Article Figures & Data
Figures
- 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).
- 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.
- 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
- Table 1.
O-GlcNAcylated peptides presented by HLA B*0702 class I MHC molecules on leukemia
# Sequence Start–Stop UniProt Tumor Source protein 1a APP(sts)AAAL 405–414 Q86TM6 ALL, CLL1 E3 Ubiquitin-protein ligase synoviolin 2b APRGnVTSL 60–68 Q9NR96 CLL1, CLL2 Toll-like receptor 9 3 APRtNGVAM 187–195 Q92567 ALL, CLL1, CLL2 Protein FAM168A 4 APTsAAAL 1116–1123 Q86Z02 ALL Homeodomain-interacting protein kinase 1 5 APVsASASV 1807–1815 Q9Y520 ALL Protein PRRC2C 6 APVsSKSSL 850–858 Q86Z02 ALL, CLL1, CLL2 Homeodomain-interacting protein kinase 1 7 EP(sst)VVSL 1076–1085 O75129 ALL Astrotactin-2 8 HPMsTASQV 345–353 Q13492 ALL Clathrin assembly lymphoid myeloid leukemia 9c HP(sss)AAVL 740–748 Q86XN7 ALL, CML Proline and serine-rich protein 1 10 HP(sst)ASTAL 3041–3050 Q96T58 ALL Msx2-interacting protein 11 IPIsLHTSL 1959–1967 Q5JSZ5 ALL Protein PRRC2B 12 IPTsSVLSL 710–718 O15027 ALL Protein transport protein Sec 16A 13d IPVsKPLSL 104–112 Q16621 AML, ALL, CLL1 Leucine zipper protein 1 14e IPVsSHNSL 147–155 Q06413 AML, ALL, CLL1, JY, S, To Myocyte-specific enhancer factor 2C 15f KPP(ts)QSSVL 411–420 Q5T6F2 ALL Ubiquitin-associated protein 2 16g KPPVsFFSL 95–103 Q6PKC3 ALL Thioredoxin domain containing protein 11 17h KPTLLYnVSL 373–381 P04220 CLL1, CLL2 Ig Mu heavy chain disease protein 18 LPRN(st)MM 335–342 Q9NPI6 ALL mRNA-decapping enzyme 1A 19 LPTsLPSSL 2464–2472 P46531 ALL Neurogenic locus notch homolog protein 1 20i MPVRPTtNTF 218–227 Q7Z3K3 ALL pogo transposable element with ZNF domain 21 NPVsLPSL 831–838 Q6VMQ6 ALL Activating transcription factor 7-interacting protein 22j PPS(ts)AAAL 405–414 Q86TM6 ALL E3 Ubiquitin-protein ligase synoviolin 23k RPPItQSSL 382–390 Q9P2N5 ALL, S RNA binding protein 27 24l RPPQsSSVSL 937–946 O15027 ALL Protein transport protein Sec 16A 25 RPP(sss)QQL 1758–1766 Q8WYB5 ALL Histone acetyltransferase KAT6B 26 RPPVtKASSF 341–350 Q9Y2K5 ALL, CLL1 R3H domain containing protein 2 27 RPVtASITTM 927–936 Q9ULH7 ALL, CLL1, CLL2, S MKL/myocardin-like protein 2 28 TPASsRAQTL 2320–2329 Q01082 CLL1 Spectrin beta chain, non-erythrocytic 1 29 TPAsSSSAL 875–883 Q9NPG3 ALL, CLL1 Ubinucleain 1 30 TPIsQAQKL 3024–3032 Q96L91 ALL E1A-binding protein p400 31 VPAsSTSTL 576–584 Q9NYV4 ALL, CLL1 Cyclin dependent kinase 12 32 VPTtSSSL 1284–1291 Q14004 ALL Cyclin dependent kinase 13 33 VPVsGTQGL 93–101 P23511 ALL Nuclear transcription factor Y subunit alpha 34 VPVsNQSSL 146–154 Q14814 ALL Myocyte-specific enhancer factor 2D 35 VPVsSASEL 596–603 Q7Z2W4 ALL Zinc finger CCCH-type, antiviral 1 36 VPVsVGPSL 1157–1164 Q86Z02 ALL Homeodomain-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.
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.
Volume 5, Issue 5
