Journal: Cancer

Article Title: Single tumor cell uptake and dosimetry of technetium-99m Fab' or minute anti-CD22 in low-grade B-cell lymphoma.

doi: 10.1002/cncr.10296

Figure Lengend Snippet: FIGURE 1. These panels show the process by which a patient with indolent, unclassifiable B-cell lymphoma and low CD22 expression was investigated with immunoscintigraphy to study the uptake of 99mTc targeted by Fab anti-CD22.

Article Snippet: Estimation of CD22 Expression Efforts were made to quantify the level of CD22 antigen expression on patient lymphoma cells and on the Raji lymphoma cell line (ATCC), which is known to express CD22.

Techniques: Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: T cell-specific non-viral DNA delivery and in vivo CAR-T generation using targeted lipid nanoparticles

doi: 10.1136/jitc-2025-011759

Figure Lengend Snippet: NCtx-dual induces robust and durable in vivo CAR-T generation, tumor control and extended survival in CD34+ HSC-engrafted NCG mice. ( a ) Schematic representation of study design: NCG mice engrafted with CD34+ HSC (NCG-His) were injected intravenously with 5×10 5 luciferase-expressing Nalm6 tumor cells, followed by IP injection of 200 ng IL-7. Mice were treated intravenously with NCtx-dual or a NCtx vehicle control encapsulating eGFP mcDNA and SB100x mRNA (vehicle control) at a total nucleic acid dose of 50 µg/kg. ( b ) CD19/CD22 dual CAR mcDNA expression was assessed by flow cytometry in circulating T cells for 40 days post-NCtx administration. n=12, data are presented as mean with individual values. ( c ) Nalm6 tumor burden was monitored by BLI. ( d ) Kaplan-Meier survival analysis. n=6 (vehicle control) or n=12 (NCtx-dual). ( e ) Expression of the exhaustion marker PD-1 in CAR+ and CAR− T cell populations over time in NCtx-dual-treated mice, analyzed by flow cytometry. n=12, data represent mean±individual values. ( f ) T cell phenotype characterization (Tnaive/Tscm, Tcm, Tem, and Teff) based on CD45RA and CD62L expression in CAR+ and CAR− T cells after NCtx-dual administration. n=12, data represent mean±SD. P values were calculated using log-rank Mantel-Cox test ( b ) or two-way ANOVA, mixed effect model ( d, e ). Significance is plotted with ns for p>0.0332 and *p<0.0332. ANOVA, analysis of variance; BLI, bioluminescent imaging; CAR, chimeric antigen receptor; HSC, hematopoietic stem cell; IL-7, interleukin 7; IP, intraperitoneal; mcDNA, minicircle DNA; mRNA, messenger RNA; ns, not significant; PD-1, programmed cell death protein-1; Tcm, central memory T cell; Teff, effector T cell; Tem, effector memory T cell; Tnaive, naïve T cell; Tscm, stem cell memory T cell.

Article Snippet: CD19/CD22 dual CAR expression was detected using CD22 CAR detection reagent (Miltenyi Biotec, REA130-126-727, 1:1000) and streptavidin-APC-Cy7 (BD, REA746, 1:200).

Techniques: In Vivo, Control, Injection, Luciferase, Expressing, Flow Cytometry, Marker, Imaging

Journal: Genes & Development

Article Title: PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes

doi: 10.1101/gad.295857.117

Figure Lengend Snippet: The absence of Phf6 promotes an altered gene expression program in B-cell leukemia. ( A ) Heat map showing differentially expressed genes (fold change >4, false discovery rate [FDR] <0.05) in pairwise comparisons between Phf6 WT ( left ) and Phf6 KO ( right ) cells as determined by RNA-seq. Each column represents a replicate sample. The scale corresponds to row-wise standardized log 2 -transformed expression values for each gene. ( B ) The top gene ontology (GO) and PANTHER terms found to be enriched in Phf6 KO cells. The P -value for each term is plotted as −log 10 ( P -value). ( C ) GSEA plot depicting significant ( P < 0.001) changes in pre-B lymphocyte signature genes upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. ( D ) Quantitative PCR (qPCR) analysis of Phf6 WT (blue) and Phf6 KO (red) cells transduced with empty vector (EV; solid) or a vector expressing Phf6 cDNA (cDNA; dotted). Relative mRNA levels for B-cell-associated genes are shown: Phf6 , Cd22 , Cd74 , Il4ra , Lyn , Ly86 , and Blk . ( E ) Schematic representation of ICA used to identify differential expression signatures (independent components [ICs]) in the integrated RNA-seq data set comprised of Phf6 WT , shPhf6, and Phf6 KO cells. Hinton diagram representation of ICA-derived signatures. Columns denote signatures, and rows denote samples. Colors denote relative directionality of gene expression ([red] up-regulation; [green] down-regulation), and the size of each square represents the magnitude of the contribution of each sample to the respective IC. Each signature is two-sided. Vertical boxes denote statistically significant ( P = 0.01, Mann-Whitney test) independent components. IC2 identified a Phf6 KO -specific gene signature. ( F ) GSEA plot depicting ( P = 0.08) enrichment in T-cell signal transduction signature upon Phf6 deletion, as compared with Phf6 WT cells. (NES) Normalized enrichment score. Data represent the mean ± SD. Statistics for these data were calculated with two-sided Student's t -test. (**) P < 0.01; (***) P < 0.001; (****) P < 0.0001; (n.s.) not significant.

Article Snippet: The TaqMan gene expression assays (Life Technologies) used were Phf6 (Mm00804415_m1), Blk (Mm00432077_m1), Cd74 (Mm00658576_m1), IL4ra (Mm01275139_m1), Ly86 (Mm00440240_m1), Lyn (Mm01217488_m1), Gapdh (Mm99999915_g1), and Cd22 (Mm00515432_m1).

