Journal: bioRxiv

Article Title: A Digital Microfluidic Platform for the Microscale Production of Functional Immune Cell Therapies

doi: 10.1101/2024.09.03.611092

Figure Lengend Snippet: a) Overview of immunotherapeutic testing assay detailing an anti-CD19 CAR T cell interacting with a CD19 negative or positive tumor cell line. Created with BioRender.com. b) Fluorescence intensity histograms showing FITC expressions for cells stained with a FITC-tagged CD19 protein from a control (grey), triDrop (green), Nucleofection (blue), and Neon (red). c) Line graphs depicting expression of an anti-CD19 CAR molecule at 6, 24, and 72 hours post EP when using the triDrop (green, 100ng of mRNA), Neon (red, 500ng of mRNA), and Nucleofection (blue, 1000ng of mRNA). Bar graphs depicting measured levels of d) TNF-α and e) IFN-γ after 24 hours (light purple and light blue) and 48 hours (dark purple and dark blue) of culture for cells electroporated using all three systems using either manufacturer recommended conditions or 0.5 x 10 cells per reaction. Engineered cells are either cultured by themselves (T), or at a 1:1 ratio with MCF-7 cells (T/M), or with Raji cells (T/R). (n = 6) f) Line graphs depicting relative killing of Raji cells cocultured with activated Pan T cells at 1:1 ratio and 4:1 ratio after 24 hours (light grey, solid line) and 48 hours (dark grey, dashed line) or with triDrop engineered CAR T cells at a 1:1 and 4:1 ratio after 24 hours (light green, solid line), and 48 hours (dark green, dashed line). Statistical significance markers indicate difference between engineered cells and activated pan T cells for each timepoint. (n = 4). All error bars represent mean +/- SD. n.s indicated no significant difference, *, **,***, and **** represent p-values below 0.05, 0.01, 0.001 and 0.0001 respectively. Statistical analysis was performed using a student’s t-test.

Article Snippet: CD19 expression was detected using a FITC Mouse anti-Human CD19 antibody (BD Bioscience, catalog #560994).

Techniques: Fluorescence, Staining, Control, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Patient-tailored design of AML cell subpopulation-selective drug combinations

doi: 10.1101/2020.07.28.222034

Figure Lengend Snippet: Gating strategy for the high-throughput flow cytometry (HTFC) assay. Live cells were gated as cells negative to 7-AAD and Annexin V markers. Leukemic stem cells were gated as CD34+ CD38-population. Lymphocytes were gated based on CD56 and CD3/CD19 to distinguish NK as well as T and B cell populations, respectively.

Article Snippet: The cells were washed with a cell staining buffer (PBS with 2% fetal bovine serum), centrifuged at 600× g for 5 min. To profile cell subpopulation responses, the cells were stained with BV605 Mouse Anti-Human CD56, BV421 Mouse Anti-Human CD19, BV421 Mouse Anti-Human CD3, FITC Mouse Anti-Human CD45, APC Mouse Anti-Human CD34, BV786 Mouse Anti-Human CD38 antibodies (all antibodies from BD Biosciences) for 30 min at room temperature in the dark.

Techniques: High Throughput Screening Assay, Flow Cytometry

Journal: Cell Transplantation

Article Title: Persistent Cytopenia After CD19 CAR T Therapy in Relapsed/Refractory DLBCL Patients Could Be a Predictor of Efficacy and Side Effects

doi: 10.1177/09636897241247951

Figure Lengend Snippet: Hematological toxicity of anti-CD19 CAR T cell therapy. (A) Grades 0–4 neutropenia, anemia, and thrombocytopenia were diagnosed after anti-CD19 CAR T cell therapy. (B) The different types and persistent times of cytopenia in the 14 patients who had Grades 3–4 persistent cytopenia. (C–E) Blood cell changes in patients with Grades 3–4 neutropenia, anemia, and thrombocytopenia. CAR: chimeric antigen receptor.

Article Snippet: Humanized CD19 CAR antibodies were provided by GenBase Biotechnology (patent owner of CD19 CAR).

