CTLs specific for hematopoietic system-restricted minor histocompatibility antigens (mHags) can serve as reagents for cellular adoptive immunotherapy after allogeneic stem cell transplantation (SCT). In the HLA-mismatched setting, CTLs specific for hematopoietic system-restricted mHags expressed solely by the non-self 'allo' HLA molecules could be used to treat relapse after HLA-mismatched SCT. The generation of mHag-specific allo-HLA-restricted CTLs requires antigen-presenting cells (APCs) expressing low numbers of endogenous peptides to avoid co-induction of undesired allo-HLA reactivities. In this study, we exploited viral evasion strategies to generate APCs expressing a controlled set of endogenous peptides. Herpesviruses persist lifelong following primary infection due to expression of viral gene products that hamper T-cell recognition of infected cells. The herpesvirus-derived proteins US6, ICP47 and UL49.5 down-regulate endogenous antigen presentation in human APCs via inhibition of the transporter associated with antigen processing. EBV-transformed B cell lines transduced with retroviral vectors encoding US6, ICP47 or UL49.5 exhibited a stable decrease in cell-surface HLA class I expression and were protected from lysis by mHag-specific CTLs. Exogenous addition of mHag peptide fully restored target cell recognition. UL49.5 showed the most pronounced inhibitory effect, reducing HLA class I expression and mHag-specific lysis up to 99%. UL49.5 also significantly diminished allo-HLA reactivities mediated by allo-HLA-specific CTLs. In conclusion, UL49.5 could be a powerful new tool to study and modulate endogenous antigen presentation.