Novel cellular microarray assay for profiling T-cell peptide antigen specificities.
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.
Yue
C
C
Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA.
Oelke
M
M
Paulaitis
M E
ME
Schneck
J P
JP
D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000032:analysis*; D015789:HLA-A2 Antigen; D057166:High-Throughput Screening Assays; D006801:Humans; D046228:Microarray Analysis; D011948:Receptors, Antigen, T-Cell; D012680:Sensitivity and Specificity; D037182:T-Cell Antigen Receptor Specificity; D013602:T-Lymphocytes, Cytotoxic; D046888:Tissue Array Analysis / Q000379:methods* / Q000592:standards
D018984:Epitopes, T-Lymphocyte; D015789:HLA-A2 Antigen; D011948:Receptors, Antigen, T-Cell