Human mesenchymal stem cells (MSC) are immunosuppressive and poorly immunogenic but may act as antigen-presenting cells (APC) for CD4(+) T-cell responses; here we have investigated their ability to serve as APC for in vitro CD8(+) T-cell responses. MSC pulsed with peptides from viral antigens evoked interferon (IFN)-gamma and Granzyme B secretion in specific cytotoxic T lymphocytes (CTL) and were lysed, although with low efficiency. MSC transfected with tumor mRNA or infected with a viral vector carrying the Hepatitis C virus NS3Ag gene induced cytokine release but were not killed by specific CTL, even following pretreatment with IFN-gamma. To investigate the mechanisms involved in MSC resistance to CTL-mediated lysis, we analyzed expression of human leukocyte antigen (HLA) class I-related antigen-processing machinery (APM) components and of immunosuppressive HLA-G molecules in MSC. The LMP7, LMP10, and ERp57 components were not expressed and the MB-1 and zeta molecules were downregulated in MSC either unmanipulated or pretreated with IFN-gamma. Surface HLA-G was constitutively expressed on MSC but was not involved in their protection from CTL-mediated lysis. MSC supernatants containing soluble HLA-G (sHLA-G) inhibited CTL-mediated lysis, whereas those lacking sHLA-G did not. The role of sHLA-G in such inhibition was unambiguously demonstrated by partial restoration of lysis following sHLA-G depletion from MSC supernatants. In conclusion, human MSC can process and present HLA class I-restricted viral or tumor antigens to specific CTL with a limited efficiency, likely because of some defects in APM components. However, they are protected from CTL-mediated lysis through a mechanism that is partly sHLA-G-dependent.