Mature thymic regulatory T cells (Tregs) are defined by dual expression of Foxp3 and CD25 on CD4SP thymocytes. However, the cellular pathways by which CD4 thymocytes differentiate into Tregs remain incompletely understood. Previous reports demonstrated that Treg development occurred via a two-step process. In step one, strong TCR signaling induces expression of CD25 leading to a CD25+ Foxp3− Treg progenitor (TRP). In step two, exposure of this TRP to IL2 results in conversion to a CD25+Foxp3+ Treg. Work from the Singer lab has defined a CD25−Foxp3lo TRP that can also be converted by IL2 into CD25+Foxp3+ Tregs. A key question is why two distinct cellular pathways involving distinct TRP populations are needed to reach the same endpoint. Using a combination of maturation marker analysis and RAG2-GFP reporter mice we observed that CD25+Foxp3− TRP arise more rapidly than the CD25−Foxp3lo TRP following positive selection. Analysis of Nur77-GFP reporter mice revealed that these two progenitor populations also display distinct affinity spectrums for self-peptide. Further, TCR sequencing has provided evidence that these 2 progenitor populations contain largely distinct TCR repertoires. Further, we have observed differential dependence on NFκB signaling and the Foxp3 regulatory element Cns3 between the 2 progenitor populations. Our preliminary data suggests that these TRPs mediate tolerance in distinct autoimmune contexts. Our results suggest a model whereby cells are driven by high affinity or high avidity interactions with self-peptide through two discrete pathways to form a comprehensive Treg repertoire capable of protecting against sterile and musical autoimmunity.

Methicillin-resistant Staphylococcus aureus (MRSA) causes skin infections in epidemic proportions among otherwise healthy individuals. Previous evidence shows that Th17 cells may be crucial to prevent localized S. aureus infections, as they occur in patients deficient in STAT3 or mice lacking IL-17A and F. In this study, we hypothesized that patients presenting to the emergency room (ER) with skin infections but no known genetic deficiency may have a reduction in Th17 differentiation, either globally or specifically to S. aureus antigens because of inhibitory factors produced by MRSA strains. Sera from 72 patients and 143 controls were collected and cytokines were measured by MultiPlex beads assay. It showed elevated cytokines in patients with skin abscesses on day 0. PBMCs from the same groups were stimulated with MRSA lysates, or anti-CD3, and supernatants were collected at 24h for detection of cytokines by ELISA. The production of IFNγ and IL-17 in response to anti-CD3 was similar in patients and controls, but MRSA stimulation triggered greater secretion of IL-6 and IFNγ in select groups of patients than in controls. These data suggest that our skin infected patients do not have a global defect in Th17 differentiation but may have a specific increase in IFNγ production in response to S. aureus antigens. Future experiments in mice will investigate whether this response may be detrimental to prevent S. aureus infections.