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: ChIP-qPCR, 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: bioRxiv

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

doi: 10.1101/2023.10.14.560755

Figure Lengend Snippet: (A) FACS analysis of H2-IA/IE (human HLA-DR) in the cell surface of B cell lymphoma models of IRF8 KO – representative histograms shown to right. (B) FACS analysis of intra-cellular (IC) CD74 in B cell lymphoma models of IRF8 KO – representative histograms shown to right. (C) FACS analysis of intra-cellular (IC) H2-DM (human HLA-DM) in B cell lymphoma models of IRF8 KO – representative histograms shown to right. (D) left panels - FACS analysis of intra-cellular (IC) CD74 and H2-DM in the IRF8 KO A20 B cell lymphoma model “rescued” with stable expression of IRF8 WT or mutants; right panels - FACS analysis of intra-cellular (IC) CD74 and H2-DM in the IRF8 KO 2PK-3 B cell lymphoma model “rescued” with stable expression of IRF8 WT or mutants - representative histograms shown at the bottom. (E) FACS analysis of intra-cellular (IC) CD74 and HLA-DM in the human DLBCL cell line stably expressing and empty vector (MSCV), IRF8 WT or mutants. (F) FACS analysis of CLIP expression in the cell surface of SU-DHL4 (IRF8 WT vs. KO) and Toledo (IRF8 WT vs mutant). (G) left , WB analysis of CD74 KO models in mouse B cell lymphoma 2PK-3 and A20; right, top and bottom , IL-2 levels in the conditioned media and percentage of CD4/CD25+ expression in DO-11.10 cells co-cultured with IRF8/CD74 WT, IRF8 KO or CD74 KO APC models. (H) left, WB analysis of CD74 with IRF8/CD74 WT, IRF8 KO or IRF8KO+CD74-FLAG in A20 models with anti-FLAG or CD74 antibodies; right, IL-2 levels in the conditioned media and percentage of CD4/CD25+ expression in DO-11.10 cells co-cultured with IRF8/CD74 WT, IRF8 KO or IRF8KO+CD74 KO APC models. (I) left , WB analysis of ectopically expressed CD74-FLAG in the IRF8 WT, IRF8 N87Y and IRF8 I424T mouse B cell lymphoma A20 models; right , IL-2 levels in the conditioned media and percentage of CD4/CD25+ expression in DO-11.10 cells co-cultured with IRF8 WTT, IRF8 N87Y and IRF8 I424T (with or without CD74 ectopic expression) APC models. In all panels data are mean ± SD of three biological replicates. FACS analysis is displayed as relative mean fluorescence intensity (MFI). P values are from ANOVA, with Bonferroni or Fisher’s LSD post-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: Anti-human HLA-DR and Anti-human HLA-DM, (Cat# 361610 and Cat# 358004, Biolegend), Anti-human CD74 (cat# FAB35901N, R&D Systems).

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Mutagenesis, Cell Culture, Fluorescence

Journal: bioRxiv

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

doi: 10.1101/2023.10.14.560755

Figure Lengend Snippet: (A) left to right , growth curve (volume) of lymphomas expressing IRF8 WT, N87Y or I424T. Data are mean ± SD of four independent cohorts; p values are from Mann-Whitney test. (B) left to right , FACS-based quantification of CD3, CD4 and CD8 T cells in the microenvironment of IRF8 WT or mutant lymphomas. (C) left to right , FACS-based quantification of T- regs and NK cells in the microenvironment of IRF8 WT or mutant lymphomas. In B) and C) data are mean ± SD of four independent cohorts; p values are from one-way ANOVA with Fisher’s LSD post-test. (D) growth curve (volume) of lymphomas expressing IRF8 WT, IRF8 N87Y or IRF8 I424T (with or without CD74 ectopic expression). Data are mean ± SD of two independent cohorts (n=84 mice); p values are from two-sided Student’s t-test – asterisk at the top is for IRF8 WT vs. N87Y or I424T, asterisk on the side is for IRF8 N87Y vs N87Y+CD74 and IRF8 I424T vs. I424T+CD74. (E) left to right , FACS-based quantification of CD3, CD4 and CD8, T-regs and NK cells in the microenvironment of lymphomas expressing IRF8 WT, IRF8 N87Y or IRF8 I424T (with or without CD74 ectopic expression). (F) left to right , Th1/Th2 ratio calculated from FACS-based quantification of Th1 and Th2 cells in the microenvironment of lymphomas expressing IRF8 WT, IRF8 N87Y or IRF8 I424T (with or without CD74 ectopic expression). (G) FACS-based quantification of Tfh cells in the microenvironment of lymphomas expressing IRF8 WT, IRF8 N87Y or IRF8 I424T (with or without CD74 ectopic expression); in E), F) and G), data are mean ± SD of multiple independent cohorts (n indicated in the figure) and the p values are from two-sided Student’s t-test. (H) left to right , growth curve (volume) of lymphomas models expressing IRF8 WT or IRF8 N87Y grown in mice treated with control antibody or anti-PDL1 antibody; CD4 and CD8 quantification in IRF8 N87Y lymphomas treated with ctrl control or anti-PDL1 antibody; tumor volume data are mean ± SEM of 40 mice, CD4 and CD8 data are mean ± SD of 14 lymphomas. P value is from two-sided Student’s t-test for IRF8 N87Y lymphomas treated with control vs anti-PDL1 antibody. For all panels, *(p≤0.05), **(p ≤ 0.01), ***(p ≤ 0.001), **** (p ≤ 0.0001).

