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anti human cd74  (R&D Systems)


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    R&D Systems anti human cd74
    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 <t>CD74</t> 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.
    Anti Human Cd74, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+human+cd74/Human+CD74+Alexa+Fluor%C2%AE+700-conjugated+Antibody/pm38996030-360-24-30
    Average 92 stars, based on 2 article reviews
    anti human cd74 - by Bioz Stars, 2026-09
    92/100 stars

    Images

    1) Product Images from "IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHCII complexes."

    Article Title: IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHCII complexes.

    Journal: Science advances

    doi: 10.1126/sciadv.adk2091

    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.
    Figure Legend 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.

    Techniques Used: Control, ChIP-qPCR, Binding Assay, Expressing, Fluorescence

    Fig. 6. IRF8 effects on B cell lymphoma aggressiveness and immune microenvironment. (A) Growth curve of lymphomas expressing IRF8 WT, N87Y, Q392X, or I424T. (B) FACS-based quantification of CD3, CD4 and CD8 T cells in the TME of IRF8 WT or mutant lymphomas. (C) FACS-based quantification of Tregs and NK cells in the TME of IRF8 WT or mutant lymphomas. (D) IHC-based quantification of T cell infiltrate in B cell lymphomas expressing IRF8 WT, N87Y, or I424T. Representative staining (B220, pink; CD3, brown) is shown to the right, scale bar is displayed. (E) Growth curve of lymphomas expressing IRF8 WT, IRF8 N87Y or IRF8 I424T (−/+ CD74 expression). (F) FACS- based quantification of CD3, CD4 and CD8 in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (G) FACS-based quantification of Tregs and NK cells in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (H) TH1/TH2 ratio, TH1, TH2, and TFH cells in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (I) TH1/TH2 ratio and TFH cells in the TME of IRF8 WT, missense (N87Y) or truncating (Q392X) mutant lymphomas. (J) Growth curve of lymphomas models expressing IRF8 WT or N87Y in mice treated with control antibody or anti–PD-L1 antibody; FACS-based quantification of CD4 and CD8 in IRF8 N87Y lymphomas treated with control or anti–PD-L1 antibody. For all panels, data are means ± SD of multiple independent cohorts (n indicated in the figure). P values are from one-way ANOVA with Fisher’s LSD posttest, Mann-Whitney test, or two-sided Student’s t test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
    Figure Legend Snippet: Fig. 6. IRF8 effects on B cell lymphoma aggressiveness and immune microenvironment. (A) Growth curve of lymphomas expressing IRF8 WT, N87Y, Q392X, or I424T. (B) FACS-based quantification of CD3, CD4 and CD8 T cells in the TME of IRF8 WT or mutant lymphomas. (C) FACS-based quantification of Tregs and NK cells in the TME of IRF8 WT or mutant lymphomas. (D) IHC-based quantification of T cell infiltrate in B cell lymphomas expressing IRF8 WT, N87Y, or I424T. Representative staining (B220, pink; CD3, brown) is shown to the right, scale bar is displayed. (E) Growth curve of lymphomas expressing IRF8 WT, IRF8 N87Y or IRF8 I424T (−/+ CD74 expression). (F) FACS- based quantification of CD3, CD4 and CD8 in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (G) FACS-based quantification of Tregs and NK cells in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (H) TH1/TH2 ratio, TH1, TH2, and TFH cells in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (I) TH1/TH2 ratio and TFH cells in the TME of IRF8 WT, missense (N87Y) or truncating (Q392X) mutant lymphomas. (J) Growth curve of lymphomas models expressing IRF8 WT or N87Y in mice treated with control antibody or anti–PD-L1 antibody; FACS-based quantification of CD4 and CD8 in IRF8 N87Y lymphomas treated with control or anti–PD-L1 antibody. For all panels, data are means ± SD of multiple independent cohorts (n indicated in the figure). P values are from one-way ANOVA with Fisher’s LSD posttest, Mann-Whitney test, or two-sided Student’s t test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

    Techniques Used: Expressing, Mutagenesis, Staining, Control, MANN-WHITNEY

    Related Articles

    Flow Cytometry:

    Article Title: IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHC CII complexes
    Article Snippet: .. Antibodies used for flow cytometry were the following: Anti-human HLA-DR and Anti-human HLA-DM, (Cat# 361610 and Cat# 358004, Biolegend), Anti-human CD74 (cat# FAB35901N, R&D Systems). .. Anti-mouse H2I-A/I-E, (Cat#10763), Anti-mouse CD3 (Cat# 100206), Anti-mouse CD8a (Cat# 100712), Anti-mouse CD11b (Cat# 101208), Anti-mouse Nkp46 (Cat# 137612), Anti-mouse CD195 (CCR5)(Cat# 107011), Anti-mouse CD279 (PD-1) (Cat# 135224), Anti-mouse CD185 (CXCR5) (Cat# 145506), Anti-mouse IL-33Rα (ST2)(Cat# 146607), Anti-mouse CD193 (CCR3) (Cat# 144527), Anti-mouse CD4 (for T-regs quantification, Cat# 116023), Anti-mouse FoxP3 (Cat# 126403), Anti-mouse CD25 (Cat# 102037), Anti-mouse CD127 (IL-7Rα) (Cat# 135011), Anti-mouse Fc block CD16/32 (Cat# 101302), all from Biolegend.

