Fig. 3: Depdc5-deficient CD8⁺ T cells display enhanced ferroptosis.

a Representative histograms showing Ki-67 levels in splenic CD4⁺ and CD8⁺ T cells from Depdc5^ncl and Depdc5^tko mice. Splenocytes from Depdc5^ncl and Depdc5^tko mice were stained with a fixable viability dye and antibodies against CD4, CD8, and Ki-67. Numbers in the gated areas show the percentage of Ki-67⁺ cells within the indicated populations. b Summary bar graph of percentage Ki-67⁺ cells within the CD4⁺ and CD8⁺ compartments of Depdc5^ncl and Depdc5^tko mice (n = 3). c Flow cytometry analysis of dead lymphocytes (Sytox green positive) among splenic CD8⁺ T cells and B cells from Depdc5^ncl and Depdc5^tko mice (after in vitro culture for 4 h with Sytox green viability dye, anti-B220 antibody, and anti-CD8 antibody). d Summary bar graphs showing the percentage of dead CD8⁺ T cells and B cells from Depdc5^ncl and Depdc5^tko mice after in vitro culture for 4 h (n = 4). e Summary bar graph showing the percentage of dead WT CD8⁺ T cells after treatment with vehicle only, pan-caspase inhibitor Z-VAD-FMK (Z-VAD), necroptosis inhibitor Necrostatin-1 (Nec-1), or ferroptosis inhibitor Deferoxamine (DFO). LN cells from Depdc5^ncl mice after in vitro culture for 4 h with vehicle only, Z-VAD, Nec-1, or DFO, then stained with anti-CD8 antibody and fixable viability dye. Total CD8⁺ T lymphocytes were gated for dead cell analysis by flow cytometry. f Summary bar graph showing the percentage of dead CD8⁺ T cells after treatment with vehicle only, Z-VAD, Nec-1, or DFO. LN cells from Depdc5^tko mice were cultured in vitro for 4 h in the presence of vehicle only, Z-VAD, Nec-1, or DFO, then stained with anti-CD8 antibody and fixable viability dye for flow cytometry analysis. Total CD8⁺ T cells were gated for dead cell analysis. Error bars indicate means ± SEM (NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired Student’s t-test).