Journal: medRxiv

Article Title: An intragenic duplication within SIRPβ1 shows a dual effect over Alzheimer’s disease cognitive decline altering the microglial response

doi: 10.1101/2022.11.19.22282342

Figure Lengend Snippet: Effect of SIRPβ1 variant on hippocampal microglia of AD patients. a , IBA1 and b , TMEM119 immunostaining of illustrative Wt/Wt and Wt/Dup from both Braak II and Braak V-VI subjects. c , confocal imaging of three Braak V-VI wild type patients and three Wt/Dup Braak V-VI subjects. A total of 29 and 47 plaques were analyzed respectively. Box plots in d and e show the IBA1 + and TMEM119 + area depending on the SIRPβ1 genotype. f , number of IBA1 + cells per plaque depending on the SIRPβ1 genotype. g , different average expression ( + SE) of TREM2 . h and i represent the average gene set scores ( + SE) estimated for microglial activation and homeostasis, respectively

Article Snippet: For activation or homeostatic set score, the expression of CD45 (Hs04189704_m1),d45 CSFsf1 (Hs00174164_m1), CDd74 (Hs00269961_m1) and PUu.1 (Hs02786711_m1) or CX3CRx3cr1 (Hs01922583_s1), P2RYry12 (Hs01881698_m1) and TMEMmem119 (Hs01938722_u1) were determined, respectively.

Techniques: Variant Assay, Immunostaining, Imaging, Expressing, Activation Assay

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: Primer/probe information

Article Snippet: Cd74 , 25599 , CD74 molecule, major histocompatibility complex, class II invariant chain , Rn00565062_m1.

Techniques: Immunopeptidomics, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: Antibody information

Article Snippet: Cd74 , 25599 , CD74 molecule, major histocompatibility complex, class II invariant chain , Rn00565062_m1.

Techniques: Binding Assay

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: Age-related induction of MHC II and inflammation-response transcripts . Bioinformatic analysis of transcriptomic expression data identified significant upregulation the MHC II antigen presentation pathway and associated inflammatory signaling factors in hippocampal synaptosomes derived from both cognitively intact and cognitively impaired aged rats compared to adults (ANOVA with SNK post hoc testing, p < 0.05). Notably, no differences were observed between aged intact and aged impaired rats, which clustered separately from adults but did not cluster by cognitive status. Log-scaled gene expression is presented relative to the adult group mean per transcript (green: decreased; black: unchanged; red: increased).

Article Snippet: Cd74 , 25599 , CD74 molecule, major histocompatibility complex, class II invariant chain , Rn00565062_m1.

Techniques: Expressing, Immunopeptidomics, Derivative Assay, Gene Expression

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: qPCR confirmation of age-related induction of MHC II pathway component genes

Article Snippet: Cd74 , 25599 , CD74 molecule, major histocompatibility complex, class II invariant chain , Rn00565062_m1.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: Gene expression levels of age and cognitive status groups, presented as group mean (% of adult mean) ± SEM

Article Snippet: Cd74 , 25599 , CD74 molecule, major histocompatibility complex, class II invariant chain , Rn00565062_m1.

Techniques: Gene Expression

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: Activation of hippocampal microglia with aging . Immunohistochemical co-localization of the microglia-specific marker Iba1 and the activation-specific marker CD74 (MHC II invariant chain) identified an increase in activated microglia in the aged hippocampus regardless of cognitive status. In adults, Iba1 + microglia were distributed throughout hippocampal subregions, with a greater population evident in CA3 than in CA1 or DG. A small fraction of microglia exhibited mild activation indicated by low levels of CD74 immunoreactivity. In both aged cognitively intact and aged cognitively impaired rats, a marked increase in the number of Iba1 + /CD74 + microglia was observed in all three subregions, in the form of both mildly-activated and moderately-activated microglia. Activated microglia were associated with both synaptic and cell body-containing hippocampal strata. Blue: Hoechst; Red: Iba1; Green: CD74; Orange: Iba1/weak CD74 co-expression (mild activation); Yellow: Iba1/high CD74 co-expression (moderate activation).

Article Snippet: Cd74 , 25599 , CD74 molecule, major histocompatibility complex, class II invariant chain , Rn00565062_m1.

Techniques: Activation Assay, Immunohistochemical staining, Marker, Expressing

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: Age-related increase in activated microglial populations . (A) High-magnification visualization of resting/surveilling, or non-activated, and activated microglia reveals differences in both immunoreactivity and morphology with aging. Non-activated microglia expressed Iba1 but not CD74, and had long, thin, branched processes. Microglia undergoing mild activation displayed increased Iba1 immunoreactivity, weak CD74 immunoreactivity, and enlarged somata, while moderately activated microglia demonstrated increased Iba1 immunoreactivity, intense CD74 immunoreactivity, enlarged somata, thickened proximal processes, and retracted distal processes. Notably, activated microglia maintained a ramified rather than ameboid morphology, indicating that microglial activation had not progressed to a reactive, phagocytic phenotype. (B) Quantitation of activated microglia (calculated as the ratio of Iba1+/CD74 + to total Iba1 + cells) revealed significant increases in all three hippocampal subregions studied. In adult rats, a small fraction of total microglia were mildly or moderately activated in CA3, with far fewer activated microglia evident in CA1 and DG. The percentage of microglia undergoing mild or moderate activation was significantly increased, by several fold, throughout the hippocampus of both aged cognitively intact and aged cognitively impaired rats compared to adults. No differences between the degree of microglial activation ( i.e ., mild versus moderate) were observed between aged intact and aged impaired rats in CA1, CA3 or DG. Increased numbers of activated microglia in aged rats were not due to proliferation/infiltration, as total populations of microglia (Iba1 + cells) were not different between groups. ***p < 0.001, one-way ANOVA with SNK post hoc testing.

Article Snippet: Cd74 , 25599 , CD74 molecule, major histocompatibility complex, class II invariant chain , Rn00565062_m1.

Techniques: Activation Assay, Quantitation 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: ( 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