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Research Articles

Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia

Stacy A. Malaker, Sarah A. Penny, Lora G. Steadman, Paisley T. Myers, Justin C. Loke, Manoj Raghavan, Dina L. Bai, Jeffrey Shabanowitz, Donald F. Hunt and Mark Cobbold
Stacy A. Malaker
1Department of Chemistry, University of Virginia, Charlottesville, Virginia.
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Sarah A. Penny
2Department of Clinical Immunology, University of Birmingham, Birmingham, United Kingdom.
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Lora G. Steadman
2Department of Clinical Immunology, University of Birmingham, Birmingham, United Kingdom.
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Paisley T. Myers
1Department of Chemistry, University of Virginia, Charlottesville, Virginia.
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Justin C. Loke
2Department of Clinical Immunology, University of Birmingham, Birmingham, United Kingdom.
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Manoj Raghavan
2Department of Clinical Immunology, University of Birmingham, Birmingham, United Kingdom.
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Dina L. Bai
1Department of Chemistry, University of Virginia, Charlottesville, Virginia.
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Jeffrey Shabanowitz
1Department of Chemistry, University of Virginia, Charlottesville, Virginia.
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Donald F. Hunt
1Department of Chemistry, University of Virginia, Charlottesville, Virginia.
3Department of Pathology, University of Virginia, Charlottesville, Virginia.
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Mark Cobbold
2Department of Clinical Immunology, University of Birmingham, Birmingham, United Kingdom.
4Massachusetts General Hospital Cancer Center and Department of Medicine, Harvard Medical School, Charlestown, Massachusetts.
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  • For correspondence: mcobbold@mgh.harvard.edu
DOI: 10.1158/2326-6066.CIR-16-0280 Published May 2017
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    Figure 1.

    The discovery of MHC class I–associated glycopeptides on primary leukemia cells. A, HCD mass spectrum of the first O-GlcNAcylated peptide detected in ALL, IPVsSHNSL. Fragment ions that define the complete amino acid sequence are labeled as b and y. Those that have lost the O-GlcNAc moiety are labeled with an asterisk. B, Fingerprint ions in the HCD spectra of O-GlcNAcylated and O-GalNAcylated peptides. Relative abundances of fragment ions derived from secondary fragmentation of the oxonium ion at m/z 204 are substantially different for O-GlcNAcylated and O-GalNAcylated peptides. C, Distribution of 36 HLA-B*07:02–restricted glycopeptides among the different leukemia and healthy cells analyzed. ALL, acute lymphoblastic leukemia; healthy cells, healthy donor tonsil/spleen cells; LCL, lymphoblastoid cell line; AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia. D, Number of copies per cell of the O-GlcNAcylated peptides identified on ALL versus healthy B cells (purified from a healthy spleen).

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    Figure 2.

    Healthy donor immunity to leukemia-associated posttranslationally modified neoantigens. A, Flow cytometry plots showing the gating strategy used in the ICS protocol to determine healthy donor immunity to the O-GlcNAcylated peptides (Supplementary Fig. S4 contains additional plots). Immunity to viral antigens was used as an internal control, for comparison. Collated results of cytokine production (B) and degranulation (C) by healthy donor T cells in response to stimulation with posttranslationally modified leukemia neoantigens. D, The correlation between the percentage of cells producing cytokine and degranulating for HD1. E, HD1 T cells that produced cytokine in response to stimulation with peptides were also stained with surface antibodies for phenotyping (CD27 and CD45RA; Supplementary Fig. S5). CM, central memory; N, naïve; EM, effector memory and TEMRA, terminal effector memory. Responses were independently verified at least twice.

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    Figure 3.

    Investigating T-cell recognition of the methylated O-GlcNAc peptide. A, Healthy donor immunity to the unmodified, O-GlcNAcylated, methylated and both O-GlcNAcylated and methylated peptide, measured by cytokine production and degranulation. B, A T-cell line was grown from HD5 against the methylated RPPItQSSL peptide. The percentage of cells recognizing the peptide was assessed by overnight stimulation with the peptide and detection of CD137 and CD107a surface markers. C, This T-cell line was using a europium release killing assay to assess killing of autologous transformed B cells pulsed with different modifications of the peptide. Responses were independently verified at least twice.

