Journal: Neuro-Oncology

Article Title: MIF-CD74 signaling drives immune modulation in medulloblastoma

doi: 10.1093/neuonc/noag020

Figure Lengend Snippet: Cell-to-cell communications within the TIME for human Group3 and Group4 MB. (A) The UMAP plot of cell type clusters show the identification of 15 distinct cell clusters from the single-cell RNA-seq data of Group3 MB samples. OPC: Oligodendrocyte Precursor Cell. APC: Astrocyte Precursor Cell. M2_1: M2 macrophages cluster 1. M2_2: M2 macrophages cluster 2. NSC: Neural stem cell. Complement-M, complement macrophage. inflam dendritic cell: inflammatory dendritic cell. (B) The violin plot of the marker gene expression for each cell cluster. (C) CellCrossTalker predicted ligand-receptor-mediated tumor-immune cell communications using scRNA-seq data from Group3 MB samples. The vertical axis represents ligands and their corresponding receptors, while the horizontal axis indicates the sender cell type associated with the ligand and the receiver cell type associated with the receptor. (D) CellCrossTalker predicted ligand-receptor-mediated communications between M2 macrophages and tumor cells, M2 macrophages and T cells, M2 macrophages and B cells, as well as M2 macrophages and myeloid cells, using scRNA-seq data from Group3 MB samples. The vertical axis represents ligands and their corresponding receptors, while the horizontal axis shows the sender cell type associated with the ligand and the receiver cell type associated with the receptor. (E) The UMAP plot of cell type clusters show the identification of 13 distinct cell clusters. Single-cell RNA-seq data from Group4 MB samples were obtained from Hendrikse et al. (F) The heatmap illustrates the expression of marker genes used to annotate each cell cluster. (G) CellCrossTalker predicted ligand-receptor-mediated cell-cell communications using scRNA-seq data from Group4 MB samples. The vertical axis represents ligands and their corresponding receptors, while the horizontal axis shows the sender cell type associated with the ligand and the receiver cell type associated with the receptor. (H) The heatmap shows the interaction strength of MIF-CD74 between various pairs of cell types, as predicted by CellCrossTalker using scRNA-seq data from Group4 MB samples. (I) CellCrossTalker predicted ligand-receptor-mediated communication co-receptors between tumor cells and immune compartment, using scRNA-seq data from Group3 and Group4 MB samples. The vertical axis represents ligands and their corresponding receptors and co-receptors, while the horizontal axis shows the sender cell type associated with the ligand and the receiver cell type associated with the receptor.

Article Snippet: In brief, tissue sections were incubated in Tris-EDTA buffer (cell conditioning 1; CC1) at 95 ̊C for 1-h to retrieve antigenicity, followed by incubation with CD74 antibody (Origene CF507339 ) at 1:500 for 1-h.

Techniques: Single Cell, RNA Sequencing, Marker, Gene Expression, Expressing

Journal: Neuro-Oncology

Article Title: MIF-CD74 signaling drives immune modulation in medulloblastoma

doi: 10.1093/neuonc/noag020

Figure Lengend Snippet: Restricted expression of CD74 and MIF in human normal tissues and MB subgroups. (A) Graph depicting the expression of CD74 (ENSG00000019582.14) in normal human tissue RNA-sequencing data obtained from the GTEx consortium. The dataset comprises 7859 samples across 31 distinct normal tissues, with sample sizes ranging from 5 to 1152 samples per tissue. Expression levels are presented as relative expression levels in transcripts per million (TPM). (B) CD74 protein expression in normal human cerebellum. Scale bar, 100 um. (C) Graph depicting the expression of MIF (ENSG00000240972.1) in normal human tissue RNA-sequencing data obtained from the GTEx consortium. The dataset comprises 7859 samples across 31 distinct normal tissues, with sample sizes ranging from 5 to 1152 samples per tissue. Expression levels are presented as relative expression levels in transcripts per million (TPM). (D) MIF protein expression in normal human cerebellum. Scale bar, 100 um. (E-J) Protein levels across human MB subgroups and subtypes (based on DNA methylome classification) from Ayrault cohort for the three selected proteins: CD74, CD68 and MIF. Boxplots show median (line), upper and lower quartiles (boxes), and lines extending to highest and lowest observations (whiskers). (K) CD74 immunohistochemistry staining analysis of paired human pediatric diagnostic (left) and relapse (right) MB samples. Black arrows depict CD74 positivity. Scale bar represents 100 µM. (L) Immunohistochemistry membrane staining depicting CD74 expression across a subgrouped human diagnostic MB tissue microarray. Scale bar represents 200 µM.