Techniques: Gene Expression, RNA Sequencing, Transformation Assay, Expressing, Real-time Polymerase Chain Reaction, Transduction, Plasmid Preparation, Quantitative Proteomics, Derivative Assay, MANN-WHITNEY

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Intersection and disjunction of 361 genes involved in phagocytosis (Haney et al., 2018) expressed (FPKM>5) by BV2 cells (PRJNA407656) and primary microglia. b , Gating scheme for FACS separation of phagocytic and non-phagocytic BV2 cells, treated with vehicle (gray) or the actin-polymerization inhibitor, cytochalasin D (red). c , Time-lapse microscopy readout of phagocytosis by BV2 cells treated with vehicle (gray) or cytochalasin D (red) (n=3, mean +/− s.e.m.). d , e , Results from CRISPR-Cas9 screen targeting 954 membrane proteins (d) or 2,015 drug targets, kinases, and phosphatases (e) in BV2 cells. Knockouts that promote phagocytosis (red) have a positive effect size and knockouts that inhibit phagocytosis (blue) have a negative effect size (screen performed in technical duplicate; dotted line, P =0.05, two-sided t-test). f , g , Distributions of negative control sgRNAs (gray) and Rab9-targeting ( f ) or CMAS-targeting ( g ) sgRNAs (blue). Positive values indicate enrichment in the phagocytic fraction, and negative values indicate enrichment in the non-phagocytic fraction. h , Statistical overrepresentation test showing enrichment of Reactome pathway annotations within phagocytosis-promoting (red) and -inhibiting (blue) hits (Fisher’s exact test). i , CD22 expression in WT (blue), CD22 KO (green), and isotype control stained (black) BV2 cells assessed by flow cytometry. j , Percent confluence of control (gray) and CD22 KO (green) BV2 cells during time-lapse microscopy phagocytosis assays (n=3, mean +/− s.e.m.). k , Number of beads ingested per cell were calculated in control and CD22 KO BV2 cells after 8 hours of phagocytosis. While CD22 KO cells display enhanced phagocytosis at a population level (n=3, * P <0.05, two-sided t-test, mean +/− s.e.m.), we observed no significant differences in the number of beads ingested per cell (two-way ANOVA). Red dot represents mean phagocytic index of the entire cell population. Data in b, c, i, j, k were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Time-lapse Microscopy, CRISPR, Membrane, Negative Control, Expressing, Control, Staining, Flow Cytometry

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Screening strategy for age-related genetic modifiers of phagocytosis. Scale bar = 50 microns. Screens were performed in technical duplicate. b , RNA-seq differential expression analysis between aged (20 m.o., n=6) and young (3 m.o., n=6) primary microglia overlaid with significant hits from CRISPR-Cas9 screen of BV2 cells. Red dots represent hits that promote phagocytosis, blue dots represent hits that inhibit phagocytosis, and gray dots are knockouts that showed no effect. Dashed line represents adjusted P -value of 0.001 (Benjamini-Hochberg method). c , qPCR analysis of CD22 expression in acutely isolated primary microglia from young (2-3 m.o.) and aged (20-22 m.o.) mice, normalized to a housekeeping gene (β-actin); data represent fold change relative to young microglia (n=4, **** P <0.00005, two-sided t-test, mean +/− s.e.m.). d , Flow cytometry quantification (mean fluorescence intensity (MFI) – fluorescence minus one (FMO) background intensity) of CD22 expression in acutely isolated primary microglia from young (2-3 m.o.) and aged (20-22 m.o.) mice (n=5, *** P <0.0005, two-sided t-test, mean +/− s.e.m.). e , Normalized phagocytosis (fluorescent area / confluence) monitored over 24 hours, imaged every hour. Control BV2 cells were infected with a “safe-targeting” sgRNA and KO BV2 cells were infected with a sgRNA targeting CD22 (n=6, **** P <0.00005, two-sided t-test; mean +/− s.e.m.). f , Representative images of young and aged cerebella probed for Tmem119 (green), CD22 (red), and nuclei (DAPI, blue). Green arrows indicate Tmem119+ microglia, and red-green arrows indicate Tmem119+CD22+ microglia. Scale bar = 30 microns. g , Percentage of Tmem119+ microglia that co-express CD22 in multiple brain regions of young (3 m.o., brown) and aged (22 m.o., gray) mice (n=3, * P <0.05, *** P <0.0005, **** P <0.00005, 2-way ANOVA with Sidak correction, mean +/− s.e.m.). Data in c - g were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: RNA Sequencing, Quantitative Proteomics, CRISPR, Expressing, Isolation, Flow Cytometry, Fluorescence, Control, Infection

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , b , c , e , f , Flow cytometry analysis of young (red) and aged (blue) microglia for expression of fluorescence minus one (FMO) background fluorescence ( a ), plant-derived lectin ligands ( b ), conserved Siglecs ( c ), mouse-specific CD33-related Siglecs ( e ), and recombinant Siglec ligands ( f ). MFI shown on a biexponential-scale. d , Microglia from WT or CD22 −/− aged mice were stained with the particular anti-CD22 clone (Ox97) used for immunophenotyping. CD22 −/− microglia show no staining relative to FMO. g , Gating strategy to immunophenotype microglia while minimizing autofluorescence. All data were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Flow Cytometry, Expressing, Fluorescence, Derivative Assay, Recombinant, Staining

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Flow cytometry gating scheme for analysis of CD22 expression in peripheral blood-derived myeloid cells (CD45+CD11b+), immature B-cells (CD45+B220+CD22lo), and mature B-cells (CD45+B220+CD22hi). Quantibrite beads are shown in the top right panel. b , Quantification of flow cytometry analysis showing the number of CD22 molecules on various cell types, interpolated from the Quantibrite bead standard curve (n=3, mean +/− s.e.m.). c , CD22 expression in various cell types of the young mouse CNS, showing exclusive expression in microglia. Data from Barres lab RNA-seq ( http://www.brainrnaseq.org/ ). d , t-SNE plot showing single cell RNA-seq (scRNA-seq) analysis of CD22 expression in microglia isolated from E14.5, P7, and adult mouse brains. CD22 is enriched in a subpopulation of P7 microglia. Data from Li et al., 2018 ( https://myeloidsc.appspot.com/ ). e , t-SNE plot showing scRNA-seq analysis of CD22 expression in cells from 20 different mouse tissues. CD22 is expressed in B-cells and microglia, but absent from non-myeloid brain cells (n=7, young mice). Data from the Tabula Muris Consortium. f , Violin plots of log-normalized CD22 counts per million reads (CPM) showing high expression in B-cells from multiple organs and in microglia (n=7, young mice). Data from the Tabula Muris Consortium. Data in a, b were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Flow Cytometry, Expressing, Derivative Assay, RNA Sequencing, Isolation

