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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
A sensitive proliferation assay to determine the specific T cell response against HLA-A2.1-binding peptides
S Mayer1, C Scheibenbogen, K H Lee
1Department of Hematology/Oncology, Medizinische Klinik V, University of Heidelberg, Germany.
Insights
This study reports a new proliferation assay to detect T cell responses to HLA class I-binding peptides. The assay shows promise for monitoring immune responses after vaccination with viral or tumor antigens.
Area of Science:
- Immunology
- Cellular immunology
- Vaccine development
Background:
- T cell responses are crucial for adaptive immunity.
- Detecting specific T cell responses to viral or tumor antigens is important for vaccine efficacy.
- HLA class I-binding peptides present antigens to cytotoxic T lymphocytes.
Purpose of the Study:
- To develop and validate a proliferation assay for detecting specific T cell responses.
- To assess the assay's sensitivity and specificity using a CMV-derived peptide.
- To evaluate the assay's utility in monitoring immune responses to vaccination.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) were cultured with a synthetic peptide derived from CMV glycoprotein B.
- Lymphocyte proliferation was measured using Bromodeoxyuridine (BrdU) uptake.
- Assay conditions including cell number, culture time, peptide concentration, and IL-2 supplementation were optimized.
- ELISPOT assay was used to confirm peptide-specific IFN-gamma secretion.
Main Results:
- The proliferation assay detected peptide-specific T cell responses in 47.8% of HLA-A2-positive, CMV IgG-positive donors.
- A lower response rate (11.1%) was observed in HLA-A2-positive, CMV IgG-negative donors (p = 0.019).
- No peptide-specific responses were detected in HLA-A2-negative individuals, indicating high specificity and reproducibility.
Conclusions:
- The developed proliferation assay can detect specific T cell responses against HLA class I-binding peptides.
- The assay demonstrates potential for monitoring immune responses induced by vaccination with tumor or viral antigens.
- Further validation is needed, but the assay shows promise for clinical applications in immunology and vaccinology.
Abstract:
A proliferation assay to detect a specific T cell response against HLA class I-binding peptides is reported. To establish the specificity and sensitivity of the assay we used a synthetic peptide derived from the CMV glycoprotein B containing the HLA-A2.1 ligand motif. Lymphocyte proliferation was measured following culture of PBMC in the presence or absence of peptide by BrdU uptake. Differing culture conditions were compared (cell number, time in culture, peptide concentration, +/- IL-2). A peptide-specific response was detected in 11 of 23 HLA-A2-positive and CMV IgG-positive donors (47.8%), and in 4 of 36 HLA-A2-positive and CMV IgG-negative donors (11.1%, p = 0.019) tested under optimized conditions. None of 22 HLA-A2-negative individuals tested showed a peptide-specific response and the reproducibility was high. Peptide-specific IFN-gamma-secreting T cells could be demonstrated in responding donors with the ELISPOT assay. This proliferation assay may be suitable for monitoring induction of a specific T cell response against known HLA class I-binding peptides following vaccination with tumor or viral antigens.
