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Loss of T Cell Antigen Recognition Arising from Changes in Peptide and Major Histocompatibility Complex Protein Flexibility

IMPLICATIONS FOR VACCINE DESIGN*
  • Francis K. Insaidoo
    Footnotes
    Affiliations
    Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556
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  • Oleg Y. Borbulevych
    Footnotes
    Affiliations
    Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556
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  • Moushumi Hossain
    Affiliations
    Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556
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  • Sujatha M. Santhanagopolan
    Footnotes
    Affiliations
    Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556
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  • Tiffany K. Baxter
    Affiliations
    Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556
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  • Brian M. Baker
    Correspondence
    To whom correspondence should be addressed: 251 Nieuwland Science Hall, Notre Dame, IN 46556. Fax: 574-631-6652
    Affiliations
    Department of Chemistry and Biochemistry and the Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556
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  • Author Footnotes
    * This work was supported in part by National Institutes of Health Grant GM067079 from the NIGMS (to B. M. B.). This work was also supported by Grant MCB0448298 from the National Science Foundation and Grant RSG-05-202-01-GMC from the American Cancer Society.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.
    1 Present address: Dept. of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032.
    2 Present address: QuantumBio, Inc., State College, PA 16801.
    3 Supported by a fellowship from the Walther Cancer Research Foundation.
Open AccessPublished:September 21, 2011DOI:https://doi.org/10.1074/jbc.M111.283564
      Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-127–35 tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-127–35 antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide·MHC complex. These results help explain how the “anchor-fixing” strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.

      Introduction

      T cell receptor (TCR)
      The abbreviations used are: TCR
      T cell receptor
      pMHC
      peptide/MHC complex
      r.m.s.
      root mean square
      BIS-TRIS
      2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol.
      recognition of an antigenic peptide bound and presented by a class I major histocompatibility complex (MHC) protein underlies the cytotoxic immune response to pathogens and cancer. Many tumor or viral antigens are poorly immunogenic, which can sometimes be attributed to weak binding of the peptide to the MHC molecule, consequently resulting in low antigen density on the surface of a presenting cell (
      • Yu Z.
      • Theoret M.R.
      • Touloukian C.E.
      • Surman D.R.
      • Garman S.C.
      • Feigenbaum L.
      • Baxter T.K.
      • Baker B.M.
      • Restifo N.P.
      ). One approach for vaccine design involves improving peptide-MHC affinity, with the goal of using the modified peptides to convert naive antigen-specific T cells into more sensitive effector T cells. This strategy is often employed when weak peptide binding results from the presence of suboptimal “anchor” residues within the peptide sequence (
      • Valmori D.
      • Fonteneau J.F.
      • Lizana C.M.
      • Gervois N.
      • Liénard D.
      • Rimoldi D.
      • Jongeneel V.
      • Jotereau F.
      • Cerottini J.C.
      • Romero P.
      ,
      • Parkhurst M.R.
      • Salgaller M.L.
      • Southwood S.
      • Robbins P.F.
      • Sette A.
      • Rosenberg S.A.
      • Kawakami Y.
      ). Many clinical trials employing anchor-modified peptides have been performed or are in progress.
      However, improving peptide binding by substituting suboptimal with optimal anchor residues does not always enhance antigenicity, complicating efforts to engineer peptide-based vaccines. A clear example of this is seen with the MART-127–35 melanoma antigen (sequence AAGIGILTV; referred to here as AAG),
      The peptides used in this study are as follows: AAG, MART-127–35 nonamer (AAGIGILTV); ALG, anchor-modified MART-127–35 nonamer (ALGIGILTV); EAA, MART-126–35 decamer (EAAGIGILTV).
      which possesses an alanine at position 2 and binds weakly to the restricting class I MHC molecule, HLA-A*0201 (HLA-A2). Valmori et al. (
      • Valmori D.
      • Fonteneau J.F.
      • Lizana C.M.
      • Gervois N.
      • Liénard D.
      • Rimoldi D.
      • Jongeneel V.
      • Jotereau F.
      • Cerottini J.C.
      • Romero P.
      ) found that when the second alanine of the AAG peptide was replaced with leucine, generating the peptide ALGIGILTV (referred to here as ALG), peptide binding to HLA-A2 was enhanced 40-fold, but antigenicity was severely curtailed. This result has been confirmed with multiple T cell clones (
      • Borbulevych O.Y.
