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Recognition of Class II MHC Peptide Ligands That Contain Β-Amino Acids Ross W

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Recognition of Class II MHC Peptide Ligands That Contain Β-Amino Acids Ross W Recognition of Class II MHC Peptide Ligands That Contain β-Amino Acids Ross W. Cheloha, Andrew W. Woodham, Djenet Bousbaine, Tong Wang, Shi Liu, John Sidney, Alessandro Sette, Samuel This information is current as H. Gellman and Hidde L. Ploegh of October 1, 2021. J Immunol published online 7 August 2019 http://www.jimmunol.org/content/early/2019/08/06/jimmun ol.1900536 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/08/06/jimmunol.190053 Material 6.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 7, 2019, doi:10.4049/jimmunol.1900536 The Journal of Immunology Recognition of Class II MHC Peptide Ligands That Contain b-Amino Acids Ross W. Cheloha,* Andrew W. Woodham,* Djenet Bousbaine,*,† Tong Wang,‡ Shi Liu,‡ John Sidney,x Alessandro Sette,x,{ Samuel H. Gellman,‡ and Hidde L. Ploegh* Proteins are composed of a-amino acid residues. This consistency in backbone structure likely serves an important role in the display of an enormous diversity of peptides by class II MHC (MHC-II) products, which make contacts with main chain atoms of their peptide cargo. Peptides that contain residues with an extra carbon in the backbone (derived from b-amino acids) have biological properties that differ starkly from those of their conventional counterparts. How changes in the structure of the peptide backbone affect the loading of peptides onto MHC-II or recognition of the resulting complexes by TCRs has not been widely explored. We prepared a library of analogues of MHC-II–binding peptides derived from OVA, in which at least one a-amino acid residue was replaced with a homologous b-amino acid residue. The latter contain an extra methylene unit in the peptide backbone Downloaded from but retain the original side chain. We show that several of these a/b-peptides retain the ability to bind tightly to MHC-II, activate TCR signaling, and induce responses from T cells in mice. One a/b-peptide exhibited enhanced stability in the presence of an endosomal protease relative to the index peptide. Conjugation of this backbone-modified peptide to a camelid single-domain Ab fragment specific for MHC-II enhanced its biological activity. Our results suggest that backbone modification offers a method to modulate MHC binding and selectivity, T cell stimulatory capacity, and susceptibility to processing by proteases such as those found within endosomes where Ag processing occurs. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/ he immune system recognizes and responds to proteina- through the action of proteases and peptidases (1, 2). The resulting ceous Ags through the production and display of short peptides are then exposed to receptive MHC molecules through a peptide Ags bound to the products of the MHC. Class I coordinated series of trafficking steps that differ for MHC-I T + MHC (MHC-I) products that stimulate CD8 T cells typically and MHC-II (3). Productive binding of peptide to MHC yields display peptides of 8–10 residues in length, derived mostly from peptide–MHC complexes that are transported for display at the intracellular proteins, whereas class II MHC (MHC-II) products surface of the APC. These complexes engage TCR complexes, that stimulate CD4+ T cells display longer peptides, mostly from stimulate TCR signaling, and drive subsequent T cell–mediated proteins originating in extracellular space or their topological immune responses. Each of these steps (proteolysis, MHC bind- by guest on October 1, 2021 equivalents. The loading of peptides onto MHC products requires ing, TCR recognition) requires recognition of the polypeptide the breakdown of the source proteins into peptide fragments Ag or some part of it. Modifications of peptide structure can affect each recognition step required for T cell engagement. Structural alterations that affect recognition by proteases can protect peptides against pro- *Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115; †Massachusetts Institute of Technology, Cambridge, MA 02142; ‡Department of teolysis, which not only generates but can also destroy the ligands x Chemistry, University of Wisconsin-Madison, Madison, WI 53706; Division of presented by MHC-II products. For example, synthetic proteins Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; and {Department of Medicine, University of California San Diego, La Jolla, composed entirely of D-amino acids are not degraded by proteases CA 92161 and cannot be processed for loading onto MHC products. Such ORCIDs: 0000-0001-9871-8333 (R.W.C.); 0000-0003-2683-9544 (D.B.); 0000- proteins therefore fail to induce immune responses that require 0003-4208-5578 (T.W.); 0000-0002-0987-405X (J.S.); 0000-0001-7013- T cell participation (4). Alternatively, the introduction of modifi- 2250 (A.S.); 0000-0001-5617-0058 (S.H.G.); 0000-0002-1090-6071 (H.L.P.). cations that inhibit extracellular proteolysis can enhance the effi- Received for publication May 9, 2019. Accepted for publication July 5, 2019. cacy of peptide vaccines (5). In some cases, endosomal proteolysis This work was supported by National Institutes of Health (NIH) National Institute of can destroy potentially antigenic peptides (6), and in this context, Allergy and Infectious Diseases Contract HHSN272201400045C (to A.S.), NIH Grant R01 GM056414 (to S.H.G.), Lustgarten Foundation Award 388167 (to H.L.P.), inhibition of proteolysis enhances immunogenicity (7, 8). These Melanoma Research Alliance Award 51009 (to H.L.P.), and NIH Grant R01-AI087879 findings suggest that carefully introduced structural modifications (to H.L.P.). R.W.C. was supported in part by a Biotechnology Training Grant from the that forestall destruction of antigenic peptides might enhance National Institute of General Medical Sciences (T32 GM008349) and a Cancer Re- search Institute Postdoctoral Fellowship. A.W.W. was supported by a Beckman Institute immunogenicity (9). Postdoctoral Fellowship. MHC-I and MHC-II bind to an enormously diverse array of Address correspondence and reprint requests to Samuel H. Gellman or Hidde L. Ploegh, peptides. The impact of side chain identities at specific sites on Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706 (S.H.G.) binding to specific allomorphs of MHC products and subsequent or Boston Children’s Hospital, 1 Blackfan Circle, RB06215, Boston, MA 02115 (H.L.P.). E-mail addresses: [email protected] (S.H.G.) or [email protected] recognition by T cells has been extensively analyzed (10, 11). (H.L.P.) The development of compounds with selectivity among these The online version of this article contains supplemental material. allomorphs could be useful to address autoimmune disorders (12). Abbreviations used in this article: DC, dendritic cell; LC/MS, liquid chromatography/ In contrast, relatively little attention has been paid to changes mass spectrometry; MHC-I, class I MHC; MHC-II, class II MHC; MOG, myelin in the peptide backbone (13). Interactions between MHC-II and oligodendrocyte glycoprotein; TFA, trifluoroacetic acid. the peptide backbone are crucial for affinity and stability of Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 the peptide-MHC-II complex (14, 15). Only a few studies have www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900536 2 MHC-II PEPTIDE LIGANDS THAT CONTAIN b-AMINO ACIDS probed the impact of peptide backbone modifications on MHC Materials and Methods binding (16–22), with diverse outcomes. TCR recognition of peptide– General MHC complexes and the ensuing T cell responses are sensitive to Cell lines were routinely tested for cell surface marker expression peptide backbone modifications as well (23–30). and mycoplasma infection. Liquid chromatography/mass spectrometry Replacement of a-residues with b-amino acid counterparts has (LC/MS) was performed on a Waters Xevo TOF system equipped with an been used to probe the importance of peptide backbone structure HPLC-C8 column. Calculations and graphing were performed using in a variety of contexts (31, 32). Each a→b replacement adds a Microsoft Excel and GraphPad Prism 6. EC50 values were generated using carbon atom to the backbone compared with the analogous con- a sigmoidal dose-response with variable slope model (four-parameter model). Statistical significance was evaluated using a two-tailed, paired ventional peptide. Such replacements can retain the original side t test. chain. The incorporation of a b-amino acid residue provides pro- tection against proteolysis, at least for peptide bonds in proximity to Flow cytometry and Abs the site of incorporation (33–35). Carefully designed a/b-peptides For cytofluorimetry, gating was performed on viable cells based on forward (i.e., peptides containing a mixture of a-andb-residues) can mimic scatter and side scatter profiles. Fluorescently labeled Abs were purchased the conformations and biological properties of a-helical peptides, from eBioscience (anti-CD45.1, A20; anti-CD4, RM4-5; anti-CD11c, N418) or BioLegend (anti-CD45.2, 104; anti–I-A/I-E, M5/114.15.2). Prolifer- but the a/b-peptides display greater resistance to proteolysis and ation was assessed using the dye CellTrace Violet (Life Technologies) have prolonged activity in vivo (36–40).
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