    Article Title: IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHCII complexes
    Article Snippet: .. Antibodies used for flow cytometry were the following: anti-human 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 T reg quantification, catalog no. 116023), anti-mouse FoxP3 (catalog no. 126403), anti-mouse CD25 (catalog no. 102037), anti-mouse CD127 (IL-7Rα) (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), anti-mouse 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), anti-mouse B220 (catalog no. 103208), anti-mouse CD69 (catalog no. 104514), all from Biolegend. .. All flow cytometry analyses, were performed on a BD LSR II (Becton Dickinson), equipped with a BD FACSDiva software v8.0.1. (BD Bioscience), except for T H 1 cells which were analyzed in a BD FACSCelesta instrument.

    Article Title: IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHCII complexes.
    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. ..

    Blocking Assay:

    Article Title: IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHCII complexes
    Article Snippet: .. Antibodies used for flow cytometry were the following: anti-human 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 T reg quantification, catalog no. 116023), anti-mouse FoxP3 (catalog no. 126403), anti-mouse CD25 (catalog no. 102037), anti-mouse CD127 (IL-7Rα) (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), anti-mouse 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), anti-mouse B220 (catalog no. 103208), anti-mouse CD69 (catalog no. 104514), all from Biolegend. .. All flow cytometry analyses, were performed on a BD LSR II (Becton Dickinson), equipped with a BD FACSDiva software v8.0.1. (BD Bioscience), except for T H 1 cells which were analyzed in a BD FACSCelesta instrument.

    Article Title: IRF8-mutant B cell lymphoma evades immunity through a CD74-dependent deregulation of antigen processing and presentation in MHCII complexes.
    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. ..



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    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 <t>CD74</t> 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.
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    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.

    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

    Fig. 6. IRF8 effects on B cell lymphoma aggressiveness and immune microenvironment. (A) Growth curve of lymphomas expressing IRF8 WT, N87Y, Q392X, or I424T. (B) FACS-based quantification of CD3, CD4 and CD8 T cells in the TME of IRF8 WT or mutant lymphomas. (C) FACS-based quantification of Tregs and NK cells in the TME of IRF8 WT or mutant lymphomas. (D) IHC-based quantification of T cell infiltrate in B cell lymphomas expressing IRF8 WT, N87Y, or I424T. Representative staining (B220, pink; CD3, brown) is shown to the right, scale bar is displayed. (E) Growth curve of lymphomas expressing IRF8 WT, IRF8 N87Y or IRF8 I424T (−/+ CD74 expression). (F) FACS- based quantification of CD3, CD4 and CD8 in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (G) FACS-based quantification of Tregs and NK cells in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (H) TH1/TH2 ratio, TH1, TH2, and TFH cells in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (I) TH1/TH2 ratio and TFH cells in the TME of IRF8 WT, missense (N87Y) or truncating (Q392X) mutant lymphomas. (J) Growth curve of lymphomas models expressing IRF8 WT or N87Y in mice treated with control antibody or anti–PD-L1 antibody; FACS-based quantification of CD4 and CD8 in IRF8 N87Y lymphomas treated with control or anti–PD-L1 antibody. For all panels, data are means ± SD of multiple independent cohorts (n indicated in the figure). P values are from one-way ANOVA with Fisher’s LSD posttest, Mann-Whitney test, or two-sided Student’s t test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

    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. 6. IRF8 effects on B cell lymphoma aggressiveness and immune microenvironment. (A) Growth curve of lymphomas expressing IRF8 WT, N87Y, Q392X, or I424T. (B) FACS-based quantification of CD3, CD4 and CD8 T cells in the TME of IRF8 WT or mutant lymphomas. (C) FACS-based quantification of Tregs and NK cells in the TME of IRF8 WT or mutant lymphomas. (D) IHC-based quantification of T cell infiltrate in B cell lymphomas expressing IRF8 WT, N87Y, or I424T. Representative staining (B220, pink; CD3, brown) is shown to the right, scale bar is displayed. (E) Growth curve of lymphomas expressing IRF8 WT, IRF8 N87Y or IRF8 I424T (−/+ CD74 expression). (F) FACS- based quantification of CD3, CD4 and CD8 in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (G) FACS-based quantification of Tregs and NK cells in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (H) TH1/TH2 ratio, TH1, TH2, and TFH cells in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). (I) TH1/TH2 ratio and TFH cells in the TME of IRF8 WT, missense (N87Y) or truncating (Q392X) mutant lymphomas. (J) Growth curve of lymphomas models expressing IRF8 WT or N87Y in mice treated with control antibody or anti–PD-L1 antibody; FACS-based quantification of CD4 and CD8 in IRF8 N87Y lymphomas treated with control or anti–PD-L1 antibody. For all panels, data are means ± SD of multiple independent cohorts (n indicated in the figure). P values are from one-way ANOVA with Fisher’s LSD posttest, Mann-Whitney test, 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: Expressing, Mutagenesis, Staining, Control, MANN-WHITNEY