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Results from CRISPR-Cas9 screen targeting 2,015 drug targets, kinases, and phosphatases in BV2 cells (screen performed in technical duplicate; dashed line, P =0.05, two-sided t-test). b, c, d, Phagocytosis of pH-sensitive fluorescent beads by control (black) vs CMAS KO (red) BV2 cells ( b ), control (black) vs PTPN6 KO (red) cells ( c ), and CMAS KO (blue) vs CMAS/CD22 double KO (green) cells ( d ) (n=3, **** P <0.00005, N.S. not significant, two-sided t-test; mean +/− s.e.m.). e , Cell-surface glycan engineering using lipid tail-functionalized glycopolymers bearing sialic acid α2,3- or α2–6-linked to N-acetyllactosamine. ITIMs = immunoreceptor tyrosine-based inhibitory motifs, SHP-1 = Src homology region 2 domain-containing phosphatase-1. f , g , Phagocytosis of pH-sensitive fluorescent beads by CMAS KO ( f ) or CMAS/CD22 double KO ( g ) BV2 cells coated with no polymer (black), α2,3-linked sialic acid (purple) or α2,6-linked sialic acid (orange) (n=3, *** P <0.0005, N.S. not significant, one-way ANOVA with Tukey’s correction; mean +/− s.e.m.). h , Western blot quantification of ratio of active phosphorylated SHP1 (p-SHP1) to total SHP1 in control (gray), CD22 KO (green), and CMAS KO (blue) BV2 cells, normalized to a loading control protein (α-tubulin) (n=3, * P <0.05, N.S. not significant, one-way ANOVA with Tukey’s correction). For raw source image, see . Data in b-h were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: CRISPR, Control, Glycoproteomics, Polymer, Western Blot

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Sambucus nigra agglutinin (SNA, recognizes α2,6-linked sialic acid) and Maackia amurensis agglutinin II (MAA II, recognizes α2,3-linked sialic acid) ligand expression in WT BV2 cells (orange), WT BV2 cells pretreated with sialidase (blue), and CMAS KO cells (red) assessed by flow cytometry. Sialidase treatment and CMAS KO reduce sialic acid ligands on the cell surface. b , Western blot showing SHP1 protein expression in WT and PTPN6 KO BV2 cells. For raw source image, see . c , Percent confluence of control (gray), CMAS KO (red), and PTPN6 KO (blue) BV2 cells during time-lapse microscopy phagocytosis assays (n=3, mean +/− s.e.m.). d , e , Phagocytosis of pH-sensitive fluorescent beads by untreated (black) and sialidase-treated (red) BV2 cells (d) or vehicle-treated and 3F ax -Neu5Ac-treated BV2 cells prior to phagocytosis (n=3, ** P <0.005, two-sided t-test; mean +/− s.e.m). f , Phagocytosis of pH-sensitive fluorescent beads by WT (black), WT + sialidase (red), CD22 KO (blue), or CD22 KO + sialidase (green) BV2 cells (n=3, * P <0.05, N.S. not significant, one-way ANOVA with Tukey’s multiple comparisons correction; mean +/− s.e.m.). g , Microglia were acutely isolated from the brains of aged (18 m.o.) WT (left) or CD22 −/− (right) mice, treated with or without sialidase, and incubated with pH-sensitive fluorescent latex beads. Microglia specific phagocytosis was measured using flow cytometry (n=6, * P <0.05, N.S. not significant, paired two-sided t-test). h , Representative images of BV2 cells coated with AlexaFluor 488 conjugated glycopolymers (green) and stained with a plasma membrane-specific dye (CellMask, red) showing overlap (yellow). Scale bar = 25 microns. i , Recombinant mouse CD22-human Fc fusion protein was pre-complexed with AF647 anti-human Fc secondary antibody, treated with various concentrations of IgG (black) or anti-CD22 (blue, red), and subsequently allowed to bind to ligands on the surface of BV2 cells or BV2 cells pretreated with sialidase (red). Binding was measured by flow cytometry. j , Internalization of IgG (black), function blocking anti-CD22 (clone Cy34.1, blue), and non-function-blocking anti-CD22 (clone OX96, green) conjugated to a pH-sensitive fluorescent dye by BV2 cells assessed by time-lapse microscopy (n=3, mean +/− s.e.m.). k , Western blot quantification of ratio of active pSHP1 to total SHP1 protein in BV2 cells pretreated with various concentrations of anti-CD22. Blue line represents the fitted variable slope inhibitor-response curve. For raw source image, see . All data were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Expressing, Flow Cytometry, Western Blot, Control, Time-lapse Microscopy, Isolation, Incubation, Staining, Clinical Proteomics, Membrane, Recombinant, Binding Assay, Blocking Assay

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Myelin debris labeled with a fluorescent dye (AF555) was stereotactically co-injected with anti-CD22 or IgG into the cortex on opposite hemispheres of the same aged (14-16 m.o.) mouse. b , Representative images of AF555-labed myelin (red, top row) overlaid with the myeloid marker Iba1 (green, bottom row) at the injection sites of IgG (left) or anti-CD22 (right) treated hemispheres of the same aged brain. Scale bar = 100 microns. c , Clearance of myelin debris in IgG (black) or anti-CD22 (green) treated hemispheres of aged mice assessed 48 hours post-injection (n=8, ** P <0.005, paired two-sided t-test). d , Flow cytometry quantification of ex vivo phagocytosis of pH-sensitive beads by aged microglia pretreated with IgG or anti-CD22 (n=6, ** P <0.005, paired two-sided t-test). e , Labeled myelin debris was stereotactically injected into the cortices of aged (12–14 m.o.) WT or CD22 −/− mice. f , Clearance of myelin debris in cortices of aged WT (black) vs CD22 −/− (green) mice assessed 48 hours post-injection (n=4, * P <0.05, two-sided t-test, mean +/− s.e.m.). g , Clearance of myelin debris in IgG (black) or anti-CD22 (green) treated hemispheres of young mice assessed 48 hours post-injection (n=4, N.S. not significant, paired two-sided t-test). h , Clearance of Aβ oligomers in IgG (black) or anti-CD22 (green) treated hemispheres of aged mice assessed 48 hours post-injection (n=8, ** P <0.005, paired two-sided t-test). i , Percent area of residual Aβ oligomers that were CypHer5E+, indicating localization to acidified lysosomes (n=8, ** P <0.005, paired two-sided t-test). All data were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Labeling, Injection, Marker, Flow Cytometry, Ex Vivo

