Rerovirus Mediated HSV-TK Gene Transfer Can Influence the Function of Human T-Lymphocyte.

Abstract

To control graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell (HSC) transplantation while maintaining the graft antileukemic effects of donor T cells, a suicide gene encoding the herpes simplex virus thymidine kinase (HSV-TK) enzyme was introduced ex vivo via a retroviral vector into donor T cells. Although previous clinical trials showed that it was an efficient tool for controlling GVHD, the gene transduction process of donor T lymphocytes may influence the immunocompetence of the T cells. In the current study, we investigated the impact that the gene manipulation in vitro had on the phenotype and functionality of human donor T lymphocytes. We engineered human T lymphocytes with HSV-TK and puromycin resistance gene with a retrovirus vector and analyzed the phenotype change of gene modified cells with flowcytometry. The secretion of gamma-interferon and interleukin-4 of T lymphocyte were detected with enzyme-linked immunospot(ELIPOT)assays. We also analyzed the lytic activity of T cells against allogeneic targets. The result showed that the number of TK gene transduced T lymphocyte was sufficient for clinical use. There was an inversion of the CD4+/CD8+ ratio in the gene modified T cell (GMC) compared with the fresh peripheral blood lymphocytes (PBLs). In addition, the GMCs showed a higher expression of HLA-DR and had more antigen-experienced cells than did the PBLs. In ELIPOT assays, significantly less gamma-interferon secretion by GMCs was observed compared to PBLs (50 vs. 115 spots/well-3×104cells, p<0.05). The results also showed that IL-4-secreting cell frequencies was similar in GMCs and in PBLs (19.3 vs. 20.3 spots/well-3×104cells, p>0.05). After stimulated by allogeneic PBMCs, the GMCs effectors showed a significant reduction in lytic activity against allogeneic targets in contrast with PBL. In conclusion, HSV-TK gene transduction and selection procedure can change the phenotype and functions of human T lymphocyte. The gene modified T lymphocytes still showed biological activities although lower than those of PBLs.