Figure 4). RORγt activity is important for maintenance of mouse and human Th17 cells.

a, b, Flow cytometry of intracellular staining for IL-17a and IFN-γ by CD4⁺ T cells. Mononuclear cells were collected from draining lymph nodes of wild-type or IL-23R-GFP knock-in heterozygous mice 7 days after MOG(35-55)/CFA injection. Cells were cultured for four more days with MOG(35-55) peptide and exogenous IL-23 or IL-12, in the presence of DMSO or 10 μM digoxin. Without pre-immunization, addition of IL-23 and MOG(35-55) peptide to culture did not lead to de novo Th17 cell differentiation (data not shown). Digoxin treatment suppressed expansion of in vivo differentiated Th17 cells, assayed by IL-23R reporter GFP expression (a) or by IL-17a production (b). c, d, Human naïve (CD45RA⁺CD3⁺CD4⁺) or memory (CD45RO⁺CD45RA⁻ CD3⁺CD4⁺CCR6⁺CD161⁺) cells were purified from healthy donor peripheral blood samples and were cultured in the presence of IL-1β, IL-23 and IL-2 for 6 days with or without 40 μM Dig(dhd). Intracellular staining for IFN-γ or IL-17a in memory CD4⁺ T cells from multiple donors (n=11) in the presence of IL-1β, IL-23, and IL-2, assessed on day 6. c, Representative FACS plots from one donor are shown. d, Each symbol indicates a separate donor. Statistical analysis was by a two-tailed unpaired Student's t test; IL-17a⁻ IFN-γ⁺, not significant and IL-17a⁺IFN-γ^+/−, p=0.02.