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

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.