Tolerogenic dendritic cells (tolDC) play a central role in regulating immune homeostasis and in promoting peripheral tolerance. These features render tolDC a promising tool for cell-based approaches aimed at inducing tolerance in T-cell mediated diseases and in allogeneic transplantation. We developed a protocol to generate genetically engineered human tolDC overexpressing IL-10 (DCIL-10) by means of a bidirectional lentiviral vector (LV) encoding for IL-10. DCIL-10promote allo-specific T regulatory type 1 (Tr1) cells, modulate allogeneic CD4+T cell responses in vitro and in vivo, and are stable in a pro-inflammatory milieu. In the present study, we investigated the ability of DCIL-10to modulate cytotoxic CD8+T cell responses. We demonstrate that DCIL-10reduces allogeneic CD8+T cell proliferation and activation in primary mixed lymphocyte reactions (MLR). Moreover, long-term stimulation with DCIL-10induces allo-specific anergic CD8+T cells without signs of exhaustion. DCIL-10-primed CD8+T cells display limited cytotoxic activity. These findings indicate that stable over-expression of IL-10 in human DC leads to a population of cells able to modulate cytotoxic allogeneic CD8+T cell responses, overall indicating that DCIL-10represent a promising cellular product for clinical applications aimed at inducing tolerance after transplantation.