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).

Techniques: Expressing, MANN-WHITNEY, Mutagenesis, Control

Journal: The Journal of investigative dermatology

Article Title: Cell Surface CD74-MIF Interactions Drive Melanoma Survival in Response to Interferon-γ

doi: 10.1038/jid.2015.204

Figure Lengend Snippet: (a) Five melanoma cell lines were analyzed by Western blot analysis for CD74 expression. (b) Representative immunohistochemical staining intensities: score 0 (negative), 1 (weak), 2 (medium) and 3 (strong). Scale bar = 100 µm. (c) TMA analysis for CD74 expression during melanoma progression. CD74 staining was scored for the percentage (upper) and intensity (lower) of positive tumor cells. n indicates the number of tissue cores in each group. LN, lymph node. Both percentage and intensity score of each melanoma group were significantly higher than those for nevus group ( P < 0.0001).

Article Snippet: Goat polyclonal anti-human actin (I-19; Santa Cruz, Carlsbad, CA), mouse monoclonal anti-human CD74 (PIN.1; Novus Biologicals, Littleton, CO), mouse monoclonal anti-human BCL-2 (124; GeneTex, San Antonio, TX), goat polyclonal anti-human MIF (R&D Systems, Minneapolis, MN) and rabbit monoclonal anti-human total AKT, phosphorylated AKT Ser473 (Cell Signaling Technology, Danvers, MA) antibodies were used as primary antibodies; horseradish peroxidase-labeled anti-mouse, anti-rabbit and anti-goat antibodies (1:1000; DAKO) were used as secondary antibodies.

Techniques: Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: The Journal of investigative dermatology

Article Title: Cell Surface CD74-MIF Interactions Drive Melanoma Survival in Response to Interferon-γ

doi: 10.1038/jid.2015.204

Figure Lengend Snippet: (a, b and c) Melanoma cell lines were treated with 0, 100 or 500 IU/ml IFN-γ for 48 hours and then assessed for CD74 expression. (a) Western blot analysis for total CD74 protein. Numbers above each band indicate relative CD74 expression levels. (b) Flow cytometric analysis for cell surface CD74. Histogram: black, isotype control; red, untreated cells stained with FITC-CD74 antibody; green, 100 IU/ml IFN-γ–treated cells with FITC-CD74 antibody; blue, 500 IU/ml IFN-γ–treated cells with FITC-CD74 antibody. (c) Immunofluorescence confocal microscopic analysis of cell surface CD74. Green (Alexa-488), CD74. Blue (DAPI), nucleus. A375 and SB2 cells showed slight expression under normal conditions (arrows). Scale bar = 30 µm. (d) Representative images of weak (left panels, score 1), medium (middle panels, score 2) and strong CD74 staining (right panels, score 3). Upper panel, Scale bar = 1 mm.; lower panel, Scale bar = 250 µm. Corresponding plasma IFN-γ levels are listed. (e) The association between CD74 staining at the tumor site and plasma IFN-γ levels in 55 patients with melanoma. Red bars, means. Error bars, standard deviations. ** P < 0.01, *** P < 0.001.