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Representative images of myelin labeled with a pH-sensitive fluorescent dye (CypHer5E, white), a constitutively fluorescent dye (AF555, red) and stained for Iba1 (green). The majority of AF555 overlapping with Iba1 is also positive for CypHer5E, indicating localization to an acidified compartment. Scale bar = 100 microns. b , 3D reconstruction of microglial cell (Iba1, green) with ingested myelin (CypHer5E and AF555, white and red, yellow arrow) near un-ingested myelin (AF555, red, white arrow). Scale bar = 5 microns. c , Microgliosis, as assessed by % Iba1+ area at the injection site, was not altered by CD22 blockade (n=8, N.S. not significant, paired two-sided t-test). d , Representative images of myelin (red) overlaid with the myeloid marker Iba1 (green) at the injection site of IgG (left) or PBS (middle) treated hemispheres of the same aged brain, or an image of a stab wound control (not injected with myelin). Scale bar = 100 microns. e , Microgliosis, as assessed by % Iba1+ area at the injection site, was not altered by IgG compared to the stab wound control (n=2, N.S. not significant, paired two-sided t-test). f , Clearance of myelin debris in the IgG (black) or PBS (blue) treated hemispheres assessed 48 hours post-injection (n=4, N.S. not significant, paired two-sided t-test). g , Representative images of Iba1 (gray), a macrophage marker, and Tmem119 (magenta), a microglia-specific marker, at the injection site in IgG (left) or anti-CD22 (right) treated hemispheres of the same aged brain. Scale bar = 100 microns. h , Percent of Iba1+ phagocytes expressing Tmem119 at the injection site (n=4, N.S. not significant, paired two-sided t-test). i , Clearance of myelin debris in young (2.5 m.o.) WT (black) or CD22 −/− (green) mice was assessed 48 hours after injection (n=4, N.S. not significant, two-sided t-test; mean +/− s.e.m.). j , Representative images of total Aβ (white,), Thioflavin S+ fibrillar Aβ (green), and Iba1 (red) in transgenic mice expressing human APP with Swedish and London familial AD mutations (left) or WT mice injected with Aβ oligomers 48 hours prior to analysis (right). k , Representative images of Aβ (red, left column) and Aβ overlaid with the myeloid marker Iba1 (green, right column) at the injection site (+/− 2mm lateral, 0mm A-P, -1.5mm D-V relative to bregma) of IgG (top row) or anti-CD22 (bottom row) treated hemispheres of the same aged brain. Scale bar = 100 microns. l , Microgliosis, as assessed by % Iba1+ area at the Aβ oligomer injection site, was not altered by CD22 blockade (n=8, N.S. not significant, paired two-sided t-test). m , Representative images of α-synuclein and Iba1 at the injection site in IgG and anti-CD22 treated mice. Scale bar = 100 microns. n , Clearance of α-synuclein fibrils in the IgG (black) or anti-CD22 (green) treated hemispheres assessed 48 hours post-injection (n=7, * P <0.05, paired two-sided t-test). o , Microgliosis, as assessed by % Iba1+ area at the α-synuclein fibril injection site, was not altered by CD22 blockade (n=7, N.S. not significant, paired two-sided t-test). All data were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Labeling, Staining, Injection, Marker, Control, Expressing, Transgenic Assay

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Schematic of long-term CNS-targeted CD22 blockade. b , c , Correlation of gene fold changes between microglia from anti-CD22 (n=7) and IgG (n=7) treated mice and between microglia from aged (22 m.o., n=3) and young (3 m.o., n=3) mice ( b ) ( R = −0.47, P =1.7e-12) or between microglia from 5xFAD (8.5 m.o., n=5) and age-matched WT (8.5 m.o., n=5) mice ( c ) ( R = −0.27, P =8.5e-6, blue line, linear regression, Spearman correlation). Genes differentially expressed ( P <0.05, two-sided t-test) in either dataset (union) are shown. d , Hierarchical clustering of gene fold changes between microglia from anti-CD22 (n=7) and IgG (n=7) treated mice, between microglia from aged (22 m.o., n=3) and young (3 m.o., n=3) mice , and between microglia from 5xFAD (8.5 m.o., n=5) and age-matched WT (8.5 m.o., n=5) mice . Genes differentially expressed ( P -value<0.05, two-tailed t-test) in all three datasets (intersection) are shown. e , f , g , h , Percentage of time spent in the novel arm of the forced alternation Y-maze ( e, g ) or displaying freezing behavior in a contextual fear conditioning test ( f, h ) for aged (18 m.o.) WT and CD22 −/− mice ( e , f , n=6) or aged (18 m.o.) WT mice treated with IgG or anti-CD22 ( g , h , independent cohorts of mice in g (n=7 IgG, 9 anti-CD22) and h (n=7 for both groups)) (** P <0.005, * P <0.05, two-sided t-test; mean +/− s.e.m.). i , Representative images of dentate gyri stained for Prox1 (white) and c-Fos (red) from IgG and anti-CD22 treated mice. White arrows indicate cFos+Prox1+ active granule neurons. Scale bar = 100 microns. j , Total number of Prox1+c-Fos+ neurons in the dentate gyrus quantified over 5 tissue sections (n=7, * P <0.05, two-sided t-test; mean +/− s.e.m.). Data in a , g , h , i , j were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Two Tailed Test, Staining

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Representative images of CD19+ B-cells (red, top row), DAPI (blue, middle row), and merged (bottom row) in the spleen (left, positive control) and hippocampus (right) of a mouse treated with anti-CD22 via intracerebroventricular osmotic pump. b , Concentration of trans-cyclooctene-labeled anti-CD22 in the plasma 7 days after administration of 200 μg anti-CD22 via IP injection (n=1) or intracerebroventricular osmotic pump infusion (n=4), assessed by in-gel fluorescence and quantification based on a standard curve (mean +/− s.e.m). For raw source image, see . c , Representative images of coronal brain sections of untreated (left column) and IgG treated (right column) mice. IgG was labeled with an AlexaFluor647 NHS-ester (top row, white) to assess antibody distribution throughout the brain (bottom row, DAPI, blue). In addition to the para-ventricular areas, antibodies penetrated the thalamus and hippocampus. d , Flow cytometry analysis of AF647-labeled antibody on microglia isolated from untreated (black), IgG (red), or anti-CD22 (blue) infused mice. Microglia from anti-CD22 treated mice display elevated AF647 signal, indicative of antibody target engagement. Data in a, c, d were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Positive Control, Concentration Assay, Labeling, Clinical Proteomics, Injection, Fluorescence, Flow Cytometry, Isolation, Drug discovery

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Venn diagram showing the lack of any intersection among 315 genes differentially expressed between IgG (n=7) and anti-CD22 (n=7) treated microglia and 40 genes differentially expressed between untreated (n=2) and IgG (n=7) treated microglia at an FDR cutoff of 10% (Benjamini-Hochberg method). b , Hierarchical clustering of normalized read counts from IgG and anti-CD22 treated microglia, normalized by row mean. The top-100 differentially expressed genes are shown (n=7). c , Enrichr gene-ontology analysis of genes upregulated (red) and downregulated (blue) by anti-CD22 treatment (Fisher’s exact test, Benjamini-Hochberg FDR). d , Gene set enrichment analysis (GSEA) showing normalized enrichment score for microglia genes modulated by anti-CD22 treatment within the gene signature for: aging microglia (this study), disease-associate microglia (DAM, Keren-Shaul, et al. 2017), microglial neurodegenerative phenotype (MGnD, Krasemann, et al. 2017), and microglia from lipopolysaccharide treated mice (LPS, Bennett, et al. 2016) (*FDR<0.05). e , f , g , h , GSEA showing enrichment distribution for microglia genes modulated by anti-CD22 treatment within the gene signature for aging microglia (e), DAM (f), MGnD (g), and LPS-activated microglia (h).

