The chemokine receptor CXCR3 is expressed on the surface of both resting and activated T- lymphocytes. We describe here a study of the endocytosis of CXCR3 using T-lymphocytes and CXCR3 transfectants. Chemokine-induced CXCR3 downregulation occurred in a rapid, dose-dependent manner, with CXCL11 the most potent and efficacious ligand. Endocytosis was mediated in part by arrestins, but appeared to occur independently of clathrin and caveolae. In contrast to other chemokine receptors, which are largely recycled to the cell surface within an hour, cell surface replenishment of CXCR3 occurred over several hours and was dependent upon mRNA transcription, de novo protein synthesis and transport through the ER and Golgi. Confocal microscopy and Western blotting confirmed the fate of endocytosed CXCR3 to be degradation, mediated in part by lysosomes and proteosomes. Site-directed mutagenesis of the CXCR3 C-terminus revealed that internalization and degradation were independent of phophorylation, ubiquitination or a conserved LL motif. CXCR3 was found to be efficiently internalized in the absence of ligand, a process involving a YXXL motif at the extreme of the C-terminus. Although freshly isolated T-lymphocytes expressed moderate cell surface levels of CXCR3, they were only responsive to CXCL11 with CXCL9 and CXCL10 only having significant activity on activated T-lymphocytes. Thus, the activities of CXCR3 are tightly controlled following mRNA translation. Since CXCR3+ cells are themselves a source of IFN-γ, which potently induces the expression of CXCR3 ligands, such tight regulation of CXCR3 may serve as a control to avoid the unnecessary amplification of activated T-lymphocyte recruitment.

T-cell development in the thymus depends on continuous supply of T-cell progenitors from bone marrow (BM). Several extrathymic candidate progenitors have been described that range from multipotent cells to lymphoid cell committed progenitors and even largely T-lineage committed precursors. However, the nature of precursors seeding the thymus under physiologic conditions has remained largely elusive and it is not known whether there is only one physiologic T-cell precursor population or many. Here, we used a competitive in vivo assay based on depletion rather than enrichment of classes of BM-derived precursor populations, thereby only minimally altering physiologic precursor ratios to assess the contribution of various extrathymic precursors to T-lineage differentiation. We found that under these conditions multiple precursors, belonging to both multipotent progenitor (MPP) and common lymphoid progenitor (CLP) subsets have robust T-lineage potential. However, differentiation kinetics of different precursors varied considerably, which might ensure continuous thymic output despite gated importation of extrathymic precursors. In conclusion, our data suggest that the thymus functions to impose T-cell fate on any precursor capable of filling the limited number of progenitor niches. (Blood. 2010;115:1137-1144) IntroductionT-cell development in the thymus depends on continuous supply of T-cell progenitors from bone marrow (BM) via the circulation. In the thymus T-cell precursors pass through a series of defined developmental stages, with the most immature thymocytes residing in the double-negative (DN) subset, characterized by the absence of the surface markers CD4 and CD8. Thymocyte differentiation then proceeds through the CD4 ϩ CD8 ϩ double-positive (DP) stage, after which thymocytes become either CD4 or CD8 single positive (SP) and leave the thymus to enter the mature T-cell pool. The most immature T-cell progenitors in the thymus are lineage negative (lin Ϫ ), CD44 ϩ , CD25 Ϫ , Sca-1 high , CD117 (c-kit) high (LSK), and CD127 Ϫ/lo (IL-7R␣) early T-lineage progenitors (ETPs), 1 which constitute a subfraction of the CD44 ϩ CD25 Ϫ DN1 population. These cells were shown to have high T but only limited B and some myeloid potential. 2,3 ETPs could be further subdivided according to their expression levels of CD135 (Fms-like tyrosine kinase receptor 3 [Flt3]) 4 and loss of CD135 expression correlated with loss of B-cell potential.Like all hematopoietic lineages T cells are ultimately derived from hematopoietic stem cells (HSCs) residing in BM. HSCs can generate CD135 ϩ multipotent progenitors (MPPs), which are likewise of the LSK phenotype, 5 as well as more committed precursors such as RAG-1-positive early lymphoid progenitors (ELPs) 6 and L-selectin-positive progenitors (LSPs), 7 both of which constitute subsets of MPPs. Common lymphoid progenitors (CLPs), 8 which are lin Ϫ Sca-1 ϩ CD117 ϩ/lo CD127 ϩ CD135 ϩ , and lin Ϫ Sca-1 ϩ CD117 Ϫ CD127 ϩ CD135 ϩ B220 ϩ CLP-2 9 differ from MPP subsets in their lack of myeloid pot...

Differentiation of T helper 17 cells (Th17) is a multistep process that involves the cytokines IL-6, TGF-β, and IL-23 as well as IL-1β, IL-21, and TNF-α. Thereby, robust induction of the capacity to produce IL-17 involves epigenetic modifications of the syntenic Il17a/f locus. Using inbred mouse strains, we identified co-regulation of gene transcription at the Il17a/f locus with the nearby microRNAs miR-133b and miR-206 that are clustered approximately 45 kb upstream of Il17a/f. Expression of these microRNAs was specific for Th17 as compared to other CD4+ T cell subsets and this was equally valid for in vitro polarized and ex vivo derived cells. From all factors analyzed, IL-23 was the most important cytokine for the in vitro induction of miR-133b and miR-206 in naive CD4+ T cells of wild type mice. However, analysis of IL-23R deficient mice revealed that IL-23R signaling was not essential for the induction of miR-133b and miR-206. Importantly, we found a similar co-regulation in CCR6+ and other γδ T cell subsets that are predisposed to production of IL-17. Taken together, we discovered a novel feature of T cell differentiation towards an IL-17-producing phenotype that is shared between αβ and γδ T cells. Notably, the specific co-regulation of miR-133b and miR-206 with the Il17a/f locus also extended to human Th17 cells. This qualifies expression of miR-133b and miR-206 in T cells as novel biomarkers for Th17-type immune reactions.

By developing a high-density murine immunophenotyping platform compatible with highthroughput genetic screening, we have established profound contributions of genetics and structure 19 co-corresponding authors. Data and Code AvailabilityThe flow cytometry files that support the findings of this study are available from www.flowrepository.

miRNAs regulate a large variety of developmental processes including development of the immune system. T cell development is tightly controlled through the interplay of transcriptional programs and cytokine-mediated signals. However, the role of individual miRNAs in this process remains largely elusive. In this study, we demonstrated that hematopoietic cell–specific loss of miR-17∼92, a cluster of six miRNAs implicated in B and T lineage leukemogenesis, resulted in profound defects in T cell development both at the level of prethymic T cell progenitors as well as intrathymically. We identified reduced surface expression of IL-7R and concomitant limited responsiveness to IL-7 signals as a common mechanism resulting in reduced cell survival of common lymphoid progenitors and thymocytes at the double-negative to double-positive transition. In conclusion, we identified miR-17∼92 as a critical modulator of multiple stages of T cell development.