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

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.

Uncovering the modified immunopeptidome reveals insights into principles of PTM-driven antigenicity

“…This suggests that unmodified lysine residues in the anchoring position are unfavorable for HLA I binding and that the modified state of a lysine residue may be preferred. In contrast, modified arginine such as di/methylated arginine and citrullination are overrepresented in positions 3 to 7, and therefore may impact the T-cell receptor recognition 44 (Figure 2F), as was previously shown for other modifications types. Interestingly, while cysteine modifications on peptides in MS analyses are considered to be introduced by sample processing, in our analysis of the HLA I landscape they have a distinct distribution motif where cysteine carbamidomethyl is enriched in positions 3-4 and cysteinylation is enriched in positions 7-8 (Figure 2C).…”

“…The most critical residues for HLA I binding are the ones that fit into the anchor pockets in the HLA I groove, typically the second and carboxy-terminal positions 43 . By contrast, T-Cell receptors recognition motif is determined by the HLA I-peptide complex and therefore most strongly influenced by the residues in position 3 to 7 of the HLA I-bound peptide 44,45 . In the presented matrix for example, known motifs, such as the tendency of serine phosphorylation modification at position 4 6,7 , were also emphasized as low correlation in this analysis (Pearson 0.41, p value = 0.21) as there was a strong deviation between the phosphorylation and underlying serine distributions (Figure 2D; F-test; p value < 2.2e-16).…”

Molecular characterization of HLA class II binding to the LAG‐3 T cell co‐inhibitory receptor

“…All experiments were performed at 25°C in TBSB buffer. Prepared biotinylated pHLA‐DR molecules were immobilized to covalently linked streptavidin coated CM5 sensor chips prepared as previously described [42]. Biotinylated pHLA‐DR molecules were bound to the chip surface at a flow rate of 10 μL/min.…”

T cell receptor alpha variable 12‐2 bias in the immunodominant response to Yellow fever virus

“…HLA A*0201 α‐chain and β2m were expressed separately, without post‐translational modification, as insoluble inclusion bodies (IBs) in competent Rosetta (DE3) Escherichia coli cells, using 0.5 M IPTG to induce expression as thoroughly described recently . Briefly, for a 1L pMHC refold, 30 mg HLA‐A*0201 α‐chain was mixed with 30 mg β2 m and 4 mg peptide at 37°C for 15 min with 10 mM DTT.…”