cd8 antibody, anti-human  (Miltenyi Biotec)

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: Development of an enrichment strategy for MR1-dependent antigen discovery by protein-metabolite cross-linking and de novo MR1 antigen discovery (A) Schematic of a recombinant platform to express fully functional, C-terminally-tagged single-chain MR1/β2M (scMR1) molecules with either lysine or alanine at position 43, developed for high-specificity MR1 enrichment. The alpha 1, 2, 3, and transmembrane (TM) domains of MR1 are depicted. (B) MR1 staining of A549 (left) and MM909.24 (right) cell lines, either wild type (WT), MR1 knockout (MR1 KO), and MR1 KO cells transduced with scMR1 (MR1 KO + scMR1-WT) and mutant scMR1 (MR1 KO + scMR1-K43A). Numbers in the left-hand corner are the MR1-specific Allophycocyanin (APC) mean fluorescence intensities (staining with anti-MR1 26.5 antibody clone). Gates are set for viable, single cells. (C) Overnight activation assay with MAIT cell TCR-T (primary CD8 + T cells transduced with A-F7 MAIT TCR) versus M. smegmatis -infected and uninfected A549 cells followed by a tumor necrosis factor (TNF) ELISA confirming MAIT cell recognition of scMR1 in the presence of endogenous antigen. Error bars depict the standard deviation of duplicate conditions. (D) Enrichment efficiency obtained from MM909.24 cells stably transduced with scMR1-K43 molecules based on protein abundances obtained by LC-MS/MS (MR1 and β2M are highlighted separately as red dots that overlap). (E) Proof of concept for the detection of MR1/Ac-6-FP cross-link in a cell-based system. scMR1-transfected MM909.24 melanoma cells pulsed with 50 μM Ac-6-FP for 16 h were subjected to the cross-linking workflow. The graph shows extracted ion chromatograms for DSVTRQ K EPRAPW and DSVTRQ K EPRAPW bound to Ac-6-FP, respectively, including the three most abundant isotopes (M, M+1, and M+2) for each of the two peptide variants. (F) Schematic of the data analysis workflow employed to detect DSVTRQ K EPRAPW cross-linked to unknown ligands. Peptide sequence ladder ions unaffected by the ligand (y1–y6 and b1–b6) were used as reporter ions. MS/MS spectra containing these ions were subsequently shortlisted. Subtraction of the theoretical DSVTRQ K EPRAPW peptide mass generates Δ-mass values for cross-linked ligands that can be corrected for the mass of the free ligand, which can be queried for candidate compounds using tools such as CEU mass mediator. (G and H) Applied to scMR1-transfected MM909.24 pulsed with 50 μM Ac-6-FP for 16 h, the data analysis pipeline successfully detected spectra indicating the presence of other ligands bound to MR1, such as 6-formylpterin (XIC, G) and methylglyoxal (XIC, H). See also and and .

Article Snippet: CD8 APC Vio770: clone BW135/80 , Miltenyi Biotec , RRID: AB_2725983.

Techniques: Recombinant, Functional Assay, Staining, Knock-Out, Transduction, Mutagenesis, Fluorescence, Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation, Stable Transfection, Liquid Chromatography with Mass Spectroscopy, Transfection, Sequencing, Tandem Mass Spectroscopy

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: CD8 APC Vio770: clone BW135/80 , Miltenyi Biotec , RRID: AB_2725983.

Techniques: Expressing, Transduction, Incubation, Staining, Fluorescence, Marker, Standard Deviation, Positive Control