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: The Journal of Biological Chemistry

Article Title: Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

doi: 10.1074/jbc.RA119.009716

Figure Lengend Snippet: AtMDLs share homology with human MIF in the MIF receptor–binding sites, bind to CD74, and activate CXCR4-mediated signaling in a yeast-based reporter system. A, multiple sequence alignment of the AtMDLs, HsMIF, and human CXCL12. Amino acid sequences of AtMDL1 (identifier Q9LU69), AtMDL2 (identifier Q9M011), AtMDL3 (identifier Q8LG92), and HsMIF (identifier P14174) were retrieved from the UniProt database and aligned by ClustalW using standard parameters in the Jalview multiple sequence alignment editor desktop application. The amino acid residues contributing to the site I and II binding interface between HsMIF and CXCR4 (41) or CXCL12 and CXCR4 (90, 91), the binding sites between HsMIF and CD74, and the predicted corresponding regions in the AtMDLs are indicated. Determinants of HsMIF contributing to CXCR2 binding, although not further examined in this study, are indicated for comparison. The degree of homology/identity of the MIF, CXCL12, or AtMDL residues in these regions is highlighted by the following color score: blue, hydrophobic; red, positively charged; magenta, negatively charged; green, polar; pink, cysteine; orange, glycine; yellow, proline; cyan, aromatic; white, unconserved. B, comparison of the in vitro binding capacity between HsMIF–6xHis and MBP–sCD74 with that of the three His-tagged AtMDLs. Binding was measured by an ELISA-type plate-binding assay. BSA, blank PBS buffer (control), and MBP alone served as negative controls as indicated to account for nonspecific binding effects. Wells were coated with BSA (2% w/v), 500 nm HsMIF, and AtMDL1–6xHis, AtMDL2–6xHis, and AtMDL3–6xHis (500 nm), followed by binding of MBP or MBP–sCD74 (500 nm). After signal development, absorbance at 450 nm was measured, and the signals were normalized by setting the absorbance of HsMIF as 1. C, curve for binding of MBP–sCD74 and AtMDL3 using increasing concentrations of MBP–sCD74 as indicated. The data in B and C are displayed as means ± S.D. (n = 3); (scatter plot with white circles indicates individual data points); ns, not significant; ***, p < 0.001; **, p < 0.01. D, CXCR4-mediated signaling in a yeast-based reporter system. In this assay, the Ste2 GPCR of the pheromone-response pathway of S. cerevisiae was substituted by the human CXCR4 receptor. Ligand binding to CXCR4 triggers signaling and expression of the lacZ gene, as assessed by β-gal activity. The concentrations of native (untagged) HsMIF, HsMIF–6xHis, and His-tagged AtMDLs were 20 μm each. The concentration of human CXCL12 was equal to 2 μm. Reporter activity is given as relative luminescence, normalized to the untreated control (Ctrl). Values shown represent means ± S.D. from three independent experiments, in which the activity of each was assessed in technical duplicates (scatter plot with white circles indicates individual data points). Statistical analysis was performed using one-way ANOVA and post-hoc Tukey's HSD test with multiple comparisons. Different letters above the bars denote a statistically significant difference between groups (p < 0.05), and groups showing the same letters are not statistically significantly different from each other.

Article Snippet: After washing, wells were incubated with 100 μl of a rabbit anti-CD74 pAb solution (1:2500 dilution in PBS) (Sinobiological, Vienna, Austria) for 30 min at room temperature.

Techniques: Binding Assay, Sequencing, In Vitro, Enzyme-linked Immunosorbent Assay, Ligand Binding Assay, Expressing, Activity Assay, Concentration Assay

Journal: Cancer cell

Article Title: Cathepsin S Regulates Antigen Processing and T Cell Activity in Non-Hodgkin Lymphoma.

