Rhesus Macaque MHC Class I Molecules Present HLA-B-Like Peptides Heather D. Hickman-Miller, Wilfried Bardet, Angela Gilb, Angela D. Luis, Kenneth W. Jackson, David I. Watkins and This information is current as William H. Hildebrand of September 24, 2021. J Immunol 2005; 175:367-375; ; doi: 10.4049/jimmunol.175.1.367 http://www.jimmunol.org/content/175/1/367 Downloaded from References This article cites 63 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/175/1/367.full#ref-list-1 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 by guest on September 24, 2021 *average 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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Rhesus Macaque MHC Class I Molecules Present HLA-B-Like Peptides1 Heather D. Hickman-Miller,2* Wilfried Bardet,* Angela Gilb,* Angela D. Luis,* Kenneth W. Jackson,* David I. Watkins,† and William H. Hildebrand3* SIV-infected Indian rhesus macaques (Macaca mulatta) are an important animal model for humans infected with HIV. Under- standing macaque (M. mulatta class I (Mamu)) MHC class I-peptide binding facilitates the comparison of SIV- and HIV-specific cellular immune responses. In this study, we characterized the endogenous peptide-binding properties of three Mamu-A (A*02, A*08, A*11) and three Mamu-B (B*01, B*03, B*12) class I molecules. Motif comparisons revealed that five of the six macaque class I molecules (A*02, A*08, A*11, B*01, and B*03) have peptide-binding motifs similar to those of human class I molecules. Of the 65 macaque endogenous peptide ligands that we sequenced by tandem mass spectroscopy, 5 were previously eluted from HLA class I molecules. Nonamers predominated among the individual ligands, and both the motifs and the individual ligands indicated P2, Downloaded from P9, and various ancillary anchors. Interestingly, peptide binding of the Mamu-A and Mamu-B molecules exhibited cross-species peptide-presentation overlap primarily with HLA-B molecules. Indeed, all of the macaque class I molecules appeared HLA-B-like in peptide presentation. Remarkably, the overlap in macaque- and HLA-peptide presentation occurred despite divergent class I peptide-binding grooves. Macaque and human class I differing by up to 42 aa (13–23%) within the ␣-1 and ␣-2 domains, including substantial divergence within specificity pockets A-F, bound the same endogenous peptide. Therefore, endogenous peptide char- acterization indicates that macaque class I molecules may be the functional equivalents of HLA-B molecules. The Journal of http://www.jimmunol.org/ Immunology, 2005, 175: 367–375. lass I MHC molecules mediate antiviral immune re- tion confirms the important role of class I MHC in mediating an- sponses by first binding short viral peptides (7–14 aa) tiviral immune responses and has led to the development of mo- C within the infected host cell and then transiting to the cell lecular class I typing methods and the application of synthetic surface where they present their peptide cargo to CTL (1–6). Vi- peptide binding assays for macaque MHC molecules (24–28). ruses have developed a myriad of means to avoid the presentation With the recent discovery of as many as 16 active rhesus MHC of viral peptides by class I MHC molecules (7–11), and as an class I gene loci, assays for determining macaque class I MHC by guest on September 24, 2021 immune countermeasure, individuals within each mammalian spe- gene types and peptide-binding specificities will play an expanded cies tend to differ in their class I MHC genotype and phenotype role in characterizing antiviral immune responses in this (12–15). The polymorphism that distinguishes one class I molecule primate (29). from the next alters the peptide ligands that can be accommodated In humans, class I MHC peptide-binding specificities have been within a class I molecule’s peptide-binding groove (16, 17). There- empirically elucidated through a combination of 1) the Edman se- fore, MHC class I molecules tend to differ functionally from in- quencing of pooled endogenous ligands, 2) the mass spectrometric dividual to individual within a species. sequencing of individual endogenous ligands, and 3) competitive Animal models represent a valuable tool for studying host- synthetic peptide-binding assays (30–32). By comparison, the pep- pathogen relationships, and the Indian rhesus macaque represents tide specificity of MHC class I molecules in the Indian rhesus an important animal model for a number of viruses that infect macaque has been derived almost exclusively from in vitro com- humans (18–23). Notably, infection of macaques with SIV results petitive-binding studies with synthetic