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-gamma 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 may be a useful tool for monitoring HCMV-specific T cell activity in immunocompromised transplanted patients.
Lozza
Laura
L
Servizio di Virologia, IRCCS Policlinico San Matteo, Pavia, Italy.
Lilleri
Daniele
D
Percivalle
Elena
E
Fornara
Chiara
C
Comolli
Giuditta
G
Revello
Maria Grazia
MG
Gerna
Giuseppe
G
D018791:CD4 Lymphocyte Count; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D018920:Coculture Techniques; D003587:Cytomegalovirus / Q000276:immunology*; D003713:Dendritic Cells / Q000502:physiology / Q000821:virology*; D005434:Flow Cytometry; D006801:Humans; D016867:Immunocompromised Host; D007371:Interferon-gamma / Q000096:biosynthesis; D008213:Lymphocyte Activation; D018655:Lymphocyte Count; D015203:Reproducibility of Results
D007371:Interferon-gamma