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T-cell Receptor Specificity Maintained by Altered Thermodynamics*

Open AccessPublished:May 22, 2013DOI:https://doi.org/10.1074/jbc.M113.464560
      The T-cell receptor (TCR) recognizes peptides bound to major histocompatibility molecules (MHC) and allows T-cells to interrogate the cellular proteome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterized by weak affinity (KD = 100 nm to 270 μm). We used phage-display to produce a melanoma-specific TCR (α24β17) with a 30,000-fold enhanced binding affinity (KD = 0.6 nm) to aid our exploration of the molecular mechanisms utilized to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, α24β17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition.
      Background: The molecular principles governing T-cell specificity are poorly understood.
      Results: High affinity binding of a melanoma-specific T-cell receptor (TCR) is mediated through new MHC contacts and distinct thermodynamics.
      Conclusion: A novel thermodynamic mechanism upholds TCR-peptide specificity.
      Significance: TCRs can maintain peptide specificity using a mechanism that may enable widespread, safe enhancement of TCR binding affinity in therapeutic applications.

      Introduction

      More than 30 different therapeutic monoclonal antibodies (mAbs) have Food and Drug Administration approval, and these soluble antigen receptors are being used in hundreds of current clinical trials for a wide range of disease states ranging from cardiovascular disease, cancer, and autoimmunity to induction of transplant tolerance (
      • Gura T.
      Therapeutic antibodies. Magic bullets hit the target.
      ,
      • Waldmann T.A.
      Immunotherapy. Past, present, and future.
      ). Therapeutic application of the other class of antigen receptor, the T-cell receptor (TCR),
      The abbreviations used are:TCR, T-cell receptor; MHCI, MHC class I; pMHC, peptide-MHC; SPR, surface plasmon resonance; Bistris propane, 1,3-bis[tris(hydroxymethyl)methylamino]propane; BSA, buried surface area; CDR, complementarity-determining region; vdW, van der Waals; TOPS, TCR/pMHC optimized protein crystallization screen.
      has lagged behind the progress made with mAbs, but several recent studies have indicated that TCRs, or TCR/mAb hybrid molecules, might have a very bright future in gene therapy or as soluble molecules (
      • Morgan R.A.
      • Dudley M.E.
      • Wunderlich J.R.
      • Hughes M.S.
      • Yang J.C.
      • Sherry R.M.
      • Royal R.E.
      • Topalian S.L.
      • Kammula U.S.
      • Restifo N.P.
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      • Nahvi A.
      • de Vries C.R.
      • Rogers-Freezer L.J.
      • Mavroukakis S.A.
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      Cancer regression in patients after transfer of genetically engineered lymphocytes.
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      Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.
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      • Carroll R.G.
      • Milicic A.
      • Mahon T.
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      • Cole D.K.
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      • June C.H.
      • Jakobsen B.K.
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      Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor.
      ). TCRs have advantages over mAbs as they can exploit the MHC class I (MHCI) peptide presentation pathway to interrogate the internal proteome and thereby access a much wider range of disease targets than are available to mAbs. Antibodies undergo somatic hypermutation and bind with strong affinity (KD = nm–pm) and long half-lives (typically hours). In contrast, TCRs are only naturally expressed at the T-cell surface and bind foreign antigens with relatively weak affinities (KD = 100 nm to 270 μm) and short half-lives (0.1–12 s) (
      • Bridgeman J.S.
      • Sewell A.K.
      • Miles J.J.
      • Price D.A.
      • Cole D.K.
      Structural and biophysical determinants of αβ T-cell antigen recognition.
      ,
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      • Gostick E.
      • Price D.A.
      • Gao G.F.
      • Sewell A.K.
      • Jakobsen B.K.
      Human TCR-binding affinity is governed by MHC class restriction.
      ) with cancer-specific TCRs at the weaker end of this scale (
      • Cole D.K.
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      • Sami M.
      • Bell J.I.
      • Gostick E.
      • Price D.A.
      • Gao G.F.
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      • Jakobsen B.K.
      Human TCR-binding affinity is governed by MHC class restriction.
      ,
      • Aleksic M.
      • Liddy N.
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      • Vuidepot A.
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      Different affinity windows for virus and cancer-specific T-cell receptors. Implications for therapeutic strategies.
