Abstract
The insertion of suicide genes in donor T lymphocytes constitutes the basis of new approaches aiming at the treatment of the graft-versus-host disease (GVHD), a frequent complication in recipients of allogeneic haematopoietic grafts. In this study we investigated the impact that the ex vivo manipulation required for the retroviral transduction of T cells had on the functionality and differentiation of these cells. Compared to fresh T cells, samples that had been subjected to standard activation (1 μg/ml of both anti-CD3i and anti-CD28i MoAbs) followed by transduction with vectors encoding for the HSV-tk and tNGFR genes maintained the proliferative response to an allogeneic stimulus. These cells, however, had a significantly lower cytotoxic response to allogeneic cells compared to fresh samples. When the concentration of anti-CD3i was reduced to up to 1000-fold (1 ng/ml), similar T-cell transductions were obtained, while the cytotoxicity of the ex vivo manipulated samples was significantly recovered, when assessed either at 7 or 14 days of culture. In all instances, a similar functionality was observed in transduced samples not subjected to immunomagnetic cell sorting, compared to purified fractions enriched in NGFR+ and NFGR− cells. The analysis of CD45RA and CCR7 markers in samples transduced under standard stimulatory conditions showed a differentiation of fresh CD8+ CD45RA+/CCR7+ naive cells to cells having a predominant central CD45RA−/CCR7+ and effector CD45RA−/CCR7− memory phenotype. However, when samples were activated with low doses of anti-CD3i, a significant population of naive cells became apparent. Although activation with high doses of anti-CD3i/anti-CD28i resulted in a similar phenotype in both NGFR+ and NFGR− populations, the naive population observed in samples activated with low concentrations of anti-CD3i was almost restricted to the NGFR− population. These results show that reducing the stimulation mediated by anti-CD3i in protocols of T-cell retroviral gene transfer significantly helps to preserve the cytotoxic capacity of these cells to allogeneic cells, without affecting the susceptibility of these cells to the retroviral vector. In addition, we observed that modulating the activation of transduced T cells implies the generation of changes in the differentiation of CD8+ cells, although we could not establish a direct relationship between the CD45RA/CCR7 phenotype of these cells and their cytotoxic reactivity to an allogeneic stimulus.
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References
Ferrara JL, Deeg HJ . Graft-versus-host disease. N Engl J Med 1991; 324: 667–674.
Kolb HJ, Holler E . Adoptive immunotherapy with donor lymphocyte transfusions. Curr Opin Oncol 1997; 9: 139–145.
Martin PJ et al. Graft failure in patients receiving T cell-depleted HLA-identical allogeneic marrow transplants. Bone Marrow Transplant 1988; 3: 445–456.
Marmont AM et al. T-cell depletion of HLA-identical transplants in leukemia. Blood 1991; 78: 2120–2130.
Broers AE et al. Increased transplant-related morbidity and mortality in CMV-seropositive patients despite highly effective prevention of CMV disease after allogeneic T-cell-depleted stem cell transplantation. Blood 2000; 95: 2240–2245.
Dazzi F, Goldman JM . Adoptive immunotherapy following allogeneic bone marrow transplantation. Annu Rev Med 1998; 49: 329–340.
Bordignon C et al. Transfer of the HSV-tk gene into donor peripheral blood lymphocytes for in vivo modulation of donor anti-tumor immunity after allogeneic bone marrow transplantation. Hum Gene Ther 1995; 6: 813–819.
Bonini C et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia [see comments]. Science 1997; 276: 1719–1724.
Cohen JL et al. Prevention of graft-versus-host disease in mice using a suicide gene expressed in T lymphocytes. Blood 1997; 89: 4636–4645.
Cohen JL, Boyer O, Klatzmann D . Suicide gene therapy of graft-versus-host disease: immune reconstitution with transplanted mature T cells. Blood 2001; 98: 2071–2076.
Tiberghien P et al. Use of donor T-lymphocytes expressing herpes-simplex thymidine kinase in allogeneic bone marrow transplantation: a phase I–II study. Hum Gene Ther 1997; 8: 615–624.
Tiberghien P et al. Administration of herpes simplex-thymidine kinase-expressing donor T cells with a T-cell-depleted allogeneic marrow graft. Blood 2001; 97: 63–72.
Lamana ML, Segovia JC, Guenechea G, Bueren JA . Systematic analysis of clinically applicable conditions leading to a high efficiency of transduction and transgene expression in human T cells. J Gene Med 2001; 3: 32–41.
Quinn ER, Lum LG, Trevor KT . T cell activation modulates retrovirus-mediated gene expression [see comments]. Hum Gene Ther 1998; 9: 1457–1467.
