producing T FH1 cells.

Bacterial, parasitic, and viral infections are well-known causes of lymphoid tissue disorganization, although the factors, both host and/or pathogen derived, that mediate these changes are largely unknown. Ehrlichia muris infection in mice causes a loss of germinal center (GC) B cells that is accompanied by the generation of extrafollicular T-bet + CD11c + plasmablasts and IgM memory B cells. We addressed a possible role for TNF-a in this process because this cytokine has been shown to regulate GC development. Ablation of TNF-a during infection resulted in an 8-fold expansion of GL7 + CD38 lo CD95 + GC B cells, and a 2.5-and 5-fold expansion of CD138 + plasmablasts and T-bet + memory cells, respectively. These changes were accompanied by a reduction in splenomegaly, more organized T and B cell zones, and an improved response to Ag challenge. CXCL13, the ligand for CXCR5, was detected at 6-fold higher levels following infection but was much reduced following TNF-a ablation, suggesting that CXCL13 dysregulation also contributes to loss of lymphoid tissue organization. T follicular helper cells, which also underwent expansion in infected TNF-a-deficient mice, may also have contributed to the expansion of T-bet + B cells, as the latter are known to require T cell help. Our findings contrast with previously described roles for TNF-a in GCs and reveal how host-pathogen interactions can induce profound changes in cytokine and chemokine production that can alter lymphoid tissue organization, GC B cell development, and extrafollicular T-bet + B cell generation.

CD11c+T-bet+ B cells are recognized as an important component of humoral immunity and autoimmunity. These cells can be distinguished from other B cells by their higher expression of the adenosine receptor 2a. Here we address whether A2A receptor activation can affect CD11c+T-bet+ B cells. We show that administration of the A2A receptor agonist CGS-21680 depletes established CD11c+T-bet+ B cells in ehrlichial-infected mice, in a B cell-intrinsic manner. Agonist treatment similarly depletes CD11c+T-bet+ B cells and CD138+ B cells and reduces anti-nuclear antibodies in lupus-prone mice. Agonist treatment is also associated with reduced kidney pathology and lymphadenopathy. Moreover, A2A receptor stimulation depletes pathogenic lymphocytes and ameliorates disease even after disease onset, highlighting the therapeutic potential of this treatment. This study suggests that targeting the adenosine signaling pathway may provide a method for the treatment of lupus and other autoimmune diseases mediated by T-bet+ B cells.

Our studies of ehrlichial infection have identified a population of CD11c+ T-bet+ IgM memory B-cells that arise within 30 days post-infection. We and others have shown that CD11c+ T-bet+B cells require for their development or function a number of factors, including T-bet, IFNγ, CD4 T-cells, IL-21, and TLRs. Here, we addressed a possible role for TNFα in the generation of CD11c+ T-bet+IgM memory B-cells, because aside from being a major inflammatory cytokine, it can regulate germinal center development, and mediate apoptosis. We observed an 8-fold increase in the number of CD11c+IgM memory B-cells (also CD73+) in TNFα-deficient mice, relative to wild-type mice, on day 16 post-infection. The B cell population persisted at much higher frequency and number on day 30 post-infection. The changes in the IgM memory cell population were associated with a concomitant 10-fold relative increase in the frequency of GL7+ CD38lo CD95+germinal center B cells in TNFα-deficient mice. These latter data contrast with published studies that have reported that TNFα is required for GC development, suggesting that the requirement we have observed for TNFα is context-dependent. These data suggest that there may be a positive relationship between GC development and generation of CD11c+ IgM memory cells, and that TNFα may act to mediate IgM memory cell homeostasis by regulating apoptosis. Ongoing studies will address mechanisms whereby TNFα contributes to the genesis and maintenance of CD11c+ T-bet+IgM memory B-cells.

T-bet+ B cells have emerged as a major B cell subset associated with both protective immunity and autoimmunity. T-bet is considered to be a master transcription factor in type I adaptive immune responses to intracellular pathogens, a response characterized by the production of interferon gamma. Our studies have shown that infection with the intracellular bacterium, E. muris, generates both extrafollicular T cell-independent T-bet+ CD11c+ IgM-secreting plasmablasts (PB), as well as T-bet+ CD11c+ IgM memory cells. Both of these B cell populations play key roles in antigen-specific humoral immunity, although the role of T-bet in their development had not been resolved. Although T-bet is often considered to define lineage in Type I B cells, we found that T-bet was dispensable for memory B cell development. Memory B cells from mice with B cell-specific T-bet deficiency exhibited nearly identical surface marker expression as those of wild-type mice, including CD11c, CD73, PD-L2, CD80, CD38, CD95, and CXCR3. In contrast, T-bet-deficient early CD11c+ splenic PB were significantly reduced in frequency, suggesting that T-bet regulates PB, but not memory cell development or differentiation. As has been reported, antigen-specific IgG2c was no longer the dominant serum antibody isotype in mice lacking B cell T-bet expression. Our data suggest that a critical function of T-bet in B cells is to promote the differentiation of short-lived antibody-secreting PB, and to restrict class switching to protective IgG2c antibodies. Our findings are relevant to how T-bet+ B cells function not only in pathogen-specific immunity, but also in autoimmune diseases, wherein the quality of the humoral response is of critical importance.

Human inborn errors of immunity (IEIs) are a class of genetic disorders caused by monogenic germline mutations that impair the function of the encoded protein. We recently described a novel IEI due to an IL2RB homozygous mutation (p.Pro222_Gln225Del) in two siblings who suffered from multi-organ autoimmunity and CMV susceptibility. This mutation resulted in decreased cell surface protein expression and increased serum IL-2/15 levels, with concomitant increased baseline STAT5 phosphorylation (pSTAT5) but poor pSTAT5 response to IL-2 and IL-15 stimulation. Counterintuitively, this IL-2Rβ hypomorphic mutation led to an expansion of memory CD8+ T and immature CD56bright NK cells, which express the highest levels of IL-2Rβ and require IL-2/15 signaling for survival and differentiation. Yet, their maturation and function appeared impaired, suggesting that both intrinsic (hypomorphic receptor) and extrinsic (serum cytokine milieu) mechanisms were at play. To further interrogate these receptor instrinsic/extrinsic mechanisms we developed a mouse model that harbors the homolog mutation (Il2rbMut/Mut). When mutant bone marrow cells reconstituted a WT host, serum IL-2/15 levels and IL-2Rβ protein surface expression partially normalized, though IL-2/15-induced signaling remained hypofunctional. Additionally, the dysregulated mutant IL-2Rβ-driven CD8+ T cell immunophenotype normalized but the NK phenotype did not. These data highlight the asymmetrical effect that a partial IL-2Rβ defect has on CD8 T and NK cell IL-2/15-dependent signaling and downstream cellular processes. These results imply that strategies to modify serum IL-2/15 levels may serve as a therapeutic approach to treating IL-2/15 signaling defects.  Supported by grants from NIH (T32 AI074491, K23 AR070897)

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