Simultaneous quantification of human cytomegalovirus (HCMV)-specific CD4+ and CD8+ T cells by a novel method using monocyte-derived HCMV-infected immature dendritic cells
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Laura Lozza1, Daniele Lilleri, Elena Percivalle
1Servizio di Virologia, IRCCS Policlinico San Matteo, Pavia, Italy.
Insights
This study developed a new assay using infected dendritic cells (DC) to measure human cytomegalovirus (HCMV)-specific T cell responses. The method effectively detects both CD4(+) and CD8(+) T cell activity, crucial for monitoring immunity in transplant patients.
Area of Science:
- Immunology
- Virology
- Cell Biology
Background:
- Human cytomegalovirus (HCMV) is a significant pathogen, particularly in immunocompromised individuals.
- Assessing HCMV-specific T cell immunity is vital for managing transplant recipients.
- Existing methods for evaluating T cell responses can be complex and time-consuming.
Purpose of the Study:
- To develop and validate a novel ex vivo assay for simultaneously measuring HCMV-specific CD4(+) and CD8(+) T cell responses.
- To investigate the efficacy of using infected dendritic cells (DCs) as a stimulus for T cell activation.
- To establish a reliable method for discriminating between immune and non-immune individuals regarding HCMV.
Main Methods:
- Immature dendritic cells (DCs) were infected with an endotheliotropic and leukotropic HCMV strain (Huv(+) Leuk(+)).
- Infected DCs were co-cultured with autologous peripheral blood mononuclear cells.
- Intracellular flow cytometry was used to analyze interferon-gamma (IFN-gamma) production, indicating T cell activation.
Main Results:
- Productively infected DCs efficiently stimulated both HCMV-specific CD4(+) and CD8(+) T cell responses.
- A negligible CD8(+) T cell response was observed with non-infectious HCMV strains or inactivated viral antigens.
- The assay successfully differentiated between HCMV-seropositive and HCMV-seronegative subjects, establishing a discrimination cut-off.
Conclusions:
- The novel ex vivo assay enables simultaneous evaluation of HCMV-specific CD4(+) and CD8(+) T cell immunity.
- This method provides a robust tool for monitoring T cell activity in immunocompromised patients, such as transplant recipients.
- The use of productively infected DCs is a key factor for efficient T cell stimulation in this assay.
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 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.
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