Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+ T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells

Immature dendritic cells (DC) infected with an endotheliotropic (Huv⁺) and leukotropic (Leuk⁺) human cytomegalovirus (HCMV) strain were used as a stimulus to determine functional HCMV-specific CD4⁺ and CD8⁺ T cells. Infected DC were co-cultured with autologous peripheral blood mononuclear cells and both arms of T cell activation were determined by intracellular flow cytometry analysis of IFN-γ production. Efficient stimulation of HCMV-specific CD4⁺ and CD8⁺ T cell responses was achieved using DC productively infected with Huv⁺ Leuk⁺ VR1814 strain. On the contrary, a negligible CD8⁺ T cell response was obtained when HCMV strains unable to infect DC, or DC pulsed with inactivated viral antigen, were used. HCMV specificity of the T cell response was confirmed in 46 HCMV-seropositive and 8 HCMV-seronegative healthy subjects. A cut-off was established to discriminate between immune and nonimmune subjects. The novel ex vivo assay enables the simultaneous evaluation of HCMV-specific CD4⁺ and CD8⁺ T cell responses and maybe a useful tool for monitoring HCMV-specific T cell activity in immunocompromised transplanted patients.