Journal Article

Comparison between enzyme-linked immunospot assay and intracellular cytokine flow cytometry assay of cytomegalovirus-specific T-cell response in healthy participants

Klinmalai, Chompunuch, Bruminhent, Jackrapong, Prasongtanakij, Somsak, Apiwattanakul, Nopporn, Srisala, Supanart

2026

Overview

Human cytomegalovirus (HCMV) usually establishes a lifelong latent infection after primary infection. Reactivation occurs sporadically and is controlled by cell-mediated immune response (CMIR). Monitoring of CMIR against CMV is mandatory in immunocompromised patients to adjust immunosuppressive drugs to prevent serious end-organ damage after CMV reactivation. Intracellular staining (ICS) and enzyme-linked immunospot (ELISPOT) quantifying CMV-specific T-cells are generally used as surrogate markers for CMIR against CMV. Whether the results of these 2 methods correlate well is not known. This study compared the numbers of CMV-specific T-cells identified by ELISPOT and ICS in healthy adult volunteers. Correlation with CMV serological status was explored. Thirty peripheral blood samples from healthy individuals were quantified for IFN-γ-producing cells after stimulation with whole CMV and IE1 using ICS, and IFN-γ-secreting cells after stimulation with whole CMV lysate and IE1 peptide pool using ICS and IFN-γ-secreting cells by ELISPOT. Anti-CMV IgG levels were analyzed concomitantly using a chemiluminescent microparticle immunoassay. There were 30 healthy participants, 15 (50%) male, with a mean age of 37.8 (± 7.6) years. Twenty-eight (93.3%) were seropositive against CMV. The CMV-specific CD3+ cells, as measured by ICS, were highly correlated with the spot numbers obtained by the ELISPOT, irrespective of CMV antigens used (whole CMV, r = 0.7677, p < 0.0001; IE1, r = 0.6516, p < 0.0001). The numbers of CMV-specific CD3+ cells quantified by IE1 stimulation by these 2 assays were statistically correlated with anti-CMV IgG levels (ICS, r = 0.5070, p = 0.004; ELISPOT, r = 0.4384, p = 0.015). Among the 30 participants, CMV-specific T cells were detected in all participants (100%), including the two seronegative individuals. The present study demonstrated that CMV-specific T-cells measured by ICS and ELISPOT assays were well correlated, suggesting that these assays could be used to monitor CMV-specific T-cells. CMV IgG levels may reflect prior CMV infection and CMV-specific cell-mediated immunity (CMIR) in immunocompetent individuals.

• Publisher: Public Library of Science,PLOS,Public Library of Science (PLoS)
• Subject: Adult / Antibodies / Antigens / Biology and Life Sciences / Care and treatment / CD3 antigen / Cell-mediated immunity / Cells / Chemiluminescence / Cloning / Comparative analysis / Correlation / Cytokines / Cytokines - metabolism / Cytomegalovirus / Cytomegalovirus - immunology / Cytomegalovirus infections / Cytomegalovirus Infections - immunology / Cytotoxicity / Damage prevention / Development and progression / Diagnosis / Enzyme-linked immunosorbent assay / Enzyme-Linked Immunospot Assay - methods / Enzymes / Female / Flow cytometry / Flow Cytometry - methods / Health aspects / Healthy Volunteers / Humans / IE1 protein / Immune response / Immune response (cell-mediated) / Immune system / Immunity (Disease) / Immunity, Cellular / Immunoassay / Immunocompromised hosts / Immunoglobulin G / Immunosuppressive agents / Infection / Infections / Interferon-gamma - metabolism / Intracellular / Latent infection / Lymphocytes / Lymphocytes T / Male / Medicine and Health Sciences / Microparticles / Middle Aged / Peptides / Peripheral blood / Physiological aspects / Research and Analysis Methods / Stimulation / T cells / T-Lymphocytes - immunology / γ-Interferon