Med. in the mesenteric lymph nodes and small intestine lamina propria of mixed bone marrow chimeras. Small clonal sizes and therefore decreased intraclonal competition were required Pipequaline hydrochloride for pTreg development. Despite bearing the same TCR, small intestine CD4IEL developed independently of precursor frequency. Both pTreg and CD4IEL development purely depended around the resident microbiota. A single clonal CD4+ T cell precursor can thus give rise to two functionally unique and anatomically Rabbit Polyclonal to MEKKK 4 segregated T cell subsets in a microbiota-dependent manner. Therefore, plasticity of the CD4 T cell compartment depends not only around the microbiota but also on specialized environmental cues provided by different tissues. INTRODUCTION Foreign antigens derived from the diet and the microbiota present a daily challenge to the gastrointestinal tract. Regulatory T cells (Tregs), abundant in the gut mucosa, prevent inflammatory bowel disease and food allergies through inhibition of harmful responses by effector T cells (1, 2). CD4+ T cells exhibit plasticity and can differentiate into unique subsets with diverse functional properties (3, 4). The intestinal lamina propria (LP), a location constantly exposed to commensal antigens, harbors many T cell subsets [e.g., T helper cell 1 (TH1), TH17, and peripheral Foxp3+ regulatory T cell (pTreg)] living in relative harmony. How these T cell fates are decided in vivo is the subject of intense study, as is the role of T cell receptor (TCR) specificity in this process. The TCR repertoire of colonic Tregs shows little, if any, overlap with the repertoire of na?ve or effector CD4+ T cells present at the same location or with Tregs isolated from organs other than the intestine (5). Similarly, the repertoire of intestinal TH17 differs substantially from that of other intestinal T cells (6). Whereas colonic commensals such as spp. favor pTreg development (5, 7), segmented filamentous bacteria (SFB) induce differentiation of standard CD4+ T cells (Tconvs) into quasi-clonal TH17 in the small intestine (6, 8). Thus, the repertoire and fate of CD4+ T cells in the Pipequaline hydrochloride intestinal LP are determined by the microbiota. Fate decisions appear to be made at the clonal level so that different TCR specificities determine different developmental outcomes. The mechanism whereby TCR specificity drives T cell fate and function is usually unclear and may depend around the relative abundance of the antigens acknowledged, environmental cues, or a combination of both. Development and growth of thymus-derived or natural Tregs (nTregs) are limited by intraclonal competition (9, 10). This process is driven by affinity for self-ligands: the higher the affinity for the antigen, the larger the nTreg niche size (11). When antigen presentation occurs under subimmunogenic or noninflammatory conditions, Tconvs may differentiate into pTregs (1). If and how the identity of the TCR dictates cell fate among Tconvs remains unsettled, as does the role of TCR specificity in determining the pTreg phenotype. To address these questions, we generated a monoclonal mouse collection cloned from a pTreg nucleus and thus bearing the prerearranged TCR of a pTreg. We found that this TCR facilitates conversion of CD4+ T cells into CD4+CD8+ intraepithelial lymphocytes (CD4IELs) while, at the same time, allowing development of pTreg in the mesenteric lymph nodes (mLNs) and small intestine LP (siLP), all in a microbiota-dependent manner. Thus, a single TCR specificity can give origin to two unique T cell phenotypes in two unique anatomical locations. RESULTS We used somatic cell nuclear transfer (SCNT) to generate a transnuclear (TN) mouse collection that carries a TCR cloned from your nucleus of a pTreg lymphocyte, isolated from your intestine-draining mLNs of a healthy, unmanipulated Foxp3-GFP (green fluorescent protein) reporter mouse (fig. S1A). To simplify the identification of the TCR in the SCNT-derived mouse, we sorted CD4+CD8- Foxp3-GFP+ pTregs that expressed V2, V8.3, or V11 (fig. S1B) by fluorescence-activated cell sorting (FACS) and used them as donors of nuclei for SCNT. Donor mLN pTregs were further identified as neuropilin-1-/low, as previously reported (12). The producing pTreg TN mouse collection carries pre-rearranged endogenous TCR and TCR loci that assemble into a functional receptor, as shown for other TN mouse lines Pipequaline hydrochloride (13, 14). Producing chimeras from SCNT were bred.