Interleukin-4 downregulates transcription factor BCL6 to promote memory B cell selection in germinal centers.

Germinal center (GC)-derived memory B cells (MBCs) are critical for humoral immunity as they differentiate into protective antibody-secreting cells during re-infection. GC formation and cellular interactions within the GC have been studied in detail, yet the exact signals that allow for the selection and exit of MBCs are not understood. Here, we showed that IL-4 cytokine signaling in GC B cells directly downregulated the transcription factor BCL6 via negative autoregulation to release cells from the GC program and to promote MBC formation. This selection event required additional survival cues and could therefore result in either GC exit or death. We demonstrate that both increasing IL-4 bioavailability or limiting IL-4 signaling disrupted MBC selection stringency. In this way, IL-4 control of BCL6 expression serves as a tunable switch within the GC to tightly regulate MBC selection and affinity maturation.

Discovery of T cell antigens by high-throughput screening of synthetic minigene libraries.

The identification of novel T cell antigens is central to basic and translational research in autoimmunity, tumor immunology, transplant immunology, and vaccine design for infectious disease. However, current methods for T cell antigen discovery are low throughput, and fail to explore a wide range of potential antigen-receptor interactions. To overcome these limitations, we developed a method in which programmable microarrays are used to cost-effectively synthesize complex libraries of thousands of minigenes that collectively encode the content of hundreds of candidate protein targets. Minigene-derived mRNA are transfected into autologous antigen presenting cells and used to challenge complex populations of purified peripheral blood CD8+ T cells in multiplex, parallel ELISPOT assays. In this proof-of-concept study, we apply synthetic minigene screening to identify two novel pancreatic islet autoantigens targeted in a patient with Type I Diabetes. To our knowledge, this is the first successful screen of a highly complex, synthetic minigene library for identification of a T cell antigen. In principle, responses against the full protein complement of any tissue or pathogen can be assayed by this approach, suggesting that further optimization of synthetic libraries holds promise for high throughput antigen discovery.

Efficient help for autoreactive B-cell activation requires CD4+ T-cell recognition of an agonist peptide at the effector stage.

T-cell recognition of peptide/MHC complexes is flexible and can lead to differential activation, but how interactions with agonist (full activation) or partial agonist (suboptimal activation) peptides can shape immune responses in vivo is not well characterized. We investigated the effect of stimulation by agonist or partial agonist ligands during initial CD4(+) T-cell priming, and subsequent T-B-cell cognate interactions, on antibody production by anti-chromatin B cells. We found that autoantibody production required TCR recognition of an agonist peptide at the effector stage of B-cell activation. However, interaction with a weak agonist ligand at this effector stage failed to promote efficient autoantibody production, even if the CD4(+) T cells were fully primed by an agonist peptide. These studies suggest that the reactivity of the TCR for a target self-peptide during CD4(+) T-B-cell interaction can be a critical determinant in restraining anti-chromatin autoantibody production.

The role of BLyS/BLyS receptors in anti-chromatin B cell regulation.

B lymphocyte stimulator (BLyS), also known as B cell-activating factor, is a key positive regulator of B cell homeostasis, and elevated levels of BLyS have been observed in systemic lupus erythematosus (SLE) patients. Given that anti-chromatin auto-antibodies are one of the hallmarks of SLE, we examined the role of BLyS and its receptors in the regulation of anti-chromatin B cells. We demonstrate that exogenous BLyS treatment leads to an increase in B cell numbers, particularly anti-chromatin B cells; yet, their localization in the spleen and auto-antibody production remain unaffected. We also examined transmembrane activator and CAML interactor (TACI), BLyS receptor 3 (BR3) and B cell maturation antigen expression on anti-chromatin B cells before and after receiving T cell help. Interestingly, in the absence of T cell help, TACI expression is greater on immature anti-chromatin B cells compared with immature Tg(-) B cells, whereas BR3 levels are comparable. After receiving T cell help, the anti-chromatin B cells that have differentiated into short-lived plasma cells no longer express BR3 but retain TACI. These data suggest a novel role for TACI in anti-chromatin B cell homeostasis and differentiation.

Exogenous and endogenous TLR ligands activate anti-chromatin and polyreactive B cells.

Autoreactive B cells may become activated in a T-independent manner via synergistic engagement of the BCR and TLRs. Using the VH3H9 Ig H chain transgene to track anti-chromatin B cells, we demonstrate that VH3H9/Vlambda1 anti-chromatin B cells proliferate in response to stimulatory oligodeoxynucleotides containing CpG motifs, suggesting that these autoreactive B cells are responsive to TLR9 signaling. Strikingly, some VH3H9 B cells, but not the well-characterized VH3H9/Vlambda1 B cells, proliferate spontaneously in culture medium. This proliferation is blocked by inhibitory CpG oligodeoxynucleotides, implicating the TLR9 (or possibly TLR7) pathway. Most hybridomas generated from the proliferating cells are polyreactive, and one exhibits binding to nuclear Ags but not to the other Ags tested. Thus, B cells carrying autoreactive and/or polyreactive specificities may be susceptible to T cell-independent activation via dual engagement of the BCR and TLRs.

The influence of effector T cells and Fas ligand on lupus-associated B cells.

Circulating autoantibodies against dsDNA and chromatin are a characteristic of systemic lupus erythematosus in humans and many mouse models of this disease. B cells expressing these autoantibodies are normally regulated in nonautoimmune-prone mice but are induced to secrete Abs following T cell help. Likewise, anti-chromatin autoantibody production is T cell-dependent in Fas/Fas ligand (FasL)-deficient (lpr/lpr or gld/gld) mice. In this study, we demonstrate that Th2 cells promote anti-chromatin B cell survival and autoantibody production in vivo. FasL influences the ability of Th2 cells to help B cells, as Th2-gld/gld cells support higher titers of anti-chromatin Abs than their FasL-sufficient counterparts and promote anti-chromatin B cell participation in germinal centers. Th1 cells induce anti-chromatin B cell germinal centers regardless of FasL status; however, their ability to stimulate anti-chromatin Ab production positively correlates with their level of IFN-gamma production. This distinction is lost if FasL-deficient T cells are used: Th1-gld/gld cells promote significant titers of anti-chromatin Abs regardless of IFN-gamma production levels. Thus, FasL from effector T cells plays an important role in determining the fate of anti-chromatin B cells.