Both CD4⁺ and CD8⁺ T cells require Gpx4 for survival and sterile expansion upon transfer into lymphopenic mice. (A and B) Mixed BM chimeras were generated by transfer of a mixture (1:1) of BM stem cells from WT (CD45.1⁺) or T^{ΔGpx4/ΔGpx4} (CD45.2⁺) mice into sublethally irradiated WT (CD45.1⁺CD45.2⁺) mice, followed by analysis at 7 wk after reconstitution. Percentages of reconstituted T cell subsets in the thymuses (A) and spleens (B) from WT and T^{ΔGpx4/ΔGpx4} donors were normalized to B220⁺ BM frequencies (n ≥ 6 per group). ****, P ≤ 0.0001 (Student’s t test). (C and D) Flow cytometry (top) and ratio (bottom) of thymocytes (C) and splenocytes (D) from mixed BM chimeras generated by transfer of a mixture (1:1) of tamoxifen-treated BM stem cells from WT (CD45.1⁺) or tam^{ΔGpx4/ΔGpx4} (CD45.2⁺) mice into sublethally irradiated WT (CD45.1⁺) mice. Analysis at 8 wk after reconstitution (n = 4 per group). **, P ≤ 0.01; ****, P ≤ 0.0001 (Student’s t test). (E) Ratios of surviving CD4⁺ and CD8⁺ T cells in the peripheral blood on days 1, 5 and 7 after adoptive transfer of equal numbers of WT (CD45.1⁺) and T^{ΔGpx4/ΔGpx4} (CD45.2⁺) donor thymocytes into Rag-1-deficient mice (n ≥ 6 per group). (F) Flow cytometry (left) and absolute numbers (right) of splenic T reg cells at 5 d after in vivo expansion of T reg cells by intraperitoneal injection with PBS or interleukin-2 (IL-2) and anti-IL-2 antibody complex into WT or T^{ΔGpx4/ΔGpx4} mice. Splenic cells were analyzed on 5 d after injection. Ns, not significant (Student’s t test). Data are representative of four (A and B), three (E), and two (C, D, and F) independent experiments.