Tab. 1

Cytokine concentrations used in the cytokine screening experiment"

Fig. 1

The combination of IL-2, IL-7 and IL-15 enhances the expansion capacity and cytotoxic phenotype of γδ T cells while reducing exhaustion A: Flowchart of the γδ T cell production process. B: Purity of γδ T cells measured by flow cytometry on day 14 (n=3 donors). C: Summary of 7-AAD- live cells at day 14 (n=3 donors). D: Fold expansion of CD3+Vδ2+ T cells from day 0 to day 14 (n=3 donors). E: Fold expansion of CD3+Vδ2+ T cells on day 14 (n=3 donors). F: Summary of flow cytometry results comparing the expansion composition on day 14 after culture with IL-2+IL-7, IL-2+IL-15, IL-2+IL-7+IL-15, or IL-2 alone (n=3 donors). G-J: Expression levels of cytotoxicity markers CD56 (G), NKG2D (H), CD16 (I), and the exhaustion marker PD-1 (J) on γδ T cells (n=3 donors). Data are presented as $\bar{x} \pm s$. *: P<0.05; ***: P<0.001; ****: P<0.000 1. NS: Not significant. Statistical significance was determined by ANOVA."

Tab. 2

Summary of expansion data from cytokine combination screening"

Tab. 3

Summary of cell expansion data from IL-7 and IL-15 cytokine concentration optimization experiments"

Tab. 4

Summary of cytotoxicity and exhaustion phenotype data from IL-7 and IL-15 cytokine concentration optimization experiments"

Fig. 2

The concentration combination of IL-2+IL-7 (10 ng/mL)+IL-15 (10 ng/mL) significantly enhances the expansion capacity and cytotoxic phenotype of γδ T cells while reducing exhaustion A: Purity of γδ T cells measured by flow cytometry on day 14 (n=3 donors). B: Fold expansion of CD3+Vδ2+ T cells on day 14 (n=3 donors). C: Summary of 7-AAD- live cells at day 14 (n=3 donors). D, E: Representative flow cytometry plots of CD16+ and PD-1+ γδ T cells on day 14. F,G: Expression levels of cytotoxicity markers CD16+ and exhaustion marker PD-1+ γδ T cells on day 14. Data are presented as $\bar{x} \pm s$. *: P<0.05; **: P <0.01. NS: Not significant. Statistical significance was as determined by ANOVA (A,B) or Student’s t-test (C, F, G)."

Fig. 3

γδ T cells activated for 96-120 h exhibit the optimal lentiviral transduction efficiency A: Flowchart of the γδ T cell production process. B: Schematic illustration of the EGFRvⅢ CAR construct. C: Representative flow cytometry plots of Strep Ⅱ-labeled CAR+ cells 6 d after transduction. D: Summary of transduction efficiency, CD25+CD69+ percentage, and CD3+Vδ2+ percentage of γδ T cells at different activation time points (n=3 donors). E: Correlation between transduction efficiency and CD25+CD69+ percentage. F: Correlation between transduction efficiency and CD3+Vδ2+ percentage in γδ T cells. Bars represent $\bar{x} \pm s$."

Fig. 4

CAR-γδ T cells transduced at MOI 5-10 exhibit optimal transduction efficiency and expansion capacity A: Representative flow cytometry plots showing Strep Ⅱ-labeled CAR+ 6 d after transduction. B: Quantification of transduction efficiency at varying MOIs (n=3 donors). C-E: Summary of purity, fold expansion and viability of EGFRvⅢ-γδ T cells on day 14. Data are presented as $\bar{x} \pm s$. *: P<0.05, **: P<0.01. NS: Not significant."

Fig. 5

CAR-γδ T cells cultured by optimal conditions demonstrate enhanced cytotoxicity with preliminarily no evidence of GVHD A: representative flow cytometry plots of CD25 and HLA-DR. B, C: Summarized results (n=3 donors) of CD25 and HLA-DR expression on γδ T or CAR-γδ T cells. D: Representative flow cytometry plots of CD107a. E: Summarized results (n=3 donors) of CD107a expression on γδ T or CAR-γδ T cells. F: Cytotoxic activity of CAR-γδ T cells against four cell lines, as determined by calcein release assay at different E∶T ratios (1∶1, 5∶1, 25∶1) after 3.5 h of co-culture (one representative donor from two is shown; n=4 independent wells). G, H: Detection of IFN-γ in the supernatants from EGFRvⅢ-γδ T and EGFRvⅢ-αβ T cells co-cultured with other donors’ PBMC and own PBMC (n=3 independent wells). I: Representative flow cytometry plots of Strep Ⅱ-labeled CAR+ 6 days after transduction. J: Summarized results (n=3 donors) of Transduction efficiency of N6-γδ T cells. K: The H9 cell line was infected with HIV-1 (NL4-3) and mixed with N6 CAR-γδ T cells at different ratios (1∶1, 5∶1, 25∶1). Direct cytotoxicity effects were detected (one representative donor from two is shown; n=4 independent wells). Data are presented as $\bar{x} \pm s$. ***: P<0.001; ****: P <0.000 1. NS: Not significant."