Journal: Science advances
Article Title: IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHCII complexes.
doi: 10.1126/sciadv.adk2091
Figure Lengend Snippet: Fig. 5. IRF8 control of components of the MHCII complex. (A) FACS analysis of H2-IA/IE (left) and HLA-DR (right) in models of IRF8 KO; WB of IRF8 in RIVA and SU-DHL2 KO models, and WB of MHCII in all IRF8 KO models in DLBCL. (B) FACS of CD74 in models of IRF8 KO. (C) FACS of H2-DM and HLA-DM in IRF8 KO models. (D) Left: FACS of CD74 and H2-DM in the IRF8 KO A20 lymphoma model “rescued” with IRF8 WT or missense or nonsense mutants (top and bottom). Right: FACS of CD74 and H2-DM in the IRF8 KO 2PK-3 lymphoma model “rescued” with IRF8 WT or missense and nonsense mutants (top and bottom). (E) Top: ChIP-qPCR of IRF8 binding to the indicated promot- ers – controls are IgG pull down, and a genomic region without a predicted IRF8 binding site (neg ctrl). Bottom: ChIP-qPCR of IRF8 WT, N87Y, or I424T binding to the Cd74, H2-Dm, Ciita, or H2-Aa promoters. (F) Top: WB of CD74 in 2PK-3 and A20 CD74-KO models. Bottom: IL-2 levels and % of CD4/CD25+ cells in IRF8/CD74 WT, IRF8 KO, or CD74 KOs models. (G) Left to right: A20, 2PK-3, and BCL1 models of IRF8 KO with CD74 ectopic expression (ee). WB of CD74-FLAG, IL-2 levels and % of CD4/CD25+ cells in IRF8/ CD74 WT, IRF8 KO, or IRF8KO + CD74. (H) Left: WB of CD74-FLAG in IRF8 WT, N87Y, and I424T A20 models. Right: IL-2 levels and % of CD4/CD25+ cells in IRF8 WT, N87Y, and I424T (−/+ CD74 ectopic expression) models. Data are means ± SD of three biological replicates. FACS displayed as relative mean fluorescence intensity (MFI). P values are from ANOVA, with Bonferroni or Fisher’s LSD posttest, or two-sided Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Article Snippet: Antibodies used for flow cytometry were the following: antihuman HLA- DR and anti- human HLA- DM, (catalog no. 361610 and catalog no. 358004, Biolegend), anti- human CD74 (catalog no. FAB35901N, R&D Systems); anti- mouse H2I- A/I- E, (catalog no. 10763), anti- mouse CD3 (catalog no. 100206), anti- mouse CD8a (catalog no. 100712), anti- mouse CD11b (catalog no. 101208), anti- mouse Nkp46 (catalog no. 137612), anti- mouse CD195 (CCR5) (catalog no. 107011), anti- mouse CD279 (PD- 1) (catalog no. 135224), anti- mouse CD185 (CXCR5) (catalog no. 145506), anti- mouse IL- 33Rα (ST2) (catalog no. 146607), anti- mouse CD193 (CCR3) (catalog no. 144527), anti- mouse CD4 (for Treg quantification, catalog no. 116023), anti- mouse FoxP3 (catalog no. 126403), anti- mouse CD25 (catalog no. 102037), anti- mouse CD127 (IL7Rα) (catalog no. 135011), anti- mouse Fc block CD16/32 (catalog no. 101302), all from Biolegend; anti- mouse H2- DM (catalog no. 624048), anti- mouse CD4 (catalog no. 563232), anti- mouse CD25 (catalog no. 553866), anti- mouse CD183 (CXCR3) (BDB755832), all from BD Biosciences; anti- mouse CD74 (FAB7478A, R&D Systems), anti- mouse Ly- 6G/Gr1 (catalog no. 17- 9668- 82, eBioscience). anti- mouse RORgt (catalog no. BDB562607, (BD Bioscience), antimouse GATA3 (catalog no. 50604769), anti–T- BET (catalog no. 50170980), anti- Human/mouse BCL6 (catalog no. 50604576), anti- mouse CD196 (catalog no. NC1898560); anti- mouse PD- 1 (catalog no. 135224), anti- mouse PD- L1 (catalog no. 17- 5982- 82), antimouse B220 (catalog no. 103208), anti- mouse CD69 (catalog no. 104514), all from Biolegend.
Techniques: Control, ChIP-qPCR, Binding Assay, Expressing, Fluorescence