      ). The weak affinity and short half-lives of natural TCR-pMHC interactions impose severe limitations on the therapeutic use of TCRs as soluble molecules. Recently, molecular engineering via phage display (
      • Varela-Rohena A.
      • Molloy P.E.
      • Dunn S.M.
      • Li Y.
      • Suhoski M.M.
      • Carroll R.G.
      • Milicic A.
      • Mahon T.
      • Sutton D.H.
      • Laugel B.
      • Moysey R.
      • Cameron B.J.
      • Vuidepot A.
      • Purbhoo M.A.
      • Cole D.K.
      • Phillips R.E.
      • June C.H.
      • Jakobsen B.K.
      • Sewell A.K.
      • Riley J.L.
      Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor.
      ,
      • Dunn S.M.
      • Rizkallah P.J.
      • Baston E.
      • Mahon T.
      • Cameron B.
      • Moysey R.
      • Gao F.
      • Sami M.
      • Boulter J.
      • Li Y.
      • Jakobsen B.K.
      Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity.
      ,
      • Li Y.
      • Moysey R.
      • Molloy P.E.
      • Vuidepot A.L.
      • Mahon T.
      • Baston E.
      • Dunn S.
      • Liddy N.
      • Jacob J.
      • Jakobsen B.K.
      • Boulter J.M.
      Directed evolution of human T-cell receptors with picomolar affinities by phage display.
      ), yeast display (
      • Kieke M.C.
      • Shusta E.V.
      • Boder E.T.
      • Teyton L.
      • Wittrup K.D.
      • Kranz D.M.
      Selection of functional T cell receptor mutants from a yeast surface-display library.
      ), and computational design (
      • Hawse W.F.
      • Champion M.M.
      • Joyce M.V.
      • Hellman L.M.
      • Hossain M.
      • Ryan V.
      • Pierce B.G.
      • Weng Z.
      • Baker B.M.
      Cutting edge. Evidence for a dynamically driven T cell signaling mechanism.
      ,
      • Irving M.
      • Zoete V.
      • Hebeisen M.
      • Schmid D.
      • Baumgartner P.
      • Guillaume P.
      • Romero P.
      • Speiser D.
      • Luescher I.
      • Rufer N.
      • Michielin O.
      Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness.
      ) have provided a route to circumvent the intrinsic weak binding affinity of TCRs. Just a few mutations within the TCR CDR loops can improve the binding affinity of a TCR to antibody-like levels and beyond (
      • Dunn S.M.
      • Rizkallah P.J.
      • Baston E.
      • Mahon T.
      • Cameron B.
      • Moysey R.
      • Gao F.
      • Sami M.
      • Boulter J.
      • Li Y.
      • Jakobsen B.K.
      Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity.
      ,
      • Li Y.
      • Moysey R.
      • Molloy P.E.
      • Vuidepot A.L.
      • Mahon T.
      • Baston E.
      • Dunn S.
      • Liddy N.
      • Jacob J.
      • Jakobsen B.K.
      • Boulter J.M.
      Directed evolution of human T-cell receptors with picomolar affinities by phage display.
      ,
      • Kieke M.C.
      • Shusta E.V.
      • Boder E.T.
      • Teyton L.
      • Wittrup K.D.
      • Kranz D.M.
      Selection of functional T cell receptor mutants from a yeast surface-display library.
      ). These developments have paved the way for the use of enhanced TCRs as soluble therapies. Indeed, we have recently shown that high affinity soluble “monoclonal” TCRs can be used to target cancer antigens at the cell surface and induce tumor regression (
      • Liddy N.
      • Bossi G.
      • Adams K.J.
      • Lissina A.
      • Mahon T.M.
      • Hassan N.J.
      • Gavarret J.
      • Bianchi F.C.
      • Pumphrey N.J.
      • Ladell K.
      • Gostick E.
      • Sewell A.K.
      • Lissin N.M.
      • Harwood N.E.
      • Molloy P.E.
      • Li Y.
      • Cameron B.J.
      • Sami M.
      • Baston E.E.
      • Todorov P.T.
      • Paston S.J.
      • Dennis R.E.
      • Harper J.V.
      • Dunn S.M.
      • Ashfield R.
