Two major areas of investigation were pursued during FY2019: 1). Foxp3+ T regulatory cells (Tregs) are critical for the maintenance of immune homeostasis. One of the major unresolved issues regarding their function is whether they can mediate antigen-specific suppression and what mechanism they utilize to perform this function. Several early in vivo studies of Treg cells suggested a role for antigen specificity, in that CD4+ T cells from mice lacking the target organ were poor suppressors of disease in those organs. Although these studies indicated the importance of antigen-mediated priming of Treg cells, they did not examine whether antigen recognition by Treg cells had any further role in suppression in vivo. Several mechanisms have been proposed for the Treg-mediated suppression that can target both Teffector cell function and antigen presentation including: production of tolerogenic molecules i.e. IL-10, TGF-beta, IL-35; consumption of IL-2 via high affinity IL-2 receptors; CTLA-4 mediated inhibition of costimulation; and contact-dependent killing of antigen presentation through Granzyme and perforin. All of these mechanisms are compatible with the paradigm of bystander suppression as suggested by the studies that Tregs primed by one antigen could subsequently suppress T cell proliferative responses to any other unrelated antigen activated in the same culture. However, these potential mechanisms for Treg suppression have been primarily derived from in vitro studies and it is clear that the mechanisms of in vivo regulation are likely to be much more complex. In this study, we aimed to analyze the fine specificity of antigen-specific Treg cell-mediated inhibition of the priming of conventional naive T cells (Tnaive cells) in vivo and to compare the results with antigen-specific Treg cell mediated suppression in vitro. To do so, we used both in vitro differentiated, antigen-specific, induced Treg cells (iTreg cells) and freshly isolated thymus-derived Treg cells (tTreg cells) from T cell antigen receptor (TCR)- transgenic mice. To determine the antigen specificity of Treg cell mediated suppression in vitro and in vivo, we stimulated Treg cells with DCs simultaneously pulsed with two distinct antigenic peptides and examined the expansion of antigen-specific Tnaive cells. In line with previous observations, antigen-specific Treg cells, following activation by such double-pulsed DCs, were capable of suppressing the expansion of Tnaive cells specific for their cognate antigen as well as that of Tnaive cells specific for an unrelated antigen in vitro. In contrast, when similar cell populations were transferred in vivo, Treg cells activated by double-pulsed DCs could suppress only Tnaive cells specific for their cognate antigen. To explore the mechanisms leading to antigen-specific suppression in vivo, we performed an in-depth analysis of the physical interaction of antigen-specific Treg cells with DCs in comparison to that of antigen-specific Tnaive cells and demonstrated that Treg cells acquired a distinct morphology after contact with DCs displaying wider membrane fusion sites, longer contact durations and larger clusters in vitro and in vivo. We demonstrated that antigen-specific Treg cells removed complexes of peptide and major histocompatibility complex class II (pMHCII) from the DC surface and thereby decreased the capacity of DCs to present antigen. Most importantly, the removal of pMHCII complexes was antigen-specific, since Treg cells captured only the pMHCII complexes that they recognize, but not any unrelated antigen expressed on the same DC. 2). A crucial aspect of T-cell dependent immunity is the formation of the germinal center (GC) within the B cell follicles of secondary lymphoid organs. These responses are initiated with the migration of T cells, more specifically T follicular helper (Tfh) cells, and B cells. Nave CD4+ T cells are localized in the T cell zone of lymphoid organs and TCR engagement with peptide/MHCII on antigen presenting cells (APC), together with the cytokines IL-21 and IL-6, results in the upregulation of CXCR5 allowing these T cells to home towards the follicle. Tfh cells express co-stimulatory molecules such as ICOS and CD40L, that are essential for B cell activation and differentiation, as their absence leads to disrupted GC responses and impaired B cell memory. A subset of CD4+ T cells has been identified within the follicle that expresses Foxp3 and has been termed T follicular regulatory (Tfr) cells. Tfr cells suppress the function of the Tfh cells, reduce the GC reaction, and inhibit antibody responses. We have developed a co-culture system using primed Tfh cells and naive B cells to explore the different suppressive mechanisms used by Tfr cells during GC responses in vitro. We demonstrate that Tfr cells can suppress Tfh cells in vitro primarily by blocking the secretion of IL-4 and to a lesser extent IL-21. In addition to the suppression of cytokine production by Tfh cells, Tfr cells also suppress the induction of CD40L expression by reducing CD40L transcripts, there by ablating the help needed by B cells to proliferate, differentiate into GC B cells, undergo class switch recombination and plasma cell differentiation. We also demonstrate that Tfr can directly suppress some aspects of B cell differentiation in a T-cell independent fashion, raising the possibility that Tfr cells can directly suppress T-independent pathways of B cell differentiation.
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