![Hypoxia alters stimulated CD8+ T-cell transcriptional profiles, inducing BNIP3 upregulation, decreased Rheb abundance, and impaired amino acid–induced mTOR activity. CD8+ T cells were cultured for 16 hours at 21% or 1% O2, then left unstimulated or stimulated via CD3/CD28 for 24 hours at these oxygen tensions prior to RNA-sequencing analysis (n = 5 independent donors). (A-B) Principal component analyses identify unstimulated samples largely cluster by donor, whereas stimulated samples cluster by oxygen tension. (C) Venn diagram, with circles representing differentially expressed genes (DEGs) between indicated conditions to identify shared and distinct DEGs when comparing cells stimulated at 21% or 1% O2 with their respective matched unstimulated controls. (D-E) Pathway analysis of genes (D) upregulated or (E) downregulated in cells stimulated at 1% O2 compared with 21% O2. (F) Volcano plot of total transcript abundance in cells stimulated at 1% O2 compared with 21% O2. (G) Example western blot (left) and summary of intracellular flow cytometry analysis (right) of BNIP3 in CD8+ T cells stimulated as above for 24 hours, or additionally treated with the HIF-1α inducer, deferoxamine (DFO) where indicated (summarized data for n = 4 independent donors). (H) Summary of intracellular flow cytometry analysis of Rheb in CD8+ T cells stimulated as above for 24 hours (n = 4 independent donors). (I) CD8+ T cells were cultured for 16 hours at 21% or 1% O2, left unstimulated or stimulated via CD3/CD28 for 24 hours, serum starved for 6 hours then provided 10 mM leucine where indicated for 30 minutes, prior to measurement of intracellular phospho-p70S6 kinase (p-p70S6K) and total p70S6K by flow cytometry (representative flow cytometry histograms and summarized data for n = 4 independent donors, shown as ratio of p-p70S6K/total p70S6K and change (Δ) in this ratio upon leucine provision.) (J-K) CD8+ T cells were transfected with control (Ctrl.) or BNIP3-targeting CRISPR guide RNAs, and green fluorescent protein (GFP)-tagged Cas9, and stimulated at 21% or 1% O2 for 24 hours. (J) BNIP3 and (K) phospho-mTOR abundance within GFP+ cells, analyzed by flow cytometry (summarized data for n = 4 independent donors, normalized to the average [mean] MFI for matched samples from each donor). P values were calculated by (G, H, J) 2-way ANOVA and Holm-Sidak’s post hoc test, or (I, K) paired t test, ∗P < .05, ∗∗ P < .01.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/9/23/10.1182_bloodadvances.2025016439/2/blooda_adv-2025-016439-gr4.jpeg?Expires=1769113272&Signature=Z3~8l7rpsj3eIivZkT0h2K-0D9vkI3R4DOPlM8Wkr7XNuayG2ELXamz9HghvwEZb1gnPslc-rML0zqdSRxTI9908IAHf0eWBhcjLdifMaz2rok8RxYltrv1yNQtH7HAL4IU2Qp7opbwwgzvqFzZGsu0BfvXfPra~aLcLTi6VYyOSRbYpNIe6OoioTGPfB5LHtjO8LqlgAPNHzaw4EQ-vK-Hu80PWyPG-uOoGH3lxh~JSenWKxDHc0DaHcG7duC8Fdr7LtHzQDIAM2ZBmv9BnUkwKFeALvRGniObAW4UmPpdbMg1P7LMlfjW4XNbEZcT37Z0Mzn9fPAFW0eaP8jQCng__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Hypoxia alters stimulated CD8+ T-cell transcriptional profiles, inducing BNIP3 upregulation, decreased Rheb abundance, and impaired amino acid–induced mTOR activity. CD8+ T cells were cultured for 16 hours at 21% or 1% O2, then left unstimulated or stimulated via CD3/CD28 for 24 hours at these oxygen tensions prior to RNA-sequencing analysis (n = 5 independent donors). (A-B) Principal component analyses identify unstimulated samples largely cluster by donor, whereas stimulated samples cluster by oxygen tension. (C) Venn diagram, with circles representing differentially expressed genes (DEGs) between indicated conditions to identify shared and distinct DEGs when comparing cells stimulated at 21% or 1% O2 with their respective matched unstimulated controls. (D-E) Pathway analysis of genes (D) upregulated or (E) downregulated in cells stimulated at 1% O2 compared with 21% O2. (F) Volcano plot of total transcript abundance in cells stimulated at 1% O2 compared with 21% O2. (G) Example western blot (left) and summary of intracellular flow cytometry analysis (right) of BNIP3 in CD8+ T cells stimulated as above for 24 hours, or additionally treated with the HIF-1α inducer, deferoxamine (DFO) where indicated (summarized data for n = 4 independent donors). (H) Summary of intracellular flow cytometry analysis of Rheb in CD8+ T cells stimulated as above for 24 hours (n = 4 independent donors). (I) CD8+ T cells were cultured for 16 hours at 21% or 1% O2, left unstimulated or stimulated via CD3/CD28 for 24 hours, serum starved for 6 hours then provided 10 mM leucine where indicated for 30 minutes, prior to measurement of intracellular phospho-p70S6 kinase (p-p70S6K) and total p70S6K by flow cytometry (representative flow cytometry histograms and summarized data for n = 4 independent donors, shown as ratio of p-p70S6K/total p70S6K and change (Δ) in this ratio upon leucine provision.) (J-K) CD8+ T cells were transfected with control (Ctrl.) or BNIP3-targeting CRISPR guide RNAs, and green fluorescent protein (GFP)-tagged Cas9, and stimulated at 21% or 1% O2 for 24 hours. (J) BNIP3 and (K) phospho-mTOR abundance within GFP+ cells, analyzed by flow cytometry (summarized data for n = 4 independent donors, normalized to the average [mean] MFI for matched samples from each donor). P values were calculated by (G, H, J) 2-way ANOVA and Holm-Sidak’s post hoc test, or (I, K) paired t test, ∗P < .05, ∗∗ P < .01.