      • Johnson A.
      • McGrath Y.
      • Plesa G.
      • June C.H.
      • Kalos M.
      • Price D.A.
      • Vuidepot A.
      • Williams D.D.
      • Sutton D.H.
      • Jakobsen B.K.
      Monoclonal TCR-redirected tumor cell killing.
      ), and this approach is now being trialed at several centers.
      Despite the promise of affinity-enhanced TCRs, concerns remain about their peptide specificity (
      • Holler P.D.
      • Chlewicki L.K.
      • Kranz D.M.
      TCRs with high affinity for foreign pMHC show self-reactivity.
      ,
      • Zhao Y.
      • Bennett A.D.
      • Zheng Z.
      • Wang Q.J.
      • Robbins P.F.
      • Yu L.Y.
      • Li Y.
      • Molloy P.E.
      • Dunn S.M.
      • Jakobsen B.K.
      • Rosenberg S.A.
      • Morgan R.A.
      High-affinity TCRs generated by phage display provide CD4+ T cells with the ability to recognize and kill tumor cell lines.
      ). Complete immune cover requires that a limited number of TCRs are able to recognize the vastly greater number of potential foreign peptides that could be encountered (
      • Sewell A.K.
      Why must T cells be cross-reactive?.
      ,
      • Wooldridge L.
      • Ekeruche-Makinde J.
      • van den Berg H.A.
      • Skowera A.
      • Miles J.J.
      • Tan M.P.
      • Dolton G.
      • Clement M.
      • Llewellyn-Lacey S.
      • Price D.A.
      • Peakman M.
      • Sewell A.K.
      A single autoimmune T cell receptor recognizes more than a million different peptides.
      ). As a result, TCRs are said to be “cross-reactive” or “poly-specific” (
      • Sewell A.K.
      Why must T cells be cross-reactive?.
      ,
      • Wucherpfennig K.W.
      • Allen P.M.
      • Celada F.
      • Cohen I.R.
      • De Boer R.
      • Garcia K.C.
      • Goldstein B.
      • Greenspan R.
      • Hafler D.
      • Hodgkin P.
      • Huseby E.S.
      • Krakauer D.C.
      • Nemazee D.
      • Perelson A.S.
      • Pinilla C.
      • Strong R.K.
      • Sercarz E.E.
      Polyspecificity of T cell and B cell receptor recognition.
      ). The ability of individual TCRs to recognize huge numbers of peptides has raised significant concerns with regard to TCRs that have undergone artificial affinity enhancement in vitro. These reservations stem from the possibility that high affinity TCRs, particularly those where interaction between the TCR and the MHC component have been increased, might recognize an even greater number of cognate peptides. As enhanced TCRs have not undergone thymic selection in vivo, there are concerns that increased peptide cross-recognition by enhanced affinity TCRs might extend to the binding of self-peptides with a high enough affinity to induce off-target pathology. To date, there has been no rigorous testing of the specificity of an enhanced affinity TCR using molecular approaches.
      Here we explored these issues using phage display to generate a high affinity TCR derived from the MEL5 TCR specific for the heteroclitic version of the HLA-A*0201-restricted primary melanoma antigen recognized by T-cells 1 (MART-126–35) peptide, ELAGIGILTV (A2-ELA) (
      • Li Y.
      • Moysey R.
      • Molloy P.E.
      • Vuidepot A.L.
      • Mahon T.
      • Baston E.
      • Dunn S.
      • Liddy N.
      • Jacob J.
      • Jakobsen B.K.
      • Boulter J.M.
      Directed evolution of human T-cell receptors with picomolar affinities by phage display.
      ,
      • Cole D.K.
      • Edwards E.S.
      • Wynn K.K.
      • Clement M.
      • Miles J.J.
      • Ladell K.
      • Ekeruche J.
      • Gostick E.
      • Adams K.J.
      • Skowera A.
      • Peakman M.
      • Wooldridge L.
      • Price D.A.
      • Sewell A.K.
      Modification of MHC anchor residues generates heteroclitic peptides that alter TCR binding and T cell recognition.
      ). This TCR, α24β17, bound to A2-ELA with an affinity 30,000 times stronger than the natural parent MEL5 TCR, resulting in picomolar levels of binding (
      • Li Y.
