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 Fy21: (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. Recent studies have shown that antigen-specific iTreg suppress CD4+ effectors T cells in vitro and in vivo by depleting peptide-MHC-II complexes from the DC surface. These, results raise questions 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 Ova323339 in association with I-Ab and determined their capacity to suppress CD8+ T cells from OT-I mice specific for Ova257264 (SIINFEKL) in association with H-2Kb. CD4+ OT-II iTreg suppressed the in vitro proliferation of OT-I T cells in the presence of dendritic cells (DCs) pulsed with both Ova323339 and Ova257264 or with DCs singly pulsed with each peptide. Surprisingly, 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 complex from the DC surface by a process of trogocytosis in vitro only in presence of their cognate antigen. Imaging experiments in vivo depict 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 and that pMHCI and pMHCII complexes may be closely associated in the immune synapse. (2) In order to determine whether Treg can inhibit memory T cell responses and their utility in suppressing ongoing diseases, we have expanded the differential sensitivity of naive and memory CD8+ T cells to Treg suppression. CD8+ T cells are major players in allogeneic graft rejection and will be principal targets of regulatory T cell therapy. We compared the response of mouse naive and memory CD8+ T cells to stimulation with allogeneic dendritic cells and to stimulation with common gamma chain cytokines in the absence of TCR activation. 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. Inhibition of CD80/CD86-CD28 interactions significantly reduced the proliferation, CD25 expression, and IL-2 mRNA transcription of both naive and memory CD8+ T cells; the inhibition could be reversed by addition of IL-2. IL-15 alone could induce robust proliferation of and moderate expression of Granzyme B and perforin by 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. Memory CD8+ T cells were more 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. (3) Adoptive cellular therapy with Treg cells is being evaluated in numerous clinical trials in GvHD and organ transplantation. Cellular biotherapy with Treg cells requires ex vivo expansion. While alloantigen stimulated Treg cells have been used in several studies, very few studies have directly compared the efficacy of alloantigen-reactive with non-stimulated Treg cells in vitro and no studies have compared their efficacy in vivo in organ transplantation. In this study, we obtained enriched populations of alloantigen-reactive Treg by stimulating proliferation dye-labeled C57BL/6 CD4+Foxp3+CD25+ Treg cells with allogeneic H-2bm12 dendritic cells in the presence of IL-2. After 5-7 days, the alloantigen-reactive Treg cells and resting Treg cells were separated by FACS. Both alloantigen-reactive and non-reactive Treg cells were co-transferred with C57BL/6 CD4+Foxp3- T cells (1:2 ratio) to immunodeficient recipients followed by transplantation of allogeneic H-2bm12 and 3rd party BALB/c skin. Recipients of alloantigen-reactive Treg cells demonstrated delayed rejection (26 days) of H-2bm12 skin, but normal rejection of 3rd party skin. Taken together, this protocol offers a simple, rapid method for enrichment of alloantigen-reactive Treg with enhanced antigen-specific suppressive function in vitro and in vivo. 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 found that when two alloantigens were presented on the same DC, the alloantigen enriched Treg suppressed both cognate and non-cognate T cell responses in vitro, but in vivo, Treg suppressed only the T cell response to cognate antigen, but did not suppress the response to the non-cognate antigen. A selective downregulation of the cognate alloantigen on DC was observed when they were cocultured with aaloantigen-specific iTreg cells. More importantly, cognate alloantigen was selectively captured by the iTreg cells. This model is consistent with our studies on Treg-specific for peptide antigen and strongly suggests that removal of antigen from the DC surface by trogocytosis is a major method by which Treg suppress immune responses in vivo.