Fig. 1

eEF2K-deficient activated CD4⁺ T cells lead to a Th17 profile and dysfunction phenotypes. CD4⁺ T cells were isolated from both wild-type (WT) and eEF2K-knockout (KO) C57BL/6 mice after three days of in vitro stimulation on plates coated with anti-CD3 monoclonal antibody (mAb), in combination with soluble anti-CD28 mAb. a Representative result of the intracellular staining analysis of IL-2 in the WT and eEF2K KO effector CD4⁺ T cells. b Result of ELISA for IL-2 in the supernatants of the CD4⁺ T cells, stimulated for 48 h, was shown in technical replicates with the standard deviation (n = 6). c Representative result of the intracellular staining analysis of IFNγ in the WT and eEF2K KO effector CD4⁺ T cells. d Result of ELISA for IFNγ in the supernatants of the CD4⁺ T cells, with stimulation for 48 h, were shown in technical replicates with the standard deviation (n = 6). e LC/MS-MS proteomics spectral analysis of inflammation-related proteins. The result of post-activated WT and eEF2K KO CD4⁺ T cells was in the format of heatmap. f Intracellular FACS analysis of IL-17F in the WT and eEF2K KO effector CD4⁺ T cells on day 3. Summarized MFI (n = 3) were shown in the bar graph. g mRNA level of RORγt from WT and eEF2K KO CD4⁺ T cells were examined through qPCR (n = 4: technical replicates). h Intracellular FACS analysis of IL-6 in the WT and eEF2K KO effector CD4⁺ T cells on day 3. The results of FACS analysis, summarized MFI (n = 3), in the histogram format were shown in the bar graph, with the standard deviations. i Effector CD4⁺ T cells were stimulated with Th17-associated cytokines for 4 days—intracellular FACS analysis of IL-17A in the WT and eEF2K KO Th17 cells. The percentages of cells were indicated in each quadrant, and the results of FACS analysis in the flow chart were shown in the bar graph, with the standard deviations. Summary data were presented as mean ± SD, derived from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001, unpaired t-test