Cytomegalovirus-Specific T Cells Isolated by IFN-γ Secretion Assay Do Not Induce Significant Graft-Versus-Host Reactions In Vitro

Background. Graft-versus-host (GvH) disease (GvHD) remains a serious concern for patients undergoing antiviral cellular therapy. Despite the major improvements in cellular immunotherapy, the immunogenicity of virus-specific Tcells has not yet been fully defined. This present study aims to examine how cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) respond to allogeneic antigen stimulation and whether they give rise to GvHD target tissue damage. Methods. Cytomegalovirus-specific CTLs were isolated by the IFN-gamma secretion assay (gamma-catch) from healthy seropositive volunteers and expanded in vitro. The levels of intracellular IFN-gamma, cytotoxic activity, IFN-gamma and granzyme B secretion, and CD25 expression were measured using flow cytometry (fluorescence-activated cell sorting). The ability of CMV-CTLs to induce GvHD target tissue damage was evaluated using the human in vitro skin explant assay (skin explant assay). Results. Cytomegalovirus-specific CTLs responded specifically to CMV-phosphoprotein 65 stimulation by secreting IFN-. and killing virus peptide loaded autologous phytohemagglutinin (PHA) blasts. Compared with unselected peripheral blood mononuclear cells, CMV-CTLs induced significantly less severe cutaneous GvH tissue damage. This observation coincided with low levels of CD25 expression, as well as IFN-. and granzyme B secretion after allogeneic antigen stimulation in both the mixed lymphocyte reaction and in the skin explant assay. Conclusions. Cytomegalovirus-specific CTLs isolated by the IFN-. secretion assay from HLA-unmatched healthy donors exhibited a high level of anti-CMV potency without inducing significant cutaneous GvH tissue damage in vitro. This finding provides novel evidence supporting the safe use of in vitro expanded CMV-CTLs as an antiviral therapy in transplant patients with refractory CMV infections.