Journal: Journal of translational medicine

Article Title: Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: a novel in vitro model and insights into target antigen dynamics.

doi: 10.1186/s12967-024-05254-z

Figure Lengend Snippet: Fig. 5 Observation of CD19-BBζ-CAR expression in relapsed Nalm-6 cells and salvage treatment. A Detection of FMC63 and CD247 transcripts and 4-1BB gene of CAR in CD19+ Nalm-6 (red) and relapsed CD19− Nalm-6 cells (blue) by qRT-PCR. Data of left bar graph represent the relative quantification using ACTB as the internal reference. Error bars represent s.d. The data are the representative of three independent experiments. B Expression of CD19 and CAR on CD19+ Nalm-6 cells and relapsed CD19− Nalm-6 cells analyzed by flow cytometry (representative of 3 experiments). Merge Graphs, the blue dots represent CD19− Nalm-6 cells and the red dots represent Nalm-6 cells. C Confocal imaging of Nalm-6 cells and relapsed CD19− Nalm-6 cells using Alexa Flour 488-conjugated anti-CD19 antibody (green), Alexa Flour 647-conjugated anti-CAR19 antibody (red), and DAPI (blue). D Lentiviral integration sites of CAR transduced Nalm-6 cells were analyzed by linear-amplification mediated PCR (LAM-PCR) and visualized with Circos plots. The integration sites across the genome and genomic features were shown from outer to inner circle: (1) cytogenetic bands; (2) genes that harbor these integration sites along with a bar chart showing the reads of integration sites; (3) the distribution of integration sites, with colored circles representing different gene functional regions of the host sequence: purple for promoter region, green for intron region, and red for distal intergenic region. E Phenotype changes of Nalm-6 cells transduced with small amount of CD19 CAR lentiviruses detected by flow cytometry over time. Gating was based on the same cells stained with isotype-matched antibody. F Dynamics of CD19− B phenotype in relapsed cells after co-culture with different ratios (5×, 20×) of Nalm-6 cells. Gating was based on the same cells stained with isotype-matched antibody. G Relapsed CD19− Nalm-6 cells were tested by qPCR specific for VSV-G sequence. H Comparison of in vitro efficacy of CD19-, CD22-, CD19/CD22- and CD22×CD19- CAR T cells. Cocultures with the relapsed cells were performed at 1:5, 1:1, and 5:1 E: T ratios, and lysis efficacies were detected by the LDH release assay Declarations

Article Snippet: The cells were then washed twice and stained with phycoerythrin (PE) streptavidin (BD bioscience, USA) for 15 min. CART-22 cells and CART-22/19 cells were washed once and incubated with CD22 Fc Alexa Fluor® 647 Protein (R&D Systems, USA) for 15 min. To detect in vitro cytotoxicity of CART-19 cells, transduced and untransduced T cells were co-cultured with Nalm-6 cells (total 1 × 106 cells) at E: T ratios (0.2:1, 0.5:1, 1:1, 5:1) for 6, 24 and 72 h. Cells were pipetted to incubate with antibodies for 30 min at room temperature in the dark and washed twice with PBS.

Techniques: Expressing, Quantitative RT-PCR, Quantitative Proteomics, Flow Cytometry, Imaging, Amplification, Functional Assay, Sequencing, Transduction, Staining, Co-Culture Assay, Comparison, In Vitro, Lysis, Lactate Dehydrogenase Assay

Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) t-distributed stochastic neighbor embedding (t-SNE) visualization of snRNA-seq data (Smart-seq) from multiple cortical areas of human brain colored by cell type. Data from Allen Brain Atlas (19). (B) t-SNE visualization of snRNA-seq data from multiple cortical areas of human brain colored by CD22 expression. (C) Representative image of human brain tissue probed for MOG (green), CD22 (magenta), and AIF1 (red) transcripts by multiplexed fluorescent RNAscope. Clustered puncta within 4′,6-diamidino-2-phenylindole–positive nuclei suggest true signal. (D) Schematic of FACS analysis of various cell types from fresh human primary cortical tissue. (E) Flow cytometry analysis of surface CD22 protein expression in CD45+ microglia (pink), MAP2+ neurons (orange), O4+MBP− OPCs (blue) and O4+MBP+ oligodendrocytes (purple) from fresh human primary cortical tissue (PCW 22). PE quantification beads are shown in gray. (F) Quantification of CD22-PE molecules bound to the surface of various human brain cell types calculated using PE bead standards (n = 2 biological replicates; PCWs 20 to 22; O4+MBP+ cells only detected at PCW 22). (G) t-SNE visualization of snRNA-seq data from multiple cortical areas of human brain colored by ST6GAL1 expression. (H) Flow cytometry analysis of human iMGLs stained with fluorophore-conjugated CD22 lacking its sialic acid–binding domain (sCD22-Δ, gray) or the full-length CD22 ECD (sCD22-ECD, red). In one condition, cells were pretreated with sialidase before sCD22-ECD staining (blue).

Article Snippet: For CD22-IGF2R kinetic analysis, anti-human Fc capture biosensors were loaded with recombinant human CD22 Fc-fusion protein (R&D Systems) and associated into recombinant His-tagged human IGF2R analyte at seven different concentrations (1000 nM, serially diluted 1:3).

Techniques: Expressing, RNAscope, Flow Cytometry, Staining, Binding Assay

Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) Schematic of human primary cortical oligodendrocyte isolation and differentiation protocol [adapted from (64)]. IHC, immunohistochemistry. (B) Representative flow cytometry of O4+ cells after 12 days in culture (d.p.c.) at the onset of antibody treatment, showing a mixed population of premyelinating O4+MBP−CD22− cells (blue) and a subpopulation of myelinating O4+MBP+CD22+ oligodendrocytes (red). (C) Representative bright-field images of isotype-, anti-CD22−, and anti-MOG–treated oligodendrocytes on day 15 after isolation (day 3 after treatment). Confluence mask overlaid in purple. Scale bar, 100 μm. (D) Quantification of confluence in isotype (gray)–, anti-CD22 (green)–, and anti-MOG (purple)–treated cells assessed by time-lapse microscopy over 3 days (n = 3 from two separate primary tissue samples, means ± SEM). (E) Representative immunofluorescence images of isotype-, anti-CD22−, and anti-MOG–treated oligodendrocytes on day 15 after isolation (day 3 after treatment), stained for MBP (green), and OLIG2 (red). Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (F) Quantification of MBP+ cells among OLIG2+ nuclei in isotype (gray)–, anti-CD22 (green)–, and anti-MOG (purple)–treated cells (n = 3 from two separate primary tissue samples, one-way ANOVA, means ± SEM).

Article Snippet: For CD22-IGF2R kinetic analysis, anti-human Fc capture biosensors were loaded with recombinant human CD22 Fc-fusion protein (R&D Systems) and associated into recombinant His-tagged human IGF2R analyte at seven different concentrations (1000 nM, serially diluted 1:3).

Techniques: Isolation, Immunohistochemistry, Flow Cytometry, Time-lapse Microscopy, Immunofluorescence, Staining

Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) Schematic of CRISPR-Cas9 screen for genetic modifiers of sCD22 binding. (B) Volcano plot of hits from CRISPR-Cas9 screen, highlighting KOs that inhibit CD22 binding (blue) and promote CD22 binding (red). (C) Flow cytometry analysis of CD22 ligand expression on U937 cells infected with a safe-targeting sgRNA (control, red) or an IGF2R-targeting sgRNA (purple). Isotype control–stained WT cells are shown in gray. AF647, Alexa Fluor 647. (D) Schematic of affinity purification LC-MS screen for direct binding partners of sCD22. (E) Volcano plot of hits from affinity purification LC-MS screen, highlighting proteins enriched in the CD22-bound fraction (red). (F) Kinetics of the CD22-IGF2R interaction determined by biolayer interferometry. Red line shows nonlinear fit of association-dissociation curve. Kd, dissociation constant.

Article Snippet: For CD22-IGF2R kinetic analysis, anti-human Fc capture biosensors were loaded with recombinant human CD22 Fc-fusion protein (R&D Systems) and associated into recombinant His-tagged human IGF2R analyte at seven different concentrations (1000 nM, serially diluted 1:3).

Techniques: CRISPR, Binding Assay, Flow Cytometry, Expressing, Infection, Control, Staining, Affinity Purification, Liquid Chromatography with Mass Spectroscopy

Journal: Science translational medicine

Article Title: The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C

doi: 10.1126/scitranslmed.abg2919

Figure Lengend Snippet: (A) Schematic of mAb generation and screening pipeline. (B) Screening results of 38 mAb clones for binding to CD22 (first column) and blocking of sCD22 to IGF2R on cell surface (second and third columns are two independent experiments). Three clones with adequate binding and potent blocking are highlighted (M22, M28, and M42). (C) Association-dissociation curves of antibody candidates binding to CD22 determined by biolayer interferometry. (D) Dose-response curves of CD22-IGF2R blockade by antibody candidates determined by flow cytometry. IC50, median inhibitory concentration. (E) Time-lapse fluorescence microscopy analysis of NPC2 trafficking to lysosomes in U937 cells treated with sCD22-Δ (gray), sCD22-ECD and an isotype control antibody (purple), or sCD22-ECD and clone M42 (green) (n = 2, ANOVA, means ± SEM). (F) Schematic of pipeline to generate isogenic WT and I1061T mutant iMGLs from iPSCs edited by CRISPR-Cas9–directed homologous recombination. After introduction of donor single-stranded DNA (ssDNA) by electroporation, a homozygous T3182C nucleotide substitution was confirmed by Sanger sequencing. NPC1 reduction was confirmed by Western blot. Mutant and isogenic control iPSCs were subsequently directed toward a hematopoietic lineage and differentiated into microglia-like cells. (G) Western blot quantification of NPC1 expression normalized to a loading control (β-actin) in WT and I1061T mutant iPSCs (n = 3, t test, means ± SEM). (H) Representative images of WT and I1061T mutant iMGLs stained for Filipin III (red, unesterified cholesterol) and IBA1 (green, microglia marker). Scale bar, 20 μm. (I) Quantification of Filipin-positive area normalized to total IBA1-positive area in WT (gray) and I1061T mutant (blue) iMGLs (n = 5 biological replicates, t test, means ± SEM). (J) Schematic of human in vitro model of microglia in NPC. Three components (iPSC-derived microglia, I1061T patient mutation, and NPC patient CSF) were combined to test the proof-of-principal in vitro efficacy of anti-CD22 in NPC. (K) Representative images of I1061T mutant iMGLs treated with NPC CSF and an isotype control antibody stained for Filipin III (red, unesterified cholesterol), LAMP2 (gray, lysosome marker), and IBA1 (green, microglia marker). Scale bars, 20 μm. (L) Representative images of I1061T mutant iMGLs treated with NPC CSF and anti-CD22 stained for Filipin III (red, unesterified cholesterol), LAMP2 (gray, lysosome marker), and IBA1 (green, microglia marker). Scale bars, 20 μm. (M) Quantification of Filipin-positive area normalized to total IBA1-positive area in isotype (gray)– and anti-CD22 (green)–treated iMGLs (n = 7 biological replicates, paired t test, means ± SEM; lines connect wells treated with the same patient’s CSF). (N) Quantification of LAMP2-positive area normalized to total IBA1-positive area in isotype (gray)– and anti-CD22 (green)–treated iMGLs (n = 7 biological replicates, paired t test, means ± SEM; lines connect wells treated with the same patient’s CSF). (O) Heatmap of normalized counts (z score) for differentially expressed genes in WT and I1061T mutant iMGLs treated with NPC CSF and isotype or anti-CD22. (P) Gene Ontology (GO) biological process enrichment analysis of differentially expressed genes between anti-CD22− and isotype–treated I1061T iMGLs. Up- or down-regulation is represented on the color scale, and the number of genes differentially expressed is indicated for each term. IRE1, inositol-requiring enzyme 1; IFN-γ, interferon-γ; UPR, unfolded protein response. (Q) GO cellular component enrichment analysis of differentially expressed genes between anti-CD22− and isotype–treated I1061T iMGLs. Up- or down-regulation is represented on the color scale, and the number of genes differentially expressed is indicated for each term. MHC-II, major histocompatibility complex class II; ER, endoplasmic reticulum.

Article Snippet: For CD22-IGF2R kinetic analysis, anti-human Fc capture biosensors were loaded with recombinant human CD22 Fc-fusion protein (R&D Systems) and associated into recombinant His-tagged human IGF2R analyte at seven different concentrations (1000 nM, serially diluted 1:3).

