Olivier Boyer

Long‐term persistence of clonally expanded T cells in patients with polymyositis

Polymyositis is a CD8+ T‐cell–mediated disease. T‐cell clonal expansions are observed at disease onset, but little is known about their persistence over time. Qualitative and quantitative spectratyping demonstrated that PM relapse features dramatically perturbed blood T‐cell repertoires but is not associated with the emergence of new T‐cell clones. It is striking that patients in remission also maintained all their T‐cell repertoire abnormalities. The clonally expanded T‐cells displayed a memory phenotype, expressed intracellular perforin, and dramatically responded to IL‐2, showing a potential to be reactivated upon appropriate conditions. These results indicate that persistent T‐cell clonal expansion is an important feature of polymyositis. Ann Neurol 2004

Would suicide gene therapy solve the ‘T-cell dilemma’ of allogeneic bone marrow transplantation?

Abstract The mature T cells that acccompany hematopoietic stem cells during allogeneic bone marrow transplantation can mediate both beneficial and deleterious clinical effects. Suicide genes that metabolize inactive prodrugs into compounds highly toxic for dividing cells, should enable elimination of genetically modified activated T cells when necessary by time-controlled prodrug administration.

In situ gene transfer into animal tendons by injection of naked DNA and electrotransfer

Degenerative or traumatic tendon injuries are extremely common but often heal poorly, not restoring the normal function of the injured tissues. Gene transfer could improve the repair process, by permitting local production of therapeutic substances, e.g. growth factors.

Long-term survival after gene therapy for a recurrent glioblastoma

Abstract—A patient presenting with a recurrent glioblastoma (GBM) survived 3 years after suicide gene therapy and finally died of a disseminated breast cancer with no indication of tumor recurrence on MRI. Postmortem analysis showed no evidence of recurrence of the GBM, neither near the initial tumor localization nor in any other area of the brain. Such an evolution is unusual in the course of this disease and may suggest in this particular case a cure of the GBM.

Suicide Gene-Mediated Modulation of Graft-Versus-Host Disease

The development of suicide genes and progress in retroviral gene transfer to T-cells open new perspectives for the treatment of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) for leukemia and lymphoma. Indeed, suicide genes that metabolize inactive prodrugs into compounds toxic for dividing cells provide a powerful means for the pharmacogenetic control of T-cell reactivity. Here, we demonstrate the selective destruction of activated TK-transgenic T-cells in vivo and develop two new transgenic lines which should be useful for preclinical studies of suicide gene therapy strategies for GVHD.

Prolonged allograft survival through conditional and specific ablation of alloreactive T cells expressing a suicide gene

BACKGROUNDnControl of antidonor activated T cells involved in allograft rejection while preserving immunocompetence is a challenging goal in transplantation. Engineered T cells expressing a viral thymidine kinase (TK) suicide gene metabolize the nontoxic prodrug ganciclovir (GCV) into a metabolite toxic only to dividing cells. We evaluated this suicide gene strategy for inducing transplantation tolerance in mice.nnnMETHODSnTransgenic mice expressing TK in mature T cells were analyzed for (i) specific T-cell depletion under GCV treatment upon various stimulations; (ii) outcome of allogeneic nonvascularized skin or heart allografts under a short 14-day GCV treatment initiated at the time of transplantation; and (iii) the capacities of T cells from such allotransplanted mice to proliferate in mixed lymphocyte reactions and to induce graft-versus-host disease in irradiated recipients with the genetic background of the donor allograft.nnnRESULTSnUpon in vitro or in vivo GCV treatment, only activated dividing TK T cells but not B cells were efficiently depleted. Acute rejection of allogeneic grafts was prevented and a significant prolongation of graft survival was obtained, although associated with signs of chronic rejection. Prolonged skin graft survival correlated with decreased in vitro and in vivo T-cell reactivities against donor alloantigens, whereas overall immunocompetence was preserved.nnnCONCLUSIONSnEfficient and specific depletion of alloreactive TK T cells can be achieved by administrating GCV. These results open new perspectives for the control of allogeneic graft rejection using suicide gene therapy.

