Fig. 7: Cell volume regulated by LRRC8A during T cell activation fine-tunes TCR proximal signal by keeping molecular density.

T cells were stimulated by OT-I peptide or antibodies in isotonic or hypotonic buffer at indicated conditions. a, b Expression of CD25 (a) and CD71 (b) measured by flow cytometry. Splenic OT-I CD8⁺ T cells from Lrrc8a^{f/f; OT-I} or Lrrc8a^{f/f, Cd4-Cre; OT-I} mice were activated by N4 peptide (1 nM) ex vivo for 6 h under isotonic or hypotonic condition and gated on TCRβ⁺CD8⁺Vα2⁺ (n = 3 replicates/mouse). c Immunoblots of TCR proximal signaling molecules in thymocytes from WT (Lrrc8a^f/f) or cKO (Lrrc8a^{f/f, Cd4-Cre}) mice, in isotonic (300 mOsm) and hypotonic (150 mOsm) solutions. d Cell volume of thymocytes from Lrrc8a^f/f or Lrrc8a^{f/f, Cd4-Cre} mice under isotonic (300 mOsm) and hypotonic (150 mOsm) condition for 15 min. n = 23–26 cells/group. e–f Immunoblots of p-Tyr (e), p-ZAP70^Y319, and p-LAT^Y171 (f) under isotonic (300 mOsm) and hypotonic (150 mOsm) in thymocytes from Lrrc8a^f/f or Lrrc8a^{f/f, Cd4-Cre} mice stimulated or not with anti-CD3ε (1 μg/ml) or anti-CD3ε (1 μg/ml) and anti-CD28 (1 μg/ml) for 2 min at 37 °C. FYN and GAPDH were used as loading control. g–i Flow cytometry of p-LCK^Y394 (g), p-ZAP70^Y319 (h) and p-LAT^Y171 (i). The OT-I T cells were stimulated with N4 (1 nM) in the indicated solutions for 30 min. n = 3 replicates/mouse for (g–i). j p-LCK^Y394 in OT-I CD8⁺ T cells stimulated as in (g) under the osmolarity ranging from 300 to 150 mOsm (n = 3 replicates/mouse). k, l Immunoprecipitation of TCRβ and followed by immunoblot of key molecules in TCR complex in the thymocytes isolated from Lrrc8a^f/f and Lrrc8a^{f/f, Cd4-Cre} mice. Representative blot in (k) and quantification in (l) (n = 7 mice/group). Representative flow cytometry histogram (left) and the quantification (right) were shown for panels (a, b, g–j). Data are representative of two (c–f, h), three (a, b, g, i), or seven (k) independent experiments. Calculation was done by the formulas: ΔX (%) = X_Iso-X_Hypo (%). Unpaired two-sided t test was used in a, b, g–i. Two-way ANOVA with Bonferroni’s post-hoc test was in (d, j). Two-sided paired t test was used in (l). Data are presented as mean ± SEM. Source data are provided as a Source Data file.