peptides (10, 26, 27, 33, in an AIDS-like illness similar to that of humans infected with HIV 34). Peptide-binding assays delineate the major peptide anchors (24). Use of the Indian rhesus macaque as a model for HIV infec- preferred by a given class I molecule and have led to predictions of peptides that are presented in vivo by a particular molecule. Sequencing of endogenous ligands complements synthetic pep- *Department of Microbiology and Immunology, University of Oklahoma Health Sci- ences Center, Oklahoma City, OK 73104; and †Wisconsin Regional Primate Center, tide-binding data by demonstrating that ligands of a particular se- University of Wisconsin, Madison, WI 53715 quence endogenously arise, traffic, survive, and are loaded into Received for publication February 11, 2005. Accepted for publication April 13, 2005. MHC molecules within a cell. The costs of publication of this article were defrayed in part by the payment of page To characterize endogenous peptide-binding specificities for In- charges. This article must therefore be hereby marked advertisement in accordance dian rhesus macaque class I molecules, and to better relate the with 18 U.S.C. Section 1734 solely to indicate this fact. peptide-binding properties of rhesus monkey and human class I 1 This work was supported by a National Institutes of Health Grant 5R24RR015371 (to D.I.W.). H.D.H.-M. was supported by National Institutes of Health Training Grant molecules, we have established endogenous-peptide motifs for six T32AI07633. macaque class I molecules. Additionally, we sequenced Ͼ60 en- 2 Current address: Laboratory of Viral Diseases, National Institute of Allergy and dogenously loaded peptide ligands, representing the first compre- Infectious Diseases, National Institutes of Health, Bethesda, MD. hensive set of naturally loaded MHC class I ligands identified in 3 Address correspondence and reprint requests to Dr. William H. Hildebrand, Depart- nonhuman primates. Like the human, the macaque class I mole- ment of Microbiology and Immunology, University of Oklahoma Health Sciences Center, 975 N.E. 10th Street, Oklahoma City, OK, 73104. E-mail address: William- cules bound primarily nonamers with distinct P2 and P9 anchor [email protected] preferences. Surprisingly, the macaque and human class I MHC Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 368 HLA-B-LIKE MACAQUE CLASS I molecules that bound similar (and in some cases identical) pep- ing identical ligands. Dashes in the alignments indicate identity. Noniden- tides did so despite divergent peptide-binding grooves. Even a sub- tities are indicated by the single letter amino acid code of the differing stantial number of nonconservative amino acid changes within residue. specificity pockets did not abrogate the binding of similar peptides Results by particular pairs of macaque and human class I molecules. Fi- Endogenous peptide motifs of six macaque molecules exhibit nally, the three Macaca mulatta class I-A (Mamu-A)4 and three properties similar to HLA molecules Mamu-B class I molecules studied here are all HLA-B functional equivalents. The implications of this similarity in peptide binding To characterize the peptides endogenously bound by Indian rhesus is discussed. macaque class I molecules and to compare their peptide repertoires to those in human MHC molecules, we produced transfectants of Materials and Methods sMamu-A*02, -A*08, -A*11, -B*01, -B*03, and -B*12 in the hu- Mamu protein production man, class I-negative cell line 721.221 (35). Through hollow-fiber bioreactor culture of stable transfectants, we produced ϳ25 mg of Cloned DNA for Mamu-A*02, -A*08, -B*01, and -B*03 was amplified using the 5MamuKE (5Ј-GGGCGAATTCCCGCCGCCACCATGGCGCCCCGAA each molecule, purified molecules by affinity chromatography, and CCC-3Ј) and 3VLDLRHIN (5Ј-CCGCAAGCTTTCAGGCAAGGTCGTCG isolated and purified ϳ0.5 mg of naturally bound peptides. We TCGGTGCTGACGACGCTCCATCTCAGGGTGAGGGG-3Ј) or 5Mamu1KE then recorded peptide motifs for each of the molecules in accor- Ј Ј (5 -GGGCGAATTCCCGCCGCCACCATGCGGGTCATGGCTCC-3 ) and dance with previously published procedures for human class I mol- 3VLDLRHIN (5Ј-CCGCAAGCTTTCAGGCAAGGTCGTCGTCGGTGCT GACGACGCTCCATCTCAGGGTGAGGGG-3Ј) for Mamu-A*11 and ecules (39) (Fig. 1). -B*12. Both primer pairs resulted in an amplicon of ϳ900 bp corresponding In terms of the location and number of anchor residues within Downloaded from to a truncated protein lacking the cytoplasmic and transmembrane domains; motifs, the macaque endogenous peptides were remarkably similar the latter primer pair also encoded the epitope tag SVVSTDDDLA which was to those found in human molecules.