Techniques: Clone Assay, Binding Assay, Blocking Assay, Flow Cytometry, Concentration Assay, Fluorescence, Microscopy, Control, Mutagenesis, CRISPR, Homologous Recombination, Electroporation, Sequencing, Western Blot, Expressing, Staining, Marker, In Vitro, Derivative Assay, Immunopeptidomics

Journal: Arthritis Research & Therapy

Article Title: TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

doi: 10.1186/ar3294

Figure Lengend Snippet: Dual immunostaining for TWEAK and cell lineage markers and cells expressing Fn14 . Dual immunostaining for TWEAK (red) and CD68 (blue) in inflamed synovial tissue from a patient with active RA ( A ). Dual immunostaining for TWEAK (red) with CD38 (blue) with co-expression of TWEAK and CD38 (purple) indicated by arrow ( B ). C ) and D ) Dual immunostaining for TWEAK (red) with CD22 (blue). E ) TWEAK expression (red) in multinucleated cells (indicated by arrows), and F ) by plasma cells in tonsil tissue. Expression of Fn14 (brown) in multinucleated cells ( G ), and blood vessels of the synovial tissue ( H ), indicated by arrows. Sections shown in E, F, G, and H were counterstained with haematoxylin. Images shown in B and C were obtained with obj ×10; image shown in A obtained with obj ×20, D, F, G, H with obj ×40 and E with obj ×60.

Article Snippet: Anti-TWEAK antibody was combined with MAbs for human cell surface markers: CD68 (macrophage; clone KP-1, Dako), CD22 (B lymphocyte; MAB1968, R&D Systems, Minneapolis, MN, USA), Tryptase G3 (mast cell; Cell Marque, Rocklin, CA, USA) and CD38 (plasma cells, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Immunostaining, Expressing, Clinical Proteomics

Journal: Arthritis Research & Therapy

Article Title: TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis

doi: 10.1186/ar3294

Figure Lengend Snippet: TWEAK expression by PBMC . PBMC from two healthy volunteers were sorted by FACS based on their expression of CD22, yielding CD22 + and CD22 - populations of greater than 94% purity based on post-sort analysis ( A ). Isolated cells were then analysed for TWEAK mRNA expression relative to that of GAPDH, by real-time RT-PCR ( B ). Data shown are means of triplicate reactions ± SD. Differences in relative expression of TWEAK mRNA between CD22 + and CD22 - populations were tested by Student's t -test (** P < 0.001).

Article Snippet: Anti-TWEAK antibody was combined with MAbs for human cell surface markers: CD68 (macrophage; clone KP-1, Dako), CD22 (B lymphocyte; MAB1968, R&D Systems, Minneapolis, MN, USA), Tryptase G3 (mast cell; Cell Marque, Rocklin, CA, USA) and CD38 (plasma cells, BD Biosciences, Franklin Lakes, NJ, USA).

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: Cell

Article Title: Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches

doi: 10.1016/j.cell.2021.12.018

Figure Lengend Snippet:

Article Snippet: Anti-Human CD22 (REA340) APC , Miltenyi Biotec , 130-120-762; RRID: AB_2752186.

Techniques: Purification, Recombinant, Staining, cDNA Synthesis, Gene Expression, Software, Microscopy

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 1A: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 indicated Nalm6 leukemia on day -3, followed by 5e6 CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day 0, as well on days 5 and 11 post-CAR. 1B: Quantification of bioluminescence data in A. 1C: ELISA measuring Granzyme B in supernatant after 16 hour co-culture of CD22-CAR T cells with the indicated leukemia. 1D: Degranulation as measured by CD107a expression after 4 hour co-culture assay. 1E: Activation as measured by CD69 expression after 6 hour co-culture assay. 1F: Activation as measured by CD25 expression after 24 hour co-culture assay. All in vitro assays performed with n=3 technical replicates, 1 experiment. In vivo assay performed with n=5 mice per group, 1 experiment. Data represent mean +/-SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: In Vivo, Injection, Imaging, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Expressing, Co-culture Assay, Activation Assay, In Vitro

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 2A: Flow cytometry plots showing IL-2 by IFNg production after 6 hour coculture of the indicated CD22-CAR T cell with the indicated leukemia. 2B: Quantification of cytokine data in A. 2C: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 indicated Nalm6 leukemia on day -3, followed by 4e6 CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR injection. Mice were monitored for survival. 2D: Quantification of bioluminescence data against WT leukemia from C. 2E: Survival of mice bearing WT leukemia. 2D: Quantification of bioluminescence data against CD22 Lo leukemia from C. 2E: Survival of mice bearing CD22 Lo leukemia. All in vitro assays performed with n=3 technical replicates, and are representative of two experiments with two independent donors. In vivo assay performed with n=5 mice per group, 1 experiment. Data represent mean +/-SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: Flow Cytometry, In Vivo, Injection, Imaging, In Vitro

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: S1A: Cell-based direct antigen-binding affinity titration assay. Indicated CARs were stained with indicated concentrations of fluorophore-conjugated CD22 Protein Fc. MFI of CAR+ Populations were measured and normalized to peak protein binding for each individual CAR. Data represents one experiment with one replicate per concentration.

