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The M33.64 cell line was activated in response to 5-OP-RU and E. coli but also exhibited robust, MR1-dependent activation in the absence of added Ag, consistent with it being stained with empty MR1 tetramer. The MAV36 TCR+ cells were activated when co-cultured with C1R.MR1 cells in the presence of E. coli or 5-OP-RU, but showed little response without added Ag. To further investigate the M33.64 TCR autoreactivity, the M33.64 TCR+ cell line was co-cultured with MR1-transduced C1R cells that were flow cytometrically sorted based on graded MR1 expression. The M33.64 TCR+ cells upregulated CD69 in an MR1-dose-dependent manner, without added Ag, confirming that in contrast to other MAIT TCRs, the M33.64 TCR is MR1 autoreactive.
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