Techniques:

Journal: Cell Transplantation

Article Title: Persistent Cytopenia After CD19 CAR T Therapy in Relapsed/Refractory DLBCL Patients Could Be a Predictor of Efficacy and Side Effects

doi: 10.1177/09636897241247951

Figure Lengend Snippet: (A) All 14 patients with Grades 3–4 persistent cytopenia obtained CR and PR, whereas no patient who obtained SD and PD had cytopenia during CAR T cell therapy. (B) The clinical response to anti-CD19 CAR T cell therapy, PD, and survival time of all 38 patients. (C) The incidence of Grades 3–4 persistent cytopenia was higher in 26 patients who obtained ORR with high tumor load than in patients with lower tumor loads. CAR: chimeric antigen receptor; CR: complete response; PR: partial remission; SD: stable disease; PD: disease progression; ORR: objective response rate.

Article Snippet: Humanized CD19 CAR antibodies were provided by GenBase Biotechnology (patent owner of CD19 CAR).

Techniques:

Journal: Cell Transplantation

Article Title: Persistent Cytopenia After CD19 CAR T Therapy in Relapsed/Refractory DLBCL Patients Could Be a Predictor of Efficacy and Side Effects

doi: 10.1177/09636897241247951

Figure Lengend Snippet: Adverse Events During Anti-CD19 CAR T Cell Therapy.

Article Snippet: Humanized CD19 CAR antibodies were provided by GenBase Biotechnology (patent owner of CD19 CAR).

Techniques:

Journal: Cell Transplantation

Article Title: Persistent Cytopenia After CD19 CAR T Therapy in Relapsed/Refractory DLBCL Patients Could Be a Predictor of Efficacy and Side Effects

doi: 10.1177/09636897241247951

Figure Lengend Snippet: Adverse events of anti-CD19 CAR T cell therapy. (A) The mean peak of IL-6 levels in patients with Grades 3–4 persistent cytopenia was higher than in patients without Grades 3–4 persistent cytopenia. (B) CRS was higher in patients with Grades 3–4 persistent cytopenia than in patients without persistent cytopenia. (C) No difference was observed in Grades 3–4 persistent cytopenia between different ICANS grades. (D) The median amplification peak of CAR T cells was higher in patients with Grades 3–4 persistent cytopenia than in patients without persistent cytopenia. (E, F) Most patients whose CAR T cells were observed at 21 and 28 days after CAR T cell infusion had Grades 3–4 persistent cytopenia. CAR: chimeric antigen receptor; IL: interleukin; CRS: cytokine release syndrome; ICANS: immune effector cell-associated neurotoxic syndrome.

Article Snippet: Humanized CD19 CAR antibodies were provided by GenBase Biotechnology (patent owner of CD19 CAR).

Techniques: Amplification

Journal: Cell Transplantation

Article Title: Persistent Cytopenia After CD19 CAR T Therapy in Relapsed/Refractory DLBCL Patients Could Be a Predictor of Efficacy and Side Effects

doi: 10.1177/09636897241247951

Figure Lengend Snippet: Follow-up after anti-CD19 CAR T cell therapy. (A, B) The progression-free and overall survival were higher in patients with Grades 3–4 persistent cytopenia than in those who did not have persistent cytopenia. CAR: chimeric antigen receptor.

Article Snippet: Humanized CD19 CAR antibodies were provided by GenBase Biotechnology (patent owner of CD19 CAR).

Techniques:

Journal: eLife

Article Title: IFNγ induces epigenetic programming of human T-bethi B cells and promotes TLR7/8 and IL-21 induced differentiation

doi: 10.7554/elife.41641

Figure Lengend Snippet: Figure 2. ASC development from BN precursors is enhanced in Th1 containing co-cultures. Cartoon (a) depicting day 6 paired co-cultures containing Th1 (Be1 co-cultures) or Th2 (Be2 co-cultures) effectors generated from the same HD, BN cells from a second allogeneic HD and exogenous IL-21 and IL-2. Flow cytometric analysis showing T-bet expression (b) on gated HD B cells (non-ASCs) from Be1 and Be2 co-cultures. Phenotyping (c) of day 6 B cell-gated Be1 cells showing T-bet expression in combination with other surface markers. (d–g) ASC development in HD day 6 paired Be1 and Be2 co-cultures showing representative flow plots (d) and frequencies (e) of CD38hiCD27+ ASCs in CD19+/lo-gated B lineage cells. Frequencies of IgM+ (f) or IgG+ (g) ASCs in day 6 paired Be1 and Be2 co-cultures. See Figure 2—figure supplement 1 for BN isolation strategy and characterization of polarized Th1 and Th2 effectors. See Figure 2—figure supplement 2 for gating strategy to identify IgG+ and IgM+ ASCs. See Figure 2—figure supplement 3 for proliferation analysis of B cells in paired day 6 HD Be1 and Be2 co-cultures. Analyses in (b–c) are from representative co-cultures (n > 30). Experiments (e–g) performed on 15 (e), 8 (f) or 6 (g) independent paired Be1 and Be2 co-cultures. Statistical analyses were performed using a non- parametric Wilcoxon paired t test (e) or paired Student’s t test (f–g). P values *<0.05, **<0.01, ****<0.0001. DOI: https://doi.org/10.7554/eLife.41641.005 The following figure supplements are available for figure 2:

Article Snippet: DOI: https://doi.org/10.7554/eLife.41641 23 of 36 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody PercP/Cy5.5 Mouse Anti-Human CD4 (clone OKT4) eBioscience 45-0048-42 (1:200) Antibody BV510 Mouse Anti-Human CD4 (clone OKT4) Biolegend 317444 (1:100) Antibody Fitc Mouse Anti-Human CD11c (clone Bu15) Biolegend 337214 (1:200) Antibody PE Mouse Anti-Human CD11c (clone Bu15) Biolegend 337205 (1:400) Antibody PercP/Cy5.5 Mouse Anti-Human CD14 (clone HCD14) Biolegend 325621 (1:200) Antibody Fitc Mouse Anti-Human CD19 (clone LT19) Miltenyi 302256 (1:100) Antibody PE Mouse Anti-Human CD19 (clone HIB19) Biolegend 302208 (1:200) Antibody PercP/Cy5.5 Mouse Anti-Human CD19 (clone HIB19) Biolegend 302230 (1:100) Antibody APC Mouse Anti-Human CD19 (clone HIB19) BD Pharmingen 555415 (1:200) Antibody APC-H7 Mouse Anti-Human CD19 (clone HIB19) BD Pharmingen 560727 (1:100) Antibody BV421 Mouse Anti-Human CD19 (clone HIB19) Biolegend 302234 (1:200) Antibody V500 Mouse Anti-Human CD19 (clone HIB19) BD Horizon 561121 (1:100) Antibody PercP/Cy5.5 Mouse Anti-Human CD21 (clone Bu32) Biolegend 354908 (1:100) Antibody Fitc Mouse Anti-Human CD23 (clone M-L23.4) Miltenyi 130-099-365 (1:100) Antibody PE Mouse Anti-Human CD23 (clone EBVCS-5) Biolegend 338507 (1:200) Antibody APC Mouse Anti-Human CD23 (clone M-L233) BD Pharmingen 558690 (1:200) Antibody Fitc Mouse Anti-Human CD27 (clone M-T271) Biolegend 356404 (1:100) Antibody PercP/Cy5.5 Mouse Anti-Human CD27 (clone M-T271) Biolegend 356408 (1:100) Antibody APC Mouse Anti-Human CD27 (clone M-T271) Biolegend 356410 (1:200) Continued on next page Zumaquero et al. eLife 2019;8:e41641.

Techniques: Generated, Expressing, Isolation

Journal: Turkish Journal of Hematology

Article Title: Cytokine Contents in Chronic Lymphocytic Leukemia: Association with ZAP70 Expression

doi: 10.4274/tjh.2014.0469

Figure Lengend Snippet: Gating strategy of chronic lymphocytic leukemia. Peripheral blood mononuclear cells from chronic lymphocytic leukemia patients (n=28) were stained for CD5, CD19, CD38, and ZAP70 mAbs and analyzed by flow cytometry. CD19+ cells were gated versus SSC (A). Representative dot-plot analyses for the expression of a patient negative (B) and positive (C) for CD38 with CD5+ in CD19+ cells are shown. In the CD19+ B cell population, CD5+ZAP70+ (D, E) and CD5+ZAP70- (F) plots from three different patients with chronic lymphocytic leukemia are also indicated. The numbers indicate the proportion of cells positive for indicated markers.

Article Snippet: Heparinized blood samples were collected from patient and healthy donors and stained with anti-CD19-fluorescein isothiocyanate (FITC), anti-CD3-allophycocyanin (APC), anti-CD5-TRI-COLOR (TC), anti-CD45-FITC, anti-CD14-phycoerythrin (PE), anti-CD23-PE, anti-CD38-PE, and PE, FITC, TC, or APC conjugated isotype control (IC) monoclonal antibodies (mAbs) (all from Caltag Laboratories, Austria) for 30 min at room temperature.

Techniques: Staining, Flow Cytometry, Expressing