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TLR9 can augment B cell activation, but also prevents autoimmune pathogenesis through an unknown mechanism. Herein we describe a cell-intrinsic mechanism that limits the extent of B cell activation by TLR9 ligands delivered by the B cell antigen receptor. This self-limiting response requires that TLR agonists be linked with B cell receptor ligands, as it is not observed with independent B cell receptor and TLR9 ligation. The underlying mechanism involves p38 dependent G1 cell cycle arrest that precedes intrinsic mitochondrial apoptosis, and is shared by all pre-immune murine B cell subsets and CD27− human B cells. B cells can be rescued from this death mechanism by survival or inflammatory cytokines, and preferentially switch to IgG2c producing cells both in vitro and in vivo. These findings reveal a previously unappreciated system of crosstalk that limits responses to antigens containing TLR9 ligands, and suggest that circumventing this response-limiting process can promote sustained autoantibody production.

Post-transcriptional regulation of gene expression is critical for the survival, differentiation, and function of B lymphocytes. To investigate roles of the RNA-binding protein HuR in B cells, we generated mice with B-cell specific deletion of HuR using Cre recombinase driven by the Mb1 promoter. These mice have modestly reduced numbers of naïve B cells, but show profound loss of serum antibodies of all isotypes and low numbers of germinal center B cells, plasma cells, and switched memory cells. Further, these mice fail to form germinal centers or undergo affinity maturation upon immunization with NP-OVA. In contrast, HuR-deficient B cells survive, proliferate, class switch, and undergo typical gene expression changes upon in vitro stimulation. We find that HuR regulates expression of CD81, CD70, and CD86, which promote the ability of B cells to physically interact with T cells. Biochemically, HuR binds to mRNAs of genes involved in cytoskeletal remodeling upon B cell activation. Because cytoskeletal reorganization is critical for antigen internalization, these data suggest that HuR-deficient B cells may have an antigen presentation defect. Consistent with the notion that B cell-intrinsic functions of HuR promote B-T communication in vivo, mice with HuR-deficient B cells have reduced numbers of T follicular helper cells, whose differentiation and maintenance are dependent on interactions with B cells. In sum, we find that HuR has critical roles in B cell function.

B lymphocyte stimulator (BLyS) signals via BR3 mediate selection and survival in the transitional, mature, and germinal center B cell pools. We have previously shown that treatment of mice with exogenous BLyS prior to immunization with HIV-1 envelope (Env) trimers improves neutralizing antibody breadth and potency. We are therefore investigating the hypothesis that broadly neutralizing B cell clonotypes are rare because of counter-selection at the transitional or germinal center checkpoints. Here we show that Env-specific murine B cells in pre-immune, germinal center, and memory B cell subsets can be visualized and tracked by flow cytometry using a biotinylated Env probe. Initial results indicate loss of Env+ B cells at both the transitional developmental stage and in the germinal center following immunization. As a first step in testing the feasibility of a BLyS pre-immunization treatment approach in nonhuman primates, we show that BLyS enhances survival of quiescent and dividing rhesus macaque B cells in vitro.

A growing literature implicates the B cell receptor (BCR), Toll-like receptors (TLR), and survival cytokines or costimulation in the etiology of SLE and other humoral autoimmune diseases. Our lab has found that BCR-delivered TLR9 ligands yield a unique post-proliferative cell death that limits responses to DNA-linked antigens. Furthermore, death via this mechanism is rescued by the survival cytokine BLyS. However, here we show that BLyS is not unique in its capacity to rescue this cell death. Indeed, T cell-derived survival signals including CD40L, IL-4, and IL-21 similarly rescue B cells from BCR delivered TLR9 agonists. All of these stimuli also induce AID, which is required for class switch recombination and somatic hypermutation. However, IL-21 uniquely upregulates T-bet in the context of TLR9 stimulation. Further, we show that IL-21 induces antibody secretion to the IgG2b and IgG2c isotypes. As these isotypes have been implicated in humoral autoimmunity, our findings suggest that IL-21 may be involved in the production of DNA autoantibodies.

We have discovered a functionally distinct mature B cell subset that accumulates with age, comprising up to 30% of all mature B cells by 22 months. Despite sharing some features with other mature B cell subsets, these cells are refractory to BCR and CD40 stimulation. Instead, they respond to TLR-9 stimulation and divide maximally upon combined BCR and TLR ligation. Although similar to follicular B cells in both BLyS receptor expression and BLyS binding capacity, these cells do not rely upon BLyS for survival. Further, they are not the result of intrinsically altered B lymphopoiesis in aged bone marrow, but instead appear to be gradually generated from a small subset of mature B cells that exhaustively expand throughout the individual’s lifetime. Thus, while the magnitude of the mature primary B cell niche is maintained with age, it is increasingly populated by cells refractory to BCR-driven activation yet responsive to innate receptor stimulation. This shift may contribute to the diminished capacity for adaptive humoral responses and increased propensity for autoimmunity associated with aging.