Article Snippet: In brief, tissue sections were incubated in Tris-EDTA buffer (cell conditioning 1; CC1) at 95 ̊C for 1-h to retrieve antigenicity, followed by incubation with CD74 antibody (Origene CF507339 ) at 1:500 for 1-h.

Techniques: Expressing, RNA Sequencing, Immunohistochemistry, Staining, Diagnostic Assay, Membrane, Microarray

Journal: Neuro-Oncology

Article Title: MIF-CD74 signaling drives immune modulation in medulloblastoma

doi: 10.1093/neuonc/noag020

Figure Lengend Snippet: Inhibition of CD74 via the lateral ventricle demonstrates immune-modulation and decreased tumor burden in primary and relapsed MB. (A) IHC of treatment-naïve primary (top row) and radiation-treated (bottom row) GTML allografts displaying H&E, CD74, and MIF expression within the tumor. Results representative of 3 independent replicates. Much of the tumor outside the cerebellum was removed for downstream RNA sequencing analyses, Scale bar, 100 µm. (B) Experimental schematic for evaluating the CD74-MIF blocking peptide C36L1. GTML primary or relapse cells expressing firefly luciferase were allografted into the cerebellum of FVBNRJ mice. C36L1 was administered either intraperitoneally or locoregionally through the lateral ventricle at time of engraftment and 1-week after, C36L1 vehicle was used as a control. Bioluminescence imaging was conducted to monitor tumor engraftment, progression, and/or regression up to 14 days post-therapy. At the endpoint, the tumor and CNS were harvested and assessed for immune infiltration and tumor burden. (C) Growth rate of mice bearing primary GTML allografts ( n = 3-4 per group) treated with vehicle control (black), C36L1 peptide through the lateral ventricle (red), or C36L1 peptide intraperitoneally (blue) was determined by calculating the slope of tumor growth between day 7 and 14 (endpoint). Line represents mean growth rate with individual points representing individual mice. Points below the dashed line indicate tumor regression. Statistical significance was calculated by two-way ANOVA with Tukey’s post-test. (D) UMAP projection of flow cytometry analysis of tumor/cerebellum of mice treated vehicle control or the C36L1 peptide via the lateral ventricle. The left panel display cells colored by experimental group (Control - black, C36L1 delivered via lateral ventricle - red, C36L1 delivered intraperitoneally - blue), highlighting the distribution and intensity target expression across different cell populations within the tumor and cerebellum of treated and control mice. The visualization provides insights into the immune cell infiltration and its association with the treatment groups. (E) UMAP projection of flow cytometry results, colored by the expression of CD74. (F) UMAP projection of flow cytometry results, colored by the expression of MHCII. (G) UMAP projection of flow cytometry results, colored by the expression of CD11b. (H) IHC of control (top row) and peptide-treated (bottom row) GTML primary tumor allografts displaying H&E, CD3, F4/80 and CD74 within the tumor. Results representative of 3 independent replicates, Scale bar, 100 um. (I) Growth rate of mice bearing recurrent GTML allografts ( n = 5 per group) treated with vehicle control (black) or C36L1 peptide through the lateral ventricle (blue) was calculated by calculating the slope of tumor growth between day 7 and 14 (endpoint). Line represents mean growth rate with individual points representing individual mice. Points below the dashed line indicate tumor regression. Statistical significance was calculated by two-way ANOVA with Tukey’s post-test. (J) Bar chart to illustrate the proportion of tumor-associated immune cells identified as microglia. A higher proportion of microglia is observed in the TME of CD36L1 peptide treatment versus scrambled control. (K) Bar chart to illustrate the level of CD74 expression in the TME of mice treated with CD36L1 versus scrambled control. (L) Bar charts displaying the proportion of CD38+ cells in the microglia population. Statistical analysis was performed using a two-way ANOVA with Tukey’s post-test to compare the groups. Error bars represent the SD. Significant differences between groups are indicated by * P < .05, ** P < .01, and *** P < .001.

Article Snippet: In brief, tissue sections were incubated in Tris-EDTA buffer (cell conditioning 1; CC1) at 95 ̊C for 1-h to retrieve antigenicity, followed by incubation with CD74 antibody (Origene CF507339 ) at 1:500 for 1-h.

Techniques: Inhibition, Expressing, RNA Sequencing, Blocking Assay, Luciferase, Control, Imaging, Flow Cytometry

Journal: Scientific Reports

Article Title: Relationship between elevated soluble CD74 and severity of experimental and clinical ALI/ARDS

doi: 10.1038/srep30067

Figure Lengend Snippet: Quantitative real-time PCR revealed a significant up-regulation of CD74 mRNA at 6, 12, 24 hrs in lipopolysaccharide induced lung injury ( A ) and cecal ligation and puncture induced lung injury ( B ). Quantitative real-time PCR data are representative of experiments performed in triplicate. n = 5 in each group. Data are presented as mean ± SEM, # p < 0.05 and **, ## p < 0.01 compared to control with Dunnett- t test after ANOVA for multiple comparisons.

