Skip to main content

The natural HLA ligandome of glioblastoma stem-like cells: antigen discovery for T cell-based immunotherapy

Abstract

Glioblastoma is the most frequent malignant primary brain tumor. In a hierarchical tumor model, glioblastoma stem-like cells (GSC) play a major role in tumor initiation and maintenance as well as in therapy resistance and recurrence. Thus, targeting this cellular subset may be key to effective immunotherapy. Here, we present a mass spectrometry-based analysis of HLA-presented peptidomes of GSC and glioblastoma patient specimens. Based on the analysis of patient samples (n = 9) and GSC (n = 3), we performed comparative HLA peptidome profiling against a dataset of normal human tissues. Using this immunopeptidome-centric approach we could clearly delineate a subset of naturally presented, GSC-associated HLA ligands, which might serve as highly specific targets for T cell-based immunotherapy. In total, we identified 17 antigens represented by 41 different HLA ligands showing natural and exclusive presentation both on GSC and patient samples. Importantly, in vitro immunogenicity and antigen-specific target cell killing assays suggest these peptides to be epitopes of functional CD8+ T cell responses, thus rendering them prime candidates for antigen-specific immunotherapy of glioblastoma.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SA, Behjati S, Biankin AV et al (2013) Signatures of mutational processes in human cancer. Nature 500:415–421. https://doi.org/10.1038/nature12477

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Andersen RS, Thrue CA, Junker N, Lyngaa R, Donia M, Ellebaek E et al (2012) Dissection of T-cell antigen specificity in human melanoma. Can Res 72:1642–1650. https://doi.org/10.1158/0008-5472.CAN-11-2614

    Article  CAS  Google Scholar 

  3. Bassani-Sternberg M, Pletscher-Frankild S, Jensen LJ, Mann M (2015) Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation. Mol Cell Proteom 14:658–673. https://doi.org/10.1074/mcp.M114.042812

    Article  CAS  Google Scholar 

  4. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P et al (2012) Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 366:2455–2465. https://doi.org/10.1056/NEJMoa1200694

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Brown CE, Alizadeh D, Starr R, Weng L, Wagner JR, Naranjo A et al (2016) Regression of glioblastoma after chimeric antigen receptor T-cell therapy. N Engl J Med 375:2561–2569. https://doi.org/10.1056/NEJMoa1610497

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Dunn GP, Dunn IF, Curry WT (2007) Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human glioma. Cancer Immunity 7:12

    PubMed  PubMed Central  Google Scholar 

  7. Dutoit V, Herold-Mende C, Hilf N, Schoor O, Beckhove P, Bucher J et al (2012) Exploiting the glioblastoma peptidome to discover novel tumour-associated antigens for immunotherapy. Brain J Neurol 135:1042–1054. https://doi.org/10.1093/brain/aws042

    Article  Google Scholar 

  8. Falk K, Rotzschke O, Stevanovic S, Jung G, Rammensee HG (1991) Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules. Nature 351:290–296. https://doi.org/10.1038/351290a0

    Article  PubMed  CAS  Google Scholar 

  9. Gunther HS, Schmidt NO, Phillips HS, Kemming D, Kharbanda S, Soriano R et al (2008) Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria. Oncogene 27:2897–2909. https://doi.org/10.1038/sj.onc.1210949

    Article  PubMed  CAS  Google Scholar 

  10. Hodi FS, O’Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB et al (2010) Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363:711–723. https://doi.org/10.1056/NEJMoa1003466

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. da Huang W, Sherman BT, Lempicki RA (2009) Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 37:1–13. https://doi.org/10.1093/nar/gkn923

    Article  CAS  Google Scholar 

  12. da Huang W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57. https://doi.org/10.1038/nprot.2008.211

    Article  CAS  Google Scholar 

  13. Hulsen T, de Vlieg J, Alkema W (2008) BioVenn—a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. BMC Genom 9:488. https://doi.org/10.1186/1471-2164-9-488

    Article  CAS  Google Scholar 

  14. Kall L, Canterbury JD, Weston J, Noble WS, MacCoss MJ (2007) Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat Methods 4:923–925. https://doi.org/10.1038/nmeth1113

    Article  PubMed  CAS  Google Scholar 

  15. Kowalewski DJ, Schuster H, Backert L, Berlin C, Kahn S, Kanz L et al (2015) HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL). Proc Natl Acad Sci USA 112:E166–E175. https://doi.org/10.1073/pnas.1416389112

