Suicide Gene Therapy of Graft-Versus-Host Disease Induced by Central Memory Human T Lymphocytes.

Abstract

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.

Understanding and harnessing the graft‐versus‐leukaemia effect

“…Clinical trials are underway. Preliminary trials describe the successful abrogation of GVHD by ganciclovir (Bondanza et al, 2006). One problem with the approach is the potential for damage to the T-cell repertoire during proliferation in culture necessary for Tk gene transfection.…”

Adoptive T‐cell transfer in cancer immunotherapy

“…44 Addition of CD28 costimulation to CD3 activation helps to preserve the immune competence of the T cells. 45,46 Alternative suicide genes based on the dimerization of Fas 47 or caspase 9 48 in the apoptotic pathway have been developed to circumvent the problem of immunogenicity of HSV-tk.…”

Regulatory T‐cell expansion and function do not account for the impaired alloreactivity of ex vivo‐expanded T cells

“…Depending on the clinical setting, gene transfer may target antigen-specific cells, using antigenspecific stimulation, 3 or polyclonal T cells, using mitogens or CD3 antibody-mediated activation. 4 We 5-7 and others [8][9][10][11][12][13][14] have shown both in vitro and in vivo, using murine and canine models of HSC transplantation, that T-cell expansion following stimulation with phytohaemaglutinin (PHA), CD3 or CD3/CD28 is associated with decreased alloreactivity. In particular, we reported that Summary CD3-and CD28-activated T cells expanded for 12 days ex vivo to produce suicide gene-modified T cells are hyporesponsive to alloantigens.…”

“…6 Assessments performed in murine GvHD models in vivo demonstrate that CD3/CD28 costimulation prevents alterations of the T-cell receptor Vb repertoire during ex vivo expansion 7,15 as well as decreases in Epstein-Barr virus (EBV) reactivity 16,17 and alloreactivity. 11,13 Thus, CD3/CD28 costimulation is preferred to CD3 or mitogen activation for producing GMC. 8,13,18 However, cells expanded with interleukin (IL)-2 after CD3/CD28 costimulation (Co CD3/CD28 cells) still have decreased alloreactivity when assessed in vitro in MLRs.…”

“…11,13 Thus, CD3/CD28 costimulation is preferred to CD3 or mitogen activation for producing GMC. 8,13,18 However, cells expanded with interleukin (IL)-2 after CD3/CD28 costimulation (Co CD3/CD28 cells) still have decreased alloreactivity when assessed in vitro in MLRs. 7 These defects are largely caused by the culture, as the longer the culture duration 14 and the greater the expansion, 8 the greater the impairment of alloreactivity.…”