2B and C). Multiplex bead immunoassays revealed increased levels of RANTES and CXCL2 in supernatants from primary cultures of T-bet−/− Th17 cells by comparison with WT Th17 cells (Fig. 2D). Conversely, the concentration of the IFN-γ-induced chemokine CXCL9 was relatively low in supernatants from T-bet−/− Th17 cell cultures. T-bet−/− cells also expressed GM-CSF at a lower frequency

than WT cells during primary culture (Fig. 2A). However, selleck chemical T-bet−/− and WT Th17 cells secreted comparable quantities of GM-CSF upon secondary challenge (Supporting Information Fig. 1). T-bet−/− and WT Th17 cells produced similar quantities of other cytokines and chemokines implicated in EAE pathogenesis, including IL-1α, IL-6, and G-CSF (Fig. 2D). The majority of T-bet−/−

Th17 cells upregulated activation markers and proliferated in response to antigen to a similar extent as their WT counterparts (Fig. 2E), indicating that their failure to acquire Th1 characteristics was not a consequence of insufficient antigen presentation or TCR engagement. The fact that a relatively high percentage of T-bet−/− cells expressed a CD44+CD69+CD25+CD62Lneg profile could reflect a less differentiated state [19]. We next compared the stability of MOG-primed, IL-23 polarized T-bet−/− and WT CD4+ CD45.2+ T cells in vivo following transfer into naïve CD45.1 congenic hosts. Spleens harvested from the recipients of T-bet−/− donor cells contained a higher frequency of MOG35–55-specific IL-17 producers and a lower frequency of MOG35–55-specific IFN-γ producers than spleens from recipients of WT donor cells (Fig. 3A). These stable T-bet−/− LY2109761 Th17 cells induced EAE in 85–90% of hosts, although disease severity was reduced compared with recipients of WT cells (Fig. 3B). Branched chain aminotransferase IL-23 polarized T-bet−/− Th17 cells did not express FoxP3 and did not mitigate EAE severity when cotransferred with WT Th17 effectors (data not shown). FACS analysis of spinal cord mononuclear cells at peak disease indicated that the majority of infiltrating CD45.2+ T-bet−/− donor cells were IL-17+IFN-γ−, while the majority of infiltrating CD45.2+ WT donor cells were IL-17−IFN-γ+ (Fig. 3C). Although T-bet−/− donor

cells were enriched for the CD4+ T-cell subset prior to transfer, we entertained the possibility that immunocompetent host T cells had been activated by contaminating donor APCs bearing MOG35–55/class II complexes. Therefore, we repeated the adoptive transfer experiments using RAG2−/− recipients. Consistent with the results obtained in immunocompetent hosts, RAG2−/− mice were susceptible to disease induced by IL-23 polarized T-bet−/− donor cells (Fig. 3D). At peak disease, a very high percent of the T-bet−/− cells that had accumulated in the CNS of RAG2−/− recipients were IL-17+IFN-γ− (Fig. 3E and F, left panel). Similarly, the frequency of IL-17+IFN-γ− T-bet−/− cells was significantly higher than that of WT donor Th17 cells in the spleen (Fig. 3F, right panel).