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: ( 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 promoters – 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: Membranes were blocked for 1 hour and probed with 5% nonfat dry milk with the following primary antibodies: anti-IRF8 [clone D20D8, catalog no. 5628, Cell Signaling Technology and clone E-9, catalog no. sc-365042, Santa Cruz Biotechnology)], anti-CD74 (sheep anti-mouse cd74, polyclonal antibody, catalog no. AF7478, R&D Systems), anti-PU1 (clone 9G7, catalog no. 2258, Cell Signaling Technology), anti-FLAG (clone 9A3, catalog no. 8146, Cell Signaling Technology), anti–MHC class II (clone LGII-612.14, catalog no. 68258, Cell Signaling Technology), anti-CIITA antibodies (rabbit polyclonal, catalog no. 3793, Cell Signaling Technology, and mouse monoclonal, clone 7-1H, sc-13556, Santa Cruz Biotechnology), and anti–β-actin (#A2228, Sigma-Aldrich).

Techniques: Binding Assay, Expressing, Fluorescence

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: ( 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 T regs 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 T regs and NK cells in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). ( H ) T H 1/T H 2 ratio, T H 1, T H 2, and T FH cells in the TME of IRF8 WT or mutant lymphomas (−/+ CD74 expression). ( I ) T H 1/T H 2 ratio and T FH 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: Membranes were blocked for 1 hour and probed with 5% nonfat dry milk with the following primary antibodies: anti-IRF8 [clone D20D8, catalog no. 5628, Cell Signaling Technology and clone E-9, catalog no. sc-365042, Santa Cruz Biotechnology)], anti-CD74 (sheep anti-mouse cd74, polyclonal antibody, catalog no. AF7478, R&D Systems), anti-PU1 (clone 9G7, catalog no. 2258, Cell Signaling Technology), anti-FLAG (clone 9A3, catalog no. 8146, Cell Signaling Technology), anti–MHC class II (clone LGII-612.14, catalog no. 68258, Cell Signaling Technology), anti-CIITA antibodies (rabbit polyclonal, catalog no. 3793, Cell Signaling Technology, and mouse monoclonal, clone 7-1H, sc-13556, Santa Cruz Biotechnology), and anti–β-actin (#A2228, Sigma-Aldrich).

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

Journal: Oncotarget

Article Title: Targeting CD74 in B-cell non-Hodgkin lymphoma with the antibody-drug conjugate STRO-001

doi: 10.18632/oncotarget.28341

Figure Lengend Snippet: Anti-GFP antibody SP7824 was used as negative control in the cell binding experiments. ( A ) SP7219 and STRO-001 showed similar high affinity cell binding on CHO cells over-expressing human and cynomolgus monkey CD74. ( B ) SP7219 and STRO-001 showed no binding to mouse B cell line A20. The mouse CD74 expression on A20 was confirmed by a commercial anti-mouse-CD74 antibody from R&D Systems.

Article Snippet: For the anti-mouse CD74 antibody conjugated to APC from R&D system (Cat. No. FAB7478A) which has no concentration information available, the antibody was mixed with cells at 1:1 volume ratio and then started 1:4 serial dilution.

Techniques: Negative Control, Binding Assay, Expressing

Journal: Oncotarget

Article Title: Targeting CD74 in B-cell non-Hodgkin lymphoma with the antibody-drug conjugate STRO-001

doi: 10.18632/oncotarget.28341

Figure Lengend Snippet: CD74 cell surface expression level and STRO-001 cytotoxicity in non-hodgkin lymphoma cell lines

Article Snippet: For the anti-mouse CD74 antibody conjugated to APC from R&D system (Cat. No. FAB7478A) which has no concentration information available, the antibody was mixed with cells at 1:1 volume ratio and then started 1:4 serial dilution.

Techniques: Expressing

Journal: Oncotarget

Article Title: Targeting CD74 in B-cell non-Hodgkin lymphoma with the antibody-drug conjugate STRO-001

doi: 10.18632/oncotarget.28341

Figure Lengend Snippet: ( A ) In vitro expanded primary B cells showed increased CD86, HLA-DR and CD74 expression compared to naïve B cells. ( B ) In vitro expanded B cell showed improved proliferation activity compared to naïve B cells. Cell proliferation was analyzed by flow cytometry using propidium iodide exclusion. ( C ) STRO-001 showed more potent cell killing of proliferating B cells (EC50 = 0.49 nM) than naïve B cells (EC50 = 11 nM).

Article Snippet: For the anti-mouse CD74 antibody conjugated to APC from R&D system (Cat. No. FAB7478A) which has no concentration information available, the antibody was mixed with cells at 1:1 volume ratio and then started 1:4 serial dilution.

Techniques: In Vitro, Expressing, Activity Assay, Flow Cytometry

Journal: BMC Cancer

Article Title: Prognostic role of CD74, CD10 and Ki-67 immunohistochemical expression in patients with diffuse malignant peritoneal mesothelioma: a retrospective study

doi: 10.1186/s12885-023-10871-w

Figure Lengend Snippet: Primary antibodies used for immunohistochemistry

Article Snippet: CD74 , Mouse anti-human monoclonal antibody , SPM523 , 1:100 , ZSGB-BIO, China.

Techniques: