Detection of human cytomegalovirus-specific T lymphocytes in human blood: comparison of two methods
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Published on: July 9, 2014
C M Schermann1, G Fischer, V Witt
1Clinical Immunology Laboratory, Intercell AG, Vienna, Austria.
Insights
Tetramer technology is a more sensitive method than cytokine secretion assay for detecting human cytomegalovirus (HCMV)-specific T cells in healthy individuals. This finding aids in monitoring HCMV infection in immunocompromised patients.
Area of Science:
- Immunology
- Virology
- Transplantation Science
Background:
- Human cytomegalovirus (HCMV) infection is a significant threat to immunocompromised patients, especially those undergoing allogeneic stem cell transplantation (SCT).
- Effective monitoring of HCMV reactivation is crucial for timely antiviral treatment and improved patient outcomes.
- Detecting virus-specific effector cells can guide therapeutic decisions in HCMV-infected patients.
Purpose of the Study:
- To compare the sensitivity and specificity of two methods for detecting HCMV-specific T lymphocytes in healthy individuals.
- To evaluate the utility of tetramer technology and cytokine secretion assay (CSA) for monitoring HCMV infection.
Main Methods:
- Blood samples from healthy, HCMV IgG-seropositive volunteers were analyzed.
- HCMV-specific CD8(+) T lymphocytes were detected using HCMV-specific tetramers and multicolor flow cytometry.
- Cytokine secretion assay (CSA) was employed to detect IFN-gamma secretion from HCMV pp65-stimulated CD4(+) and CD8(+) T cells.
Main Results:
- Both tetramer technology and CSA demonstrated high specificity (100%) for detecting HCMV-specific T cells.
- Tetramer technology identified HCMV-specific T cells in 86.5% of individuals, outperforming CSA, which detected 65.4% (CD8+) and 78.8% (CD4+/CD8+).
- A good correlation was observed between the two assays, despite differences in T cell type detection.
Conclusions:
- Tetramer technology offers superior sensitivity for detecting HCMV-specific CD8(+) T cells compared to CSA.
- While CSA detects IFN-gamma secretion across all T cell types, it is more time-consuming and involves indirect detection.
- Tetramer analysis is rapid and reliable but limited by HLA type restrictions, whereas CSA is broadly applicable but less sensitive.
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)-gamma 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-gamma 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.
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