Figure 6
From: Alternative splicing of MR1 regulates antigen presentation to MAIT cells
Double positive thymocyte expression of surface MR1A is associated with transcript expression and MAIT frequency in the thymus. MR1 + thymocytes were isolated using magnetic sorting based on MR1 expression (A) and used as antigen presenting cells following infection with M.smegmatis for 1 h at an MOI of 3 to stimulate MAIT production of IFN-γ, Error bars represent mean and standard deviation of duplicate wells from one experiment. T cell production of IFN-γ is represented as spot forming units (SFU). (B) Gating strategy for sorting MR1 + CD4 + CD8 + (DP) thymocytes from human thymus. Singlet cells were gated on live CD3 + cells. Live CD3 + cells (left) were gated on CD4 and CD8 DP positives (middle). Gate for MR1 + cells was set based on an IgG2a isotype control for MR1(third). An example plot of MR1 + cells based on this gating strategy is shown (right) (C) Absolute (left, middle), and relative (right) amounts of MR1A and MR1B transcript in DP MR1 + thymocytes versus surface expression of MR1 in 9 thymus donors. Pearson’s correlation was calculated and R2 and statistical significance is reported. (D) Gating strategy to identify MAIT cells in human thymus. Gates were set based on a negative control (FMO). Briefly, cells were stained with markers for viability, CD3, and the MR1/5-OPRU tetramer or the MR1/6-FP control tetramer (not shown). Singlet cells were gated on live CD3 + (left) and gates for MR1/5-OPRU tetramer + were set based on a negative control (FMO-middle). An example donor is shown on the right (Th97) (E). Frequency of MR1/5-OPRU tetramer + MAIT cells (from D) plotted versus surface MR1A expression in DP thymocytes (from B) from 9 donors. Pearson’s correlation was calculated and R2 and statistical significance is reported. (F) Same as (C), Expression of MR1A and MR1B transcript in DP thymocytes (B,C) is plotted versus frequency of MR1/5-OPRU tetramer staining MAIT cells (D). Pearson’s correlation was calculated and R2 and statistical significance is reported. Error bars for all represent mean and standard deviation of triplicate wells from one experiment.
