T-lymphocyte function after retroviral-mediated thymidine kinase gene transfer and G418 selection

Generation of an efficient graft-versus-leukemia (GVL) effect in patients w ith hematological malignancies who relapse after allogeneic bone marrow tra nsplantation depends in pari upon the number of infused T lymphocytes. Curr ently, a GVL reaction cannot be achieved without inducing concomitant graft -versus-host disease (GVHD); thus, one strategy is to try to modulate this GVL/GVHD ratio. We engineered human T lymphocytes with herpes simplex virus -thymidine kinase and neomycin resistance genes, with an LXSN-derived vecto r that confers a ganciclovir-specific sensitivity to the transduced T cells . We analyzed proliferation, interleukin-2 production, alloreactivity in a mixed lymphocyte culture, and clonogenicity during the different sta of ret roviral infection and G418 selection. Our results confirm that a sufficient number of transduced T lymphocytes can be obtained after selection for cli nical studies. Their proliferative activity, alloresponsiveness, and abilit y to produce and respond to interleukin-2 were retained. Compared with cont rol populations, their clonogenicity, as assessed by limiting dilution assa ys, was reduced after retroviral infection and G418 selection by 1.6 and 2. 9 logs, respectively, with both viral supernatant incubation and coculture procedures. This study shows that infection and selection with the thymidin e kinase-neomycin resistance gene retroviral vector significantly reduces t he number of functional T lymphocytes. This finding should be taken into ac count when establishing the dose of T lymphocytes necessary to trigger a mo dulated GVL/GVHD effect.