Control of Autoimmunity by Regulatory T Cells

National Institute Of Allergy And Infectious Diseases

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Abstract

Advances were made in a number of different areas during FY22: 1. T Regulatory cells (Tregs) plays an important role in regulating immune mediated responses against self and foreign antigens by various known and unknown mechanisms. T Regulatory cells (Tregs) regulate antigen-specific immune responses by using a variety of mechanisms. Recent studies have shown that antigen-specific Treg can suppress CD4+ effector T cells in vitro and in vivo by depleting peptide-MHC-II complexes from the DC surface. It remains unclear as to how Treg suppress CD8+ T effectors, particularly in vivo. To explore Treg-mediated suppression of CD8+ T cells, we generated CD4+ iTregs from OT-II mice specific for Ova323-339 in association with I-Ab and determined their capacity to suppress CD8+ T cells from OT-I mice specific for Ova257-264 (SIINFEKL) in association with H-2Kb. CD4+ OT-II iTreg suppressed the in vitro proliferation of OT-I T cells in the presence of DCs pulsed with both Ova323-339 and Ova257-264 or with DCs singly pulsed with each peptide. Interestingly, the expansion of OT-I CD8+ T cells in vivo was not suppressed by OT-II iTreg when the two peptides were presented on separately pulsed DCs, but was suppressed when both peptides were presented on the same DCs. OT-II Tregs depleted the Kb-SIINFEKL complexes from the DC surface in vitro by a process of trogocytosis only in the presence of their cognate antigen. The uptake of Kb-SIINFEKL complexes by OT-II Treg was not secondary to leakage of free SIINFEKL from the DC surface as OT-II Treg from MHC-I deficient mice were as efficient in uptake of pMHC-I as Treg from wild type mice. Multiphoton imaging microscopy in vivo reveals a close interaction between Tregs and CD8+ T cells suggesting that suppression of CD8+ T cells in vivo requires close proximity between Treg and responder CD8+ T cell may involve an artificial synapse between Treg and CD8+ T cells. 2. While antigen-specific Treg obtained from TCR transgenic mice appear to mediate a component of their suppressor function by capturing peptide MHC-II complexes from the surface of DC and thereby reducing their antigen-presenting capacity, it remains to be determined whether polyclonal Treg or alloantigen-specific Treg also capture their target peptide MHC-II complexes. To address this issue, we have focused on alloantigen-specific iTreg generated in culture in response to allogeneic DC. We have also examined whether the enriched population of alloantigen-specific Treg function in a manner similar to peptide-antigen specific Treg by depleting their target antigen from the dendritic cell surfaces. We developed a highly efficient protocol for the generation of alloantigen-specific iTreg and demonstrated that marked suppression of responder cells in MLR was only observed when both the responder T cells and the iTreg cells were stimulated with the same alloantigen used to generate the iTreg cells. One of the major unresolved questions relating to the use of polyclonal or even antigen-specific Treg cells is whether Treg cells will also suppress irrelevant antigen specific-responses such as pathogen- or tumor antigen-specific responses in addition to their target antigen-specific response. When the alloantigen and peptide antigen were both presented on the same DC in vitro, but not on separate DC populations, both responses could be suppressed by iTreg specific for either the alloantigen or for the peptide antigen. To determine whether a similar situation occurred in vivo, we developed a number of different short-term foot-pad assays to examine the specificity of iTreg suppression in vivo. In contrast to the in vitro studies, both polyclonal allo-antigen-specific and monoclonal antigen-specific iTregs only suppressed the response to their cognate antigen and no bystander suppression was observed in vivo even when both allo-antigen and peptide antigen were present on the same DC. To examine whether alloantigen-specific iTregs mediated their suppressive function by removal of their target antigen from DC by a trogocytosis-like process, we demonstrated that the cognate alloantigen, but not non-cognate alloantigen, is reduced on allogeneic DC after co-culture with allo-iTreg cells. Similarly, we were able to demonstrate that allo-iTreg capture their target alloantigen from the DC surface and that allo-iTreg cells fail to capture non-cognate MHC even if the non-cognate MHC is co-expressed together with cognate MHC on the same DC. Taken together with our previous studies on peptide antigen-specific Treg from TCR transgenic mice, these studies with allo-antigen-specific Treg support the concept that antigen-capture by Treg is a general mechanism of Treg-mediated suppression. 3. In order to determine whether Treg can inhibit memory T cell responses and their utility in suppressing ongoing diseases, we have examined the differential sensitivity of naive and memory CD8+ T cells to Treg suppression. CD8+ T cells are major players in allogeneic graft rejection and are the principal targets of regulatory T cell therapy in transplantation. We have characterized the susceptibility to Treg suppression of mouse naive CD8+ T cells, virtual memory polyclonal CD8+ T cells, and bona fide memory antigen-specific CD8+ T cells generated by priming in vivo with antigen-pulsed DC. Higher percentages of naive CD8+ T cells proliferated in response to allogeneic DCs, but expressed lower levels of CD25 than CD8+ memory cells. Addition of IL-2 to the co-cultures enhanced the expression of CD25 on divided naive CD8+ T cells and enhanced the proliferation of memory CD8+ T cells. IL-15 alone could induce robust proliferation and expression of Granzyme B and perforin by both types of memory CD8+ T cells. IL-2 alone induced less proliferation of memory CD8+ T cells than IL-15, but higher levels of Granzyme and perforin expression. The capacity of memory CD8+ T cells to respond to cytokine stimulation was likely secondary to elevated basal levels of IL-2R beta-chain on memory CD8+ T cells compared to naive CD8+ T cells and anti-IL-2R beta abolished the proliferation of memory CD8+ T cells to stimulation with IL-15. Memory CD8+ T cells were highly resistant to suppression by Treg and were completely resistant in the presence of IL-2 or IL-15. In conclusion, naive and memory CD8+ T cells have totally different response profiles when stimulated with alloantigen, IL-2 and IL-15. This difference may result in a lack of response to Treg therapy for graft rejection or GVHD.