Fig. 7. TH17 cells are resilient to pyroptosis despite GSDME plasma membrane pores.
a, Representative electropherogram obtained with a Jess Simple Western System after normalization to total protein. TH17 cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for 48 h and then transfected with RNPs containing an NTC or crGSDME (KO). The TH17 cells were then expanded for another 7 d. The data represent three experiments. b, Heatmap with gene sets constructed based on the fold-changes of the genes (see Supplementary Fig. 10). All annotation terms significant in at least three gene sets (FDR ≤ 5%) are shown. The observed −log10(FDR) values were capped at 10 for ease of visualization. In addition, all cell death-associated annotation terms are shown. c, Gene set expression comparison using a GO term in TH17 cells analyzed by scRNA-seq after grouping into GSDME+ and GSDME– TH17 cells (Wilcoxon’s rank-sum test). d, CytoTox 96 Non-Radioactive Cytotoxicity Assay from TH17 cells with and without CRISPR–Cas9 gene editing for GSDME stimulated with anti-CD3 and anti-CD28 monoclonal antibodies or from monocytes stimulated with or without LPS and nigericin (24 h). Supernatants from washed TH17 cell cultures were collected between days 4 and 5 of stimulation or from monocytes 24 h after stimulation (paired Student’s t-test). e, Cloning efficiency of TH17 cell clones with varying degrees of IL-1α expression (top) and of control TH cell clones with varying degrees of IFN-γ expression, but lacking IL-1α coexpression (bottom) as assessed by intracellular cytokine staining. f, Intracellular staining and flow cytometric analysis of TH17 cell clones after repetitive restimulation with anti-CD3 and anti-CD28 monoclonal antibodies (n = 5 individual TH17 cell clones). g, GSEA of IL-1α+ compared with IL-1α– TH17 cell clones. h, Gene set expression comparison after scRNA-seq as in c. i, Flow cytometric analysis of TH17 cells stimulated for 5 d with anti-CD3 and anti-CD28 monoclonal antibodies. Left, representative experiment. Right, cumulative data (n = 3, two-tailed, paired Student’s t-test). Each circle indicates an independent blood donor.