Fig. 9: HIF-1 stabilization impairs CD4 T cell responses in vitro.

a, b The FSC of blasts (a) and the % cell size increase relative to unstimulated cells in the blast population (b) were depicted. c, d The % of S-phase (c) and G2/M (d) of CD4 T cells are shown. e CFSE profiles of Vhl Hif1a dcKO and WT CD4 T cells 3 days after stimulation with anti-CD3/CD28 are shown. f–m RNA-seq was performed in independent cultures of WT (n = 4), Vhl cKO (n = 3) and Vhl Hif1a dcKO CD4 T (n = 4) cells before or 24 h after anti CD3/CD28 stimulation. f The Pearsson R² correlation matrix of the expression of all genes in each sample is shown. g Heat map of the log₂ counts for each gene standardized to the mean. h, i Volcano plots showing the log₂ fold change in gene expression (x-axis) and p-value (y-axis), of differentially upregulated or downregulated genes (p ≤ 0.05 and log₂ fold change > 1) (i). j, k The log₁₀ p value of the most enriched KEEG-pathway terms in Vhl cKO vs WT (j) or in WT vs Vhl cKO CD4 T cells (k). l, m The MFI (l) and a representative histogram (m) of phospho-rpS6 on WT and mutant CD4 T cells 2 h after anti-CD3/CD28 or PMA/I stimulation are illustrated. n The frequency of IFN-γ expressing WT, Vhl cKO and Vhl Hif1a dcKO CD4 T cells 6 h after PMA/I or 72 h after anti-CD3/CD28 stimulation are shown. A group was stimulated with anti-CD3/CD28 for 65 h and then incubated for 6 h with PMA/I. a–d, l, n Each symbol represents one independent sample (n = 3 per group), and the data are presented as the mean ± SEM. p-values were calculated using a two-tailed unpaired t test with Welch’s correction (a–d) and 2-way ANOVA test, with Sidak adjustment for multiple comparisons (l, n). Source data are provided as a Source Data file. o VHL promotes the cell cycle progression, growth responses, proliferation, IL-2 secretion and expression of activation markers of TCR-stimulated CD4 T cells, the differentiation and the protective function of T cells against M. tuberculosis infection as well as the responses to immunization by impairing HIF-1 stabilization.