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques:

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Western blot for Sall1 and α-Tubulin (loading control) in whole hippocampus lysates from IgG and anti-CD22 treated mice. For raw source image, see . b , Quantification of (a) showing upregulation of Sall1 protein in anti-CD22 hippocampi (n=3, * P <0.05, two-sided t-test, mean +/− s.e.m.). c , Protein concentration of CCL3 in the supernatant of acutely isolated aged microglia treated for 8 hours with IgG or anti-CD22 in the absence or presence of Aβ oligomers (n=4, N.S. not significant, *P<0.05, ANOVA with Sidak’s multiple hypothesis correction, mean +/− s.e.m.). d , Representative images of p-CREB expression (red) in the dentate gyrus of IgG (left) and anti-CD22 (right) treated mice. e , Quantification of p-CREB mean intensity in the dentate gyrus of IgG (black) and anti-CD22 (green) treated mice (n=7, two-sided t-test, mean +/− s.e.m.). f , Quantification of total doublecortin-positive cells in three equally-spaced dentate gyrus sections of IgG (black) and anti-CD22 (green) treated mice (n=3, N.S. not significant, two-sided t-test, mean + s.e.m.). g , h , i , Quantification of C1q (g), synaptophysin (h, pre-synaptic marker), and PSD-95 (i, post-synaptic marker) mean intensity in the hippocampus of IgG (black) and anti-CD22 (green) treated mice (n=3, N.S. not significant, two-sided t-test, mean + s.e.m.). All data were replicated in at least 2 independent experiments.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Western Blot, Control, Protein Concentration, Isolation, Expressing, Marker

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: a , Working memory and exploratory behavior in aged (18 m.o.) mice treated with IgG (black) or anti-CD22 (green) via IP injection weekly for one month as assessed by % of time spent in the novel arm in a forced alternation Y-maze test (n=6, N.S. not significant, two-sided t-test, mean +/− s.e.m.). b , Contextual memory aged (18 m.o.) mice treated with IgG (black) or anti-CD22 (green) via IP injection weekly for one month as assessed by % of time displaying freezing behavior in a contextual fear conditioning test (n=6, N.S. not significant, two-sided t-test, mean +/− s.e.m.).

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Injection

Journal: Nature

Article Title: CD22 blockade restores homeostatic microglial phagocytosis in aging brains

doi: 10.1038/s41586-019-1088-4

Figure Lengend Snippet: Microglial phagocytosis declines with age, accompanied by increased CD22 expression. CD22 inhibition restores phagocytosis, promotes a homeostatic transcriptional state, and improves cognitive function in aged mice.

Article Snippet: The complex was then incubated with various concentrations of a mouse monoclonal anti-CD22 antibody (Cy34, BioXCell) or a mouse IgG1 isotype control antibody (MOPC21, BioXCell) on ice for 30 minutes.

Techniques: Expressing, Inhibition

Journal: Blood Cancer Discovery

Article Title: Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies

doi: 10.1158/2643-3230.BCD-21-0087

Figure Lengend Snippet: Alternative splicing of CD22 in human B-ALL. A, Quantification of LSVs across transcripts encoding major B-cell immunotherapeutic targets from pediatric B-ALL samples from the NCI TARGET consortium. B, CD22 splice graph depicting splicing events across the 14 exons of CD22 in B-ALL, with specific depiction of CD22 Δex5–6 and CD22 Δex2* variants. C, Relative frequencies of reads originating in exon 1 (blue numbers) or terminating in exon 7 (green numbers) in normal B-cell precursors (top) and B-ALL (bottom). D and E, Stack plots depicting relative abundance of CD22 isoforms including/skipping exon 5 and 6 across TARGET dataset ( n = 219) and normal B-cell subtypes ( n = 25, from 11 individuals), respectively. BP, datasets corresponding to B-cell precursors obtained through the BLUEPRINT project; ped, pediatric samples. F and G, Stack plots depicting relative abundance of CD22 Δex2* variants across TARGET dataset and normal B-cell subtypes, respectively. H, RT-PCR analysis validating CD22 isoforms in the Nalm6 cell line. I, ONT-based long-read RNA-seq of CD22 transcripts in cells from a TCF3–HLF B-ALL PDX model (ALL1807). CD22 Δex5–6 and Δex2* variant transcripts are highlighted in yellow and purple, respectively.

Article Snippet: For detection of murine and human proteins, primary human anti-CD22 antibodies (Boster Bio, PB9691; R&D Systems, MAB19681) were used in combination with anti-rabbit or anti-mouse horseradish peroxidase–linked secondary antibodies (Cell Signaling Technology) and Amersham Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Life Sciences).

Techniques: Alternative Splicing, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing, Variant Assay

Journal: Blood Cancer Discovery

Article Title: Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies

doi: 10.1158/2643-3230.BCD-21-0087

Figure Lengend Snippet: Biochemical and functional characterization of the CD22 Δex5–6 isoform. A, RT-PCR detection of CD22 Δex5–6 across 18 diagnostic de novo B-ALL samples driven by various genetic alterations. B, Western blotting using N-terminus–directed anti-CD22 antibody performed on CD22 -deleted OCI-Ly8 cells engineered to express CD22 Δex5–6 or FL isoforms. KO, knockout. C, Amino acid sequence of the peptide used for mAb production. D, RT-PCR detection of CD22 Δex5–6 in OCI-Ly8 cell lines from B , human B-ALL cell lines, and B-ALL PDXs. E, Western blotting using the 11F11 mAb performed on cells from D . F, Flow cytometric detection of CD22 KO, CD22 FL, and CD22 Δex5–6. FSC-A, forward scatter area. G, Western blotting demonstrating electrophoretic mobilities of CD22 isoforms treated with deglycosylating enzymes with or without denaturation. H, Western blotting detecting CD22 and total and phosphorylated (p) BLNK in derivatives from B treated with an anti-IgM antibody for indicated time intervals. I, Quantitation of pBLNK bands from H . The experiment was replicated twice with concordant results. J and K, In vitro killing assays performed on cells from B using HA22- and m971-based CD22 CAR T cells (CART22), respectively ( n = 2 technical replicates). Data in both panels are represented as mean values ± SD error bars.

Article Snippet: For detection of murine and human proteins, primary human anti-CD22 antibodies (Boster Bio, PB9691; R&D Systems, MAB19681) were used in combination with anti-rabbit or anti-mouse horseradish peroxidase–linked secondary antibodies (Cell Signaling Technology) and Amersham Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Life Sciences).

