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

Abstract

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 cocultured with autologous peripheral blood mononuclear cells and both arms of T cell activation were determined by intracellular flow cytometry analysis of IFN-c 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.

Follicular helper T-cells and virus-specific antibody response in primary and reactivated human cytomegalovirus infections of the immunocompetent and immunocompromised transplant patients

“…Absolute CD3 + CD4 + and CD3 + CD8 + T-cell counts were measured in whole blood samples by flow cytometry (TruCOUNT tubes, BD Biosciences, San Jose, CA, USA). For determination of HCMV-specific T-cells (defined as cells producing IFN-g in response to the HCMV stimulus), PBMC were incubated with autologous monocyte-derived HCMV VR1814-infected dendritic cells (iDC) (Lozza et al, 2005), then washed and incubated with Live/Dead Fixable Violet Dye (Invitrogen, Frederick, MD, USA) and V500-conjugated anti-CD8 (clone RPA-T8) for cell surface staining. Cells were then washed and permeabilized (FACS Permeabilizing Solution, BD Biosciences) and incubated with an intracellular mix of the following mAbs: PerCP-Cy5.5-conjugated anti-CD3 (clone UCHT1), APC-Cy7-conjugated anti-CD4 (clone RPA-T4), PECy7-conjugated anti-IFN-g (clone B27) (BD Biosciences).…”

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

“…As for adaptive T cell immunity, in our laboratory HCMVspecific T cell responses to HCMV infection were routinely measured using an in-house developed procedure [136]. This method, which does not require HLA typing and measures both HCMV-specific CD4 + and CD8 + T cells, is based on the incubation of PBMCs with autologous monocytederived HCMV-infected dendritic cells, followed by determination of multiple membrane/intracellular parameters by cytokine flow cytometry [137].…”

A Randomized Trial of Hyperimmune Globulin to Prevent Congenital Cytomegalovirus

“…Surface phenotype (in the case of CD4+ and CD8+ lymphocytes) as well as interferon-γ-producing CMV-specific T cells were determined retrospectively by means of flow cytometry, as described previously. 16 …”