Immunological Defects after Suicide Gene Therapy of Experimental Graft-versus-Host Disease

Donor T cells are beneficial for engraftment, immune reconstitution, and antileukemic effects after allogeneic marrow transplantation, but they also cause graft-versus-host disease. Treatment with ganciclovir can control graft-versus-host disease if donor T cells are genetically engineered to express viral thymidine kinase. Clinical protocols with thymidine kinase-expressing T cells currently prescribe the curative use of ganciclovir for genetic immunosuppression only after clinical manifestations of graft-versus-host disease have appeared. The aim of this work was to compare early/preventive versus delayed/curative treatment of GVHD. Here, we found that ganciclovir administered early after experimental marrow transplantation was highly effective in preventing graft-versus-host disease caused by thymidine kinase-expressing T cells, and surviving recipient mice were able to mount a T cell-dependent B cell response. In contrast, curative ganciclovir administration later after transplantation was much less effective in treating graft-versus-host disease and surviving recipients had markedly impaired immune function. These findings should be considered in the development of future clinical trials using thymidine kinase-expressing T cells; to date, such trials have envisaged the use of GCV to treat only declared graft-versus-host disease. The use of thymidine kinase-expressing T cells for conditional elimination of activated T cells after allogeneic marrow transplantation offers a promising new approach for the control of graft-versus-host disease. The versatility of the thymidine kinase/ganciclovir system offers clinical options depending on whether thymidine kinase-expressing T cells are infused at the time of bone marrow transplantation or in a delayed manner, and depending on whether GCV is administered in an early/preventive or delayed/curative manner. The rationale underlying these options is more complex than it may appear and is likely to have a profound impact on the efficacy of such treatments. In the present work, we analyze the immunological impact when GCV is administered in an early/preventive or delayed/curative manner. Our results demonstrate that the delayed/curative strategy is clearly associated with severe immunological defects. To our knowledge, this is the first report of immunodeficiency subsequent to suicide gene therapy for GVHD.

T cell repertoire in patients with stable scleroderma

At onset of systemic sclerosis (SSc), T cells have been found to oligoclonally expand in the skin, presumably in response to auto‐antigens, but the T cell repertoire has not been evaluated at a later stage. To determine whether a perpetuating immune response contributes to the pathogenesis of stable SSc, the T cell repertoire was analysed in patients with diffuse (d) or limited (l) SSc, and compared to patients with primary Raynauds phenomenon (RP) or healthy volunteers (Ctrl). The T cell repertoire (total, CD4 or CD8 sorted blood T cells) was analysed by qualitative and quantitative immunoscope (14 BV families analysed) in 11 untreated dSSc and 11 untreated lSSc, 10 RP and 11 Ctrl. To better detect in vivo activated cells, repertoire analysis was also performed on sorted CD4 T cells after in vitro culture with IL‐2. In parallel, 6 skin biopsies from SSc patients were analysed. After 7–8u2003years of disease evolution, SSc patients did not show detectable clonal T cell expansions in the skin, even after tentative expansion from the biopsy with IL‐2. Total T cell, sorted CD4 and CD8 T cell repertoires from the blood of patients with SSc did not show significant perturbation as compared to patients with RP and Ctrl. After IL‐2 culture for 7u2003days, blood CD4 T cells from the patients did not preferentially expand as compared to RP and Ctrl. These findings suggest that antigen‐driven immune responses may play a lesser role in established SSc than at disease onset.

Efficient transduction and selection of human T-lymphocytes with bicistronic Thy1/HSV1-TK retroviral vector produced by a human packaging cell line.

T‐cells expressing the HSV1‐TK suicide gene can be used for the control of graft‐versus‐host disease following allogeneic stem cell transplantation. To develop clinical trials based on such a strategy, we have generated under good manufacturing procedures a novel ‘split genome’ human packaging cell line (1704 cells).

Restricted BV gene usage by factor VIII-reactive CD4+ T cells in inhibitor-positive patients with severe hemophilia A

In the present study, we have analyzed the T cell receptor (TCR) repertoires of CD4+ T cells isolated from peripheral blood of 10 inhibitor-positive patients with severe hemophilia A. The distribution of complementarity determining region (CDR3) lengths of the beta chain of the TCRs was analyzed by spectratyping prior to and following in vitro stimulation of the cells with human factor VIII (FVIII). The repertoires of CD4+ T cells of patients were perturbed when compared to those of healthy blood donors. The perturbations of T cell repertoires were heterogeneous among patients with respect to the number and the nature of V-beta (BV) families that exhibited expansion following incubation with FVIII. Some patients showed alterations in one or two BV families, others exhibited more perturbed repertoires affecting 5 to 8 of the 14 BV families tested. Alterations of BV2, BV5 and/or BV9 were consistently found after incubation of CD4+ T cells in the presence of FVIII in 80% of the patients. These findings indicate that the presence of FVIII inhibitors in patients with severe hemophilia A is associated with measurable perturbations of the CD4+ T cell repertoire that results from oligoclonal expansion of FVIII-specific cells and may be relevant for the design of strategies aimed at modulating the anti-FVIII immune responses by T cell-targeted therapy