human cd34 cell enriched blood  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Structural surfaceomics reveals an AML-specific conformation of Integrin-β2 as a CAR-T therapy target

doi: 10.1101/2022.10.10.511511

Figure Lengend Snippet: a. Identified cross-linked peptides mapped on to the crystal structure of integrin-αL/integrin-β2 heterodimer (PDB: 5E6R). b. Flow cytometry histogram plot showing expression of total and activated integrin-β2 on AML and B-cell lines (BV and Namlwa). The y-axis represents percent count normalized to mode. Gating strategy shown in . Representative plots from n = 3 independent experiments. c. Flow cytometry plot showing absence of active Integrin-β2 on CD34+ HSPCs from GM-CSF mobilized peripheral blood. Gating strategy shown in . Deidentified patient samples were used for this analysis ( n = 5 independent donors). Representative of 1-2 independent experiments. d. Representative flow cytometry histogram plots showing expression of active Integrin-β2 on primary AML cells. The y-axis represents percent count normalized to mode. Gating strategy shown in . (Representative of n = 10 total deidentified samples, performed in single assay each). e. Heat map showing inverse expression pattern of ITGB2 against other AML targets in publicly available primary AML RNA-seq data. Color bar represents maximum expression in each row based on normalized read counts. Sample size of BEAT AML (adult), TARGET (pediatric) and TCGA were 510, 255 and 150 respectively. f. Aggregated single cell RNA-seq data showing essentially exclusive expression of ITGB2 in hematopoietic tissue, obtained from the Human Protein Atlas .

Article Snippet: Fully de-identified human CD34 cells enriched blood samples were obtained from Bone Marrow and Transplantation Laboratory at UCSF and were MACS sorted using CD34 MicroBead Kit (Miltenyi Biotec, 130-046-702) and incubated with CD3 antibody (Biolegend, 317302, clone – OKT3) for T cell depletion10 minutes prior to injection in mice, to limit any possible development of graft-vs-host disease.

Techniques: Flow Cytometry, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: Structural surfaceomics reveals an AML-specific conformation of Integrin-β2 as a CAR-T therapy target

doi: 10.1101/2022.10.10.511511

Figure Lengend Snippet: a. Cartoon diagram showing proposed inactive and active conformations of ITGB2. b. Flow cytometry gating strategy for ( , , ). c. Flow cytometry plot showing presence of total Integrin-β2 on CD34+ HSPCs from GM-CSF mobilized peripheral blood. Cells were gated on singlet cells for analysis. Deidentified patient samples were used for this analysis ( n = 5, independent donors). Representative of 1-2 independent experiments. d. Flow cytometry gating strategy for (c) and . e. Flow cytometry gating strategy for . f. Flow cytometry analysis showing expression of active ITGB2 in PDX models of AML (PDX-A and PDX-B). The y-axis represents percent count normalized to mode. Cells were gated on human CD45+ population cells for analysis. Representative plot from n = 2 separate PDX models of AML. g. Flow cytometry gating strategy for (f).

Article Snippet: Fully de-identified human CD34 cells enriched blood samples were obtained from Bone Marrow and Transplantation Laboratory at UCSF and were MACS sorted using CD34 MicroBead Kit (Miltenyi Biotec, 130-046-702) and incubated with CD3 antibody (Biolegend, 317302, clone – OKT3) for T cell depletion10 minutes prior to injection in mice, to limit any possible development of graft-vs-host disease.

Techniques: Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Structural surfaceomics reveals an AML-specific conformation of Integrin-β2 as a CAR-T therapy target

doi: 10.1101/2022.10.10.511511

Figure Lengend Snippet: a. Representative flow cytometry-based cytotoxicity assay showing specificity of aITGB2 CAR-T against activated peripheral blood T cells which harbors activated integrin-β2 (focus on lower right quadrant, with CAR-negative, CD3-positive T-cells). Both resting and activated conditions performed in overnight co-culture assays with aITGB2 CAR-T cells. (Gating strategy similar to shown .) b. Representative flow cytometry analysis showing successful activation of T cells and partial abundance of activated integrin-β2 on activated T cells. (Gating strategy similar to shown in .) c. Quantitative analysis of active T-cell depletion data in (a); n = 3 technical replicates. d. Representative flow cytometry analysis showing no discernible impact of aITGB2 CAR-T against CD34+ HSPCs from GM-CSF mobilized peripheral blood. The y-axis represents percent count normalized to mode. (Gating strategy similar to that shown in ) (n = 1 donor) and similarly for e. T cells and B cells. (Gating strategy similar to shown in .) (n = 3 technical replicates and representative of 2 independent experiments). Also see . f. Schematic flow diagram for generation of humanized immune system (HIS) mice. g. Representative flow cytometry data from HIS mice data showing apparent non-toxicity of aITGB2 CAR-T against myeloid cells (CD14+). All events were used for gating and analysis. (Representative plot from n = 4 – 6 mice and 6 days post CAR-T treatment). h. Quantification of hCD45+ data in (g). Gating strategy similar to shown . p -value by two-tail t -test. *** p < 0.005. i. Complete blood count profiling of HIS mice treated with aITGB2 CAR-T at day 5 (data from n = 4 mice). All the statistical data in this figure are represented as mean ± SEM. For all the in vitro cytotoxicity assays, E:T ratio was 1:1 with overnight incubation time.

