absence of TCR -activation maintain an intact immune competence (original) (raw)

Gene transfer into T lymphocytes is currently tested for the treatment of lymphohematologic disorders. We previously showed that suicide gene transfer into donor lymphocytes infused to treat leukemic relapse after allogeneic hematopoietic stem cell transplantation allowed control of graft-versus-host disease. However, the T-cell receptor activation and sustained proliferation required for retroviral vector transduction may impair the half-life and immune competence of transduced cells and reduce graft-versus-leukemia activity. Thus, we tested lentiviral vectors (LV) and stimulation with cytokines involved in antigen-independent T-cell homeostasis, such as IL-7, IL-2 and IL-15. Late-generation LV transduced efficiently nonproliferating T cells which had progressed from G 0 to the G 1 phase of the cell cycle upon cytokine treatment. Importantly, IL-2 and IL-7, but not IL-15 stimulation preserved physiologic CD4/CD8 and naïve/memory ratios in transduced cells with only minor induction of some activation markers. Functional analysis of immune response to cytomegalovirus (CMV) showed that, although CMV-specific T cells were preserved by all conditions of transduction, proliferation and specific killing of autologous cells presenting a CMV epitope were higher for IL-2 and IL-7 than IL-15. Thus, LV transduction of IL-2 or IL-7 pre-stimulated cells overcome the limitations of retroviral vectors and may significantly improve the efficacy of T cell-based gene therapy. (Word count 202) For personal use only. by guest on June 10, 2013. bloodjournal.hematologylibrary.org From recently shown that cytokines, such as IL-7, IL-2 and IL-15, able to promote longterm survival in vitro of memory and naïve T lymphocytes (24-25), can render T cells susceptible to LV transduction in the absence of TCR-activation (22-23) (26). In this study, we confirmed and extended these findings using late-generation LV with advanced performance and safety profiles and showing efficient transduction of T cells which have progressed from G 0 into the G 1 phase of the cell cycle upon cytokine treatment without becoming committed to proliferation. Most importantly, we showed that culture conditions allowing for LV transduction of T cells in the absence of TCR-triggering result in preservation of full immune competence, thus allowing to overcome the intrinsic functional limitations of retroviral-mediated transduction. Methods Cell culture HeLa and 293T human embryonic kidney cell lines were cultured in Iscove's Modified Dulbecco's Medium (IMDM, Sigma Chemical Co, Milan, Italy) supplemented with 10% Fetal Bovine Serum (FBS, Gibco BRL, Inchinnam, Scotland UK) and a combination of Penicillin (100IU/ml), Streptomycin (100µg/ml) and L-Glutamine (2mM). Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors by leukapheresis and Ficoll-Hypaque gradient separation (Pharmacia, Uppsala, Sweden). Cells were cultured in IMDM supplemented with 10% Fetal Calf Serum (FCS, Gibco BRL) at a density of 1x10 6 cells/ml in the presence of different cytokines: human recombinant IL-2 10U/ml (EuroCetus Italia S.r.l., Milan, Italy); human recombinant IL-7 5ng/ml (Boehringer Mannheim-Roche GmbH, Mannheim, Germany); human recombinant IL-15 10ng/ml (R&D system or PeproTech Inc,Rocky Hill, NH). TCR