Fig 3. “IRI”-Treated EC Selectively Expand T_PH Cells In Vitro.

HUVEC were subjected to “IRI” in the presence or absence of normal human sera as a source of complement (C’) prior to co-culture with CD4+CD45RO+ T cells (T_mem) for 7–10 days prior to T cell analysis for activation (a, top) and proliferation (a, bottom). ***p<0.001, **p<0.005, *p<0.05, N.S. not significant (p>0.05). T_mem cocultured with HUVEC subjected to “IRI” in the presence of WT C’, C1q-deficient C’, or C6-deficient C’ (b). T_mem were cocultured with IRI-treated EC and analyzed for CD4+CD45RO+PD-1+CXCR5-CCR2+ and CD4+CD45RO+PD-1+CXCR5+CCR2- T cells (c). FACS-sorted T cells were cocultured with IRI-treated EC as indicated and analyzed by FACS (d). T_mem were cocultured with EC for 7–10 days and bcl-6:BLIMP ratios were assessed (e). T_mem were cocultured with EC for 14 days and intracellular cytokines were analyzed (f). “IRI”-treated EC were cocultured with FACS-sorted T cells in the presence of autologous B cells for 14 days, and supernatant titers of anti-class I (g, left) and anti-class II (g, right) HLA Ab specific for cultured EC, i.e., dnDSA, were quantified (g) along with dnDSA IgG subclasses (h). Total Ig isotypes were quantified by ELISA (i). Student’s t-test was used for Fig 3c. One-way ANOVA followed by Tukey’s pairwise comparison was used for Fig 3g, 3h, and 3i. Two-way ANOVA followed by Tukey’s pairwise comparison was used for Fig 3a, 3b, 3d, and 3f. Experiments were repeated 2–8 times using ≥2 PBMC donors and ≥2 HUVEC donors.