Th17 cells fail to control VV encephalitis due to lack of Prf1 expression. (A) CD3+CD4+ T cells were highly purified by FACS sorting from the CNS of MVA/CFA immunized wild type or α4 CKO mice on day 4 after intrathecal infection. Fold change in relative expression of the indicated genes in α4 deficient vs. wild type control CD4+ T cells (log scale, means + SD, n = 4). (B) Flow cytometrically purified naïve CD3+CD4+CD44-Foxp3- T cells were stimulated without exogenous cytokines (Th0) or differentiated into Th1 cells (IL-12 + anti-IL-4), or Th17 cells (TGF-β + IL-6). Levels of Prf1 RNA expression were measured by quantitative RT-PCR (left in B, means + SD, Student’s t test, n = 4). Time course of Prf1 protein expression was determined by Western Blot in Th0, Th1, and Th17 cells from day 3 to day 5 of culture (right in B). (C) RNA levels of Eomes were measured in wild type and α4 integrin deficient T cells isolated from the CNS of VV infected mice (means + SD, n = 4). (D) Naïve T cells were differentiated into Th17 cells in vitro and transduced retrovirally with a control vector (pMIG) or an Eomes GFP vector (pMIG Eomes). GFP+ cells were purified by flow cytometry and RNA levels of Eomes and Prf1 were measured in control transduced Th17 cells vs Th17 cells that ectopically expressed Eomes (means + SD, n = 4).