      • Insaidoo F.K.
      • Baxter T.K.
      • Powell Jr., D.J.
      • Johnson L.A.
      • Restifo N.P.
      • Baker B.M.
      ,
      • Derré L.
      • Ferber M.
      • Touvrey C.
      • Devevre E.
      • Zoete V.
      • Leimgruber A.
      • Romero P.
      • Michielin O.
      • Lévy F.
      • Speiser D.E.
      ) as well as staining experiments with pMHC tetramers (
      • Derré L.
      • Ferber M.
      • Touvrey C.
      • Devevre E.
      • Zoete V.
      • Leimgruber A.
      • Romero P.
      • Michielin O.
      • Lévy F.
      • Speiser D.E.
      ). Interestingly, however, T cells that recognize the ALG peptide poorly (or not at all) readily cross-react with a decameric form of the native MART-1 peptide possessing a glutamic acid at the N terminus (EAAGIGILTV; referred to as EAA) (
      • Valmori D.
      • Fonteneau J.F.
      • Lizana C.M.
      • Gervois N.
      • Liénard D.
      • Rimoldi D.
      • Jongeneel V.
      • Jotereau F.
      • Cerottini J.C.
      • Romero P.
      ,
      • Borbulevych O.Y.
      • Insaidoo F.K.
      • Baxter T.K.
      • Powell Jr., D.J.
      • Johnson L.A.
      • Restifo N.P.
      • Baker B.M.
      ,
      • Derré L.
      • Ferber M.
      • Touvrey C.
      • Devevre E.
      • Zoete V.
      • Leimgruber A.
      • Romero P.
      • Michielin O.
      • Lévy F.
      • Speiser D.E.
      ). Previously, these results were interpreted to indicate that anchor modification of the nonameric peptide altered the conformation away from one shared by the native nonamer and the decamer (
      • Sliz P.
      • Michielin O.
      • Cerottini J.C.
      • Luescher I.
      • Romero P.
      • Karplus M.
      • Wiley D.C.
      ,
      • Romero P.
      • Valmori D.
      • Pittet M.J.
      • Zippelius A.
      • Rimoldi D.
      • Lévy F.
      • Dutoit V.
      • Ayyoub M.
      • Rubio-Godoy V.
      • Michielin O.
      • Guillaume P.
      • Batard P.
      • Luescher I.F.
      • Lejeune F.
      • Liénard D.
      • Rufer N.
      • Dietrich P.Y.
      • Speiser D.E.
      • Cerottini J.C.
      ).
      However, when the structures of the native and anchor-modified nonamers bound to HLA-A2 were determined, it was observed that the alanine-to-leucine modification at position 2 had only a minor effect on the conformation of the peptide (
      • Borbulevych O.Y.
      • Insaidoo F.K.
      • Baxter T.K.
      • Powell Jr., D.J.
      • Johnson L.A.
      • Restifo N.P.
      • Baker B.M.
      ). Indeed, the structural differences between the AAG and ALG nonamers were substantially less than the differences between the AAG nonamer and the EAA decamer, leading us to question how multiple T cell clones could on one hand be highly sensitive to a very small structural perturbation while on the other tolerant of a much more dramatic structural difference. This question reflects the well appreciated yet poorly understood dichotomy of high specificity together with high cross-reactivity that is characteristic of the T cell arm of the immune system.
      Here we investigated the consequences of anchor modification of the MART-127–35 AAG peptide, aiming to understand how the “anchor-fixing” strategy fails despite achieving the goal of enhancing peptide binding affinity with only minor conformational consequences. We found that the alanine-to-leucine substitution at position 2 increases the flexibility of the N-terminal half of the MART-1 peptide, leading to the subtle structural effects seen crystallographically. Anchor modification also increases the flexibility of the helices that form the peptide binding domain. The increased flexibility and thus higher entropy of the complex with the modified ALG peptide contributes to the improvement in peptide binding to HLA-A2. However, it will also raise the entropic cost for TCR binding to the pMHC complex, contributing to a general loss in affinity with MART-1-specific TCRs. These results have implications not only for vaccine design and other efforts to alter immunogenicity through peptide modification but also for our understanding of the mechanisms of viral and tumor escape from immune destruction and the origins of T cell receptor specificity in general.

      DISCUSSION

      Anchor modification of peptides is a common strategy for enhancing peptide-MHC binding affinity and is frequently used in the design of peptide-based vaccine candidates for infectious disease and cancer. However, anchor modification is not always successful at enhancing peptide immunogenicity. Failures are often attributed to the high sensitivity of the T cell receptor toward subtle chemical changes in antigenic peptides, although the underlying mechanisms are usually not well understood.
      To examine how subtle peptide modifications can negatively impact TCR recognition, we explored one of the most dramatic failures of the anchor modification strategy reported: despite substantially improving the interaction with HLA-A2, replacement of the suboptimal alanine at position 2 of the nonameric MART-127–35 melanoma antigen with leucine considerably reduces or abolishes immunogenicity with a wide range of clonally diverse T cells (
      • Valmori D.
      • Fonteneau J.F.
      • Lizana C.M.
      • Gervois N.
      • Liénard D.
      • Rimoldi D.
      • Jongeneel V.
      • Jotereau F.
      • Cerottini J.C.
      • Romero P.
      ,
      • Borbulevych O.Y.
      • Insaidoo F.K.
      • Baxter T.K.
      • Powell Jr., D.J.
      • Johnson L.A.
      • Restifo N.P.
      • Baker B.M.
      ,
      • Derré L.
      • Ferber M.
      • Touvrey C.
      • Devevre E.
      • Zoete V.
      • Leimgruber A.
      • Romero P.
      • Michielin O.
      • Lévy F.
      • Speiser D.E.
      ). This negative outcome has led to the use of other MART-1 antigens in clinical trials for the immunological treatment of melanoma (e.g. Ref.
      • Phan G.Q.
      • Touloukian C.E.
      • Yang J.C.
      • Restifo N.P.
      • Sherry R.M.
      • Hwu P.
      • Topalian S.L.
      • Schwartzentruber D.J.
      • Seipp C.A.
      • Freezer L.J.
      • Morton K.E.
      • Mavroukakis S.A.
      • White D.E.
      • Rosenberg S.A.
      ) despite evidence that the nonamer is the clinically relevant antigen in HLA-A2+ individuals (
      • Skipper J.C.
      • Gulden P.H.
      • Hendrickson R.C.
      • Harthun N.
      • Caldwell J.A.
      • Shabanowitz J.
      • Engelhard V.H.
      • Hunt D.F.
      • Slingluff Jr., C.L.
      ).
      The loss in immunogenicity with the modified MART-127–35 nonamer is attributable to weaker TCR binding, as demonstrated here in a direct binding experiment with the DMF5 TCR. However, anchor modification has only a minor structural effect on the peptide, leading to the partial occupancy of an alternative flipped conformation for the central glycine at position 5 of the peptide. In the structures of two different TCRs bound to the MART-127–35 nonamer (
      • Borbulevych O.Y.
      • Santhanagopolan S.M.
      • Hossain M.
      • Baker B.M.
      ), there are no interactions with the region of the peptide that is altered. Further, as T cells that are sensitive to anchor modification also recognize the decameric MART-126–35 peptide, which possesses substantially greater structural variation in this region (
      • Borbulevych O.Y.
      • Insaidoo F.K.
      • Baxter T.K.
      • Powell Jr., D.J.
      • Johnson L.A.
      • Restifo N.P.
      • Baker B.M.
      ), structural aspects alone cannot easily explain the poor recognition of the modified nonamer.
      The data presented here suggest a more general effect: rather than altering the structure per se, anchor modification of the MART-127–35 peptide enhances ligand flexibility. In addition to inducing heterogeneity in the peptide, the enhanced flexibility extends to the HLA-A2 α1 and α2 helices, resulting in greater breathing of the peptide binding domain. The fluctuations in the peptide and the peptide binding domain appear linked, with the enhanced flexibility of the helices reducing steric barriers that otherwise hinder peptide movement. As protein flexibility translates into conformational entropy, the result is that the complex with the modified peptide possesses higher entropy.
