Journal: Cell Reports Methods
Article Title: MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens
doi: 10.1016/j.crmeth.2025.101120
Figure Lengend Snippet: The B6 vitamers pyridoxal and PLP activate Jurkat cells expressing the A-F7 MAIT TCR and the MC.7.G5 TCR, as well as primary CD8 + T cells expressing the A-F7 MAIT TCR (A) Jurkat cells with no TCR or transduced with A-F7 MAIT TCR were co-incubated overnight with A549 WT and A549 MR1 KO cell lines treated with pyridoxal at 100, 10, and 1 μg/mL or loaded with M. smegmatis (MOI: 1:300). Cells were stained for CD69 expression with mean fluorescence intensity (MFI) displayed. Background MFI of Jurkat cells with A549 WT or MR1 KO alone with no pyridoxal or M. smegmatis was subtracted. Jurkat cells with A-F7 were gated on co-marker rCD2 + . Data display duplicate conditions ( E). (B) Jurkat cells with no TCR or transduced with A-F7 MAIT TCR were co-incubated overnight with A549 WT and the following compounds: 5-A-RU (converts to MAIT ligand 5-OP-RU in cells and was added in the absence of exogenously applied methylglyoxal, which increases potency), pyridoxal and PLP at 100, 10, 1, 0.1, 1 × 10 −2 , 1 × 10 −3 , and 1 × 10 −4 μg/mL. Cells were stained for CD69 expression with MFI displayed. Background MFI of Jurkat cells with A549 cells alone with no pyridoxal was subtracted. Jurkat cells expressing the A-F7 TCR were gated on the rCD2 co-marker. Assay was performed in triplicate ( F), and curves were fitted using a four-parameter logistic model. Points indicate mean values, with error bars depicting standard deviation. EC 50 values with a 95% confidence interval (CI) and R 2 are indicated, with the results reproducible over two assays ( G). (C) Primary CD8 + T cells from three healthy donors with no TCR transduction or expression of the A-F7 TCR to generate TCR-T cells, were co-incubated for 4 h with A549 WT cells ± pre-treatment with 100 μg/mL of pyridoxal, and then reactivity measured via T107 assay. T cells were also incubated alone or with CD3/CD28 Dynabeads, with the latter acting as a positive control. Cells were gated on lymphocytes, viable CD3 + , single cells, rCD2 + /CD8 + (or CD8 + for the untransduced), and then TNF + versus CD107a + for reactivity. For the pyridoxal conditions, background reactivity toward A549 cell lines with no pyridoxal has been subtracted. For reactivity toward CD3/CD28 Dynabeads, the reactivity for the T cell-alone condition has been subtracted ( H). (D) Jurkat cells with no TCR or transduced with MC.7.G5 TCR were co-incubated overnight with C1R cells ± pyridoxal at 100, 10, 1, 0.1, and 1 × 10 −2 μg/mL. Cells were stained for CD69 expression with MFI displayed. Background MFI of Jurkat cells alone with no pyridoxal was subtracted. Jurkat cells with MC.7.G5 TCR were gated on co-marker rCD2 + . Assay was performed in triplicate ( I), and curves were fitted using a four-parameter logistic model. Points indicate mean values, with error bars depicting standard deviation. EC 50 values with a 95% CI and R 2 are indicated ( J). See also and .
Article Snippet: B-cell lymphoblastoid cell line C1R (ATCC information, CRL-1993) were sourced locally and engineered to overexpress MR1∗01 using lentivirus.
Techniques: Expressing, Transduction, Incubation, Staining, Fluorescence, Marker, Standard Deviation, Positive Control
