Figure 3.

Effect of HLA-G expression on MAIT cell function. B-LCLs were infected with S. Typhi for 3 hours. Uninfected cells were used as controls. (A) PBMC were exposed to either uninfected (UN) or S. Typhi-infected (INF) B-LCL cells treated with Abs to HLA-G (HLA-G) or isotype (ISO) control (2.5 μg/ml). After 16-18 hours of co-culture, cells were collected, and the levels of MAIT cells expressing IFN-γ were evaluated by flow cytometry. Data are representative of two independent experiments. Bar graphs extend from the 25^th to 75^th percentiles; the line in the middle represents the median of the pooled data. The whiskers delineate the smallest to the largest value. (B) Relationship between the levels of IFN-γ+ MAIT cells and HLA-G expression on B-LCLs evaluated by mass cytometry. Four individual volunteers were studied under 3 different conditions (UN, ISO, and HLA-G). (C) Pearson’s correlations among levels of HLA-G and S. Typhi antigen (CSA) expression on B-LCLs and IFN-γ production by MAIT cells. Data are representative of at least five individual experiments. (D) Kinetics of HLA-G expression on B cells cultured alone (UN and INF) or cocultured with PBMC (UN & PBMC, and INF & PBMC). Data are representative of 3 individual experiments with 2-3 replicates Student’s t-test was performed to compare HLA-G+ B-LCL cell levels after exposure to MAIT cells. *, represent significant differences (P<0.05) between the uninfected () and infected B-LCLs () in the presence of MAIT cells at the same timepoint. (E) Dose effect of 3 concentrations of recombinant IFN-γ (0.5, 5, or 25ng/ml) on HLA-G expression on B cells cocultured for 18 hours with PBMC (UN & PBMC, and INF & PBMC). Co-cultures in the absence of IFN-γ (i.e., 0) were used as controls. Data are representative of 1 individual experiment with 2 replicates. (F) Expression of HLA-G vs. CSA on live (upper panel) and dead (lower panel) INF B-LCLs after 16-18 hours of co-culture with PBMC.