      The higher entropy of the complex with the modified peptide will have two consequences. First, as the overall entropic penalty for binding will be reduced, binding of the peptide to HLA-A2 will be improved to a degree beyond that expected solely from the tighter packing and burial of hydrophobic surface associated with an alanine-to-leucine substitution at the P2 anchor position. The peptide binding data are consistent with this; anchor modification of the MART-127–35 peptide increases peptide binding to HLA-A2 by 40-fold (
      • Valmori D.
      • Fonteneau J.F.
      • Lizana C.M.
      • Gervois N.
      • Liénard D.
      • Rimoldi D.
      • Jongeneel V.
      • Jotereau F.
      • Cerottini J.C.
      • Romero P.
      ). For comparison, the same alanine-to-leucine P2 anchor modification in the MART-126–35 EAA decamer leads to a much smaller 2-fold enhancement in binding affinity (
      • Valmori D.
      • Fonteneau J.F.
      • Lizana C.M.
      • Gervois N.
      • Liénard D.
      • Rimoldi D.
      • Jongeneel V.
      • Jotereau F.
      • Cerottini J.C.
      • Romero P.
      ). Importantly, there are no indications that the modification in the decamer alters pMHC structure or dynamics, and it does not negatively impact recognition by MART-1-specific T cells (
      • Borbulevych O.Y.
      • Insaidoo F.K.
      • Baxter T.K.
      • Powell Jr., D.J.
      • Johnson L.A.
      • Restifo N.P.
      • Baker B.M.
      ,
      • Sliz P.
      • Michielin O.
      • Cerottini J.C.
      • Luescher I.
      • Romero P.
      • Karplus M.
      • Wiley D.C.
      ).
      However, in addition to favorably impacting peptide binding to HLA-A2, a higher entropy for the complex with the modified MART-127–35 peptide will negatively impact TCR binding. TCRs contact both peptide and MHC, and the majority of TCRs whose structures have been determined “focus” on the center of the peptide and the α1/α2 helices (
      • Rudolph M.G.
      • Stanfield R.L.
      • Wilson I.A.
      ). This is true for the DMF5 TCR used here, as well as the unrelated MART-127–35-specific DMF4 TCR (
      • Borbulevych O.Y.
      • Santhanagopolan S.M.
      • Hossain M.
      • Baker B.M.
      ). As the formation of TCR-pMHC contacts in this region will necessitate a reduction in binding site flexibility, the enhanced dynamics of the modified pMHC complex will result in a less favorable entropy change for TCR binding, translating into reduced affinity. This effect has been seen with the A6 TCR, which recognizes the Tax and Tel1p peptides presented by HLA-A2. The Tel1p peptide induces greater flexibility in the HLA-A2 α2 helix, and as anticipated, receptor binding occurs with a less favorable binding entropy change (ΔΔS° = −3 cal/K/mol) (
      • Borbulevych O.Y.
      • Piepenbrink K.H.
      • Gloor B.E.
      • Scott D.R.
      • Sommese R.F.
      • Cole D.K.
      • Sewell A.K.
      • Baker B.M.
      ).
      In the case studied here, without a high affinity MART-127–35-specific TCR to quantify binding thermodynamics, the extent of the increased entropic penalty for TCR binding resulting from anchor modification of the peptide cannot be quantified. Further, as different TCRs will require different contacts to pMHC, the overall impact will likely vary with different receptors. However, the DMF5 TCR used here provides some guidance. Anchor modification to the MART-127–35 peptide weakens the KD of the DMF5 TCR from 68 μm to greater than 3.3 mm, amounting to an unfavorable loss in binding free energy of at least 2.3 kcal/mol at 25 °C. If we assume that this value is solely attributable to conformational entropy, it translates into a minimum entropic penalty of 8 cal/K/mol. In absolute terms, this is not large as reported ΔS° values for TCR recognition of pMHC span a 100 cal/K/mol range (
      • Armstrong K.M.
      • Insaidoo F.K.
      • Baker B.M.
      ). However, given the relatively weak affinities and thus binding free energies associated with TCR recognition of pMHC (and self-antigens in particular) (
      • Davis M.M.
      • Boniface J.J.
      • Reich Z.
      • Lyons D.
      • Hampl J.
      • Arden B.
      • Chien Y.
      ), the consequence of even a small gain in the entropic penalty for binding will be significant. A 5 cal/K/mol increase in binding ΔS° will convert TCR binding affinities occurring in the range of 50–100 μm (a range typical for TCR recognition of self-antigens) to 600–1200 μm, weaker than that typically expected to result in a functional T cell response.
