This movie depicts extravasation of adoptively transferred allospecific (anti-H2d) T regs in the cremasteric vasculature.of CBA (H2k) mice. ncomms4436-s3.avi (11M) GUID:?6B7AAD6D-B3B3-4ECD-B7BA-EB34B0291B04 Abstract Localization of CD4+CD25+Foxp3+ regulatory T (Treg) cells to lymphoid and non-lymphoid tissue is instrumental for the effective control of immune responses. were pooled (to ensure that the same number of Tregs in the cell preparation) and injected intravenously (i.v.) in IFN–treated or untreated recipients. As it is shown in Fig. 1a,b, significantly larger numbers of green fluorescent protein (GFP)-tagged Tregs migrated to the peritoneal cavity of syngeneic recipients previously exposed to IFN-, compared with those detected in the lavage of IFN–treated irrelevant (CBA/Ca) mice and non-treated syngeneic recipients. Notably, 50% of the Tregs recruited in the peritoneal cavity and draining LNs (dLNs) upregulated CD69 expression, indicative of recent T-cell receptor (TCR) engagement in this cell population (Fig. 1c). Similar observations were Cortisone made when irrelevant BALB/c (H2d) recipients were used (Supplementary Fig. 1). Treg cells also preferentially accumulated Cortisone in (mesenteric) dLNs of IFN–treated syngeneic, but not irrelevant mice, suggesting that antigen presentation also affects their localization to secondary lymphoid tissue, as previously suggested17. Open in a separate window Figure 1 Antigen recognition facilitates Treg trafficking.Total CD4+ T cells from FoxP3-eGFP reporter mice (107/mouse) were injected intravenously into syngeneic C57BL/6 or irrelevant CBA (H2k) mice that had received an intraperitoneal injection of 600?U IFN- 72?h earlier. Some C57BL/6 recipient received saline solution alone. The presence of GFP+ Tregs in lavage, dLN and spleen was analysed by CD140b flow cytometry 16?h later. Tregs were identified by gating on the CD4+GFP+ population. Representative dot plots are shown in panel a. The mean number of Treg cells (in the total CD4+ population) of Treg cells detected in the peritoneal cavity and lymphoid organs is shown in panel b. Error bars represent s.d. Statistical significance was calculated with unpaired Students culture with BALB/c-derived immature dendritic cells (DCs) and IL-2 (ref. 21). culture did not affect Treg phenotype and regulatory activity, which however shifted towards the alloantigen with time (Fig. 2aCc). We then compared the recruitment of circulating allospecific Tregs into the peritoneum of IFN–treated (i.p.) allogeneic BALB/c, syngeneic C57BL/6 and irrelevant CBA recipients. As shown in Fig. 2d,e, allospecific Tregs migrated more efficiently to the peritoneal Cortisone cavity of allogeneic BALB/c mice compared with that of syngeneic C57BL/6 and irrelevant CBA mice. Similar to what Cortisone we observed in the experiments with freshly isolated Tregs, this effect was accompanied by enhanced allospecific Treg recruitment in the dLNs of IFN–treated alloantigen-expressing recipients. Open in a separate window Figure 2 Allospecific Tregs migrate more efficiently to the peritoneal cavity of allogeneic mice.Allospecific Tregs (H2b) were expanded by culture with BALB/c-derived, (H2d) immature DCs and IL-2. Key phenotypic markers are depicted in panels a (CD25 and FoxP3) and b (CCR7 and CD62L). In panel c, increasing numbers of allospecific Tregs were added to co-cultures of C57BL/6 conventional na?ve T cells (105) stimulated with BALB/c-derived DCs (103), or CD3/CD28 beads. T-cell proliferation was measured as 3HTdR incorporation in triplicate cultures ((5 105/well) were seeded onto IFN–treated allogeneic BALB/c, syngeneic B6 and irrelevant CBA EC monolayers grown Cortisone on transwells. The mean percentage migration measured at 6?h from three experiments of identical design is shown. Error bars represent s.d. **by measuring migration of Tregs through antigen-expressing EC monolayers. Treg were isolated from FoxP3-eGFP reporter mice20 by cell sorting and seeded onto IFN–treated syngeneic (self) EC monolayers. As a control, syngeneic untreated EC and IFN–treated monolayers EC derived from irrelevant CBA/Ca mice were used. As shown in Fig. 7b, Treg migration through B6-derived EC was significantly enhanced by exposure of syngeneic EC to IFN- and compared with migration through IFN–treated CBA/Ca EC. As expected,.
This movie depicts extravasation of adoptively transferred allospecific (anti-H2d) T regs in the cremasteric vasculature