Sandra A Calarota1, Antonella Chiesa1, Lucia Scaramuzzi2, Kodjo M G Adzasehoun1, Giuditta Comolli3, Filippo Mangione2, Pasquale Esposito2, Fausto Baldanti4. 1. Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy. 2. Nephrology, Dialysis and Transplantation Unit, Fondazione IRCCS Policlinico San Matteo, Viale Golgi 19, 27100 Pavia, Italy. 3. Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy; Experimental Research Laboratories, Biotechnology Area, Fondazione IRCCS Policlinico San Matteo, Viale Golgi 19, 27100 Pavia, Italy. 4. Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy. Electronic address: f.baldanti@smatteo.pv.it.
Abstract
BACKGROUND: Human cytomegalovirus (HCMV) is the most common opportunistic virus infection in solid organ transplant recipients. The analysis of HCMV-specific T-cell immunity after organ transplant is of relevant clinical interest. OBJECTIVES: To analyze HCMV-specific CD4(+) and CD8(+) T-cell responses in healthy subjects and kidney transplant recipients (KTR). STUDY DESIGN: HCMV-specific T-cell responses were evaluated by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) using overlapping 15-mer peptide pools of immediate early (IE)-1, IE-2, phosphoprotein 65 (pp65) (for stimulation of both CD4(+) and CD8(+) T-cell responses) and a pool of 34 short peptides (8-12 amino acids in length, for stimulation of CD8(+) T-cell responses). ELISPOT results were normalized to T-cell subset counts and their correlations with a reported dendritic cell (DC)-based assay, which simultaneously quantifies HCMV-specific CD4(+) and CD8(+) T-cell responses, were analyzed. RESULTS: HCMV-seropositive KTR showed higher ELISPOT responses compared to HCMV-seropositive healthy subjects. IE-1 and pp65 ELISPOT responses were mediated mainly by CD8(+) T-cells and, to a lesser extent, CD4(+) T cells; IE-2 peptides appear to stimulate CD56(+) cells (natural killer cells). In HCMV-seropositive healthy subjects, ELISPOT results (expressed either as net spots/million cells or normalized to the corresponding T-cell count) significantly correlated with the DC assay. However, in HMCV-seropositive KTR, only normalized ELISPOT responses to overlapping 15-mer peptide pools significantly correlated with DC-assay responses. CONCLUSIONS: The normalized ELISPOT represents a novel and simple approach for quantifying and monitoring HCMV-specific CD4(+) and CD8(+) T-cell responses in KTR.
BACKGROUND:Human cytomegalovirus (HCMV) is the most common opportunistic virus infection in solid organ transplant recipients. The analysis of HCMV-specific T-cell immunity after organ transplant is of relevant clinical interest. OBJECTIVES: To analyze HCMV-specific CD4(+) and CD8(+) T-cell responses in healthy subjects and kidney transplant recipients (KTR). STUDY DESIGN:HCMV-specific T-cell responses were evaluated by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) using overlapping 15-mer peptide pools of immediate early (IE)-1, IE-2, phosphoprotein 65 (pp65) (for stimulation of both CD4(+) and CD8(+) T-cell responses) and a pool of 34 short peptides (8-12 amino acids in length, for stimulation of CD8(+) T-cell responses). ELISPOT results were normalized to T-cell subset counts and their correlations with a reported dendritic cell (DC)-based assay, which simultaneously quantifies HCMV-specific CD4(+) and CD8(+) T-cell responses, were analyzed. RESULTS:HCMV-seropositive KTR showed higher ELISPOT responses compared to HCMV-seropositive healthy subjects. IE-1 and pp65 ELISPOT responses were mediated mainly by CD8(+) T-cells and, to a lesser extent, CD4(+) T cells; IE-2 peptides appear to stimulate CD56(+) cells (natural killer cells). In HCMV-seropositive healthy subjects, ELISPOT results (expressed either as net spots/million cells or normalized to the corresponding T-cell count) significantly correlated with the DC assay. However, in HMCV-seropositive KTR, only normalized ELISPOT responses to overlapping 15-mer peptide pools significantly correlated with DC-assay responses. CONCLUSIONS: The normalized ELISPOT represents a novel and simple approach for quantifying and monitoring HCMV-specific CD4(+) and CD8(+) T-cell responses in KTR.