Background and purpose:In recent years,chimeric antigen receptor T(CAR-T)cell therapy has achieved breakthrough progress in cancer treatment.γδ T cells,with their non-major histocompatibility complex(MHC)-restricted antigen recognition,broad antitumor activity,and low risk of graft-versus-host disease(GVHD),have garnered significant interest in CAR-γδ T cell therapy.However,critical challenges including suboptimal in vitro expansion and low viral transduction efficiency severely hinder the research and clinical application of CAR-γδ T cells.This study aimed to establish an efficient platform for preparing CAR-γδ T cells by optimizing the in vitro expansion conditions of γδ T cells and refining lentiviral transduction strategies.Methods:We first optimized the expansion protocol for γδ T cells by screening various cytokine combinations and the concentrations of individual cytokines within combination,and evaluating cell purity,viability,fold expansion,and expressions of cytotoxicity and exhaustion markers to identify the optimal culture conditions.Subsequently,the transduction conditions for CAR-γδ T cells were improved by determining the optimal activation duration of γδ T cells prior to gene transfer,as well as the optimal multiplicity of infection(MOI)for lentiviral transduction.Finally,CAR-γδ T cells were successfully generated using the optimized protocol,and their cytotoxic activity against target cells was validated via calcein-release assay and flow cytometry,with a preliminary assessment of the potential risk of GVHD induction.Results:Experimental data demonstrated that,compared with the interleukin(IL)-2-only culture,the IL-2+IL-7+IL-15 combination significantly enhanced the expansion capacity of γδ T cells(876.50±238.35-fold vs 1 627.50±472.15-fold),cell purity(73.67%±1.53%vs 90.69%±2.00%),and cell viability(63.01%±7.05%vs 89.00%±3.61%).It also increased the expression of the cytotoxicity marker CD16(4.20%±1.73%vs 14.66%±0.58%)and reduced the expression of the exhaustion marker programmed death-1(PD-1)(35.67%±6.26%vs 21.10%±6.49%).A cytokine concentration gradient orthogonal assay further identified 10 ng/mL IL-7 and 10 ng/mL IL-15 as the optimal concentrations within the IL-2+IL-7+IL-15 combination.Gene transduction performed 96-120 h after activation using a multiplicity of infection(MOI)of 5-10 resulted in the highest transduction efficiency for CAR-γδ T cells(96 h:12.87%±4.35%;120 h:11.37%±2.35%).CAR-γδ T cells generated using the optimized system exhibited specific cytotoxic effects against tumor cells expressing the target antigen,and no evidence of GVHD induction was observed.Conclusion:CAR-γδ T cells produced using the IL-2+IL-7(10 ng/mL)+IL-15(10 ng/mL)regimen combined with a 96-120 h activation period prior to transduction using a multiplicity of infection(MOI)of 5-10 significantly outperformed conventional methods in terms of expansion efficiency,cell purity,and transduction efficiency.The synergistic antitumor effects mediated by both natural immune receptors and CAR-specific recognition,along with the initial absence of GVHD risk,provide critical technical support for the clinical translation of CAR-γδ T cell therapy,establishing a solid theoretical and practical foundation.

Background and purpose:In recent years,chimeric antigen receptor T(CAR-T)cell therapy has achieved breakthrough progress in cancer treatment.γδ T cells,with their non-major histocompatibility complex(MHC)-restricted antigen recognition,broad antitumor activity,and low risk of graft-versus-host disease(GVHD),have garnered significant interest in CAR-γδ T cell therapy.However,critical challenges including suboptimal in vitro expansion and low viral transduction efficiency severely hinder the research and clinical application of CAR-γδ T cells.This study aimed to establish an efficient platform for preparing CAR-γδ T cells by optimizing the in vitro expansion conditions of γδ T cells and refining lentiviral transduction strategies.Methods:We first optimized the expansion protocol for γδ T cells by screening various cytokine combinations and the concentrations of individual cytokines within combination,and evaluating cell purity,viability,fold expansion,and expressions of cytotoxicity and exhaustion markers to identify the optimal culture conditions.Subsequently,the transduction conditions for CAR-γδ T cells were improved by determining the optimal activation duration of γδ T cells prior to gene transfer,as well as the optimal multiplicity of infection(MOI)for lentiviral transduction.Finally,CAR-γδ T cells were successfully generated using the optimized protocol,and their cytotoxic activity against target cells was validated via calcein-release assay and flow cytometry,with a preliminary assessment of the potential risk of GVHD induction.Results:Experimental data demonstrated that,compared with the interleukin(IL)-2-only culture,the IL-2+IL-7+IL-15 combination significantly enhanced the expansion capacity of γδ T cells(876.50±238.35-fold vs 1 627.50±472.15-fold),cell purity(73.67%±1.53%vs 90.69%±2.00%),and cell viability(63.01%±7.05%vs 89.00%±3.61%).It also increased the expression of the cytotoxicity marker CD16(4.20%±1.73%vs 14.66%±0.58%)and reduced the expression of the exhaustion marker programmed death-1(PD-1)(35.67%±6.26%vs 21.10%±6.49%).A cytokine concentration gradient orthogonal assay further identified 10 ng/mL IL-7 and 10 ng/mL IL-15 as the optimal concentrations within the IL-2+IL-7+IL-15 combination.Gene transduction performed 96-120 h after activation using a multiplicity of infection(MOI)of 5-10 resulted in the highest transduction efficiency for CAR-γδ T cells(96 h:12.87%±4.35%;120 h:11.37%±2.35%).CAR-γδ T cells generated using the optimized system exhibited specific cytotoxic effects against tumor cells expressing the target antigen,and no evidence of GVHD induction was observed.Conclusion:CAR-γδ T cells produced using the IL-2+IL-7(10 ng/mL)+IL-15(10 ng/mL)regimen combined with a 96-120 h activation period prior to transduction using a multiplicity of infection(MOI)of 5-10 significantly outperformed conventional methods in terms of expansion efficiency,cell purity,and transduction efficiency.The synergistic antitumor effects mediated by both natural immune receptors and CAR-specific recognition,along with the initial absence of GVHD risk,provide critical technical support for the clinical translation of CAR-γδ T cell therapy,establishing a solid theoretical and practical foundation.