Journal: bioRxiv

Article Title: Delivery of loaded MR1 Monomer Results in Efficient Ligand Exchange to Host MR1 and Subsequent MR1T cell activation

doi: 10.1101/2022.07.11.499573

Figure Lengend Snippet: A . Biotinylated MR1 monomer, recombinant TCR, or HLA-A2 were immobilized on a traptavidin-coated plate before incubation with PBS at the indicated pH overnight at 4C. Binding of either anti-MR1 (clone 26.5) or anti-b2m (clone B2M-01) antibody was assess with an HRP-conjugated secondary antibody and TMB substrate. Error bars denote standard deviation. B . MR1T clone (5e3) IFN-γ response to BEAS-2B (1e4) pre-treated with Bafilomycin A1 for 2 hours, followed by addition of the MR1/5-OP-RU monomer or 5-OP-RU prodrug. Data in B are representative of three independent experiments.

Article Snippet: After removal of the acidic buffer and washing with PBS pH 7.2, either anti-human MR1 (unlabeled, clone 26.5, BioLegend 361102) or anti-human B2m (unlabeled, clone B2M-01, Novus Biologicals NB500-317) antibody was applied to the plate, followed by a rabbit anti-mouse IgG H+L, HRP-conjugated secondary antibody (Abcam ab6728).

Techniques: Recombinant, Incubation, Binding Assay, Standard Deviation

Journal: Nature Communications

Article Title: Combinatorial immunotherapies overcome MYC-driven immune evasion in triple negative breast cancer

doi: 10.1038/s41467-022-31238-y

Figure Lengend Snippet: a – d Scatter plot correlation tested between the expression of ( a ) B2M gene and MYC_BC signature in TCGA TNBC patients, Pearson’s coefficient (Rp), adjusted p -value (Benjamini-Hochberg FDR corrected). b B2M and the MYC_BC signature in TONIC Trial patients, Spearman’s r (ρ), exact p -value. c NLRC5 gene and MYC_BC signature in TCGA TNBC patients, Pearson’s coefficient (Rp), adjusted p -value (Benjamini–Hochberg FDR for corrected). d NLRC5 gene and the MYC signature in the TONIC Trial patients, Spearman’s r (ρ), exact p -value. e Antigen presentation pathway genes in MCF10A-MYC cells compared to MCF10A-vector cells. Data displayed as fold change with values < 1 (dotted line) equating to gene repression. Displayed n = 4 independent cell passage replicates. Mean ± S.E.M, two-sided, unpaired t -test, exact p -values. f Differential expression analysis for RNA-seq data in MTB/TOM tumors published by Rohrberg et al. Left: MYC-ON compared to normal mammary tissue (Ctrl), Right: 3 days off-doxycycline (MYC-OFF) compared to animals on doxycycline (MYC-ON). Adjusted p -values. g Representative histogram for MHC-I expression by flow cytometry in MYC-ON and MYC-OFF states. Adjacent bar graph displaying geometric mean fluorescence intensity (GMFI) for MHC-I in MYC-ON ( n = 6) and MYC-OFF ( n = 7). Line represents median, data points represent individual animals, two-sided, Mann–Whitney test, exact p -value. h Immunohistochemistry staining of immune cell markers within tumor sections in MYC-ON and MYC-OFF state. Images are at 20X. Adjacent bar graphs displaying cumulative counts per field of view (FOV). n = 3 animals per group, 3 FOV analyzed per tumor, mean ± S.E.M. two-sided unpaired t -test, exact p -values. i Flow cytometry analysis of immune cells found in MYC-ON tumors or MYC-OFF tumors (off doxycycline chow for 3 days) ( n = 6 per group). Line represents median, data points are individual animals, outliers removed, two-sided Mann–Whitney test, exact p -values. Source data are provided in the Source Data file.

Article Snippet: Proteins were resolved with the Bolt 4–12% Bis-Tris gel and buffer system (Invitrogen) and transferred onto nitrocellulose membranes using iBlot2 (Invitrogen) on program P0 for all proteins, except for B2M, which was transferred at 20 V for 6 min. Membranes were blocked with 5–10% non-fat milk (Rockland) in TBST and incubated with primary antibodies diluted in 5% non-fat milk TBST overnight on a 4 °C shaker.

Techniques: Expressing, Immunopeptidomics, Plasmid Preparation, Quantitative Proteomics, RNA Sequencing, Flow Cytometry, Fluorescence, MANN-WHITNEY, Immunohistochemistry, Staining