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In allogeneic hematopoietic cell transplantation (allo-HCT), the immune recognition of host antigens by donor T lymphocytes leads to a beneficial graft-versus-leukemia (GvL) effect as well as to life-threatening graft-versus-host disease (GvHD). Genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK ؉ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK ؉ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM)TK IntroductionAllogeneic hematopoietic cell transplantation (allo-HCT) is the curative option for many hematologic malignancies. Moreover, it is being investigated to treat solid tumors. In allo-HCT, the recognition of host antigens by donor T lymphocytes evokes a graft-versusleukemia (GvL) effect. 1 This is associated with the risk of graft-versus-host disease (GvHD). T-cell depletion prevents GvHD but increases the probability of leukemia relapse. Suicide gene therapy offers a pragmatic solution to the "T-cell dilemma" of allo-HCT. 2 A suicide gene codifies for a protein able to convert, at a cellular level, a nontoxic prodrug into a toxic product. Suicide gene modification of donor lymphocytes aims at exploiting allo-HCT for a GvL effect, while providing a selective "switch" to GvHD. 3 The thymidine kinase of herpes simplex virus (TK) is a cell cycledependent suicide gene, that is, it catalyzes the generation of triphosphate ganciclovir (GCV), which is toxic to proliferating cells. 4 In HLA-identical allo-HCT, delayed infusions of TK ϩ lymphocytes were effective against relapsing leukemia, lymphoma, multiple myeloma, and Epstein-Barr virus (EBV)-related lymphoproliferative disorders. 5 When administered at the time of transplantation, TK ϩ lymphocytes facilitated the engraftment of hematopoietic cells. 6,7 In patients developing GvHD, GCV administration eliminated circulating TK ϩ cells and controlled the disease. [5][6][7] Despite these encouraging results, dissemination of the TK technology has been limited by the difficulty in defining optimal conditions for cell manipulation. Current protocols for genetic modification of T lymphocytes with retroviral vectors (RVs) may reduce antigen responsiveness in vitro 8 and antihost reactivity in vivo. 9,10 Given the strict clinical association between GvL and GvHD, it may be postulated that the therapeutic efficacy of TK ϩ lymphocytes will be improved by protocols designed to maximize antihost reactivity in vivo.Genetic modification of T lymphocytes with RVs relies on cell proliferation and leads to a memory phenotype. 11,12 Different models have been proposed to describe mature T-cell differentia...

Suicide gene therapy (SGT) is a powerful approach to exploit anti-host reactivity following allogeneic hemopoietic cell transplantation (allo-HCT) for a full graft-versus-leukemia (GvL) effect, while controlling graft-versus-host disease (GvHD). This is accomplished through genetic modification of donor lymphocytes with a suicide gene. The herpes simplex thymidine kinase (TK) suicide gene converts at a cellular level the pro-drug ganciclovir (GCV) into tri-phosphate toxic derivatives, thence conferring selective sensitivity. Clinical studies as well as animal models have substantiated the concept that a time-wise GCV administration is able to actually separate the therapeutic GvL effect from life-threatening GvHD. Genetic modification of lymphocytes with TK is currently pursued through retroviral vectors (RV). In vitro RV genetic modification requires proliferation, which is easily accomplished by polyclonal stimulation. Polyclonal stimulation with anti-CD3 antibodies (aCD3) has been show to reduce anti-host reactivity of gene-modified lymphocytes. This possibly limits the impact of the suicide gene strategy in allo-HCT. In this study we tackled the rules governing anti-host reactivity of TK+ human lymphocytes. We found that TK+ lymphocytes generated with aCD3 are mainly CD45RA−CCR7− effector memory (EM) cells (84,6±6,6%). Upon re-stimulation they produce interferon-γ, perforin B and granzyme A, but fail to up-regulate CD40L. EM TK+ lymphocytes have a mixed phenotype in regard to CD28/CD27 co-expression and displayed a limited ability to engraft and cause GvHD in a xenogeneic model using conditioned NOD/scid mice (take: 11%). In sharp contrast, TK+ lymphocytes generated with novel protocols taking advantage of anti-CD3 and anti-CD28 antibodies conjugated to para-magnetic cell-sized beads are enriched for CD45RA−CCR7+ central memory (CM) cells (65,3±6,2%) that are able to produce IL-2 and to strongly up-regulate CD40L. CM TK+ lymphocytes are homogenously CD28+CD27+ (91,1,3±2,5%). When infused in conditioned NOD/scid mice CM TK+ lymphocytes persistently engrafted and caused lethal GvHD in a significant fraction of mice (take: 55%, P=0,0017 vs EM TK+ cells). GCV administration to diseased animals resulted in the elimination of TK+ cells in blood and in target organs. Treated animals were rescued with survival up to 120 days (P=0,009 vs saline-treated mice). These results demonstrate that CM TK+ lymphocytes retain significant anti-host reactivity and provide a clue to their in vitro generation. CM TK+ lymphocytes are promising candidates for safe and effective allo-HCT for the cure of hematological malignancies.

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Studies have demonstrated that the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta suppress human immunodeficiency type 1 (HIV-1) replication in vitro. Infection with HIV-1 requires expression of CD4 antigen and the chemokine receptor CXCR4 (X4) or CCR5 (R5) on the surface of target cells. The engagement of these receptors with the viral surface proteins is essential for the membrane fusion process. This study investigated the anti-HIV-1 activity of a derivative of RANTES, the CCR5 antagonist aminooxypentane (AOP)-RANTES, on R5 HIV-1 isolates in peripheral blood mononuclear cells. In drug exposure experiments, AOP-RANTES efficiently inhibited viral replication of HIV-1 R5 strains, with a viral breakthrough observed after the withdrawal of the compound. The HIV-1-specific proliferative capacity was maintained under all conditions when compared with controls. An increase in IFN-gamma production accompanied by a parallel decrease in the generation of IL-10 was observed following the in vitro exposure of cells to AOP-RANTES in the presence of three of four HIV-1 R5 isolates. These experiments confirmed that the chemokine receptor antagonist AOP-RANTES was effective as an inhibitor of HIV-1 R5 strain infectivity in peripheral blood mononuclear cells. The capacity of this compound to maintain HIV-1-specific proliferative activity with a shift toward a type 1 cytokine profile makes this compound a unique molecule, one adopting an immunological pathway to limit HIV-1 infection.