Figure 8. Splenic Stat3^–/– donor T cells do not have significant inhibition of GSH/Myc pathway activity.

Lethally irradiated BALB/c recipients were engrafted with TCD-BM (5 × 10⁶) from WT C57BL/6 donors and CD90.2⁺ T cells (1 × 10⁶) from WT or STAT3^–/– C57BL/6 donors. (A) On days 0, 3, 5, and 6 after HCT, CD90.2⁺ T cells from spleen were isolated for Seahorse analysis. Normalized Glyco-ATP, Mito-ATP, and total ATP production rates are shown, using mean STAT3^–/– values as the reference. n = 2 per group combined from 2 replicates; each sample contained lymphocytes from 3 recipients. (B–L) On day 6 after HCT, lymphocytes from spleen were isolated for RNA-Seq, immunoblotting, and flow cytometry analysis. (B) NES of KEGG pathway activity of CD4⁺ and CD8⁺T cells are shown, setting the WT as the reference. (C) MYC target V2 pathway–related gene set expression in CD4⁺ (top) and CD8⁺ (bottom) T cells were compared between WT and STAT3^–/– donors. GSEA plots are shown. (D) Myc protein in Thy1.2⁺ T cells was measured by immunoblotting. (E–J) Means ± SEM of MFI of CD98 reduced GSH (E), CD36 (F), CD98 (G), GLUT1 (H), HK2 (I), and CPT1A (J) of CD4⁺ and CD8⁺ T cells in the spleen. n = 5–7 per group combined from 2 replicates. (K and L) Representative flow cytometry patterns and means ± SEM of percentages of MitoSOX^hiMitoGreen^hi and percentages of MitoRed^loMitoGreen^hi of CD4⁺ (K) and CD8⁺ T cells (L) from the spleen of different groups are compared, n = 8–9 per group combined from 3 replicates. P values were calculated by unpaired Student’s t tests. NS, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.