Normalizing ELISPOT responses to T-cell counts: A novel approach for quantification of HCMV-specific CD4(+) and CD8(+) T-cell responses in kidney transplant recipients

作者：Calarota Sandra A; Chiesa Antonella; Scaramuzzi Lucia; Adzasehoun Kodjo M G; Comolli Giuditta; Mangione Filippo; Esposito Pasquale; Baldanti Fausto^*

来源：Journal of Clinical Virology, 2014, 61(1): 65-73.

DOI：10.1016/j.jcv.2014.05.017

• Human cytomegalovirus
• ELIS POT
• T-cell counts
• Interferon-gamma
• Kidney transplantation

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-gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) using overlapping 15-mer peptide pools of immediate early (IE)-1, IE-2, phosphoprotein 65 (pp 65) (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; 1E-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.

• 出版日期2014-9