During EAE, IFN-γ drives local expression of CXCL10, a ligand for CXCR3, in the inflamed CNS [[13]]. CNS T cells showed elevated expression of T-bet and CXCR3 which was particularly high in CNS-Treg cells (Fig. 3A). CXCR3 expression correlated with the absence of CD126 on CD4+ cells from naïve spleen (Fig. 3B) suggesting that the CXCR3+ Treg cells which arrive at the CNS early after the onset of inflammation will be drawn from a pool mostly lacking CD126 expression. The model that develops from these data is that, in vivo, Treg cells might be susceptible to IL-6-driven diversion to an IL-17-producing phenotype when expressing CD126 and gp130 (i.e.

in the lymphoid organs, as can be seen by the ability of splenic Treg cells from KPT-330 cell line mice with EAE to Cobimetinib order produce IL-17

upon in vitro exposure to an IL-6-containing cocktail (Fig. 1B). However, upon arrival in the organ under autoimmune attack, Treg cells have lost this capacity because they have down-regulated CD126 and gp130. Of course, this loss of receptors was not restricted to Treg cells; they were also low/absent on CNS GFP− cells (Fig. 2B and C) and pSTAT1 and pSTAT3 were absent in all CNS CD4+ cells exposed to either IL-6 or HDS. However, CNS GFP− cells (but not GFP+ cells) are clearly able to produce large quantities of IL-17 (Fig. 1A). This is most likely maintained because effector cells, initially triggered in the presence of IL-6, are induced to express the IL-23R [[14]]. IL-23 is readily available in the inflamed CNS during EAE [[15]], but the

IL-23R Tau-protein kinase is not expressed by Treg cells [[16]]. Therefore, we propose that although both CNS T effectors and Treg cells are insensitive to IL-6 signaling, their differential sensitivity to IL-23 allows T effectors to maintain IL-17 production. Lack of CD126 should therefore serve as a marker of preactivated Treg and T effectors. We sorted splenic GFP+ and GFP− cells, that either did or did not express CD126, from naïve Foxp3-GFP mice and found that CD126+ cells produced IL-17 only if IL-6 was included in the culture while GFP−CD126− cells would produce IL-17 in IL-23-containing medium without IL-6 (Fig. 3C). Furthermore, GFP+CD126− cells could not be provoked to produce IL-17, consistent with the reported absence of IL-23R from Treg cells [[16]]. CNS-Treg cells express T-bet, CXCR3 and have lost CD126 (Fig. 3). Expression of CXCR3 is T-bet dependent [[12]]. However, CXCR3 expression was not a surrogate marker identifying IL-6-insensitive Treg cells. Sorted CXCR3+ splenic Treg cells from naïve mice maintained the ability to produce IL-17 (Supporting Information Fig. 3), correlating with ∼20% of Foxp3+CXCR3+ cells expressing CD126 (as shown in Fig. 3B).