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Transduction efficiencies and phenotype differences between expanded cells (A) Transduction efficiencies for <t>pepmix-stimulated</t> vs. anti-CD3/CD28 dynabeads-stimulated T cells for bulk CD3+, CD4+, and CD8+ cells, respectively; n = 6, shown are medians with range. (B) CD4 vs. CD8 proportions within different populations of expanded pepmix-stimulated T cells; n = 6, shown are means with SD. (C) Memory phenotypes and (D) exhaustion marker expression of expanded pepmix-stimulated T cells among wild-type (WT), RNP-only transfected and transduced bulk CD3+, CD4+, and CD8+ cells; n = 4. For (B)–(D), asterisks represent statistically significant differences (∗p < 0.05, ∗∗p < 0.005, two-way ANOVA, Tukey’s multiple comparisons test). ANOVA, analysis of variance; <t>EBV,</t> Epstein-Barr virus; RNP, ribonucleoprotein; AAV, adeno-associated virus; Temra, terminally differentiated; Tem, effector memory; Tcm, central memory; Tscm, stem cell memory T cells.
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Transduction efficiencies and phenotype differences between expanded cells (A) Transduction efficiencies for pepmix-stimulated vs. anti-CD3/CD28 dynabeads-stimulated T cells for bulk CD3+, CD4+, and CD8+ cells, respectively; n = 6, shown are medians with range. (B) CD4 vs. CD8 proportions within different populations of expanded pepmix-stimulated T cells; n = 6, shown are means with SD. (C) Memory phenotypes and (D) exhaustion marker expression of expanded pepmix-stimulated T cells among wild-type (WT), RNP-only transfected and transduced bulk CD3+, CD4+, and CD8+ cells; n = 4. For (B)–(D), asterisks represent statistically significant differences (∗p < 0.05, ∗∗p < 0.005, two-way ANOVA, Tukey’s multiple comparisons test). ANOVA, analysis of variance; EBV, Epstein-Barr virus; RNP, ribonucleoprotein; AAV, adeno-associated virus; Temra, terminally differentiated; Tem, effector memory; Tcm, central memory; Tscm, stem cell memory T cells.

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A method for polyclonal antigen-specific T cell-targeted genome editing (TarGET) for adoptive cell transfer applications

doi: 10.1016/j.omtm.2023.06.007

Figure Lengend Snippet: Transduction efficiencies and phenotype differences between expanded cells (A) Transduction efficiencies for pepmix-stimulated vs. anti-CD3/CD28 dynabeads-stimulated T cells for bulk CD3+, CD4+, and CD8+ cells, respectively; n = 6, shown are medians with range. (B) CD4 vs. CD8 proportions within different populations of expanded pepmix-stimulated T cells; n = 6, shown are means with SD. (C) Memory phenotypes and (D) exhaustion marker expression of expanded pepmix-stimulated T cells among wild-type (WT), RNP-only transfected and transduced bulk CD3+, CD4+, and CD8+ cells; n = 4. For (B)–(D), asterisks represent statistically significant differences (∗p < 0.05, ∗∗p < 0.005, two-way ANOVA, Tukey’s multiple comparisons test). ANOVA, analysis of variance; EBV, Epstein-Barr virus; RNP, ribonucleoprotein; AAV, adeno-associated virus; Temra, terminally differentiated; Tem, effector memory; Tcm, central memory; Tscm, stem cell memory T cells.

Article Snippet: PBMCs were stimulated with either anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s instructions or with EBV pepmix (PepTivator EBV Consensus peptide pool [Miltenyi Biotec, Bergisch Gladbach, Germany]), at a final concentration of 60 pmol/peptide/mL in CTLm supplemented with 400 U/mL IL-4 and 10 ng/mL IL-7 (R&D Systems, Minneapolis, MN) for 3 days.

Techniques: Transduction, Marker, Expressing, Transfection, Virus

Specificity and functionality of pepmix-stimulated and expanded transduced T cells (A) Production of cytotoxicity markers and cytokines (CD107a, Granzyme B, IFNγ, and TNFα) among bulk CD3+, CD4+, and CD8+ populations in response to EBV-pepmix-restimulation, n = 4, means with standard deviation (SD). Asterisks represent statistically significant differences (∗p < 0.05, ∗∗p < 0.005, two-way ANOVA, Tukey’s multiple comparisons test). (B) Six-hour cytotoxicity assay against autologous EBV-transformed LCLs (effector/target = 20:1), means of triplicates with SD for three donors, two-way ANOVA mixed effects analysis, ∗∗ = 0.0043, ∗ = 0.0405, α = 0.05. ANOVA, analysis of variance; EBV, Epstein-Barr virus; WT, wild type; RNP, ribonucleoprotein; AAV, adeno-associated virus; GrB, granzyme B.

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A method for polyclonal antigen-specific T cell-targeted genome editing (TarGET) for adoptive cell transfer applications

doi: 10.1016/j.omtm.2023.06.007

Figure Lengend Snippet: Specificity and functionality of pepmix-stimulated and expanded transduced T cells (A) Production of cytotoxicity markers and cytokines (CD107a, Granzyme B, IFNγ, and TNFα) among bulk CD3+, CD4+, and CD8+ populations in response to EBV-pepmix-restimulation, n = 4, means with standard deviation (SD). Asterisks represent statistically significant differences (∗p < 0.05, ∗∗p < 0.005, two-way ANOVA, Tukey’s multiple comparisons test). (B) Six-hour cytotoxicity assay against autologous EBV-transformed LCLs (effector/target = 20:1), means of triplicates with SD for three donors, two-way ANOVA mixed effects analysis, ∗∗ = 0.0043, ∗ = 0.0405, α = 0.05. ANOVA, analysis of variance; EBV, Epstein-Barr virus; WT, wild type; RNP, ribonucleoprotein; AAV, adeno-associated virus; GrB, granzyme B.

Article Snippet: PBMCs were stimulated with either anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific) according to the manufacturer’s instructions or with EBV pepmix (PepTivator EBV Consensus peptide pool [Miltenyi Biotec, Bergisch Gladbach, Germany]), at a final concentration of 60 pmol/peptide/mL in CTLm supplemented with 400 U/mL IL-4 and 10 ng/mL IL-7 (R&D Systems, Minneapolis, MN) for 3 days.

Techniques: Standard Deviation, Cytotoxicity Assay, Transformation Assay, Virus