Pollok KE et al. Costimulation of transduced T lymphocytes via T cell receptor–CD3 complex and CD28 leads to increased transcription of integrated retrovirus. Hum Gene Ther 1999; 10: 2221–2236.
Ferrand C et al. Retrovirus-mediated gene transfer in primary T lymphocytes: influence of the transduction/selection process and of ex vivo expansion on the T cell receptor beta chain hypervariable region repertoire. Hum Gene Ther 2000; 11: 1151–1164.
Di Ianni M et al. T-lymphocyte function after retroviral-mediated thymidine kinase gene transfer and G418 selection. Cancer Gene Ther 2000; 7: 920–926.
Duarte RF et al. Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy. Gene Therapy 2002; 9: 1359–1368.
Sauce D et al. Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti-Epstein–Barr virus potential through both culture-dependent and selection process-dependent mechanisms. Blood 2002; 99: 1165–1173.
Berger C, et al., CD28 costimulation and immunoaffinity-based selection efficiently generate primary gene-modified T cells for adoptive immunotherapy. Blood 2002; 22: 22.
van Dijk AM et al. Determination of helper T-cell precursor frequencies against non-haemopoietic cells: comparison of co-stimulation provided by anti-CD28 antibody versus the cellular ligand B7-1. Br J Haematol 2000; 110: 322–326.
Dong H, Zhu G, Tamada K, Chen L . B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med 1999; 5: 1365–1369.
June CH, Ledbetter JA, Linsley PS, Thompson CB . Role of the CD28 receptor in T-cell activation. Immunol Today 1990; 11: 211–216.
Contassot E et al. In vivo alloreactive potential of ex vivo-expanded primary T lymphocytes. Transplantation 1998; 65: 1365–1370.
Drobyski WR, Majewski D, Ozker K, Hanson G . Ex vivo anti-CD3 antibody-activated donor T cells have a reduced ability to cause lethal murine graft-versus-host disease but retain their ability to facilitate alloengraftment. J Immunol 1998; 161: 2610–2619.
Lenardo MJ . Interleukin-2 programs mouse alpha beta T lymphocytes for apoptosis. Nature 1991; 353: 858–861.
Nagata S, Golstein P . The Fas death factor. Science 1995; 267: 1449–1456.
Weijtens M et al. Reduced graft-versus-host disease-inducing capacity of T cells after activation, culturing, and magnetic cell sorting selection in an allogeneic bone marrow transplantation model in rats. Hum Gene Ther 2002; 13: 187–198.
Marktel S et al. Immunologic potential of donor lymphocytes expressing a suicide gene for early immune reconstitution after hematopoietic T cell-depleted stem cell transplantation. Blood 2003; 101: 1290–1298.
Deeths MJ, Kedl RM, Mescher MF . CD8+ T cells become nonresponsive (anergic) following activation in the presence of costimulation. J Immunol 1999; 163: 102–110.
Miller DG, Adam MA, Miller AD . Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection [published erratum appears in Mol Cell Biol 1992 Jan; 12(1): 433]. Mol Cell Biol 1990; 10: 4239–4242.
Sallusto F et al. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999; 401: 708–712.
Champagne P et al. Skewed maturation of memory HIV-specific CD8T lymphocytes. Nature 2001; 410: 106–111.
Dardalhon V et al. Highly efficient gene transfer in naive human T cells with a murine leukemia virus-based vector. Blood 2000; 96: 885–893.
Pollok KE et al. High-efficiency gene transfer into normal and adenosine deaminase-deficient T lymphocytes is mediated by transduction on recombinant fibronectin fragments. J Virol 1998; 72: 4882–4892.
Fehse B et al. Highly-efficient gene transfer with retroviral vectors into human T lymphocytes on fibronectin. Br J Haematol 1998; 102: 566–574.
Acknowledgements
The authors wish to thank Sergio García for excellent technical assistance in the generation and tittering of infective supernatants. Also the authors thank Paloma López for the preparation of peripheral blood samples. This work was supported by grants of the Comisión Interministerial de Ciencia y Tecnología (SAF 2002-030234) and Programa Redes de Investigación Cooperativa del Ministerio de Sandidad y Consumo (G03/073).
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Lamana, M., Bueren, J., Vicario, J. et al. Functional and phenotypic variations in human T cells subjected to retroviral-mediated gene transfer. Gene Ther 11, 474–482 (2004). https://doi.org/10.1038/sj.gt.3302188
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DOI: https://doi.org/10.1038/sj.gt.3302188
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