Efficacy of gene therapy for X-linked severe combined immunodeficiency.

BACKGROUND: The outcomes of gene therapy to correct congenital immunodeficiencies are unknown. We reviewed long-term outcomes after gene therapy in nine patients with X-linked severe combined immunodeficiency (SCID-X1), which is characterized by the absence of the cytokine receptor common gamma chain. METHODS: The nine patients, who lacked an HLA-identical donor, underwent ex vivo retrovirus-mediated transfer of gamma chain to autologous CD34+ bone marrow cells between 1999 and 2002. We assessed clinical events and immune function on long-term follow-up. RESULTS: Eight patients were alive after a median follow-up period of 9 years (range, 8 to 11). Gene therapy was initially successful at correcting immune dysfunction in eight of the nine patients. However, acute leukemia developed in four patients, and one died. Transduced T cells were detected for up to 10.7 years after gene therapy. Seven patients, including the three survivors of leukemia, had sustained immune reconstitution; three patients required immunoglobulin-replacement therapy. Sustained thymopoiesis was established by the persistent presence of naive T cells, even after chemotherapy in three patients. The T-cell-receptor repertoire was diverse in all patients. Transduced B cells were not detected. Correction of the immunodeficiency improved the patients' health. CONCLUSIONS: After nearly 10 years of follow-up, gene therapy was shown to have corrected the immunodeficiency associated with SCID-X1. Gene therapy may be an option for patients who do not have an HLA-identical donor for hematopoietic stem-cell transplantation and for whom the risks are deemed acceptable. This treatment is associated with a risk of acute leukemia. (Funded by INSERM and others.)

Adenovirally transduced dendritic cells induce bispecific cytotoxic T lymphocyte responses against adenovirus and cytomegalovirus pp65 or against adenovirus and Epstein-Barr virus EBNA3C protein: a novel approach for immunotherapy.

Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (Ad) cause significant morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. We have established a procedure to generate polyclonal cytotoxic T lymphocyte (CTL) populations with specificity against Ad and CMV or against Ad and EBV. Healthy donor-derived dendritic cells (DCs) were transduced with recombinant adenovirus encoding either CMV pp65 or EBV EBNA3C and used to stimulate autologous T cells. Stimulated T lymphocytes displayed specific simultaneous cytotoxicity against CMV and adenovirus and to a lesser extent against adenovirus and EBV. Recombinant vaccinia virus encoding individual adenovirus proteins showed that the T cell response to the adenovirus was directed mainly against the capsid protein hexon. The frequency of IFN-gamma-secreting T cells was 0.02% for adenovirus alone, and 0.05 and 0.14% for adenoviruses encoding EBNA3C and pp65, respectively. pp65-specific CTLs killed autologous fibroblasts infected with the laboratory strain CMV AD169. The culture conditions were specific as alloreactive T cells were not expanded. Therefore, this approach could be considered in order to generate efficient virus cytolytic T cells to be used as adoptive immunotherapy in transplanted patients.

Cryopreservation of Dendritic Cells Grown in Vitro from Monocytes for Their Future Clinical Use.

Dendritic cells are professional antigen presenting cells which are being used as adjuvants in tumor vaccination trials. Most clinical protocols currently include 4 to 10 weekly infusions of doses > 10(6) cells, each inoculum coming from a simple culture of blood monocytes. In the present study, several millions of dendritic cells from a single leukapheresis were produced; monocytes were isolated by elutriation and then cultured in Teflon bags in presence of 800 U/ml GM-CSF + 100 micro g/ml IL-13 + 10% fetal calf serum (FCS). The dendritic cells from this single batch were aliquoted in many doses for potential multiple infusions and cryopreserved in 10% DMSO + 2% human albumin in Teflon-kapton Fresenius bags either at -1 degrees C/min using a controlled rate freezer, or putting the bags directly in a -80 degrees C mechanical freezer without controlling the temperature rate. Six experiments were carried out. After one month of cryopreservation, the cells were thawed in a 40 degrees C water bath. Before and after freezing, cells were evaluated for immunophenotype (CD1a, CD14, CD40, CD80, CD83, CD86, CD54, CD58, CD16, CD32, CD64 and HLA-DR) and for their capacity to stimulate allogenic (MLR) or autologous (antigen presentation tests) lymphocytes. The results demonstrated that the mean recovery rates after freezing in liquid nitrogen or at -80 degrees C were (67 +/- 14)% and (71 +/- 13)% respectively, without any significant difference between the two techniques. The immunophenotype was not modified by the freezing-thawing procedure, as well as the lymphocyte stimulating capacities. In conclusion, our study showed that substantial numbers of functional DCs can be derived from peripheral blood monocytes using Teflon bags. DCs can be cryopreserved in a good laboratory practice setting for further clinical trials with an acceptable loss of cells and without modification of their functions.