      • Moysey R.
      • Molloy P.E.
      • Vuidepot A.L.
      • Mahon T.
      • Baston E.
      • Dunn S.
      • Liddy N.
      • Jacob J.
      • Jakobsen B.K.
      • Boulter J.M.
      Directed evolution of human T-cell receptors with picomolar affinities by phage display.
      ). Comparison of the crystallographic structure of the α24β17 TCR and wild type MEL5 TCR (
      • Cole D.K.
      • Yuan F.
      • Rizkallah P.J.
      • Miles J.J.
      • Gostick E.
      • Price D.A.
      • Gao G.F.
      • Jakobsen B.K.
      • Sewell A.K.
      Germ line-governed recognition of a cancer epitope by an immunodominant human T-cell receptor.
      ) in complex with A2-ELA showed that this remarkable enhancement in binding was attributable to new or altered contacts with the MHC protein. Surprisingly though, α24β17 remained exquisitely peptide-specific, mimicking the specificity of the wild type receptor. Structural and thermodynamic investigations highlighted the role of solvent in determining peptide specificity, a novel finding that sheds light on the molecular rules that govern TCR specificity in general. Overall, we provide a new molecular mechanism by which TCRs maintain peptide specificity and show that it is possible to affinity mature TCRs for therapeutic use as soluble molecules without concomitant loss of peptide specificity.

      DISCUSSION

      The clonotypic TCR, expressed on the surface of CD8+ T-cells, allows recognition of peptide fragments from endogenous proteins presented at the cell surface by MHCI. TCRs discriminate between peptides and permit T-cell-mediated elimination of any cell expressing potentially dangerous intracellular proteins. Exploitation of the TCR offers exciting new possibilities for disease-specific therapies. Unlike antibodies that bind with a relatively strong affinity, TCRs bind with a weak affinity (KD = 100 nm to 270 μm) and short half-lives (0.1–12 s) (
      • Bridgeman J.S.
      • Sewell A.K.
      • Miles J.J.
      • Price D.A.
      • Cole D.K.
      Structural and biophysical determinants of αβ T-cell antigen recognition.
      ,
      • Cole D.K.
      • Pumphrey N.J.
      • Boulter J.M.
      • Sami M.
      • Bell J.I.
      • Gostick E.
      • Price D.A.
      • Gao G.F.
      • Sewell A.K.
      • Jakobsen B.K.
      Human TCR-binding affinity is governed by MHC class restriction.
      ). This disparity is magnified during cancer-specific T-cell responses due to self-specific TCRs binding at the weaker end of this scale (
      • Cole D.K.
      • Pumphrey N.J.
      • Boulter J.M.
      • Sami M.
      • Bell J.I.
      • Gostick E.
      • Price D.A.
      • Gao G.F.
      • Sewell A.K.
      • Jakobsen B.K.
      Human TCR-binding affinity is governed by MHC class restriction.
      ). The weak affinity and short half-lives of natural TCR-pMHC interactions impose severe constraints on the use of soluble TCRs for targeting cell surface-expressed pMHCs. This limitation has recently been overcome using phage display (
      • Li Y.
      • Moysey R.
      • Molloy P.E.
      • Vuidepot A.L.
      • Mahon T.
      • Baston E.
      • Dunn S.
      • Liddy N.
      • Jacob J.
      • Jakobsen B.K.
      • Boulter J.M.
      Directed evolution of human T-cell receptors with picomolar affinities by phage display.
      ), yeast display (
      • Kieke M.C.
      • Shusta E.V.
      • Boder E.T.
      • Teyton L.
      • Wittrup K.D.
      • Kranz D.M.
      Selection of functional T cell receptor mutants from a yeast surface-display library.
      ), and computational design (
      • Hawse W.F.
      • Champion M.M.
      • Joyce M.V.
      • Hellman L.M.
      • Hossain M.
      • Ryan V.
      • Pierce B.G.
      • Weng Z.
      • Baker B.M.
      Cutting edge. Evidence for a dynamically driven T cell signaling mechanism.
      ,
      • Irving M.
      • Zoete V.
      • Hebeisen M.
      • Schmid D.
      • Baumgartner P.
      • Guillaume P.
      • Romero P.
      • Speiser D.
