HIF1α deficiency results in diminished T_H17 differentiation. (A) Naive T cells from WT and HIF1α^−/− mice were differentiated under T_H17 conditions, followed by intracellular staining of IL-17. Data represent six independent experiments. (B) Effector T_H17 cells, as described in A, were restimulated with 5 µg/ml of plate-bound anti-CD3) overnight, and supernatants were collected and subjected to Bioplex measurement of IL-17 and IL-2. (C) Effector T_H17 cells, as described in A, were stimulated with anti-CD3/CD28 for 5 h for RNA and expression analyses. Data represent three independent experiments. (D and E) Naive T cells from WT and HIF1α^−/− mice were differentiated under T_H17 conditions for 2.5 d, and IL-23R (D) and Foxp3 (E) expression was examined by real-time PCR analyses. Data represent three independent experiments. (F and G) Naive T cells from WT and HIF1α^−/− mice were differentiated under T_H17 conditions for 2.5 d, and RNA was analyzed by microarrays to compare expression profiles between WT and HIF1α^−/− cells (F) with selected genes in metabolism, transcription factors/signaling pathways, and surface/secreted molecules presented (G). Heat maps display gene expression relative to the WT mean on a log₂ scale. (H) WT and HIF1α^−/− mice were immunized with MOG/CFA and, 9 d later, DLN cells were stimulated with MOG for 5 d, followed by intracellular staining of IL-17. Data represent two independent experiments. (I–K) WT and HIF1α^−/− mice were immunized with MOG/CFA and, 9 d later, DLN cells were expanded with MOG and IL-23 for 5 d, followed by adoptive transfer into C57BL/6 mice to induce T_H17-polarized transfer EAE. Disease scores were recorded daily (I) and, 11–12 d after transfer, mice were euthanized for histological analysis of spinal cord inflammation (J) with pathology scores calculated (K). **, P < 0.01. Bars, 100 µm. Data represent two independent experiments. Data represent the mean ± SEM.