Fig. 6

VISTA suppressed antigen-specific TCR activation. (A) Schematic illustration of the luminescence assay with antigen-specific TCR Jurkat NFAT-luminescence reporter cells. In brief, E7 TCR Jurkat NFAT cells were co-cultured with THP-1 derived macrophages that were pulsed with HPV-16 E7 (10 µg/mL). Luciferase activity was detected after 6 h in Relative Light Units (RLU). (B) RLU after co-culture of Jurkat NFAT cells with THP-1 derived M2-like macrophages at the indicated effector:target (E:T) ratios. Fold increase of normalized RLU activity in Jurkat cells co-cultured with HPV-16 E7 treated cells was calculated over the values from untreated co-cultures. ^*P < 0.05 and ^**P < 0.01 by two-way ANOVA with post hoc intergroup comparisons using Tukey’s test. (C) RLU loss in Jurkat cells co-cultured with VISTA or ECD transduced THP-1 cells compared to that co-cultured with THP1^EV cells. ^*P < 0.05 by student’s t-test. (D) RLU in a co-culture of Jurkat NFAT cells with CD34⁺ CB cell derived M2-like macrophages at the indicated E:T ratios. Fold increase of normalized RLU activity in Jurkat cells co-cultured with HPV-16 E7 treated cells was calculated over the values from that co-cultured with untreated cells. ^*P < 0.05 and ^***P < 0.001 by two-way ANOVA with post hoc intergroup comparisons using Tukey’s test. (E) RLU loss in Jurkat cells co-cultured with VISTA or ECD transduced CD34⁺ CB cell derived macrophages compared to co-cultures with EV transduced CD34⁺ CB cell derived macrophages. F-G. Expression of HLA-ABC (F) and HLA-A2 (G) in VISTA, ECD, or EV transduced THP-1 derived M2-like macrophages. H-I. Expression levels of HLA-ABC (H) and HLA-A2 (I) in VISTA-, ECD-, or EV-transduced CD34⁺ CB cell derived M2-like macrophages. J. Binding of recombinant (His tagged) human VISTA protein to VISTA- or EV-transduced Jurkat cells measured using APC-conjugated anti-His antibody. K. Visual representation of the most optimal molecular binding model between VISTA and HLA-ABC using the HDOCK Server. L. Co-IP western blot for VISTA and HLA-ABC in THP-1^EV, THP-1^VISTA, HL60^EV, and HL60^VISTA cells. M. Log10-transformed IBAQ intensities of quantified membrane proteins identified through mass spectrometry, obtained from anti-HLA-ABC immunoprecipitation (IP) of HL60^VISTA cells. The dashed horizontal line represents the Log10 IBAQ threshold of 3. The dotted vertical lines enclose a region where the log2 fold change of protein quantities, obtained from anti-HLA-ABC IP of HL60^VISTA cells compared to anti-HLA-ABC IP of HL60^EV cells, ranges from − 1 to 1. N. Log10-transformed IBAQ intensities of quantified membrane proteins identified through mass spectrometry, obtained from anti-HLA-ABC IP of THP-1^VISTA cells. The dashed horizontal line represents the Log10 IBAQ threshold of 3. The dotted vertical lines enclose a region where the log2 fold change of protein quantities, obtained from anti-HLA-ABC IP of THP-1^VISTA cells compared to anti-HLA-ABC IP of THP-1^EV cells, ranges from − 1 to 1. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 by one-way ANOVA with post hoc intergroup comparisons using Tukey’s test