The GARP KO mice that were constructed for these studies had two features. First, the gene expressing M-cherry was knocked into the second exon of the GARP gene so that all cells that express GARP could be identified in flow-cytometric studies by the expression of a fluorochrome, M-cherry. Second, the 2nd exon of the GARP gene was floxed so that breeding of the GARP KO mice with mice expressing cell-specific Cre would lack GARP in the Cre-expressing cells . The studies to be described involve mice that express M-cherry in their GARP gene but have not yet been bred with Cre-expressing mice;thus these mice retain GARP expression. In initial studies it was shown that about 3% of thymocytes are M-cherry positive and that about 11% of spleen cells are M-cherry positive. These percentages closely match the known proportion of Foxp3+ cells in these tissues and indeed, 94-97% of Foxp3+ cells are also M-Cherry (and GARP) positive. Thus it is clear that M-cherry is an excellent marker of Foxp3+ cells in our Knock-In mice. Importantly, we also found that whereas nTregs (thymus-derived)Foxp3+ cells express intracellular and surface M-cherry, iTregs (TGF-beta-induced Foxp3+ cells)express intra-cellular M-cherry but not surface M-cherry. We next conducted function studies of M-cherry expressing cells by assessing their capacity to suppress the proliferation of naive CD4+ T cells derived from mice whose Foxp3 gene has a knocked in GFP gene; thus we could determine if the target cell population expresses Foxp3 (i.e., becomes a suppressor cells) during the culture. We found that surface M-cherry+ cells were excellent suppressor cells and did not induce the target cell population to express Foxp3. Thus their suppressor function was not dependent on their ability to induce suppressor cells in the naive T cell population by virture of their expression of GARP (TGF-beta). We also found that M-cherry positive cells whether they expressed surfact M-cherry or not are able to be converted into IL-17-producing effector cells by culture in the presence of IL-6;this illustrated the plasticity of GARP+ regulatory cells. In summary, these studies establish that M-cherry+ (GARP-positive) cells in our knock-in mice are Foxp3+ regulatory cells with regulatory function and IL-17 plasticity. However, whereas nTregs express both intra-cellular and surface M-Cherry, iTregs express only intra-cellular M-Cherry. Thus, GARP is a faithful marker of nTregs and can be used to distinuish nTregs from iTregs in inflamed tissue.
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