Figure 1. Hypoxia and TGF-β1 synergize to induce a CD69⁺CD103⁺ population that expresses human T_RM-associated markers.

Naive CD8⁺ T cells sorted from PBMCs were activated in 20% O₂ (AtmosO₂) or 2% O₂ (hypoxia) for 4 days and then for an additional 2 days with the addition of recombinant human TGF-β1 (rhTGF-β1). Expression levels of T_RM-associated genes were analyzed via quantitative real-time PCR. (A–C) Fold change of gene transcript levels in 2% O₂ + TGF-β1 over 20% O₂ + TGF-β1. (D) The frequency of the CD69⁺CD103⁺ T_RM-like population and (E) expression of T_RM-associated markers were assessed by flow cytometry. Representative results from 1 donor are shown (D and E). Blue histograms represent CD69⁺CD103⁺ cells from 20% O₂ + TGF-β1, red histograms represent CD69⁺CD103⁺ cells from 2% O₂ + TGF-β1, and gray histograms represent fluorescence minus one (FMO). (A–C) n = 7, 3 independent experiments; paired t test with Benjamini, Krieger, and Yekutieli correction for multiple comparisons; **q < 0.01, ***q < 0.001, ****q < 0.0001; FDR < 0.05, data are mean ± SEM. (D) n = 8, 3 independent experiments; ratio paired t test; ****P < 0.0001. Naive CD8⁺ T cells were activated in 20% O₂ or 2% O₂ for 4 days and then for an additional 2 days with or without rhTGF-β1. (F) Frequency of the CD69⁺CD103⁺ population and (G) expression of T_RM-associated markers on CD69^–CD103^– cells (20% O₂), CD69⁺CD103^– cells (2% O₂), and CD69⁺CD103⁺ cells (2% O₂ + TGF-β1) were assessed by flow cytometry; representative pseudocolor plots shown for 1 donor, n = 8. Two-way ANOVA (F) or repeated measures 1-way ANOVA (G), *P adj < 0.05, **P adj < 0.01, ***P adj < 0.001, ****P adj < 0.0001; data are mean ± SEM.