Article Snippet: The anti-CD74 antibody (Ii) for western blotting, dot blotting and immunohistochemistry was purchased from BD Pharmingen (San Diego, CA, USA), and anti-CD74 antibodies (catalog number: AF7478 and MAB7478) for enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction, Ligation, Control

Journal: Scientific Reports

Article Title: Relationship between elevated soluble CD74 and severity of experimental and clinical ALI/ARDS

doi: 10.1038/srep30067

Figure Lengend Snippet: Immunoblotting using 30 μg lung proteins revealed marked up-regulation of CD74 at certain time post injury in lungs from lipopolysaccharide and cecal ligation and puncture induced acute lung injury models compared to control. Two bands of 31 and 41 kDa corresponding to two different isoforms of CD74 (p31 and p41) are shown ( A,B ). The amount of p31 expression was much higher than p41. Relative protein levels were quantified by densitometry and expressed as optical density ratio ( C–F ) with GAPDH serving as internal standards. Immunoblotting data are representative of experiments performed in triplicate and statistical differences are noted (*p < 0.05, **, ## p < 0.01 compared to control with Dunnett- t test after ANOVA for multiple comparisons).

Article Snippet: The anti-CD74 antibody (Ii) for western blotting, dot blotting and immunohistochemistry was purchased from BD Pharmingen (San Diego, CA, USA), and anti-CD74 antibodies (catalog number: AF7478 and MAB7478) for enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Ligation, Control, Expressing

Journal: Scientific Reports

Article Title: Relationship between elevated soluble CD74 and severity of experimental and clinical ALI/ARDS

doi: 10.1038/srep30067

Figure Lengend Snippet: Immunohistochemical examination was performed for CD74 in control mouse lung tissue and lipopolysaccharide induced acute lung injury model (6 h, 12 h, 24 h post lipopolysaccharide instillation). CD74 is indicated by brown staining and nuclei are counterstained in blue. Limited CD74 staining was observed in control mouse lung tissue. Increased CD74 expression in lung tissue of acute lung injury was observed. Some CD74 staining was localized on cell membrane of nucleated cells (arrow). Left magnification ×400, Right magnification ×800.

Article Snippet: The anti-CD74 antibody (Ii) for western blotting, dot blotting and immunohistochemistry was purchased from BD Pharmingen (San Diego, CA, USA), and anti-CD74 antibodies (catalog number: AF7478 and MAB7478) for enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunohistochemical staining, Control, Staining, Expressing, Membrane

Journal: Scientific Reports

Article Title: Relationship between elevated soluble CD74 and severity of experimental and clinical ALI/ARDS

doi: 10.1038/srep30067

Figure Lengend Snippet: Immunofluorescence examination was performed for CD74 in control mouse lung tissue and lipopolysaccharide induced acute lung injury model. Increased surface CD74 expression (green) in lung tissue of acute lung injury was observed compared with control ( A,B ). Surface CD74-positive cells were observed mainly on the alveolar septa, and colocalize with F4/80-positive cells (macrophage cells; red) ( B ). Arrows, positive staining of macrophage cells; arrowhead, positive type II alveolar epithelial cells. Scale bar represents 100 μm.

Article Snippet: The anti-CD74 antibody (Ii) for western blotting, dot blotting and immunohistochemistry was purchased from BD Pharmingen (San Diego, CA, USA), and anti-CD74 antibodies (catalog number: AF7478 and MAB7478) for enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Control, Expressing, Staining

Journal: Scientific Reports

Article Title: Relationship between elevated soluble CD74 and severity of experimental and clinical ALI/ARDS

doi: 10.1038/srep30067

Figure Lengend Snippet: RAW264.7 cells were treated with different concentrations of MIF (0, 10, 50, 100, 200 ng/ml) for 24 hrs. In experiment of ISO-1 treatment, RAW264.7 cells were pre-treated with 100 μg/ml ISO-1 for 30 min, following stimulated with 100 ng/ml MIF for 24 hrs. After stimulation, immunofluorescence examination was performed for CD74. Surface CD74 expression was observed to increase in a concentration-dependent manner.

Article Snippet: The anti-CD74 antibody (Ii) for western blotting, dot blotting and immunohistochemistry was purchased from BD Pharmingen (San Diego, CA, USA), and anti-CD74 antibodies (catalog number: AF7478 and MAB7478) for enzyme-linked immunosorbent assay (ELISA) and immunofluorescence were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Immunofluorescence, Expressing, Concentration Assay

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

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

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: