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Using X-ray Crystallography, Biophysics, and Functional Assays to Determine the Mechanisms Governing T-cell Receptor Recognition of Cancer Antigens

DOI: 10.3791/54991 DOI Help

Authors: Bruce J. Maclachlan (Cardiff University) , Alexander Greenshields Watson (Cardiff University) , Georgina H. Mason (Cardiff University) , Andrea J. Schauenburg (Cardiff University; University Hospital of Lausanne (CHUV); University of Lausanne) , Valentina Bianchi (Cardiff University; University Hospital of Lausanne (CHUV); University of Lausanne) , Pierre J. Rizkallah (Cardiff University) , Andrew K. Sewell (Cardiff University) , Anna Fuller (Cardiff University) , David K. Cole (Cardiff University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Visualized Experiments

State: Published (Approved)
Published: March 2017
Diamond Proposal Number(s): 6232

Open Access Open Access

Abstract: Human CD8+ cytotoxic T lymphocytes (CTLs) are known to play an important role in tumor control. In order to carry out this function, the cell surface-expressed T-cell receptor (TCR) must functionally recognize human leukocyte antigen (HLA)-restricted tumor-derived peptides (pHLA). However, we and others have shown that most TCRs bind sub-optimally to tumor antigens. Uncovering the molecular mechanisms that define this poor recognition could aid in the development of new targeted therapies that circumnavigate these shortcomings. Indeed, present therapies that lack this molecular understanding have not been universally effective. Here, we describe methods that we commonly employ in the laboratory to determine how the nature of the interaction between TCRs and pHLA governs T-cell functionality. These methods include the generation of soluble TCRs and pHLA and the use of these reagents for X-ray crystallography, biophysical analysis, and antigen-specific T-cell staining with pHLA multimers. Using these approaches and guided by structural analysis, it is possible to modify the interaction between TCRs and pHLA and to then test how these modifications impact T-cell antigen recognition. These findings have already helped to clarify the mechanism of T-cell recognition of a number of cancer antigens and could direct the development of altered peptides and modified TCRs for new cancer therapies.

Journal Keywords: Immunology; CD8+ T cells; T-cell receptor (TCR); peptide-human leukocyte antigen (pHLA); surface plasmon resonance; X-ray crystallography; cancer; gp100; melanoma; heteroclitic peptides

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography