Molecular basis for enhanced T-cell recognition and cross-reactivity

T cells of the adaptive immune system recognize and kill infected cells by interacting with pathogenic peptides displayed on major histocompatibility complexes (MHC) through its T-cell receptor (TCR). TCR interactions with peptide/MHC complexes occur throughout the lifespan of T cells, from development to activation, with the origin of the peptide varying between self and foreign. The vast universe of foreign peptides that could appear on infected cells presented by polymorphic MHCs is by far outnumbering the size of the T-cell repertoire. Thus, a property that characterizes T cells is cross-reactivity which enables weak recognition of self-peptides and activation by foreign peptides. Despite being cross-reactive T cells are known to be highly specific for cognate antigens, which might seem like a paradox. Therefore, dual recognition of MHC and peptide with the need to be cross-reactive yet specific presents challenges to T cells and occurs through mechanisms that are not entirely understood.

The studies within this thesis made use of the P14 T-cell system to investigate how the TCR P14 can differentiate between peptides to learn more about the molecular basis dictating specificity, sensitivity and cross-reactivity. P14 is sensitive to the mutation Y4F at the main TCR-recognition site p4Y of the viral peptide gp33, which is utilized by the Lymphocytic choriomeningitis virus (LCMV) to escape P14 T cells. Surprisingly, alanine substitution at p4 (Y4A) of gp33 is weakly recognized by P14 through the use of a different thermodynamic signature.

At the center of these studies was a peptide modification where position 3 had been substituted to proline (p3P), enhancing both TCR affinity and peptide/MHC stability through independent mechanisms. Importantly, p3P did not alter the peptide conformation, which is important for T-cell cross-recognition. Weak interactions between p3P and the conserved H-2Db residue Y159 accounted for the increased MHC stability, whereas enhanced TCR affinity seemed to arise from decreased binding entropy. The p3P modification was applied to the viral escape mutant Y4F, which reestablished P14 recognition. Co-crystal structures of P14 in complex with gp33/H-2Db and the p3P-modifed gp33/H-2Db visualized the structural basis for enhanced TCR affinity as well as the central role of p4Y of gp33 explaining its sensitivity to mutations.

Finally, P14 also weakly recognizes the self-peptide mDBM which under certain circumstances can lead to auto-reactivity. The p3P modification was also applied to mDBM, which increased TCR affinity and MHC stability, facilitating crystallization. mDBM was found to be a structural mimic of gp33, despite a moderate sequence homology. The ternary structure of P14 in complex with mDBM(3P)/H-2Db demonstrated that flexibility in the CDR3β loop mediated cross-reactivity between gp33 and mDBM.

In conclusion, the results here visualize the different faces of TCRs; sensitivity, specificity and cross-reactivity on a molecular basis. Certain peptide substitutions are tolerated through TCR flexibility and peptide adjustments, whereas others are not. Finally, p3P substitution increased the immunogenicity of our H-2Db-restricted peptides and could present a novel strategy for designing peptide vaccines with increased MHC binding properties.

List of scientific papers

I. Unexpected T-cell recognition of an altered peptide ligand is driven by reversed thermodynamics. Allerbring EB, Duru AD, Uchtenhagen H, Madhurantakam C, Tomek MB, Grimm S, Mazumdar PA, Friemann R, Uhlin M, Sandalova T, Nygren PA, Achour A. Eur J Immunol. 2012 Nov; 42(11):2990-3000.
https://doi.org/10.1002/eji.201242588

II. Structural and thermodynamic basis underlying reestablishment of P14 T-cell recognition of a viral escape mutant. Allerbring EB, Duru AD, Markov N, Uchtenhagen H, Popov A, Madhurantakam C, Sandalova T, Nygren PA and Achour A. [Manuscript]

III. Proline substitution independently enhances H-2Db-complex stabilization and TCR recognition of melanoma-associated peptides. Uchtenhagen H, Abualrous ET, Stahl E, Allerbring EB, Sluijter M, Zacharias M, Sandalova T, van Hall T, Springer S, Nygren PA, Achour A. Eur J Immunol. 2013 Aug 13.
https://doi.org/10.1002/eji.201343456

IV. Structural basis for CD8+ T-cell auto-reactivity in LCMV infection Allerbring EB, Duru AD, Nygren PA, Sandalova T and Achour A. [Manuscript]