cd19 variants  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28

doi: 10.3389/fimmu.2021.639818

Figure Lengend Snippet: Anti-CD28 stimulation of CD19-chimeric antigen receptors (CAR) T cells is TMD dependent. (A) Designs of five CAR against CD19 bearing a 4-1BB costimulatory domain and differing by their hinge domain (HD) and their transmembrane domain (TMD). (B) Fluorescence-activated cell sorting (FACS) -sorted CD4 + CD127 + CD25 low T cells were electroporated with a CRISPR-Cas9 ribonucleoprotein complex (RNP) targeting the constant region of the TCR β chain gene (TRBC), followed by stimulation with anti-CD3/CD28 beads (1:1 ratio). (C) Representative results of flow cytometric analysis of the CD3 expression over time of cells electroporated with or without RNP. Percentages of residual CD3 + population and fold-expansion after 9 days of culture of CD4 + T cells electroporated with or without RNPs targeting TRBC are shown. Results from four independent experiments. (D) A representative example of carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution of a mixed population of CD3 +/− CAR +/− T restimulated with anti-CD3/28 beads. (E) The normalized CFSE mean fluorescence intensity (MFI) ratio for CD3 − mCherry + , CD3 + mCherry − , and CD3 + mCherry + cells was calculated by dividing CFSE MFI of these populations with the MFI of the CD3 − mCherry − cells in the same culture. Two-way ANOVA was used for statistical analysis (bold line set as reference). (F) Percentages of CD3 + and mCherry + cells before and 5 days after restimulation of edited T cells with anti-CD3/CD28 beads. The Unpaired t -test was performed by comparing CD8-TMD and CD28-TMD-containing CARs on D14. For (E,F) , the results shown are a summary of two independent experiments using T cells from five unrelated donors for each construct. *** p < 0.001.

Article Snippet: For generating CD19 − variants of Raji cells, Raji cells (ATCC® CCL-86TM, Manassas, VA) were electroporated (pulse code EH140) with RNP targeting CD19 , and the CD19- negative fraction was purified by FACS after culturing the cells for more than 1 week.

Techniques: Fluorescence, FACS, CRISPR, Expressing, Construct

Journal: Frontiers in Immunology

Article Title: The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28

doi: 10.3389/fimmu.2021.639818

Figure Lengend Snippet: CAR-CD28 heterodimers are B7-unresponsive. (A) A representative example showing CD71 upregulation in CAR T cells containing an IgG 4 -HD/CD28-TMD co-cultured for 48 h with irradiated (4,000 Rad) CD19-wild type or deficient Raji cells with or without CTLA-4 Ig. (B) CD25 + CD71 + T cells were analyzed in low, intermediate (int), or high mCherry-expressing CAR T cells using the gating strategy described in . Data were pooled from four independent experiments using T cells from four to five unrelated donors. The two-way ANOVA was used for statistical analysis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For generating CD19 − variants of Raji cells, Raji cells (ATCC® CCL-86TM, Manassas, VA) were electroporated (pulse code EH140) with RNP targeting CD19 , and the CD19- negative fraction was purified by FACS after culturing the cells for more than 1 week.

Techniques: Cell Culture, Irradiation, Expressing

Journal: Frontiers in Immunology

Article Title: The CD28-Transmembrane Domain Mediates Chimeric Antigen Receptor Heterodimerization With CD28

doi: 10.3389/fimmu.2021.639818

Figure Lengend Snippet: CAR-CD28 heterodimers reduce CD28 expression. (A) Editing strategy and homology-directed repair-mediated integration into the TRAC locus of various CD19 CARs using an AAV-6 transduction protocol. (B) The expression of Myc and CD28 in a representative example was analyzed 6 days after editing and the removal of beads. (C) The CD28 MFI ratio was calculated by dividing CD28 MFI of Myc + cells by Myc − cells in the same culture. Pooled data from three to four independent experiments across five unrelated donors are shown. Each dot represents one independent editing condition. The two-way ANOVA was used for statistical analysis. *** p < 0.001.

Article Snippet: For generating CD19 − variants of Raji cells, Raji cells (ATCC® CCL-86TM, Manassas, VA) were electroporated (pulse code EH140) with RNP targeting CD19 , and the CD19- negative fraction was purified by FACS after culturing the cells for more than 1 week.

