Fig. 5: Rapamycin treatment increases blood CD8⁺ T cell frequency in Depdc5^tko mice.

a Representative histograms showing phosphorylated S6-S235/236 levels in splenic CD8⁺ T cells from Depdc5^ncl and Depdc5^tko mice after treatment with vehicle only or rapamycin (100 μg/kg daily for 4 weeks). After treatment, splenocytes were isolated and stained with a fixable viability dye, anti-mouse CD8 antibody, and anti-S6-S235/236 phosphorylation antibody for analysis by flow cytometry. b Summary bar graph showing MFI of phosphorylated S6-S235/236 in splenic CD8⁺ T cells from Depdc5^ncl and Depdc5^tko mice (n = 3) as described as a. c–e Representative flow cytometry plots (c) and summary bar graphs showing percentage of CD8⁺ T cells (d) and CD4⁺ T cells (e) in LN from Depdc5^ncl and Depdc5^tko mice treated with rapamycin or vehicle only as in a. LN single cell suspensions were prepared from Depdc5^ncl and Depdc5^tko mice treated daily with vehicle only or 100 μg/kg rapamycin for 4 weeks prior to staining with a fixable viability dye and antibodies against CD4 and CD8 prior to flow cytometry analysis. f, g Representative histogram (f) and a summary bar graph (g) showing relative lipid ROS levels in splenic CD8⁺ T cells from Depdc5^ncl and Depdc5^tko mice treated with vehicle only or rapamycin for 4 weeks. Splenocytes from the same mice in a were cultured in the presence of 2 μM BODIPY™ 581/591 C11 for 4 h, washed twice in PBS, then stained with a fixable viability dye and antibodies against CD4 and CD8 for flow cytometry analysis. Error bars indicate means ± SEM. (NS, not significant, **P < 0.01 by unpaired Student’s t-test).