Figure 4.

Activation of mutant SKW3.BV20 cells by S. typhimurium. 11 mutant α chain (A) and 14 mutant β chain (B) SKW3.TRBV20 cell lines were incubated with C1R cells infected with S. typhimurium at a MOI of 100. Shaded bars show the fold increase in CD69 surface expression (fold increase in MFI) of mutant SKW3.TRBV20 cells co-incubated with C1R cells infected with S. typhimurium compared with SKW3.BV20 cells co-incubated with uninfected C1R cells (open bars). A positive control, wild-type SKW3.TRBV20 (TRBV20.WT), and a negative control, SKW3.LC13 (LC13.WT), were included. The mutant CDR1β Leu26A cell line was included as an internal control where activation was expected to remain intact. (C) MAIT.TRBV6-1 TCRs with six separate solvent-exposed residues of the β chain mutated to alanine were transduced into SKW3 cells and tested in the same manner as the mutant SKW3.TRBV20 cell lines in A and B. (D) SKW3 cells transduced with MAIT TCRs containing CDR3β regions of the MAIT.TRBV6-1 TCR or the MAIT.TRBV20 TCR exchanged with the CDR3β regions of known functional TCRs using TRBV6-1 or TRBV20, respectively, were then tested in the same manner as the mutant SKW3.TRBV20 cell lines in A and B. The following SKW3-transduced cell lines were tested: wild-type SKW3.TRBV6-1 cells (TRBV6-1.WT); SKW3.TRBV6-1 cells with a TCR containing the SB27 TRBV6-1 CDR3β region (TRBV6-1/SB27β); wild-type SKW3.TRBV20 cells (TRBV20.WT); SKW3.TRBV20 cells with a TCR containing the ABC TRBV20 CDR3β region (TRBV20/ABCβ; to be described elsewhere); SKW3 cells with a TCR containing the MAIT invariant α chain paired with the LC13 TCR β chain (MAIT.LC13β); and SKW3 cells with the wild-type LC13 TCR (LC13αβ.WT). The experiments shown in A–D were also performed at an MOI of 1 and 10 and yielded similar results (not depicted). The experiments shown in A and B and C and D were done three times and twice, respectively, with similar results.