![Hypoxia does not affect initial CD8+ T-cell cytotoxicity elicited by BCMA×CD3 bispecific antibody, but limits CD25 and IFN-γ induction, proliferation, and alters memory differentiation. (A-B) CD8+ T cells were cultured with BCMA-expressing, CTV-labeled JJN3 target cells and BCMA×CD3 bispecific antibody where indicated for 24 hours at either 21% O2 or 1% O2, prior to assessment of target cell viability (live/dead probe exclusion). (A) Representative flow cytometry plots, and (B) summarized data for n = 5 independent donors of CD8+ T cells across indicated T cell:target ratios. (C-D) CD8+ T cells and target JJN3 cells were cultured as above, additionally in the presence of brefeldin A/monensin and CD8+ T cells assessed for CD107a trafficking, intracellular granzyme B (GzmB), and perforin A (PrfA), by flow cytometry (C) representative flow cytometry plots/histograms, and (D) summarized data for n = 5 independent donors of CD8+ T cells at 2.5 T cells:1 target cell ratio. (E-F) CD8+ T cells were cultured as in panels C-D with indicated myeloma cell lines, and assessed for (E) surface CD25 and (F) intracellular IFN-γ by flow cytometry. Data are normalized to the average (mean) MFI for the 4 matched samples from each donor. (G-H) CD8+ T cells were activated as in panels A-B, and assessed after 7 days for (G) proliferation (carboxyfluorescein succinimidyl ester [CFSE] dilution), and (H) frequency of indicated populations as defined by surface expression of CD45RA and CD62L (summarized data for n = 4 independent donors of CD8+ T cells at 1 T cell:1 target cell ratio). P values were calculated by 2-way ANOVA and Holm-Sidak’s post hoc test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/9/23/10.1182_bloodadvances.2025016439/2/blooda_adv-2025-016439-gr5.jpeg?Expires=1769113272&Signature=opxzCPx-OUC5YNSWUziiKLyonSZnFQP9ebDqWPzAF37uOffC8j3vu9afwJg-jH4MuHugbw1cyGns~rQYhyCt3BUfS9E8kmz1Nf5CNe07hamcgqPW260Fh1qKCLzXJrCeRsoaZLR7KoUYcTrCEKADzNtREygOuUwd1tkfYTnwbDUhFzalYE8E1dGB4Sl9PsKLdimY2HKxX0mAOH9zFq9NUvBhCNkuP0erDCRHvRDeaEN--uloUnckuxUZ5CXc9b6ii4mBxp1d4PBjdKiL1Yr0AceZUhed3tOvvTIC8yF5dibEl40RnzmHKvrjAmxBfj5uOb1o3VOcIZVBXUbpL42uvg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Hypoxia does not affect initial CD8+ T-cell cytotoxicity elicited by BCMA×CD3 bispecific antibody, but limits CD25 and IFN-γ induction, proliferation, and alters memory differentiation. (A-B) CD8+ T cells were cultured with BCMA-expressing, CTV-labeled JJN3 target cells and BCMA×CD3 bispecific antibody where indicated for 24 hours at either 21% O2 or 1% O2, prior to assessment of target cell viability (live/dead probe exclusion). (A) Representative flow cytometry plots, and (B) summarized data for n = 5 independent donors of CD8+ T cells across indicated T cell:target ratios. (C-D) CD8+ T cells and target JJN3 cells were cultured as above, additionally in the presence of brefeldin A/monensin and CD8+ T cells assessed for CD107a trafficking, intracellular granzyme B (GzmB), and perforin A (PrfA), by flow cytometry (C) representative flow cytometry plots/histograms, and (D) summarized data for n = 5 independent donors of CD8+ T cells at 2.5 T cells:1 target cell ratio. (E-F) CD8+ T cells were cultured as in panels C-D with indicated myeloma cell lines, and assessed for (E) surface CD25 and (F) intracellular IFN-γ by flow cytometry. Data are normalized to the average (mean) MFI for the 4 matched samples from each donor. (G-H) CD8+ T cells were activated as in panels A-B, and assessed after 7 days for (G) proliferation (carboxyfluorescein succinimidyl ester [CFSE] dilution), and (H) frequency of indicated populations as defined by surface expression of CD45RA and CD62L (summarized data for n = 4 independent donors of CD8+ T cells at 1 T cell:1 target cell ratio). P values were calculated by 2-way ANOVA and Holm-Sidak’s post hoc test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.