Techniques: Expressing, Transduction

Journal: Cytotechnology

Article Title: Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

doi: 10.1007/s10616-020-00433-4

Figure Lengend Snippet: Agarose gel electrophoresis of SC, OC and linear forms of four plasmids. Lane 1, native plasmid DNA of VSVG; lane 2, 7-day treatment at 50 °C for plasmid DNA of VSVG; lane 3, a mixture of native VSVG plasmid DNA and heat-treated VSVG plasmid DNA at ratio of 1:1; lane 4, native plasmid DNA of MDK; lane 5, 7-day treatment at 50 °C for plasmid DNA of MDK; lane 6, a mixture of native MDK plasmid DNA and heat-treated MDK plasmid DNA at ratio of 1:1; lane 7, native plasmid DNA of RSK; lane 8, 7-day treatment at 50 °C for plasmid DNA of RSK; lane 9, a mixture of native RSK plasmid DNA and heat-treated RSK plasmid DNA at ratio of 1:1; lane 10, native plasmid DNA of pLenti6.3/V5-CD19 CAR; lane 11, 7-day treatment at 50 °C for plasmid DNA of pLenti6.3/V5-CD19 CAR; lane 12, a mixture of native pLenti6.3/V5-CD19 CAR plasmid DNA and heat-treated pLenti6.3/V5-CD19 CAR plasmid DNA at ratio of 1:1

Article Snippet: Forty-eight hours post transduction, the CAR expressing cells were stained with a phycoerythrin-labeled antibody that recognized the single chain fragment variant of the CD19 CAR (Immunochina Pharmaceuticals, Beijing, China), and analyzed using fluorescent activated cell sorting assay.

Techniques: Agarose Gel Electrophoresis, Plasmid Preparation

Journal: Cytotechnology

Article Title: Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

doi: 10.1007/s10616-020-00433-4

Figure Lengend Snippet: Groups with different SC proportion of plasmid DNA

Article Snippet: Forty-eight hours post transduction, the CAR expressing cells were stained with a phycoerythrin-labeled antibody that recognized the single chain fragment variant of the CD19 CAR (Immunochina Pharmaceuticals, Beijing, China), and analyzed using fluorescent activated cell sorting assay.

Techniques: Plasmid Preparation, Concentration Assay

Journal: Cytotechnology

Article Title: Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

doi: 10.1007/s10616-020-00433-4

Figure Lengend Snippet: Agarose gel electrophoresis of SC, OC and linear forms of four plasmids after heat or enzyme treatment. Lane 1, native plasmid DNA of RSK; lane 2, 7-day treatment at 50 °C for plasmid DNA of RSK; lane 3, 10-day treatment at 50 °C for plasmid DNA of RSK; lane 4, RSK plasmid DNA was digested with Xho I; lane 5, native plasmid DNA of VSVG; lane 6, 7-day treatment at 50 °C for plasmid DNA of VSVG; lane 7, 10-day treatment at 50 °C for plasmid DNA of VSVG; lane 8, VSVG plasmid DNA was digested with Hand III; lane 9, native plasmid DNA of MDK; lane 10, 7-day treatment at 50 °C for plasmid DNA of MDK; lane 11, 10-day treatment at 50 °C for plasmid DNA of MDK; lane 12, MDK plasmid DNA was digested with Mun I; lane 13, native plasmid DNA of pLenti6.3/V5-CD19 CAR; lane 14, 7-day treatment at 50 °C for plasmid DNA of pLenti6.3/V5-CD19 CAR; lane 15, 10-day treatment at 50 °C for plasmid DNA of pLenti6.3/V5-CD19 CAR; lane 16, pLenti6.3/V5-CD19 CAR plasmid DNA digested with Sal I

Article Snippet: Forty-eight hours post transduction, the CAR expressing cells were stained with a phycoerythrin-labeled antibody that recognized the single chain fragment variant of the CD19 CAR (Immunochina Pharmaceuticals, Beijing, China), and analyzed using fluorescent activated cell sorting assay.

Techniques: Agarose Gel Electrophoresis, Plasmid Preparation

Journal: Cytotechnology

Article Title: Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

doi: 10.1007/s10616-020-00433-4

Figure Lengend Snippet: The information of four plasmids for lentivirus packaging

Article Snippet: Forty-eight hours post transduction, the CAR expressing cells were stained with a phycoerythrin-labeled antibody that recognized the single chain fragment variant of the CD19 CAR (Immunochina Pharmaceuticals, Beijing, China), and analyzed using fluorescent activated cell sorting assay.

Techniques: Plasmid Preparation