    Article  PubMed  CAS  Google Scholar 

  16. Kowalewski DJ, Stevanovic S (2013) Biochemical large-scale identification of MHC class I ligands. Methods Mol Biol 960:145–157. https://doi.org/10.1007/978-1-62703-218-6_12

    Article  PubMed  CAS  Google Scholar 

  17. Kreiter S, Vormehr M, van de Roemer N, Diken M, Lower M, Diekmann J et al (2015) Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520:692–696. https://doi.org/10.1038/nature14426

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Kvistborg P, Shu CJ, Heemskerk B, Fankhauser M, Thrue CA, Toebes M et al (2012) TIL therapy broadens the tumor-reactive CD8(+) T cell compartment in melanoma patients. Oncoimmunology 1:409–418

    Article  PubMed  PubMed Central  Google Scholar 

  19. Mohme M, Neidert MC, Regli L, Weller M, Martin R (2014) Immunological challenges for peptide-based immunotherapy in glioblastoma. Cancer Treat Rev 40:248–258. https://doi.org/10.1016/j.ctrv.2013.08.008

    Article  PubMed  CAS  Google Scholar 

  20. Neidert MC, Schoor O, Trautwein C, Trautwein N, Christ L, Melms A et al (2013) Natural HLA class I ligands from glioblastoma: extending the options for immunotherapy. J Neurooncol 111:285–294. https://doi.org/10.1007/s11060-012-1028-8

    Article  PubMed  CAS  Google Scholar 

  21. Neumann A, Horzer H, Hillen N, Klingel K, Schmid-Horch B, Buhring HJ et al (2013) Identification of HLA ligands and T-cell epitopes for immunotherapy of lung cancer. Cancer Immunol Immunother 62:1485–1497. https://doi.org/10.1007/s00262-013-1454-2

    Article  PubMed  CAS  Google Scholar 

  22. Rammensee H, Bachmann J, Emmerich NP, Bachor OA, Stevanovic S (1999) SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50:213–219

    Article  PubMed  CAS  Google Scholar 

  23. Rapp C, Warta R, Stamova S, Nowrouzi A, Geisenberger C, Gal Z et al (2017) Identification of T cell target antigens in glioblastoma stem-like cells using an integrated proteomics-based approach in patient specimens. Acta Neuropathol. https://doi.org/10.1007/s00401-017-1702-1

    PubMed  Article  Google Scholar 

  24. Robbins PF, Lu YC, El-Gamil M, Li YF, Gross C, Gartner J et al (2013) Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med 19:747–752. https://doi.org/10.1038/nm.3161

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Rudolf D, Silberzahn T, Walter S, Maurer D, Engelhard J, Wernet D et al (2008) Potent costimulation of human CD8 T cells by anti-4-1BB and anti-CD28 on synthetic artificial antigen presenting cells. Cancer Immunol Immunother 57:175–183. https://doi.org/10.1007/s00262-007-0360-x

    Article  PubMed  CAS  Google Scholar 

  26. Schumacher T, Bunse L, Pusch S, Sahm F, Wiestler B, Quandt J et al (2014) A vaccine targeting mutant IDH1 induces antitumour immunity. Nature 512:324–327. https://doi.org/10.1038/nature13387

    Article  PubMed  CAS  Google Scholar 

  27. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T et al (2004) Identification of human brain tumour initiating cells. Nature 432:396–401. https://doi.org/10.1038/nature03128

    Article  PubMed  CAS  Google Scholar 

  28. Sturm T, Leinders-Zufall T, Macek B, Walzer M, Jung S, Pommerl B et al (2013) Mouse urinary peptides provide a molecular basis for genotype discrimination by nasal sensory neurons. Nat Commun 4:1616. https://doi.org/10.1038/ncomms2610

    Article  PubMed  CAS  Google Scholar 

  29. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF et al (2012) Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366:2443–2454. https://doi.org/10.1056/NEJMoa1200690

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Tran E, Turcotte S, Gros A, Robbins PF, Lu YC, Dudley ME et al (2014) Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 344:641–645. https://doi.org/10.1126/science.1251102

    Article  PubMed  CAS  Google Scholar 

  31. van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B et al (1991) A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254:1643–1647

    Article  PubMed  Google Scholar 

  32. van Rooij N, van Buuren MM, Philips D, Velds A, Toebes M, Heemskerk B et al (2013) Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma. J Clin Oncol Off J Am Soc Clin Oncol 31:e439–e442. https://doi.org/10.1200/JCO.2012.47.7521

