Figure 4. Surface IL-15 on MAC-activated ECs is presented in trans and enhances allogeneic CD8⁺ Tem cell activation, proliferation, and differentiation.

(A) Proliferation of CD4⁺ Tem cells after coculture for 7 days with IFN-γ–primed ECs pretreated with NLRP3 inflammasome inhibitor MCC950, IL-1 receptor antagonist, anti–IL-15 blocking antibody, or DMSO before PRA sera or vehicle treatment for 6 hours. CFSE dilution was assessed on day 7 by flow cytometry. FACS plots show a representative experiment (n = 3). (B) CD8⁺ Tem cell proliferation by CFSE dilution, activation by HLA-DR surface expression, and differentiation by granzyme B and perforin expression were assessed after coculture with IFN-γ–primed ECs pretreated with MCC950, z-YVAD-FMK, IL-1Ra, anti–IL-15 blocking antibody, or DMSO before PRA sera or vehicle treatment for 6 hours. Flow cytometry analysis was performed after 7 days of coculture. FACS plots show a representative experiment (n = 3). (C) IFN-γ production by allogeneic memory CD8⁺ T cells after coculture with IFN-γ–primed ECs transfected with control or p65 siRNA before addition of exogenous IL-1β or mock treatment. IFN-γ production was assessed by ELISA after 24 hours for coculture (72 hours after siRNA transfection). (D) Proliferation, activation, and granzyme B and perforin expression of CFSE-labeled CD8⁺ Tem cells after coculture with IFN-γ–primed ECs transfected with p65 or control siRNA and PRA or vehicle treatment for 6 hours. Flow cytometry analysis was performed on day 7. Data represent mean ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA and Tukey’s multiple comparisons test. Representative of 3 to 4 independent experiments using 3 HUVEC donors.