Fig. 6: Sestrins regulate DAP12 and NKG2D expression in CD8⁺ T cells.

a) Expression of DAP12, sestrin 2 and p-Jnk (T183/Y185) in lysed CD28⁺CD8⁺ T cells and CD28⁻CD8⁺ T cells immunoprecipitated with NKG2D. Loading control: IgG light chain (IgGL). Results are representative of two independent experiments. b) Cellular localization of Sesn2 (AF488, green), DAP12 (PE, red) and P-Jnk (T183/Y185, AF647, yellow) in CD27⁺CD28⁺CD8⁺ and CD27⁻CD28⁻CD8⁺ T cells. “Sesn2”, “DAP12”, and “p-Jnk” denote single stain controls in CD27⁻CD28⁻CD8⁺. Nuclei are stained with DAPI (blue). Scale bars – 7 μm. c) Overlap of Sesn2 and DAP12 or P-Jnk in CD27⁺CD28⁺CD8⁺ and CD27⁻CD28⁻CD8⁺ T cells. Bright detail similarity scores exceeding 2 were considered to be overlapping. Data are normalized to the CD27⁺CD28⁺CD8⁺ T cell subset for each donor (means and s.d., n = 6). d-e) Isolated human CD8⁺CD28⁻ T cells were transduced with control (shCtrl) or anti-sestrin (shSesn) vectors. d) Representative immunoblot for Sesn2 and DAP12 (representative of two experiments). e) Representative contour plots and summary data of NKG2D expression. Results are presented relative to cells transduced with shCtrl for each donor, set as 1 (means and s.d., n = 3 donors). f) Frequency of NKG2D and CD28 in isolated CD28⁻CD8⁺ T treated with siJnk (means and s.d., n = 6 donors). g) Lck phosphorylation in CD28⁻CD8⁺ T cells pre-treated with Jnk inhibitor (SP-600125, 10 μM) prior to anti-CD3 stimulation (OKT3, 10 μg/ml, 15 minutes; means and s.d., n = 8 donors). Two-tailed paired Student’s t tests in c,f-g) (*p <0.05, **p <0.01, ***p <0.001).