Novel Cellular Microarray Assay for Profiling T-Cell Peptide Antigen Specificities

• Published in: Journal of proteome research 2010-11, Vol.9 (11), p.5629-5637
• Main Authors: Yue, C, Oelke, M, Paulaitis, M. E, Schneck, J. P
• Format: Article
• Language: English
• Subjects: CD8-Positive T-Lymphocytes - immunology; Epitopes, T-Lymphocyte - analysis; High-Throughput Screening Assays; HLA-A2 Antigen; Humans; Microarray Analysis; Receptors, Antigen, T-Cell; Sensitivity and Specificity; T-Cell Antigen Receptor Specificity; T-Lymphocytes, Cytotoxic; Tissue Array Analysis - methods; Tissue Array Analysis - standards
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr100447b

We present a novel cellular microarray assay using soluble peptide-loaded HLA A2-Ig dimer complexes that optimizes the avidity of peptide-HLA binding by preserving the molecular flexibility of the dimer complex while attaining much higher concentrations of the complex relative to cognate T-cell receptors. A seminal advance in assay development is made by separating the molecular T-cell receptor recognition event from the binding interactions that lead to antigen-specific cell capture on the microarray. This advance enables the quantitative determination of antigen-specific frequencies in heterogeneous T-cell populations without enumerating the number of cells captured on the microarray. The specificity of cell capture, sensitivity to low antigen-specific frequencies, and quantitation of antigenic T-cell specificities are established using CD8 T-cell populations with prepared antigen-specific CTL frequencies and heterogeneous T cells isolated from peripheral blood. The results demonstrate several advantages for high-throughput broad-based, quantitative assessments of low-frequency antigen specificities. The assay enables the use of cellular microarrays to determine the stability and flux of antigen-specific T-cell responses within and across populations.