doi: 10.1016/j.ccell.2020.03.016

Figure Lengend Snippet: Figure 1. Functional Characterization of CTSS Y132D Mutation in FL (A) Distribution of CTSS mutations in 299 FL patients. (B) The total number of FL and DLBCL patients and the frequency of the Y132D mutation in the reported studies. (C) Stacked barplot indicating the percentage of patients with CTSS WT and mutated (Y132D/N). (D) Schematic representation of tyrosine (Y) 132 amino acid localization on CTSS binding site, and tridimensional structure of the proteins, colored according to the amino acid electrostatic potential. (E) Western blot of recombinant of CTSS-His WT and mutated CTSS-His Y132D using an anti-His-tag antibody. M, marker. (F) Quantification of western blot signals of CTSS mature protein normalized to the pro-CTSS signal (n = 3). (G) Graphical representation of CD74 sequence between amino acids 89 and 121 and fluorescence resonance energy transfer (FRET) experimental design. The CTSS substrate is highlighted in blue, the arrow indicates the exact cleavage site, and the CLIP sequence is shown in green. (H) Representative FRET experiment with recombinant CTSS-His-WT and CTSS-His-Y132D. Signal Intensity normalized to the background (n = 3). (I) Quantification FRET emission normalized to CTSS WT signals (n = 6). The p value was calculated using a paired t test. (F, H, and I) Data are presented as mean ± standard deviation. See also Figure S1 and Tables S1, S2, and S3.

Article Snippet: After blocking in 5% milk (Applichem, cat #A0830) in PBS-0.1%Tween (Fisher scientific, cat #BP337), membranes were incubated overnight in 5% milk in PBS-Tween with the following primary antibodies: goat anti-human Cathepsin S (RD, cat #AF1183, 1:2000), Cathepsin B (D1C7Y) XP Rabbit (Cell signaling mAb #31718), rabbit anti-mouse Cathepsin S (Sino Biological, cat #50769-R054, 1:1000), mouse anti-human CD74 (PIN.1, StressMarq, cat #SMC-116, diluted 1:1000), rat-anti-mouse CD74 (clone ln-1, BD biosciences, cat #555317, 1:1000), rabbit anti-his-Tag (Cell Signaling, cat #D3I10, dilute 1:1000), mouse anti-human alpha-Tubulin (clone B-5-1-2, Sigma Aldrich, cat#T6072, diluted 1:10000), anti-beta actin (clone 8H10D10, ThermoFisher Scientific, cat# MA5-15452, 1:1000).

Techniques: Functional Assay, Mutagenesis, Binding Assay, Western Blot, Recombinant, Marker, Sequencing, Förster Resonance Energy Transfer, Standard Deviation

Journal: Journal of Neuroinflammation

Article Title: D-dopachrome tautomerase activates COX2/PGE 2 pathway of astrocytes to mediate inflammation following spinal cord injury

doi: 10.1186/s12974-021-02186-z

Figure Lengend Snippet: Interference of CD74 receptor decreased D-DT-induced astrocyte production of PGE 2 . a–b Immunofluorescence showed colocalization of CD74 with S100β-positive astrocytes in the spinal cord. Rectangle indicates region magnified. Arrowheads indicate the positive signals. c Western blot analysis of CD74, COX2, and mPGES-1 following siRNA2 knockdown of CD74 receptor for 48 h, prior to stimulation with 1 μg/ml recombinant D-DT protein for 24 h. d–f Quantification data as shown in ( c ). Quantities were normalized to endogenous β-actin. g , h ELISA determination of PGE 2 in supernatant and lysate following astrocytes transfected with CD74 siRNA2 for 48 h, followed by treatment with 1 μg/ml recombinant D-DT for 24 h. Experiments were performed in triplicates. Error bars represent the standard deviation (* P < 0.05)

Article Snippet: The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, and then an overnight incubation at 4 °C with primary antibodies: D-DT (1:500, Abcam); MIF (1:500, Abcam); COX1 (1:1000, CST); COX2 (1:1000, Cayman); mPGES-1 (1:200, Cayman); mPGES-2 (1:200, Cayman); cPGES (1:1000, Abcam); CD74 (1:1000, Biorbyt).

Techniques: Immunofluorescence, Western Blot, Knockdown, Recombinant, Enzyme-linked Immunosorbent Assay, Transfection, Standard Deviation