      • Luescher I.
      • Rufer N.
      • Michielin O.
      Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness.
      ) techniques that enhance TCR affinity by vastly extending the half-life of TCRs for cognate pMHC. These new developments enable cellular targeting of diseased tissue with enhanced TCR in soluble form (
      • Sewell A.K.
      Why must T cells be cross-reactive?.
      ).
      We previously generated several high affinity TCRs directed against a range of antigens using phage display and directed evolution (
      • Varela-Rohena A.
      • Molloy P.E.
      • Dunn S.M.
      • Li Y.
      • Suhoski M.M.
      • Carroll R.G.
      • Milicic A.
      • Mahon T.
      • Sutton D.H.
      • Laugel B.
      • Moysey R.
      • Cameron B.J.
      • Vuidepot A.
      • Purbhoo M.A.
      • Cole D.K.
      • Phillips R.E.
      • June C.H.
      • Jakobsen B.K.
      • Sewell A.K.
      • Riley J.L.
      Control of HIV-1 immune escape by CD8 T cells expressing enhanced T-cell receptor.
      ,
      • Li Y.
      • Moysey R.
      • Molloy P.E.
      • Vuidepot A.L.
      • Mahon T.
      • Baston E.
      • Dunn S.
      • Liddy N.
      • Jacob J.
      • Jakobsen B.K.
      • Boulter J.M.
      Directed evolution of human T-cell receptors with picomolar affinities by phage display.
      ,
      • Liddy N.
      • Bossi G.
      • Adams K.J.
      • Lissina A.
      • Mahon T.M.
      • Hassan N.J.
      • Gavarret J.
      • Bianchi F.C.
      • Pumphrey N.J.
      • Ladell K.
      • Gostick E.
      • Sewell A.K.
      • Lissin N.M.
      • Harwood N.E.
      • Molloy P.E.
      • Li Y.
      • Cameron B.J.
      • Sami M.
      • Baston E.E.
      • Todorov P.T.
      • Paston S.J.
      • Dennis R.E.
      • Harper J.V.
      • Dunn S.M.
      • Ashfield R.
      • Johnson A.
      • McGrath Y.
      • Plesa G.
      • June C.H.
      • Kalos M.
      • Price D.A.
      • Vuidepot A.
      • Williams D.D.
      • Sutton D.H.
      • Jakobsen B.K.
      Monoclonal TCR-redirected tumor cell killing.
      ). Here, we investigated the interaction of one of these mutants, α24β17, derived from the MEL5 TCR that is specific for the HLA-A*0201-restricted MART-126–35 antigen (
      • Garboczi D.N.
      • Utz U.
      • Ghosh P.
      • Seth A.
      • Kim J.
      • VanTienhoven E.A.
      • Biddison W.E.
      • Wiley D.C.
      Assembly, specific binding, and crystallization of a human TCR-αβ with an antigenic Tax peptide from human T lymphotropic virus type 1 and the class I MHC molecule HLA-A2.
      ). The α24β17 TCR bound to A2-ELA with an affinity 30,000 times stronger than the MEL5 TCR (
      • Li Y.
      • Moysey R.
      • Molloy P.E.
      • Vuidepot A.L.
      • Mahon T.
      • Baston E.
      • Dunn S.
      • Liddy N.
      • Jacob J.
      • Jakobsen B.K.
      • Boulter J.M.
      Directed evolution of human T-cell receptors with picomolar affinities by phage display.
      ), primarily attributed to a longer off-rate. To better understand the mechanism of high affinity TCR binding, we solved the structure of a high affinity TCR, α24β17, in complex with A2-ELA. Although α24β17 used a similar overall binding mode to MEL5 during engagement, finer examination of the structure demonstrated just three new contacts between α24β17 and the ELA peptide, compared with 41 new MHC contacts. Thus, the enhanced affinity was mediated primarily through additional interactions with the surface of the MHC molecule. This observation raised the possibility that the α24β17 TCR might exhibit reduced peptide specificity, an outcome with significant implications for the development and widespread use of high affinity TCRs. To investigate the peptide specificity of α24β17, we performed an investigation of Ala substitutions across the peptide backbone. Other investigations have shown that peptide substitutions can have a range of effects on TCR binding. Usually, substitutions in the center of the peptide have the largest effect on TCR binding, whereas substitutions at the ends of the peptide have a smaller or no effect (
      • Bulek A.M.
      • Cole D.K.
      • Skowera A.
      • Dolton G.
      • Gras S.
      • Madura F.
      • Fuller A.
      • Miles J.J.
      • Gostick E.
      • Price D.A.
      • Drijfhout J.W.
      • Knight R.R.
      • Huang G.C.
      • Lissin N.
      • Molloy P.E.
      • Wooldridge L.
      • Jakobsen B.K.
      • Rossjohn J.
      • Peakman M.
      • Rizkallah P.J.
      • Sewell A.K.
      Structural basis for the killing of human beta cells by CD8+ T cells in type 1 diabetes.
      ,
      • Miles J.J.
      • Bulek A.M.
      • Cole D.K.
      • Gostick E.
      • Schauenburg A.J.
      • Dolton G.
      • Venturi V.
      • Davenport M.P.
      • Tan M.P.
      • Burrows S.R.
      • Wooldridge L.
      • Price D.A.
      • Rizkallah P.J.
      • Sewell A.K.
      Genetic and structural basis for selection of a ubiquitous T cell receptor deployed in Epstein-Barr virus infection.
      ,
      • Wu L.C.
      • Tuot D.S.
      • Lyons D.S.
      • Garcia K.C.
      • Davis M.M.
      Two-step binding mechanism for T-cell receptor recognition of peptide MHC.
      ). Surprisingly, we observed that the α24β17 TCR was highly sensitive to single Ala substitutions at all positions in the peptide with some mutations capable of completely abrogating binding.
      Although the finding that α24β17 was highly sensitive to single Ala peptide mutations could be system-specific (i.e. may not be observed for other high affinity TCRs), it was unexpected and warranted further investigation into the molecular mechanisms underlying this effect. We solved the structure of α24β17 in complex with two Ala mutants, A2-ELA4A and A2-ELA7A. Additionally, we solved the structure of the unligated α24β17 TCR and unligated A2-ELA1A, A2-ELA4A, and A2-ELA8A molecules. Collectively, the structures demonstrated that the α24β17 used an almost identical binding strategy to engage the Ala mutants compared with the native A2-ELA and that the Ala mutations did not alter the peptide conformation of the unligated pMHCs. The total number of contacts made in the α24β17-A2-ELA complex was similar to the mutated α24β17-A2-ELA4A and α24β17-A2-ELA7A complexes and could not explain the substantially weaker binding affinity to the α24β17 TCR.
      Previous analyses have shown TCRs can use a range of different thermodynamic strategies to bind to pMHC, although favorable enthalpy, most likely mediated through the formation of new bonds during ligation, is the most common driving force (
      • Armstrong K.M.
      • Insaidoo F.K.
      • Baker B.M.
      Thermodynamics of T-cell receptor-peptide/MHC interactions. Progress and opportunities.
      ). This energetic diversity reflects the flexible binding strategies implemented by the TCR during pMHC engagement. Although conformational plasticity in the TCR CDR loops upon pMHC binding is the most common mechanism deployed (
      • Wu L.C.
      • Tuot D.S.
      • Lyons D.S.
      • Garcia K.C.
      • Davis M.M.
      Two-step binding mechanism for T-cell receptor recognition of peptide MHC.
      ,
      • Garcia K.C.
      • Degano M.
      • Pease L.R.
      • Huang M.
      • Peterson P.A.
      • Teyton L.
      • Wilson I.A.
      Structural basis of plasticity in T cell receptor recognition of a self- peptide-MHC antigen.
      ,
      • Scott D.R.
      • Borbulevych O.Y.
      • Piepenbrink K.H.
      • Corcelli S.A.
      • Baker B.M.
      Disparate degrees of hypervariable loop flexibility control T-cell receptor cross-reactivity, specificity, and binding mechanism.
      ), a number of studies have also shown that the TCR can remain rigid (
      • Borbulevych O.Y.
      • Santhanagopolan S.M.
      • Hossain M.
      • Baker B.M.
      TCRs used in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens via distinct mechanisms.
      ,
      • Bulek A.M.
      • Cole D.K.
      • Skowera A.
