Abstract
The advent in recent years of the application of tetrameric arrays of class I peptide-MHC complexes now enables us to detect and study rare populations of Ag-specific CD8(+) T cells. However, available methods cannot visualize or determine the number and distribution of these TCR ligands on individual cells nor detect APCs in tissues. In this study, we describe for the first time studies of human class I peptide-MHC ligand presentation. These studies were facilitated by applying novel tools in the form of peptide-specific, HLA-A2-restricted human recombinant Abs directed toward a viral epitope derived from human T cell lymphotropic virus type I. Using a large human Ab phage display library, we isolated a large panel of recombinant Fab Abs that are specific for a particular peptide-MHC class I complex in a peptide-dependent, MHC-restricted manner. We used these Abs to visualize the specific complex on APCs and virus-infected cells by flow cytometry, to quantify the number of, and visualize in situ, a particular complex on the surface of APCs bearing complexes formed by naturally occurring active intracellular processing of the cognate viral Ag. These findings demonstrate our ability to transform the unique fine specificity, but low intrinsic affinity of TCRs into high affinity soluble Ab molecules endowed with a TCR-like specificity toward human viral epitopes. These molecules may prove to be crucial useful tools for studying MHC class I Ag presentation in health and disease as well as for therapeutic purposes in cancer, infectious diseases, and autoimmune disorders.
Highlights
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We describe the isolation of a large panel of human recombinant Abs with Ag-specific, MHC-restricted specificity of T cells binding with high affinity to HLA-A2 complexes that display the HTLV-1-derived Tax11–19 peptide
We have demonstrated our ability to select from a large nonimmune human Ab phage library a panel of recombinant Fab Abs directed against a human viral T cell epitope derived from HTLV-1
Summary
ER, endoplasmic reticulum; 2m, 2-microglobulin; hTERT, human telomerase reverse transcriptase; HTLV-I, human T cell lymphotropic virus type I; IPTG, isopropyl -D-thiogalactoside; RT, room temperature; sc, single chain. Only recently, by using large human Ab phage libraries, such unique Abs were generated toward human class I MHC molecules complexed with tumor-associated peptides (16 –18) We used this strategy to isolate human TCR-like Abs directed toward a human viral epitope and show that such Abs can be used for phenotypic analysis of MHC Ag presentation on APCs by direct visualization and in situ detection of the particular specific peptideMHC complex. We demonstrate the experimental utility of such human recombinant Abs in direct phenotypic analysis of the specific class I MHC complex on the surface of APCs and virus-infected cells, including visualization and enumeration of the number of complexes on APCs after naturally occurring active intracellular processing of the Ag as well as in situ detection of the specific peptide-MHC complex on the surface of APCs
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