Techniques: Functional Assay, Reverse Transcription Polymerase Chain Reaction, Diagnostic Assay, Western Blot, Knock-Out, Sequencing, Quantitation Assay, In Vitro

Journal: Blood Cancer Discovery

Article Title: Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies

doi: 10.1158/2643-3230.BCD-21-0087

Figure Lengend Snippet: CD22 protein expression in B-cell malignancies is limited by exon 2 inclusion. A, Flow cytometric detection of exogenously expressed CD22 protein in CD22 -deleted OCI-Ly8 cells using anti-CD22 antibodies directed toward either the extreme N-terminus (S-HCL-1) or the C-terminal region (RFB-4) of the extracellular domain. KO, knockout. B, Western blotting detection of CD22 in the same cell lines and parental controls using anti-CD22 antibodies directed toward the N-terminus (R&D Systems, MAB19681) and the C-terminus (Boster Bio, PB9691). C, Schematic annotating the CD22 exon splice junctions (gray arches) assayed using junction-spanning qPCR primers following treatment with Ex2In2 morpholino. Bottom right, morpholino sequence is shown in red, with complementary exon–intron junction sequence shown in blue/white. D, qRT-PCR detection of various CD22 mRNA isoforms in Reh B-ALL cells transfected for 48 hours with Ex2In2 (10 μmol/L and 100 μmol/L). Constitutive expression of ex13–14 junction was used as a measure of total CD22 expression ( n = 6, 2 independent experiments with 3 technical replicates each). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ns, not significant. E and F, Detection of CD22 protein in the morpholino-treated Reh cells by Western blotting and flow cytometric site density assay, respectively ( n = 2 independent experiments). G, Viability assay performed on CD22 -deleted OCI-Ly8 cells reconstituted with indicated CD22 isoforms and treated for 24 hours with indicated concentrations of inotuzumab ( n = 3 technical replicates). IC 50 95% confidence intervals (CI) were as follows: 54–119 ng/mL for CD22 KO, 25–85 ng/mL for CD22 Δex2, and 5–11 ng/mL for CD22 Δex5–6. H, Bar graph representing IC 50 values from G . P values were determined using an unpaired t test. I, Viability assay performed on Ex2In2-treated (48 hours) and inotuzumab-treated (24 hours) Reh cells ( n = 3 technical replicates). Cell viability was assessed using the WST-1 assay. IC 50 95% CIs were 29–72 ng/mL for Ctrl treatment and 119–333 ng/mL for Ex2In2 treatment. J, Bar graph representing IC 50 values from I on the log scale. P values were determined using an unpaired t test. Data in D , F , H , and J are presented as individual and mean values ± SD error bars. Data in G and I are presented as mean values ± SD error bars.

Article Snippet: For detection of murine and human proteins, primary human anti-CD22 antibodies (Boster Bio, PB9691; R&D Systems, MAB19681) were used in combination with anti-rabbit or anti-mouse horseradish peroxidase–linked secondary antibodies (Cell Signaling Technology) and Amersham Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Life Sciences).

Techniques: Expressing, Knock-Out, Western Blot, Sequencing, Quantitative RT-PCR, Transfection, Viability Assay, WST-1 Assay

Journal: Blood Cancer Discovery

Article Title: Modulation of CD22 Protein Expression in Childhood Leukemia by Pervasive Splicing Aberrations: Implications for CD22-Directed Immunotherapies

doi: 10.1158/2643-3230.BCD-21-0087

Figure Lengend Snippet: CD22 protein expression is limited by exon 2 inclusion in B-ALL cells. A, Correlation analysis of CD22 site density versus CD22 mRNA levels in pretreatment primary B-ALL bone marrow or peripheral blood specimens obtained from children enrolled on the COG AALL1621 phase II clinical trial. CD22 mRNA levels were measured by qRT-PCR using primers specific for the exon 13–14 (left) or exon 1–2 (right) junctions. CD22 expression was normalized to that of β-actin. Regression coefficients and P values are shown for each comparison. B, Relative expression of CD2 2 exon 2–containing and exon 2–skipping splice variants within baseline AALL1621 B-ALL specimens. Each stack plot represents a single patient (designated by the COG unique specimen identifier). Yellow arrow highlights PAYYZW as a sample apparently devoid of protein-coding CD22 mRNA isoforms. The legend shows color-coded CD22 splice variants. C, Flow cytometric quantitation of CD22 molecules in paired pre– and post–inotuzumab treatment (pre-ino/post-ino) bone marrow specimens from AALL1621 patient PAWUXD with multiply relapsed B-ALL. D, CD22 mutational analysis of the paired PAWUXD samples. E, CD22 exon 2 splicing analysis of the paired PAWUXD samples. For color coding, refer to legend in B . F, Flow cytometric quantitation of CD22 molecules in paired pre- and posttreatment (pre-ino/post-ino) bone marrow specimens from AALL1621 patient PAVDRV with multiply relapsed B-ALL. G, CD22 mutational analysis of the paired PAVDRV samples. H, CD22 exon 2 splicing analysis of the paired PAVDRV samples. For color coding, refer to legend in B .

Article Snippet: For detection of murine and human proteins, primary human anti-CD22 antibodies (Boster Bio, PB9691; R&D Systems, MAB19681) were used in combination with anti-rabbit or anti-mouse horseradish peroxidase–linked secondary antibodies (Cell Signaling Technology) and Amersham Enhanced Chemiluminescence Western Blotting Detection Reagent (GE Life Sciences).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Quantitation Assay

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: EC 50 values in dose–response ELISA for clones identified through fishing.

Article Snippet: For blocking, Raji or CLL cells were incubated with a polyclonal antibody (rabbit anti-CD22 (Sino Biological, 11958-T26-50), goat anti-FCRL5 (Invitrogen, PA5-48003), and goat anti-ROR1 (R&D Systems, AF2000)), 100 µg/ml, 25 µl/well, or buffer, for 1 h at +4°C.

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: Binding analysis of antibodies generated through fishing with recombinant CD22, ROR1, and FCRL5 proteins. (A) Dose–response ELISA of one representative antibody (human IgG1) per protein. Each antibody was analyzed for binding to the specific protein used for fishing (target protein) and a non-related protein carrying the same tag (non-target protein). Binding was detected using an HRP-labeled anti-human antibody and a luminescent substrate. (B) Flow cytometry analysis of one representative antibody (human IgG1 or scFv) per target showing the binding to target cells, that is, chronic lymphocytic leukemia (CLL) cells (anti-FCRL5) or Raji cells (anti-CD22 and anti-ROR1), with or without a prior blocking step with a commercial polyclonal antibody of the same specificity as the tested antibody. Binding was detected using an APC-labeled anti-human antibody (anti-CD22 and anti-ROR1) or an AF647-labeled anti-tag antibody (anti-FCRL5).

Article Snippet: For blocking, Raji or CLL cells were incubated with a polyclonal antibody (rabbit anti-CD22 (Sino Biological, 11958-T26-50), goat anti-FCRL5 (Invitrogen, PA5-48003), and goat anti-ROR1 (R&D Systems, AF2000)), 100 µg/ml, 25 µl/well, or buffer, for 1 h at +4°C.

Techniques: Binding Assay, Generated, Recombinant, Enzyme-linked Immunosorbent Assay, Protein Binding, Labeling, Flow Cytometry, Blocking Assay

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: Protein and gene expression levels in chronic lymphocytic leukemia (CLL) cells (target cells) and CD19− PBMC from healthy donors (non-target cells) for receptors successfully used in fishing.

Article Snippet: For blocking, Raji or CLL cells were incubated with a polyclonal antibody (rabbit anti-CD22 (Sino Biological, 11958-T26-50), goat anti-FCRL5 (Invitrogen, PA5-48003), and goat anti-ROR1 (R&D Systems, AF2000)), 100 µg/ml, 25 µl/well, or buffer, for 1 h at +4°C.

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Deep Mining of Complex Antibody Phage Pools Generated by Cell Panning Enables Discovery of Rare Antibodies Binding New Targets and Epitopes

doi: 10.3389/fphar.2019.00847

Figure Lengend Snippet: Frequency, based on NGS analysis, of discovered clones in the different strategies including the input phage pool. Median frequency is shown with red bars. (A) Antibodies, previously discovered through direct screening with corresponding specificities, CD23 ( n = 107), CD72 ( n = 132), and CD200, CD21, CD32, or HLA-DR ( n = 34). (B) Antibodies, discovered through fishing, binding FCRL5, ROR1, or CD22 ( n = 17). For statistical analysis, Friedman’s test with Dunn’s multiple comparison was done using GraphPad Prism.

Article Snippet: For blocking, Raji or CLL cells were incubated with a polyclonal antibody (rabbit anti-CD22 (Sino Biological, 11958-T26-50), goat anti-FCRL5 (Invitrogen, PA5-48003), and goat anti-ROR1 (R&D Systems, AF2000)), 100 µg/ml, 25 µl/well, or buffer, for 1 h at +4°C.

Techniques: Clone Assay, Binding Assay

Journal: Journal of neuroinflammation

Article Title: CD22 blockade exacerbates neuroinflammation in Neuromyelitis optica spectrum disorder.

doi: 10.1186/s12974-024-03305-2

Figure Lengend Snippet: Fig. 1 Expression profile of CD22 in circulating immune cell subsets from patients with NMOSD and controls. A Single-cell sequencing analysis of im mune cells from human peripheral blood samples from patients with NMOSD and healthy controls (total: 90,381 cells from 5 NMOSD patients and 5 controls; control: 43,985 cells; NMOSD: 46,396 cells). B, C CD22 expression profiles of B cells B and B-cell subsets C from patients with NMOSD and healthy controls; n = 5 per group. D CD22 expression profiles across various immune cell subsets in patients with NMOSD and healthy controls at the indi vidual patient level; n = 5 per group. E Gating strategy for human circulating immune cell subsets, including neutrophils (CD45+CD3−CD16+), monocytes (CD45+CD16−CD14+), B cells (CD45+CD3−CD19+), CD4+ T cells (CD45+CD3−CD4+), CD8+ T cells (CD45+CD3−CD8+) and NK cells (CD45+CD3−CD56+). F Summarized bar graph showing CD22 expression in monocytes, neutrophils, B cells, CD4+ T cells, CD8+ T cells and NK cells; n = 6 per group. G Visualization of circulating exosomes from patients with NMOSD and controls. H Flow cytometry gating strategy and bar graph showing microglia-derived exosomes (CD22+TMEM119+); n = 8 per group. The data are presented as the mean ± SEM. **p < 0.01

Article Snippet: An anti-CD22 monoclonal antibody (clone ID: CY34.1; BioXcell, West Lebanon, NH) was given to NMOSD mice via intrastriatal injection at a dose of 100 μg/mouse to deplete CD22-expressing cells [24].

Techniques: Expressing, Sequencing, Control, Flow Cytometry, Derivative Assay

Journal: Journal of neuroinflammation

Article Title: CD22 blockade exacerbates neuroinflammation in Neuromyelitis optica spectrum disorder.

doi: 10.1186/s12974-024-03305-2

Figure Lengend Snippet: Fig. 2 CD22 expression profile in microglia and leukocytes from NMOSD mice. A Flow cytometry gating strategy for microglia (CD45+CD11bint), monocytes (CD45highCD11b+Ly6C+), neutrophils (CD45highCD11b+Ly6G+), B cells (CD45highCD3−CD19+), CD4+ T cells (CD45highCD3+CD4+), CD8+ T cells (CD45highCD3+CD8+) and NK cells (CD45highCD3−NK1.1+). B Histograms showing CD22-expressing cell subsets in sham and NMOSD mice. C, D Bar graphs showing the percentage of each cell type expressing CD22 in brain and spleen tissues from NMOSD mice; n = 8 per group. The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01

Article Snippet: An anti-CD22 monoclonal antibody (clone ID: CY34.1; BioXcell, West Lebanon, NH) was given to NMOSD mice via intrastriatal injection at a dose of 100 μg/mouse to deplete CD22-expressing cells [24].

Techniques: Expressing, Flow Cytometry

Journal: Journal of neuroinflammation

Article Title: CD22 blockade exacerbates neuroinflammation in Neuromyelitis optica spectrum disorder.

doi: 10.1186/s12974-024-03305-2

Figure Lengend Snippet: Fig. 3 CD22 blockade exacerbates NMOSD pathology in mice. A T2WI scans revealed demyelinating lesions in the indicated groups of NMOSD mice. The lesion areas are marked with red lines. Scale bar: 2 mm. B Bar graphs depicting the volume of demyelinating lesions in the indicated groups of NMOSD mice; n = 10 per group. C Immunostaining of the indicated markers (GFAP, AQP4, or MBP) in brain tissue sections from NMOSD mice receiving the anti- CD22 mAb or IgG control on day 3 after NMOSD induction. The white lines indicate areas with loss of AQP4, GFAP or MBP. Scale bar: 3,000 μm (left), 100 μm (right). D Bar graphs illustrating demyelination in NMOSD mice receiving anti-CD22 mAb or IgG control; n = 10 per group. The data are expressed as the mean ± SEM. ** p < 0.01

Article Snippet: An anti-CD22 monoclonal antibody (clone ID: CY34.1; BioXcell, West Lebanon, NH) was given to NMOSD mice via intrastriatal injection at a dose of 100 μg/mouse to deplete CD22-expressing cells [24].