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: Binding Assay, Titration, Staining, Protein Binding, Concentration Assay

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: Figures S2A to S2F quantify indicated metrics by flow cytometry after coculture of indicated CD22-CAR with indicated leukemia after 6 hour coculture. S2A: %+ and MFI for IFNg production against WT leukemia. S2B: %+ and MFI for IL2 production against WT leukemia. S2C: %+ of cells making IFNg and IL-2 against WT leukemia. S2D: %+ and MFI for IFNg production against CD22 Lo leukemia. S2E: %+ and MFI for IL2 production against CD22 Lo leukemia. S2F: %+ of cells making IFNg and IL-2 against CD22 Lo leukemia. Figures S2G to S2J quantify CAR and leukemia counts relative to a starting 5:1 ratio of leukemia and CAR to fluorescent counting beads. Aliquots were taken from each condition and analyzed by flow cytometry at each of the indicated time points. S2G: Quantification of CAR Count/Bead Count ratio against WT leukemia. S2H: Quantification of Leukemia Count/Bead Count ratio for WT leukemia. S2I: Quantification of CAR Count/Bead Count ratio against CD22 Lo leukemia. S2J: Quantification of Leukemia Count/Bead Count ratio for CD22 Lo leukemia. All in vitro assays performed with n=3 technical replicates. are representative of two experiments with two independent donors. are representative of one experiment. Data represent mean +/- SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: Flow Cytometry, In Vitro

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 3A: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 WT Nalm6 leukemia on day-3, followed by 2e6 of indicated CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR. 3B: Quantification of average bioluminescence data for each group in A. 3C: Quantification of individual bioluminescence data for each group in A. 3D: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 WT Nalm6 leukemia on day -3, followed by 4e6 of indicated CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR. 3E: Quantification of average bioluminescence data for each group in D. 3F: Survival of mice treated with 4e6 of the indicated CAR T cells. In vivo assay performed with n=5 mice per group, 1 experiment (3A to 3C) or 3 experiments with independent donors (3D to 3F). 3D to 3E are representative data from one experiment. 3F is pooled data, SA-SL (n=15), HA-SL (n=15), HA-LL (n=15). Data represent mean +/- SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: In Vivo, Injection, Imaging

Journal: bioRxiv

Article Title: Rational redesign of antigen binding domain improves in vivo efficacy of CD22-CAR T cells

doi: 10.1101/2025.03.13.643183

Figure Lengend Snippet: 4A: Schematic: Timeline for in vivo experiment. NSG mice were injected with 1e6 CD22 Lo Nalm6 leukemia on day -3, followed by 4e6 of indicated CD22-CAR T cells on day 0. Bioluminescent imaging was performed before CAR dosing on day -1, and biweekly post-CAR. 4B: Quantification of average bioluminescence data for each group in A. 5C: Quantification of individual bioluminescence data for each group in A. For 4D to 4E, bone marrow was analyzed by flow cytometry at day 18 post-CAR for indicated cell population. 4D: % CAR+ of live marrow. 4E: % leukemia of live marrow. 4F: Survival of mice treated with 4e6 of indicated CAR T cells. In vivo assay performed with n=5 mice per group, 4 experiments with independent donors. Data in 4A to 4C is representative data from one experiment. Survival is pooled from 3 experiments with independent donors: Mock (n=10), SA-SL (n=15), HA-SL (n=10), HA-LL (n=15). Data represent mean +/- SD. * p<0.05, ** p<0.01, *** p<0.001, ****

Article Snippet: Following expansion, transduction efficiency of the CD22-CAR was evaluated by flow cytometry staining with CD22-Protein Fc (R&D Systems) and CAR T cells were cryopreserved or used immediately for in vitro or in vivo assays.

Techniques: In Vivo, Injection, Imaging, Flow Cytometry