Control of Autoimmunity by Regulatory T Cells

National Institute Of Allergy And Infectious Diseases

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Abstract

Advances were made in a several different areas during FY23: 1. Tregs suppress T cell responses in vivo and in vitro by using different mechanisms. Our studies have demonstrated that one mechanism utilized by antigen-specific CD4+Foxp3+ Treg involves capture of their target peptide-MHC-II (pMHC-II) antigen from the dendritic cell (DC) cell surface thereby limiting antigen presentation. It remains unclear how this mechanism would result in suppression of CD8+ T cell responses. We have demonstrated that MHC-II restricted OT-II Tregs could suppress OT-I CD8+ T cells in vivo only when challenged with DC pulsed with both OVA239-250 and OVA268-280. Under these conditions, we found that OT-II Tregs could uptake pMHC-I complexes from DC and could also form clusters with OT-I cells, thereby acting as antigen presenting cells (APC) for OT-I T cells. The amount of uptake of pMHC-I complexes is directly dependent on the strength of TCR-MHC-II interaction in vitro and OT-II Tregs could also uptake pMHC-I in vivo. When peptide-pulsed Treg were injected in vivo with OT-I T cells, they induced a partial response as manifest by induction of CD44, but lack of induction of CD25, and a deficient proliferative response. Taken together, MHC II-restricted Treg form a high avidity interaction with DC resulting in removal of both their target pMHC-II complex as well as pMHC-I complexes from DC expressing both peptides. Treg expressing pMHC-I complexes form a synapse with responder CD8+ T cells resulting in aberrant T cell activation. 2. Foxp3+ Treg cells use multiple pathways to mediate suppression and maintain immunologic homeostasis. Different pathways may be operative in short term-, long term-regulation, or organ specific- or disease specific-regulation. The goals of the present study were to examine in detail the fine specificity of the suppressive function of allo-iTreg and to begin to dissect their suppressive mechanisms. Our previous studies using monoclonal populations of antigen-specific iTreg demonstrated that their suppressive function in vitro was completely antigen-non-specific in that they would suppress both the responses to non-cognate antigen expressed on a separate population of dendritic cells or the responses to non-cognate antigens co-expressed with the cognate antigens on the same DC population. Allo-iTreg behaved somewhat differently in vitro in that they only exhibited bystander suppression when the non-cognate alloantigen and the cognate antigen were expressed on the same DC population, while suppression was restricted to the cognate alloantigen when the two alloantigens were expressed on the different populations of DC. Nevertheless, in vivo both allo- and antigen-specific iTreg behaved similarly in that they only suppressed responses to their cognate alloantigen when both the cognate and the non-cognate alloantigen (or a foreign peptide antigen) were expressed on the same populations of DC. We developed a novel method to detect capture of alloantigens by allo-iTregs and demonstrated that at least in vitro allo-iTregs specifically capture their target alloantigen from the DC surface and express the captured alloantigen both on the surface and intracellularly. These results have important implications for the use of allo-iTreg therapeutically in humans both for prevention/prolongation of graft rejection and for treatment of GVHD without unwanted immune suppression such as increased infections or decreased tumor immunity. 3. Studies in mice expressing the diphtheria toxin receptor (DTR) exclusively on regulatory Treg cells (Foxp3-DTR mice) demonstrated the critical importance of Treg cells in maintaining normal immune homeostasis. Adult Foxp3-DTR mice injected with DT begin to die from day 10 after DT treatment. Marked expansion of almost all immune cell types was observed prior to death. The purpose of the present studies was to examine in-depth changes in lymphocytes and antigen-presenting cells at early time points after Treg depletion to determine the most critical cell types controlled by Treg in the steady state. Complete depletion of Treg was observed 2 days after treatment, but profound activation of CD4(+) and CD8(+) T cells as measured by induction of CD44 expression and vigorous proliferation as measured by Ki-67 incorporation were not seen on day 2, but were detected on day 6 of DT treatment. Curiously, most of the CD44(hi)Ki- 67(+) T cells were CD25(-) and plasma levels of IL-2 or intracellular levels of IL-2 were not increased suggesting that early activation of CD4(+) and CD8(+) T cells was not the result of a burst of IL-2 production. Increased expression of CD80 and CD86 could be detected on CD11c(+) dendritic cells, and anti-CD80/CD86 mAbs reversed the activation of T cells provoked by the depletion of Treg cells. Taken together, these studies suggest that disruption of the regulation of Treg-mediated control of DC activation represents the first step in the complex autoimmune disease seen after Treg depletion.