Techniques: Immunostaining, Control

Journal: Journal of neuroinflammation

Article Title: CD22 blockade exacerbates neuroinflammation in Neuromyelitis optica spectrum disorder.

doi: 10.1186/s12974-024-03305-2

Figure Lengend Snippet: Fig. 4 CD22 blockade augments the inflammatory activity of microglia in NMOSD mice. A Flow cytometry gating strategy for inflammatory markers (CD86, IL-1β and TNF-α) and immune regulatory markers (CD206, IL-10 and TGF-β) in microglia. B Bar graph showing the effects of CD22 blockade on the counts of microglia, brain-infiltrating monocytes, neutrophils, B cells, CD4+ T cells and CD8+ T cells in NMOSD mice; n = 10 per group. C Flow cytometry results showing the effects of CD22 blockade on the expression of inflammatory markers (CD86, IL-1β and TNF-α) and immunoregulatory markers (CD206, IL-10 and TGF-β) in microglia from NMOSD mice; n = 6 per group. D Immunostaining showing Iba1+ cells in the indicated groups. The white lines delineate the areas with an accumulation of Iba1+ cells. Scale bar: 100 μm. E Bar graph showing that CD22 blockade enhanced the accumulation of Iba1+ cells. n = 10 per group. F, G Skeletal analysis showing that the lengths of microglial processes were reduced on day 3 in mice treated with the anti-CD22 mAb; n = 6 per group. H Sholl analysis summarizing the results of microglial processes in NMOSD mice receiving the anti-CD22 mAb or IgG control. The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01

Article Snippet: An anti-CD22 monoclonal antibody (clone ID: CY34.1; BioXcell, West Lebanon, NH) was given to NMOSD mice via intrastriatal injection at a dose of 100 μg/mouse to deplete CD22-expressing cells [24].

Techniques: Activity Assay, Flow Cytometry, Expressing, Immunostaining, Control

Journal: Journal of neuroinflammation

Article Title: CD22 blockade exacerbates neuroinflammation in Neuromyelitis optica spectrum disorder.

doi: 10.1186/s12974-024-03305-2

Figure Lengend Snippet: Fig. 5 Microglia contribute to exacerbated NMOSD pathology in mice receiving anti-CD22 mAb. A Flow chart depicting drug administration and the indicated assessment. On day 14 after microglial depletion via PLX5622, wild-type mice received intrastriatal injections of anti-CD22 mAb after NMOSD induction. B Assessment of microglia following PLX5622 administration; n = 7 per group. C T2WI scans showing brain lesions in the indicated groups of NMOSD mice. Red lines outline lesion areas. Scale bar: 2 mm. D Bar graph showing lesion volume in the indicated groups of NMOSD mice; n = 6 per group. The data are presented as the mean ± SEM. **p < 0.01

Article Snippet: An anti-CD22 monoclonal antibody (clone ID: CY34.1; BioXcell, West Lebanon, NH) was given to NMOSD mice via intrastriatal injection at a dose of 100 μg/mouse to deplete CD22-expressing cells [24].

Techniques:

Journal: Journal of neuroinflammation

Article Title: CD22 blockade exacerbates neuroinflammation in Neuromyelitis optica spectrum disorder.

doi: 10.1186/s12974-024-03305-2

Figure Lengend Snippet: Fig. 6 Gr-1+ myeloid cells contribute to exacerbating NMOSD pathology in mice receiving anti-CD22 mAb. A Flow chart depicting the drug administra tion and experimental procedures. Mice received anti-Gr-1 mAb before and one day after NMOSD induction. B Assessment of Gr-1+ myeloid cells in mice receiving anti-Gr-1 mAb or IgG control; n = 6 per group. C T2WI scans showing brain lesions in the indicated groups of NMOSD mice. Red lines mark the lesion areas. D Bar graph depicting lesion volume in the indicated groups of NMOSD mice; n = 6 per group. The data are presented as the mean ± SEM. **p < 0.01

Article Snippet: An anti-CD22 monoclonal antibody (clone ID: CY34.1; BioXcell, West Lebanon, NH) was given to NMOSD mice via intrastriatal injection at a dose of 100 μg/mouse to deplete CD22-expressing cells [24].

Techniques: Control

Journal: Journal of neuroinflammation

Article Title: CD22 blockade exacerbates neuroinflammation in Neuromyelitis optica spectrum disorder.

doi: 10.1186/s12974-024-03305-2

Figure Lengend Snippet: Fig. 7 CD22 blockade exacerbated NMOSD pathology in mice receiving anti-CD20 mAb. A Flow chart depicting the experimental procedures. The mice received anti-CD20 mAb three days prior to NMOSD induction. B Assessment of B cells in mice receiving anti-CD20 mAb; n = 6 per group. C T2WI scans showing brain lesions in the indicated groups of NMOSD mice. Red lines mark the lesion areas. D Bar graph showing lesion volume in the indicated groups of NMOSD mice; n = 6 per group. The data are presented as the mean ± SEM. **p < 0.01

Article Snippet: An anti-CD22 monoclonal antibody (clone ID: CY34.1; BioXcell, West Lebanon, NH) was given to NMOSD mice via intrastriatal injection at a dose of 100 μg/mouse to deplete CD22-expressing cells [24].

Techniques:

Journal: Journal of neuroinflammation

Article Title: CD22 blockade exacerbates neuroinflammation in Neuromyelitis optica spectrum disorder.

doi: 10.1186/s12974-024-03305-2

Figure Lengend Snippet: Fig. 8 The detrimental effects of CD22 blockade on NMOSD pathology involve SYK-GSK3β signaling. A Flow chart depicting the experimental pro cedures. The mice received R406 starting from the onset of modeling until they were sacrificed. B, C Assessment of the phosphorylation levels of SYK and GSK3β in the indicated groups of NMOSD mice; n = 4 per group. D T2WI scans showing brain lesions in the indicated groups of NMOSD mice. Bar graph showing lesion volume in the indicated groups of NMOSD mice. Red lines mark the lesion areas; n = 6 per group. The data are presented as the mean ± SEM. *p < 0.05

Article Snippet: An anti-CD22 monoclonal antibody (clone ID: CY34.1; BioXcell, West Lebanon, NH) was given to NMOSD mice via intrastriatal injection at a dose of 100 μg/mouse to deplete CD22-expressing cells [24].

Techniques: Phospho-proteomics