Importantly, the proportion of Sirp+ DC was also expanded in perinatal p4 thymi from mice (Figure 6E)

Importantly, the proportion of Sirp+ DC was also expanded in perinatal p4 thymi from mice (Figure 6E). enabling selection of Treg with non-overlapping specificities (Leventhal et al., 2016; Perry et al., 2014). In addition to displaying self-antigens, DC express other molecules essential for Treg generation, including CD80/CD86, CD70 and IL-2 (Coquet et al., 2013; Salomon et al., 2000; Weist et al., 2015). Thymocytes must enter the medulla and scan mTEC and DC efficiently to encounter the full spectrum of self-antigens that enforce central tolerance. The chemokine receptor CCR7 is critical for medullary accumulation and rapid motility Cobicistat (GS-9350) of SP thymocytes (Ehrlich et al., 2009; Ueno et al., Cobicistat (GS-9350) 2002). In CCR7 deficient mice, SP thymocytes do not efficiently encounter self-antigens displayed by medullary APC (Nitta et al., 2009), auto-reactive thymocytes are not effectively deleted, and autoimmunity ensues (Davalos-Misslitz et al., 2007a; Kurobe et al., 2006). Because the medulla is important for Treg generation (Coquet et al., 2013; Cowan et al., 2013), we anticipated that impaired medullary entry would inhibit Treg generation; however, we find that Treg cellularity increases in mice, increased thymic Treg cellularity could be accounted for by re-entry of peripheral Treg, consistent with a previous report (Cowan et al., 2016). Surprisingly, however, intrathymic generation of Treg was enhanced in neonates and in lympho-deficient bone marrow chimera recipients. Treg generated during the neonatal period and following recovery from lymphodepletion are particularly critical for maintaining self-tolerance (Guerau-de-Arellano et al., 2009; Yang et al., 2015). To investigate the mechanism by which CCR7 deficiency results in increased Treg generation, we analyzed mixed bone marrow chimeras and found that CCR7 deficiency in thymic DCs was responsible for increased Treg cellularity. CCR7 deficiency selectively impaired survival of mature Sirp?MHC-IIhi DCs, resulting in an increased frequency of Sirp+MHCIIlo DCs, a subset that efficiently promotes Treg generation. Thus, CCR7 deficiency promotes an increase in thymic Treg cellularity both by enhancing peripheral recruitment of Treg in the adult and by skewing the thymic DC compartment to favor Treg generation in the neonate and following lymphodepletion. Results Treg cellularity is increased in thymi. Instead, both the percentage and absolute number of Cobicistat (GS-9350) FOXP3+ CD25+ Treg cells were increased in thymi (Figures 1AC1C), consistent with a recent report (Cowan et al., 2016). Treg arise from both CD25?FOXP3+ and FOXP3?CD25+ Treg progenitors (Tai et al., 2013). The number of FOXP3?CD25+ Treg progenitors was increased in (Figure S1A). Open in a separate window Figure 1 CCR7 deficiency results in an TEF2 increased number and percentage of thymic Treg in adult and neonatal mice(A) Representative flow cytometry plots showing the percentage of FOXP3? CD25+ and FOXP3+ CD25? Treg progenitors and FOXP3+ CD25+ Treg cells within the CD4SP population of thymi was due to recirculation or thymic Treg generation, we bred mice to a RAG2 promoter-driven GFP reporter strain (RAG2p-GFP), in which progressive loss of GFP signal after positive selection reflects the age of non-dividing thymocytes (Boursalian et al., 2004). CCR7 deficiency resulted in an increased percentage of GFP? Treg (GFP? CD25+CD4SP) that had recirculated into the thymus from the periphery. However, the percentage of newly generated Treg (GFP+ CD25+ CD4SP) did not increase (Figures S1B and C), consistent with a recent report (Cowan et al., 2016). These data indicate that increased thymic Treg cellularity in adult mice can be accounted for by enhanced recirculation of Treg into the thymus. Because re-entered Treg suppress differentiation of new Treg in the thymus (Thiault et al., 2015), and because the number of Treg progenitors was elevated in thymi (Figures 1DC1F). Relative to the adult, there were very few splenic Treg at P4 available to recirculate to the thymus (Figure 1G), indicating that increased thymic Treg in mice reflected enhanced Treg generation. To determine whether CCR7 deficiency also enhances thymic Treg generation in the absence of recirculation in the adult, we transferred or bone marrow into lethally irradiated recipients, which are devoid of peripheral Treg. Thymic Treg chimerism was analyzed after 3 weeks, when the first wave of SP thymocytes had differentiated, but had not significantly emigrated to the periphery (Krueger et al., 2017; Serwold et.