Fig. 4: Transcriptional profiles relying on TCR stimulation strength require LRRC8A-mediated cell volume regulation.

Transcriptomic analysis of RNA samples of OT-I CD8⁺ T cells from WT (Lrrc8a^f/f) and KO (Lrrc8a^{f/f, Cd4-Cre}) mice, stimulated with OT-I peptides at indicated concentration for 6 h ex vivo. N4^low or NL (10 pM), G4 (1000 nM), N4^high or NH (1000 pM). n = 3 mice per group. a Principal component analysis (PCA) of the normalized expression data demonstrated distinct clusters. b The correlation analysis between FSC/SSC and RNA reads by sequencing (total transcripts per million, TPM). The error bands were mean ± SD. c–e Differentially expressed genes (DEGs) identified between different groups. Stimulation (stim.) (NL, G, NH) groups versus unstimulated (unstim.) group (c), WT versus KO in each treatment (d), and G4 versus N4 (e). f Heatmap of DEGs across all the groups. I, genes switched on by TCR signal; II, genes switched off by TCR signal; III, genes sensitive to LRRC8A-deficiency. g KEGG pathway analysis of DEGs between WT (Lrrc8a^{f/f; OT-I}) versus KO (Lrrc8a^{f/f, Cd4-Cre; OT-I}) under various stimulations. h–i Heatmap of DEGs associated with TCR signaling pathways (h) and transcription factors (i). One-way ANOVA test was performed to evaluate the statistical significance in (b). Source data are provided as a Source Data file.