Article Snippet: Fully de-identified human CD34 cells enriched blood samples were obtained from Bone Marrow and Transplantation Laboratory at UCSF and were MACS sorted using CD34 MicroBead Kit (Miltenyi Biotec, 130-046-702) and incubated with CD3 antibody (Biolegend, 317302, clone – OKT3) for T cell depletion10 minutes prior to injection in mice, to limit any possible development of graft-vs-host disease.

Techniques: Flow Cytometry, Cytotoxicity Assay, Co-Culture Assay, Activation Assay, In Vitro, Incubation

Journal: Clinical and Translational Medicine

Article Title: Direct targeted therapy for MLL‐fusion‐driven high‐risk acute leukaemias

doi: 10.1002/ctm2.933

Figure Lengend Snippet: Effects of MLL‐fusion depletion by disulfiram (DSF) treatment. (A) Effects of DSF–Cu on target gene expression in MLL ‐rearranged cells. The expressions of HOXA10 , MEIS1 and MYB were examined after 16 h with .3‐μM DSF or .6‐μM diethyldithiocarbamate (DDC) in the presence of 1 μM of Cu treatment through RT‐quantitative PCR (qPCR). The relative expression was normalized to 18 s. Bar charts are mean ± SD ( t ‐test, * p < .05, ** p < .01, *** p < .001) from three independent experiments. (B) CFU assay of acute myeloid leukaemia (AML) or CD34 + cord blood cells treated with DMSO or .3‐μM DSF in the presence of 1 μM of Cu. Data represent the mean of three independent experiments. Bar charts are mean ± SD (C) CFU assay of two MLL ‐rearranged patient–derived xenograft samples treated with DMSO or .3‐μM DSF in the presence of 1 μM of Cu. Data represent the mean of two independent patient samples repeated twice. Bar charts are mean ± SD and t ‐test performed. (D) Anti‐proliferative activity of DSF and DDC against MLL ‐rearranged AML. Cells were exposed to DMSO, DSF (.3 μM) or DDC (.6 μM) supplemented with 1 μM of Cu for periods varying from 0 to 72 h. Data illustrate a representative experiment of three independent replicates. Error bars are ±SD (E) western blot analysis of DSF effect on patient derived ALL MLL‐AF9 cells. MLL‐AF9 ALL cells were treated with .3‐μM DSF and 1‐μM Cu for 16 h. The proteins were detected using antibodies against N‐terminal MLL and vinculin

Article Snippet: CD34 + cord blood–derived cells were plated and cultured in HSC005 (R&D system) for 2 weeks.

Techniques: Targeted Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Colony-forming Unit Assay, Derivative Assay, Activity Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: Direct targeted therapy for MLL‐fusion‐driven high‐risk acute leukaemias

doi: 10.1002/ctm2.933

Figure Lengend Snippet: Disulfiram (DSF) blocks leukaemic stem cell activity and reduces leukaemic burden in vivo. (A) DSF treatment blocks the engraftment of MLL ‐rearranged leukaemia. A million MLL‐AF9 rearranged THP‐1‐luciferase cells either treated with DSF/Cu or vehicle control for 16 h were injected intravenous (i.v.) into NSG mice. Kaplan–Meier survival curves in each group as indicated ( n = 6 for Con and n = 4 for DSF). (B) Outline of treatment of MLL ‐rearranged acute myeloid leukaemia (AML) with DSF. NSG mice ( n = 6 mice/group) were either orally or intraperitoneal (i.p.) treated with DSF or vehicle 10 days after i.v. injection of THP‐1‐luciferase cells (5 × 10 5 cells/mouse). DSF was subsequently administered 5 days/week. Leukaemia engraftment was tracked by serial luciferase measurements. Examples and quantification of leukaemic burden shows a statistically significant difference for both oral and i.p. administration of DSF as indicated (* p < .05; ** p < .001). (C) C57BL/6J mice ( n = 5 mice/group) were either i.p. treated with DSF or vehicle 2 days after i.v. injection of murine MLL–ENL overexpressing leukaemia (1 × 10 5 cells/mouse). DSF was subsequently administered for 5 days and CD11b expression was assessed by flow cytometry. Example and quantification show a statistically significant up‐regulation of CD11b expression ( p < .001).

Article Snippet: CD34 + cord blood–derived cells were plated and cultured in HSC005 (R&D system) for 2 weeks.

Techniques: Activity Assay, In Vivo, Luciferase, Control, Injection, Expressing, Flow Cytometry