Article Snippet: Goat polyclonal anti-human actin (I-19; Santa Cruz, Carlsbad, CA), mouse monoclonal anti-human CD74 (PIN.1; Novus Biologicals, Littleton, CO), mouse monoclonal anti-human BCL-2 (124; GeneTex, San Antonio, TX), goat polyclonal anti-human MIF (R&D Systems, Minneapolis, MN) and rabbit monoclonal anti-human total AKT, phosphorylated AKT Ser473 (Cell Signaling Technology, Danvers, MA) antibodies were used as primary antibodies; horseradish peroxidase-labeled anti-mouse, anti-rabbit and anti-goat antibodies (1:1000; DAKO) were used as secondary antibodies.

Techniques: Expressing, Western Blot, Control, Staining, Immunofluorescence, Clinical Proteomics

Journal: The Journal of investigative dermatology

Article Title: Cell Surface CD74-MIF Interactions Drive Melanoma Survival in Response to Interferon-γ

doi: 10.1038/jid.2015.204

Figure Lengend Snippet: (a and b) Cells were treated with ISO-1 for 48 hours and then analyzed by Western blot analysis for AKT Ser473 phosphorylation (a) and BCL-2 expression (b). Numbers above each band indicate the relative level of phosphorylated AKT Ser473 (pAKT) to total AKT (AKT) or BCL-2 to actin. (c, d and e) mRNA expression levels of BCL-2 (c), IL-6 (d) and IL-8 (e) in melanoma cells treated with ISO-1 for 24 hours were measured using qRT-PCR. (f) CD74 knockdown A375 cells were analyzed for mRNA expression of the indicated genes by qRT-PCR. shNT, non-target shRNA. * P < 0.05, ** P < 0.01.

Article Snippet: Goat polyclonal anti-human actin (I-19; Santa Cruz, Carlsbad, CA), mouse monoclonal anti-human CD74 (PIN.1; Novus Biologicals, Littleton, CO), mouse monoclonal anti-human BCL-2 (124; GeneTex, San Antonio, TX), goat polyclonal anti-human MIF (R&D Systems, Minneapolis, MN) and rabbit monoclonal anti-human total AKT, phosphorylated AKT Ser473 (Cell Signaling Technology, Danvers, MA) antibodies were used as primary antibodies; horseradish peroxidase-labeled anti-mouse, anti-rabbit and anti-goat antibodies (1:1000; DAKO) were used as secondary antibodies.

Techniques: Western Blot, Phospho-proteomics, Expressing, Quantitative RT-PCR, Knockdown, shRNA

Journal: The Journal of investigative dermatology

Article Title: Cell Surface CD74-MIF Interactions Drive Melanoma Survival in Response to Interferon-γ

doi: 10.1038/jid.2015.204

Figure Lengend Snippet: Cell surface CD74-negative MeWo cells were subcutaneously injected into the flank of SCID Beige mice. Six days after cell injection, mice were randomly assigned to treatment groups (6 mice per group) and started on daily intraperitoneal treatment with ISO-1 (500 µg/mouse) and/or IFN-γ (1000 IU/mouse). ISO-1 and IFN-γ were administered for 21 days, and tumor growth was monitored. (a) Fold changes of tumor size. (b) Tumor weight on day 21. Error bars, standard deviations. * P < 0.05. (c) Representative mouse from each group (day 21). (d) Immunohistochemical staining for CD74 (upper) and MIF (lower) on day 21. Scale bar = 100 µm. ; AEC chromogen counterstained with hematoxylin.

Article Snippet: Goat polyclonal anti-human actin (I-19; Santa Cruz, Carlsbad, CA), mouse monoclonal anti-human CD74 (PIN.1; Novus Biologicals, Littleton, CO), mouse monoclonal anti-human BCL-2 (124; GeneTex, San Antonio, TX), goat polyclonal anti-human MIF (R&D Systems, Minneapolis, MN) and rabbit monoclonal anti-human total AKT, phosphorylated AKT Ser473 (Cell Signaling Technology, Danvers, MA) antibodies were used as primary antibodies; horseradish peroxidase-labeled anti-mouse, anti-rabbit and anti-goat antibodies (1:1000; DAKO) were used as secondary antibodies.

Techniques: Injection, Immunohistochemical staining, Staining