ABSTRACT
Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR-peptide-MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)-A2 tumor epitope NY-ESO-1(157-165)-SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine-tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA-A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials.
Subject(s)
Antigens, Neoplasm/chemistry , Cancer Vaccines/pharmacology , Epitopes, T-Lymphocyte/chemistry , Membrane Proteins/chemistry , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccines, Synthetic/pharmacology , Animals , Antigens, Neoplasm/immunology , Cancer Vaccines/chemistry , Cell Line, Tumor , Chemokine CCL4 , Crystallography, X-Ray , Epitopes, T-Lymphocyte/immunology , HLA-A2 Antigen/chemistry , HLA-A2 Antigen/immunology , Immunization , Interferon-gamma/analysis , Macrophage Inflammatory Proteins/analysis , Major Histocompatibility Complex/immunology , Membrane Proteins/immunology , Mice , Mice, Transgenic , Peptides/chemistry , Peptides/immunology , Protein Binding/immunology , Protein Conformation , Receptors, Antigen, T-Cell/chemistry , Spleen/cytology , Spleen/drug effects , Spleen/immunology , Transfection , Vaccines, Synthetic/chemistryABSTRACT
Tetrameric MHC/peptide complexes are important tools for enumerating, phenotyping, and rapidly cloning Ag-specific T cells. It remains however unclear whether they can reliably distinguish between high and low avidity T cell clones. In this report, tetramers with mutated CD8 binding site selectively stain higher avidity human and murine CTL capable of recognizing physiological levels of Ag. Furthermore, we demonstrate that CD8 binding significantly enhances the avidity as well as the stability of interactions between CTL and cognate tetramers. The use of CD8-null tetramers to identify high avidity CTL provides a tool to compare vaccination strategies for their ability to enhance the frequency of high avidity CTL. Using this technique, we show that DNA priming and vaccinia boosting of HHD A2 transgenic mice fail to selectively expand large numbers of high avidity NY-ESO-1(157-165)-specific CTL, possibly due to the large amounts of antigenic peptide delivered by the vaccinia virus. Furthermore, development of a protocol for rapid identification of high avidity human and murine T cells using tetramers with impaired CD8 binding provides an opportunity not only to monitor expansion of high avidity T cell responses ex vivo, but also to sort high avidity CTL clones for adoptive T cell transfer therapy.
Subject(s)
CD8 Antigens/analysis , Cytotoxicity, Immunologic , Epitopes, T-Lymphocyte/analysis , Membrane Proteins , Staining and Labeling , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Animals , Antigens, Neoplasm/analysis , Antigens, Neoplasm/metabolism , Binding Sites/genetics , Binding Sites/immunology , CD8 Antigens/genetics , CD8 Antigens/metabolism , Cell Line , Cell Line, Tumor , Clone Cells , Cytotoxicity, Immunologic/genetics , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/metabolism , H-2 Antigens/genetics , H-2 Antigens/metabolism , HLA-A2 Antigen/genetics , HLA-A2 Antigen/metabolism , Humans , Immunization, Secondary , Jurkat Cells , Lymphocyte Activation/genetics , Mice , Mice, Transgenic , Plasmids/administration & dosage , Proteins/analysis , Proteins/genetics , Proteins/metabolism , T-Lymphocytes, Cytotoxic/chemistry , Vaccinia/genetics , Vaccinia/immunology , beta 2-Microglobulin/analysis , beta 2-Microglobulin/metabolismABSTRACT
Recombinant vaccines encoding strings of virus- or tumor-derived peptides and/or proteins are currently being designed for use against both cancer and infectious diseases. These vaccines aim to induce cytotoxic immune responses against several Ags simultaneously. We developed a novel tetramer-based technique, based on chimeric HLA A2/H-2K(b) H chains, to directly monitor the CTL response to such vaccines in HLA-A2 transgenic mice. We found that priming and boosting with the same polyepitope construct induced immune responses that were dominated by CTL of a single specificity. When a mixture of viruses encoding single proteins was used to boost the polyepitope primed response, CTL of multiple specificities were simultaneously expanded to highly effective levels in vivo. In addition, we show that a preexisting response to one of the epitopes encoded within a polyepitope construct significantly impaired the ability of the vaccine to expand CTL of other specificities. Our findings define a novel vaccination strategy optimized for the induction of an effective polyvalent cytotoxic response.
