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Analysis of CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides
F Kern1, N Faulhaber, C Frömmel
1Institut für Medizinische Immunologie, Charité, Berlin, Germany. florian.kern@charite.de
Insights
This study developed a sensitive method to detect human cytomegalovirus (HCMV) protein-specific CD8 T cells using comprehensive peptide pools. The approach accurately identifies T cell responses to HCMV pp65 and IE-1 proteins, independent of HLA type.
Area of Science:
- Immunology
- Virology
- Vaccinology
Background:
- Human cytomegalovirus (HCMV) infection is common, and T cell responses are crucial for control.
- Accurate measurement of HCMV-specific T cells is important for understanding immunity and vaccine efficacy.
Purpose of the Study:
- To develop and validate a method for measuring human cytomegalovirus (HCMV) protein-specific CD8 T cell frequencies.
- To assess the immunogenicity of HCMV pp65 and IE-1 proteins in healthy donors and transplant patients.
Main Methods:
- Peripheral blood mononuclear cells (PBMC) were stimulated with comprehensive peptide pools covering HCMV pp65 and IE-1 proteins.
- Intracellular IFN-gamma production was measured by flow cytometry to quantify CD8 T cell responses.
- Testing was designed to be independent of donor HLA type by using overlapping peptides spanning entire proteins.
Main Results:
- The method demonstrated 100% sensitivity and specificity in predicting recognized epitopes in HCMV pp65 and IE-1 proteins.
- HCMV IE-1 was confirmed as a significant target for CD8 T cell responses.
- The approach showed high accuracy in both healthy HCMV-seropositive donors and renal transplant patients.
Conclusions:
- Comprehensive peptide pools offer a robust and HLA-independent method for quantifying HCMV-specific CD8 T cells.
- This technique has broader applications for studying immune responses to other pathogens, autoimmune diseases, and vaccine efficacy.
Abstract:
The frequencies of human cytomegalovirus (HCMV) protein-specific CD8 T cells, identified by the presence of intracellular IFN-gamma, were measured by flow cytometry following stimulation of freshly isolated peripheral blood mononuclear cells (PBMC) with comprehensive peptide pools. These pools spanned the entire amino acid sequences of the HCMV pp65 and major immediate early (IE-1) proteins and consisted of 15-amino acid peptides with at least nine overlaps between neighboring peptides. As a result all potential CD8 T cell epitopes contained in these proteins were provided by the complete pools and, therefore, unlike with single epitopes, testing was independent of donor HLA type. Individual stimulating peptides from the same pools were identified in parallel experiments. Thus we found that our results with the complete pools using PBMC from 26 healthy HCMV-seropositive donors were 100% sensitive and specific with respect to predicting the presence of recognized epitopes in the respective proteins. In addition, cells from 15 renal transplant patients were tested with complete pools alone. While our results confirmed our previous contention that HCMV IE-1 is an important CD8 T cell target, the technical improvement we made in order to address this question has clearly wider implications. Similar pools may be applied to examine the role of proteins from other pathogens, in autoimmune disease or following vaccination.
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