468. Cellular Therapy with Transgene Expressing APC Activates CD4 + CD25+ Regulatory T Cells Which Modulate the Immune Response to Gene Therapy Derived Products in Immunocompetent Mice
Authors: Andrea Annoni, Manuela Battaglia, Antonia Follenzi, Angelo Lombardo, Luigi Naldini, Maria Grazia Roncarolo
Journal: Molecular Therapy (2005)

Stable gene replacement by in vivo administration of lentiviral vectors (LV) has important therapeutic applications. However, successful gene therapy is often limited by the immune response to the transgene products, which leads to the clearance of transduced cells. Induction of tolerance to a transgene is highly desirable for successful gene therapy trials. We developed a model in which immunocompetent B6 mice were injected with LV expressing GFP under the CMV promoter (LV-GFP). Peak of GFP expression was detected two weeks after injection and a rapid decrease thereafter. Infiltration of CD8+ T cells was observed in the liver two weeks after LV-GFP injection. GFP-specific IFN-g production by splenic CD8+ T cells derived from LV-GFP injected mice was detected by ELISPOT assay starting from the first week until fifteen weeks post injection. Anti-GFP Abs were also present two weeks after injection and gradually decreased over time. Overall these data indicate that immunocompetent mice injected with LV-GFP develop an Ag-specific CD8+ T cell response that leads to clearance of the transgene expressing cells. CD4+CD25+ T regulatory (Tr) cells play a key role in peripheral tolerance and they have been extensively characterized in preclinical models of T cell mediated diseases. In the present model we investigated whether Tr cells could prevent GFP clearance in vivo. B6 mice were co-injected with LV-GFP and Tr cells isolated from syngeneic wt mice (wt Tr). Clearance of GFP expressing cells and anti-GFP immune response was unmodified in LV-GFP+ wt Tr cells co-injected mice, suggesting that injection of wt Tr cells and consequent increase in the Tr cells pool did not protect from the immune response to the transgene. To investigate whether CD4+CD25+ Tr cells need to be GFP specific in order to modulate the anti-GFP immune response, B6 mice were co-injected with LV-GFP and GFP expressing cells obtained from splenocytes of GFP-Tg mice (GFP Tg cells). Interestingly, transfer of GFP Tg cells was able to down-modulate expansion of splenic GFP-specific CD8+ T cells. Since the GFP Tg cells comprise of both CD4+CD25+ Tr (13,5%) and GFP Tg antigen presenting cells (GFP Tg APC) we next investigated the respective role of these two cell populations. We first co-injected mice with LV-GFP and highly purified GFP Tg Tr (99,1%). As with the administration of wt Tr cells, the transfer of highly purified GFP Tg Tr cells did not modulate the immune response to GFP in vivo. On the contrary, the co-administration of LV GFP and highly purified GFP Tg APC significantly modulated both the cellular and humoral anti-GFP immune responses. These results demonstrate that APC isolated from GFP tolerant mice can reduce the immune response to GFP when transferred in vivo. We hypothesize that after LV-GFP injection the transgene expressing GFP Tg APC activate the endogenous CD4+CD25+ Tr in the absence of danger signals leading to down regulation of the anti-GFP response. Transgene presentation by APC is therefore crucial to trigger an immune-regulatory response mediated by CD4+CD25+ Tr cells.