Final Activity Report Summary - SGT FOR GVL (Safe and effective immunotherapy of leukaemia with suicide gene-modified human T lymphocytes retaining anti-minor histocompatibility antigen reactivity)
The most important scientific achievement made in this project is the demonstration that retroviral (RV) gene transfer into human T lymphocytes, after stimulation with cell-sized beads covered with anti-CD3 and anti CD28 antibodies (CD3/CD28-beads) and culture with interleukin (IL-) 7 and IL-15, fully preserves their anti-leukaemic potential for minor histocompatibility antigens (mHag). This finding is not only novel but also of crucial importance for the optimisation of suicide gene therapy for the management of graft-versus-host disease (GvHD after allogeneic hemopoietic cell transplantation (allo-HCT). At the start of the project, it was unknown whether RV gene transfer with existing protocols retained the reactivity of human T lymphocytes against alloantigens (alloreactivity). In allo-HCT, alloreactive donor T cells critically participate to an anti-leukaemic effect, also known as the graft versus leukaemia (GvL) effect. Suicide gene transfer into donor T cells aims at taking advantage of the GvL effect, while controlling a generalised immune attack against the patient, the so called graft-versus-host disease (GvHD). The problem is that due to decreased alloreactivity, the GvL effect mediated by TK+ cells is suboptimal. To preserve the alloreactivity of TK+ cells, I firstly designed novel protocols for RV gene transfer. The two major changes over existing protocols were: i) using polyclonal stimulation with CD3/CD28-beads, instead of soluble anti-CD3 antibodies (OKT3), ii) culturing cells with homeostatic cytokines, such as IL-7 and IL-15, instead of IL-2.
mHag are HLA-restricted immunogenic peptides derived from intracellular polymorphic proteins. mHag differences between patient and donor dictate both the GvL effect and GvHD after HLA-matched allo-HCT. Prof. E. Goulmy, the head of my hosting laboratory, is a world-recognised authority in the field of mHag. To demonstrate that the newly designed protocols offer a solution to the problem of decreased alloreactivity of TK+ lymphocytes, I used the HLA-A2-restricted mHag HA-1 and H-Y as model alloantigens.
Below a brief summary of the results achieved within the specific objectives of the project is provided:
1) Ex vivo generation of TK+ lymphocytes with novel and existing protocols. Starting from peripheral blood mononuclear cells (PBMC) of HLA-A2+ HA-1- or H-Y-, I generated TK+ cells with protocols that included polyclonal stimulation with CD3/CD28-beads and culture with low-dose IL-2, IL-7 or IL-15, alone or in combination. Regardless of the cytokine used for culture, TK+ cells generated after stimulation with CD3/CD28-beads were mainly central memory (CM). TK+ cells generated with aCD3 and high dose-IL-2 were enriched for effector memory (EM) cells.
2) In vitro evaluation of anti-mHag reactivity of TK+ lymphocytes. Upon exploring different protocols, TK+ lymphocytes were pulsed with autologous dendritic cells loaded with the HLA-A2-restricted mHag HA-1 and H-Y peptides. HA-1- or H-Y-specific TK+ high-affinity cytotoxic T cell lines (CTL) were generated only in the case RV gene transfer was previously performed with CD3/CD28-beads and a combination of IL-7 and IL-15. In vitro expansion of high-affinity HA-1- and H-Y-specific CTL was associated with IL-7 receptor expression.
3) In vivo demonstration of GvL and GvHD activity of TK+ lymphocytes. After expansion, HA-1- or H-Y-specific TK+ high-affinity CTL were infused into cohorts of immunodeficient mice previously grafted with HLA-A2+, HA-1+, H-Y+ human leukaemia. When compared to control animals infused with saline or virus-specific CTL, HA-1- or H-Y-specific TK+ high-affinity CTL were able to significantly delay leukaemic outgrowth. In collaboration with my home laboratory, I verified that TK+ cells generated with CD3/CD28-beads cause significant GvHD in an in vivo model based on the grafting of fully mismatched human skin onto immunodeficient mice and that this can be controlled upon GCV administration.
mHag are HLA-restricted immunogenic peptides derived from intracellular polymorphic proteins. mHag differences between patient and donor dictate both the GvL effect and GvHD after HLA-matched allo-HCT. Prof. E. Goulmy, the head of my hosting laboratory, is a world-recognised authority in the field of mHag. To demonstrate that the newly designed protocols offer a solution to the problem of decreased alloreactivity of TK+ lymphocytes, I used the HLA-A2-restricted mHag HA-1 and H-Y as model alloantigens.
Below a brief summary of the results achieved within the specific objectives of the project is provided:
1) Ex vivo generation of TK+ lymphocytes with novel and existing protocols. Starting from peripheral blood mononuclear cells (PBMC) of HLA-A2+ HA-1- or H-Y-, I generated TK+ cells with protocols that included polyclonal stimulation with CD3/CD28-beads and culture with low-dose IL-2, IL-7 or IL-15, alone or in combination. Regardless of the cytokine used for culture, TK+ cells generated after stimulation with CD3/CD28-beads were mainly central memory (CM). TK+ cells generated with aCD3 and high dose-IL-2 were enriched for effector memory (EM) cells.
2) In vitro evaluation of anti-mHag reactivity of TK+ lymphocytes. Upon exploring different protocols, TK+ lymphocytes were pulsed with autologous dendritic cells loaded with the HLA-A2-restricted mHag HA-1 and H-Y peptides. HA-1- or H-Y-specific TK+ high-affinity cytotoxic T cell lines (CTL) were generated only in the case RV gene transfer was previously performed with CD3/CD28-beads and a combination of IL-7 and IL-15. In vitro expansion of high-affinity HA-1- and H-Y-specific CTL was associated with IL-7 receptor expression.
3) In vivo demonstration of GvL and GvHD activity of TK+ lymphocytes. After expansion, HA-1- or H-Y-specific TK+ high-affinity CTL were infused into cohorts of immunodeficient mice previously grafted with HLA-A2+, HA-1+, H-Y+ human leukaemia. When compared to control animals infused with saline or virus-specific CTL, HA-1- or H-Y-specific TK+ high-affinity CTL were able to significantly delay leukaemic outgrowth. In collaboration with my home laboratory, I verified that TK+ cells generated with CD3/CD28-beads cause significant GvHD in an in vivo model based on the grafting of fully mismatched human skin onto immunodeficient mice and that this can be controlled upon GCV administration.