Detection of human cytomegalovirus-specific T lymphocytes in human blood: comparison of two methods

Background Human cytomegalovirus (HCMV) infection remains a major cause of morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation (SCT). In the case of HCMV reactivation, the well-defined detection of virus-specific effector cells in patients might positively impact antiviral treatment.

Methods We examined blood samples from healthy volunteers serologically typed for HCMV IgG. Based on multicolor flow cytometry analysis, we addressed HCMV-specific CD8⁺ effector T lymphocytes using HCMV-specific tetramers for the respective major histocompatibility complex (MHC) class I type. As a second approach, we employed the cytokine secretion assay (CSA), which allows the indirect detection of target-specific CD4⁺ and CD8⁺ T cells via their interferon (IFN)-γ secretion upon HCMV pp65 in vitro stimulation.

Results We hypothesized the detection of HCMV-specific lymphocytes in >50% of healthy Caucasians that were IgG-seropositive for HCMV. In terms of specificity, both assays showed comparably good results (specificity 100%, confidence interval >95%). Regarding sensitivity, both assays met the zero hypothesis. However, with 45/52 (86.5%) the tetramer technology was superior to the CSA, which detected 34/52 (65.4%) based on CD8⁺ T cells and 41/52 (78.8%) based on both CD4⁺ and CD8⁺ T cells.

Discussion A good correlation was observed between both assays, although the tetramers addressed only CD8⁺ HCMV-specific T cells, whereas IFN-γ secretion was detected on all T-cell types. Disadvantages of the CSA are the time-consuming stimulation, the extensive cell washing steps and the fact that the target cells are detected indirectly. The analysis with tetramers is rapid and reliable but their general use is hampered because of the restriction to a few HLA types.