Abstract: Objective To explore the anti-tumor effect of genetically CDR3δ2- modified γδ T lymphocytes which were generated by co-transfection of the full-length γ9 mRNA and δ2 mRNA with tumor-reactive CDR3δ into anti-CD3 antibody stimulated human peripheral blood mononuclear cells (PBMC). Methods By PCR, specific δ2 sequences containing tumor-reactive CDR3δ2 (OT3 and OT10) and γ9 chain were amplified to make recombinant plasmids pGEM4Z / δ2 (OT3) / A64, pGEM4Z / δ2 (OT10) / A64 and pGEM4Z/γ9/A64. Linearized plasmids were used as templates for the synthesis of δ2 (OT3), δ2 (OT10) and γ9 mRNAs in vitro. Synthesized δ2 (OT3) mRNA and δ2 (OT10) mRNA were co-transfected with γ9mRNA respectively into anti-CD3 antibody stimulated human PBMC. Flow cytometry and sorting were performed to isolate γ9δ2 (OT3) T cells and γ9δ2 (T10) T cells. MTT assay and ELISA were applied to detect the anti-tumor effect of these CDR3δ2 genetically modified γδ T lymphocytes. Results CDR3δ2 genetically modified γ9δ2 (OT3) T cells and γ9δ2 (T10) T cells secreted high levels of IFN-γ and TNF-α and displayed significant cytotoxicity to a variety of tumor cells. Conclusion The generation of genetically modified tumor-reactive γ9δ2 T cells is an efficient approach to enhance the anti-tumor activity of lymphocytes, which provides a novel strategy for adoptive immunity against tumors.

Key words: T cell, CDR3δ2, γδTCR, adoptive immune therapy, OT3, OT10