Tables

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  • Table 1.

    O-GlcNAcylated peptides presented by HLA B*0702 class I MHC molecules on leukemia

    #SequenceStart–StopUniProtTumorSource protein
    1aAPP(sts)AAAL405–414Q86TM6ALL, CLL1E3 Ubiquitin-protein ligase synoviolin
    2bAPRGnVTSL60–68Q9NR96CLL1, CLL2Toll-like receptor 9
    3APRtNGVAM187–195Q92567ALL, CLL1, CLL2Protein FAM168A
    4APTsAAAL1116–1123Q86Z02ALLHomeodomain-interacting protein kinase 1
    5APVsASASV1807–1815Q9Y520ALLProtein PRRC2C
    6APVsSKSSL850–858Q86Z02ALL, CLL1, CLL2Homeodomain-interacting protein kinase 1
    7EP(sst)VVSL1076–1085O75129ALLAstrotactin-2
    8HPMsTASQV345–353Q13492ALLClathrin assembly lymphoid myeloid leukemia
    9cHP(sss)AAVL740–748Q86XN7ALL, CMLProline and serine-rich protein 1
    10HP(sst)ASTAL3041–3050Q96T58ALLMsx2-interacting protein
    11IPIsLHTSL1959–1967Q5JSZ5ALLProtein PRRC2B
    12IPTsSVLSL710–718O15027ALLProtein transport protein Sec 16A
    13dIPVsKPLSL104–112Q16621AML, ALL, CLL1Leucine zipper protein 1
    14eIPVsSHNSL147–155Q06413AML, ALL, CLL1, JY, S, ToMyocyte-specific enhancer factor 2C
    15fKPP(ts)QSSVL411–420Q5T6F2ALLUbiquitin-associated protein 2
    16gKPPVsFFSL95–103Q6PKC3ALLThioredoxin domain containing protein 11
    17hKPTLLYnVSL373–381P04220CLL1, CLL2Ig Mu heavy chain disease protein
    18LPRN(st)MM335–342Q9NPI6ALLmRNA-decapping enzyme 1A
    19LPTsLPSSL2464–2472P46531ALLNeurogenic locus notch homolog protein 1
    20iMPVRPTtNTF218–227Q7Z3K3ALLpogo transposable element with ZNF domain
    21NPVsLPSL831–838Q6VMQ6ALLActivating transcription factor 7-interacting protein
    22jPPS(ts)AAAL405–414Q86TM6ALLE3 Ubiquitin-protein ligase synoviolin
    23kRPPItQSSL382–390Q9P2N5ALL, SRNA binding protein 27
    24lRPPQsSSVSL937–946O15027ALLProtein transport protein Sec 16A
    25RPP(sss)QQL1758–1766Q8WYB5ALLHistone acetyltransferase KAT6B
    26RPPVtKASSF341–350Q9Y2K5ALL, CLL1R3H domain containing protein 2
    27RPVtASITTM927–936Q9ULH7ALL, CLL1, CLL2, SMKL/myocardin-like protein 2
    28TPASsRAQTL2320–2329Q01082CLL1Spectrin beta chain, non-erythrocytic 1
    29TPAsSSSAL875–883Q9NPG3ALL, CLL1Ubinucleain 1
    30TPIsQAQKL3024–3032Q96L91ALLE1A-binding protein p400
    31VPAsSTSTL576–584Q9NYV4ALL, CLL1Cyclin dependent kinase 12
    32VPTtSSSL1284–1291Q14004ALLCyclin dependent kinase 13
    33VPVsGTQGL93–101P23511ALLNuclear transcription factor Y subunit alpha
    34VPVsNQSSL146–154Q14814ALLMyocyte-specific enhancer factor 2D
    35VPVsSASEL596–603Q7Z2W4ALLZinc finger CCCH-type, antiviral 1
    36VPVsVGPSL1157–1164Q86Z02ALLHomeodomain-interacting protein kinase 1
    • NOTE: Thirty-six peptides, often with multiple forms of glycosylation, were isolated from class I MHC molecules on several leukemias, cell lines, and healthy tissue. These sources are indicated as follows: CML; chronic myeloid leukemia, 1 and 2; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; J, JY cell line; S, spleen; To, tonsil; see Supplementary Table S1. Small letters, s, t, and n specify Ser, Thr, and Asn residues that are modified by O-GlcNAc unless otherwise indicated in a footnote. Parentheses enclose s and t residues that could be a site of GlcNAcylation. Samples were independently analyzed by MS at least 3 times.