      • Dolton G.
      • Gras S.
      • Madura F.
      • Fuller A.
      • Miles J.J.
      • Gostick E.
      • Price D.A.
      • Drijfhout J.W.
      • Knight R.R.
      • Huang G.C.
      • Lissin N.
      • Molloy P.E.
      • Wooldridge L.
      • Jakobsen B.K.
      • Rossjohn J.
      • Peakman M.
      • Rizkallah P.J.
      • Sewell A.K.
      Structural basis for the killing of human beta cells by CD8+ T cells in type 1 diabetes.
      ,
      • Chen J.L.
      • Stewart-Jones G.
      • Bossi G.
      • Lissin N.M.
      • Wooldridge L.
      • Choi E.M.
      • Held G.
      • Dunbar P.R.
      • Esnouf R.M.
      • Sami M.
      • Boulter J.M.
      • Rizkallah P.
      • Renner C.
      • Sewell A.
      • van der Merwe P.A.
      • Jakobsen B.K.
      • Griffiths G.
      • Jones E.Y.
      • Cerundolo V.
      Structural and kinetic basis for heightened immunogenicity of T cell vaccines.
      ,
      • Gras S.
      • Saulquin X.
      • Reiser J.B.
      • Debeaupuis E.
      • Echasserieau K.
      • Kissenpfennig A.
      • Legoux F.
      • Chouquet A.
      • Le Gorrec M.
      • Machillot P.
      • Neveu B.
      • Thielens N.
      • Malissen B.
      • Bonneville M.
      • Housset D.
      Structural bases for the affinity-driven selection of a public TCR against a dominant human cytomegalovirus epitope.
      ,
      • Tynan F.E.
      • Reid H.H.
      • Kjer-Nielsen L.
      • Miles J.J.
      • Wilce M.C.
      • Kostenko L.
      • Borg N.A.
      • Williamson N.A.
      • Beddoe T.
      • Purcell A.W.
      • Burrows S.R.
      • McCluskey J.
      • Rossjohn J.
      A T cell receptor flattens a bulged antigenic peptide presented by a major histocompatibility complex class I molecule.
      ), enabling a “lock and key”-like interaction. A thermodynamic investigation of α24β17 binding to A2-ELA, compared with A2-ELA4A and A2-ELA7A, generated some highly unanticipated results. The α24β17 TCR used a distinct thermodynamic signature to engage A2-ELA compared with A2-ELA4A and A2-ELA7A (we observed a marked decrease in favorable entropy for α24β17 binding to A2-ELA4A and A2-ELA7A). This entropy drop suggested that differences in the ordering of solvent molecules involved during α24β17 binding could have an important role in governing antigen specificity. In support of this notion, we observed a reduction in the number of water bridges for α24β17 binding to A2-ELA4A and A2-ELA7A compared with A2-ELA. These data support the idea that peptide specificity can be mediated almost solely through changes in solvent.
      In summary, we show that major improvements to TCR affinity can be gained by increasing interactions between the TCR and the MHC. Despite predictions to the contrary (
      • Holler P.D.
      • Chlewicki L.K.
      • Kranz D.M.
      TCRs with high affinity for foreign pMHC show self-reactivity.
      ,
      • Zhao Y.
      • Bennett A.D.
      • Zheng Z.
      • Wang Q.J.
      • Robbins P.F.
      • Yu L.Y.
      • Li Y.
      • Molloy P.E.
      • Dunn S.M.
      • Jakobsen B.K.
      • Rosenberg S.A.
      • Morgan R.A.
      High-affinity TCRs generated by phage display provide CD4+ T cells with the ability to recognize and kill tumor cell lines.
      ), however, such an outcome need not abrogate the exquisite peptide specificity characteristic of TCR recognition. Our biophysical and thermodynamics analyses of the α24β17 TCR suggest that altered interactions with solvent molecules were the major contributor to the maintenance of peptide specificity. This observation broadens our understanding of T-cell antigen recognition and provides a new mechanism by which TCRs maintain peptide specificity.

      Acknowledgments

      We thank the staff at Diamond Light Source for providing facilities and support. Results shown in this report are also derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the United States Department of Energy, Office of Biological and Environmental Research under Contract DE-AC02–06CH11357.

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