Figure 4.

EC-induced CD69 expression on memory CD8+ T cells is partly mediated by synergistic action of IL-15, ICAM-1 and VCAM-1. (A+B+C) HMEC were left unstimulated (resting) or stimulated with 10 ng/mL TNFα and/or IFNγ for 3 days (activated) before addition of FACS-sorted memory CD3+ T cells to the co-culture or HMEC cultured medium. CD69 expression and proliferation were assessed at various time points of the co-culture. (A) Percentage of CD69+ cells (left panel) and median fluorescent intensity (MFI) of CD69 (right panel) within CD8+ T cells after co-culture with TNFα and IFNγ-stimulated HMEC, resting HMEC, their cultured medium (sup), or TNFα and IFNγ alone (cyt only). N=3, mean+SEM. 2-Way-ANOVA with Sidak post-hoc test. */#/⁰P < 0.05, **/##/⁰⁰P < 0.01, ***/###/⁰⁰⁰P < 0.001,****/####/⁰⁰⁰⁰P < 0.0001. (B) Percentage of CD69+ cells (left panel) and median fluorescent intensity (MFI) of CD69 (right panel) within CD8+ T cells after culture with activated HMEC cultured medium (sup), transwell co-culture or direct co-culture with activated HMEC. N = 5, median. 2-Way ANOVA with Sidak’s multiple comparison test; *p < 0.05, ns = not significant. (C) Percentage of CD69+ cells (left panel) and median fluorescent intensity (MFI) of CD69 (right panel) within CD8+ T cells after co-culture with TNFα- and/or IFNγ-stimulated HMEC or their cultured medium (sup). N = 3, mean+SEM. 2-Way-ANOVA with Sidak post-hoc test. */#/⁰P < 0.05, **/##/⁰⁰P < 0.01, ***/###/⁰⁰⁰P < 0.001,****/####/⁰⁰⁰⁰P < 0.0001. (D+E) Levels of soluble ICAM-1 and VCAM-1 (D) and IL-15 and TGF-β (E) measured in cultured medium of resting or TNFα- and/or IFNγ-stimulated HMEC after 3 days, by multiplex immunoassay. N = 3, mean+SEM. Kruskal-Wallis with Dunn’s post-hoc test versus resting EC. *P < 0.05, **P < 0.01. (F+G) Co-culture of TNFα and IFNγ-stimulated HMEC with FACS-sorted memory CD3+ T cells in the presence of (increasing concentrations) of monoclonal antibodies blocking IL-15, TGF-β, ICAM-1 and/or VCAM. (F) The percentage of CD69+ cells was measured by flow cytometry after 18 hours and normalized to the percentage of CD69+ cells in the condition with isotype control (set to 100). N = 3, median. Kruskal-Wallis with Dunn’s post-hoc test versus isotype. (G) The percentage of CD69+ cells was measured by flow cytometry after 18 and 90 hours and normalized to the condition with isotype control (set to 100). N = 4, median. 2-Way-ANOVA with Sidak post-hoc test versus isotype. *P < 0.05, **P < 0.01. (H) Transwell and direct co-culture of TNFα and IFNγ-stimulated HMEC with FACS-sorted memory CD3+ T cells in the presence of monoclonal antibodies blocking IL-15, ICAM-1 and/or VCAM. Percentage of CD69+ cells within CD8+ T cells was measured by flow cytometry after 90 hours and normalized to the condition with isotype control (set to 100). N = 4, median. 2-Way-ANOVA with Sidak post-hoc test versus isotype. (I) Co-culture of TNFα and IFNγ-stimulated HMEC with FACS-sorted memory CD3+ T cells in the presence of 35 μg/mL monoclonal antibody blocking HLA-ABC or isotype control. The percentage of CD69+ cells and median fluorescent intensity (MFI) of CD69 was measured by flow cytometry after 18 and 90 hours. N = 4. Dotted lines indicate paired measurements. 2-Way-ANOVA with Sidak post-hoc test versus isotype. *P < 0.05, ***P < 0.001.