      This analysis leads us to consider a more refined view of TCR specificity. Receptor specificity, typically defined by changes in peptide composition translating into a loss of recognition in functional assays, is usually thought to arise from either the disruption of favorable interactions or the introduction of unfavorable interactions between TCR and peptide. This interpretation evokes the commonly held view that TCRs are highly tuned toward the structure of a particular antigen. We propose that in the case of TCRs that recognize MART-1 antigens, this is not the case. The majority of these TCRs can in fact tolerate substantial structural variation, as seen by the widespread cross-reactivity between the 27–35 AAG nonamer and the structurally divergent 26–35 EAA decamer (
      • Valmori D.
      • Fonteneau J.F.
      • Lizana C.M.
      • Gervois N.
      • Liénard D.
      • Rimoldi D.
      • Jongeneel V.
      • Jotereau F.
      • Cerottini J.C.
      • Romero P.
      ,
      • Borbulevych O.Y.
      • Insaidoo F.K.
      • Baxter T.K.
      • Powell Jr., D.J.
      • Johnson L.A.
      • Restifo N.P.
      • Baker B.M.
      ,
      • Derré L.
      • Ferber M.
      • Touvrey C.
      • Devevre E.
      • Zoete V.
      • Leimgruber A.
      • Romero P.
      • Michielin O.
      • Lévy F.
      • Speiser D.E.
      ), recently visualized in crystallographic structures of the DMF4 and DMF5 TCRs (
      • Borbulevych O.Y.
      • Santhanagopolan S.M.
      • Hossain M.
      • Baker B.M.
      ). Rather, “highly specific” TCR recognition of the MART-1 nonamer revealed by anchor modification may arise because moderate TCR affinity allows what may be a relatively small increase in the entropic cost of binding to weaken TCR affinity to levels that substantially weaken or fully ablate the immunological response.
      Could this alternative view of specificity be a more general phenomenon? It is notable that, at least for class I MHC molecules, the flexibility of the peptide binding domain seems to be readily tunable via peptide or MHC alterations. Complementing the data presented here, as noted above, we recently demonstrated that the flexibility of the HLA-A2 α2 helix differs when the Tax and Tel1p peptides are bound (
      • Borbulevych O.Y.
      • Piepenbrink K.H.
      • Gloor B.E.
      • Scott D.R.
      • Sommese R.F.
      • Cole D.K.
      • Sewell A.K.
      • Baker B.M.
      ). Fabian et al. (
      • Fabian H.
      • Huser H.
      • Narzi D.
      • Misselwitz R.
      • Loll B.
      • Ziegler A.
      • Böckmann R.A.
      • Uchanska-Ziegler B.
      • Naumann D.
      ,
      • Fabian H.
      • Huser H.
      • Loll B.
      • Ziegler A.
      • Naumann D.
      • Uchanska-Ziegler B.
      ,
      • Fabian H.
      • Loll B.
      • Huser H.
      • Naumann D.
      • Uchanska-Ziegler B.
      • Ziegler A.
      ) have demonstrated that allelic variations as well as peptide modifications induce different degrees of flexibility in HLA-B27 subtypes. It is thus possible that alterations in peptide and MHC flexibility stemming from changes to antigenic peptides could explain other cases where T cells have been shown to “sense” modifications to antigenic peptides despite any clear indications of structural changes (e.g. Refs.
      • 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.
      and
      • Ding Y.H.
      • Baker B.M.
      • Garboczi D.N.
      • Biddison W.E.
      • Wiley D.C.
      ).
      Altogether, these results suggest that further studies of the dynamical consequences of peptide modifications are needed, particularly in the design and optimization of peptide-based vaccines. Although predicting dynamical effects from structural alterations is challenging, the use of molecular simulations in conjunction with structural information and other tools for quantifying protein dynamics may be useful in such endeavors. Lastly, alterations of pMHC flexibility that weaken TCR binding could also serve as a mechanism for viral and tumor escape from immune destruction.

      Acknowledgments

      We thank Cynthia Piepenbrink for outstanding technical assistance and Daniel R. Scott for critical comments on the manuscript. Results shown in this study are derived from work performed at the Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. The Argonne National Laboratory 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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