    • ↵aPeptide was detected in a total of five forms: single GlcNac, double GlcNAc, single hexose-GlcNAc, single GlcNAc (S6) + hexose- GlcNac (T5), and double hexose-GlcNAc.

    • ↵bN5 is modified by N-linked hexose-GlcNAc.

    • ↵cPeptide was detected in two forms, GlcNAc on S4 and two GlcNAcs on S4 and S5.

    • ↵dPeptide was detected in two forms: GlcNAc (S4) and hexose-GlcNAc (S4).

    • ↵ePeptide was detected in four forms: GlcNAc (S4), double GlcNAc (S4, S5), single hexose-GlcNAc (S4), and an acetyl-GlcNAc (S4).

    • ↵fPeptide was detected in two forms: GlcNAc and hexose-GlcNAc (T4).

    • ↵gS5 is modified by O-linked hexose-GlcNAc.

    • ↵hN7 is modified by N-linked hexose-GlcNAc.

    • ↵iPeptide was detected in two forms: hexose-GlcNAc and asymmetric di-methyl (R4) + hexose-GlcNAc (T7).

    • ↵jT4 or S5 is modified by O-linked hexose-GlcNAc.

    • ↵kPeptide was detected in four forms: GlcNAc (T5), mono-methyl (R1) + GlcNAc (T5), asymmetric di-methyl (R1) + GlcNAc (T5), and asymmetric di-methyl (R1) + acetyl-GlcNAc (T5).

    • ↵lS5 is modified by O-linked hexose-GlcNAc.

Additional Files

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  • Supplementary Data

    • Supplementary Tables 1 and 2 and Supplementary Figures 1 through 7 - Table S1. Summary of samples used for this study. Table S2. Antibodies used in the study. Figure S1. Mass spectra of all peptides referenced in figures 2 and 3. Figure S2. ETD Mass spectrum of RPPItQSSL containing an asymmetrically dimethylated Arg residue. Figure S3. ICS flow cytometry plots for HD5. Figure S4. Gating strategy for phenotyping shown in Fig 2E. Figure S5. Healthy donor immunity to O-GlcNAc peptides measured using IFNgamma ELISpot. Figure S6. ICS flow cytometry plots for HD5. Figure S7. Positional analysis of O-GlcNAc peptides.
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Cancer Immunology Research: 5 (5)
May 2017
Volume 5, Issue 5
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Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia
Stacy A. Malaker, Sarah A. Penny, Lora G. Steadman, Paisley T. Myers, Justin C. Loke, Manoj Raghavan, Dina L. Bai, Jeffrey Shabanowitz, Donald F. Hunt and Mark Cobbold
Cancer Immunol Res May 1 2017 (5) (5) 376-384; DOI: 10.1158/2326-6066.CIR-16-0280

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Identification of Glycopeptides as Posttranslationally Modified Neoantigens in Leukemia
Stacy A. Malaker, Sarah A. Penny, Lora G. Steadman, Paisley T. Myers, Justin C. Loke, Manoj Raghavan, Dina L. Bai, Jeffrey Shabanowitz, Donald F. Hunt and Mark Cobbold
Cancer Immunol Res May 1 2017 (5) (5) 376-384; DOI: 10.1158/2326-6066.CIR-16-0280
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