Fig 4. IL-18 Directly Expands IL-18R1+ T_PH Cells.

EC were treated with Ac-YVAD-FMK (a) or MCC950 (b) during “IRI” prior to coculture with CD4+CD45RO+ T cells (T_mem) in the presence or absence of exogenous IL-18 (0.5μg) as indicated for 10 days prior to FACS analysis. T_PH cells and T_FH cells were gated among T_mem cocultured with “IRI”-treated ECs in the presence IL-18-depleting antibody (10μg/mL, c, left) or exogenous IL-18 as indicated (c, right). Mean fluorescent intensities (MFI) of bcl-6 and BLIMP1 were assessed in T_mem with αIL-18 Ab (d, left) or exogenous IL-18 (d, right) following coculture with IRI-treated EC. T_mem were stimulated with “IRI”-treated EC (e). IL-18R1 expression after gating on T_PH and T_FH cell populations following 7 day coculture with “IRI”-treated HUVEC (f). T_PH and T_FH cells were analyzed after gating on IL-18R1 (g). T_mem were stimulated with αCD3/CD28 for 24h prior in the presence or absence of IL-18 (h,i). IL-18R1 shRNA was transduced into “IRI”-treated HUVEC, T_mem, or both prior to EC:T cell coculture for 7 days (j, **p<0.001, N.S., not significant). Student’s t-test was used for Fig 4e and 4g. Two-way ANOVA followed by Tukey’s pairwise comparison was used for Fig 4a, 4b, 4c, 4d, 4f, 4h, 4i, and 4j. Experiments were repeated 2–6 times using ≥3 PBMC donors and ≥2 HUVEC donors.