    Article  Google Scholar 

  33. Verdegaal EM, de Miranda NF, Visser M, Harryvan T, van Buuren MM, Andersen RS et al (2016) Neoantigen landscape dynamics during human melanoma-T cell interactions. Nature. https://doi.org/10.1038/nature18945

    PubMed  Article  Google Scholar 

  34. Vigneron N, Stroobant V, Van den Eynde BJ, van der Bruggen P (2013) Database of T cell-defined human tumor antigens: the 2013 update. Cancer Immunity 13:15

    PubMed  PubMed Central  Google Scholar 

  35. Walter S, Herrgen L, Schoor O, Jung G, Wernet D, Buhring HJ et al (2003) Cutting edge: predetermined avidity of human CD8 T cells expanded on calibrated MHC/anti-CD28-coated microspheres. J Immunol 171:4974–4978

    Article  PubMed  CAS  Google Scholar 

  36. Warde-Farley D, Donaldson SL, Comes O, Zuberi K, Badrawi R, Chao P et al (2010) The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function. Nucleic Acids Res 38:W214–W220. https://doi.org/10.1093/nar/gkq537

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Weller M, van den Bent M, Tonn JC, Stupp R, Preusser M, Cohen-Jonathan-Moyal E et al (2017) European Association for Neuro-Oncology (EANO) guideline on the diagnosis and treatment of adult astrocytic and oligodendroglial gliomas. Lancet Oncol 18:e315–e329. https://doi.org/10.1016/S1470-2045(17)30194-8

    Article  PubMed  Google Scholar 

  38. Westphal M, Lamszus K (2011) The neurobiology of gliomas: from cell biology to the development of therapeutic approaches. Nat Rev Neurosci 12:495–508. https://doi.org/10.1038/nrn3060

    Article  PubMed  CAS  Google Scholar 

  39. Widenmeyer M, Griesemann H, Stevanovic S, Feyerabend S, Klein R, Attig S et al (2012) Promiscuous survivin peptide induces robust CD4+ T-cell responses in the majority of vaccinated cancer patients. Int J Cancer 131:140–149. https://doi.org/10.1002/ijc.26365

    Article  PubMed  CAS  Google Scholar 

  40. Wolpert F, Tritschler I, Steinle A, Weller M, Eisele G (2014) A disintegrin and metalloproteinases 10 and 17 modulate the immunogenicity of glioblastoma-initiating cells. Neuro-oncology 16:382–391. https://doi.org/10.1093/neuonc/not232

    Article  PubMed  CAS  Google Scholar 

  41. Yadav M, Jhunjhunwala S, Phung QT, Lupardus P, Tanguay J, Bumbaca S et al (2014) Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing. Nature 515:572–576. https://doi.org/10.1038/nature14001

    Article  PubMed  CAS  Google Scholar 

  42. Yawata T, Nakai E, Park KC, Chihara T, Kumazawa A, Toyonaga S et al (2010) Enhanced expression of cancer testis antigen genes in glioma stem cells. Mol Carcinogen 49:532–544. https://doi.org/10.1002/mc.20614

    Article  CAS  Google Scholar 

Download references

Funding

This project was funded by a grant of the Canton of Zurich (HSM-2) to LR, PR and MiW. MN received financial support from the Filling-The-Gap Program (University of Zurich), the Hartmann Müller-Foundation and the EMDO Foundation. HGR received financial support from the ERC Grant 339842, MUTAEDITING.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Marian Christoph Neidert.

Ethics declarations

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Conflict of interest

DK is an employee and HGR is a shareholder of Immatics Biotechnologies GmbH. HGR is also shareholder of Curevac AG. MiW has received research grants from Acceleron, Actelion, Bayer, Isarna, MSD, Merck & Co, Novocure, Piqur and Roche and honoraria for lectures or advisory board participation or consulting from BMS, Celldex, Immunocellular Therapeutics, Isarna, Magforce, MSD, Merck & Co, Northwest Biotherapeutics, Novocure, Pfizer, Roche, Teva and Tocagen. FW has received travel support from Roche. PR has received honoraria from Roche, MSD, Novocure, Novartis and BMS for advisory board participation or lectures. The other authors declare no competing financial interests.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Neidert, M.C., Kowalewski, D.J., Silginer, M. et al. The natural HLA ligandome of glioblastoma stem-like cells: antigen discovery for T cell-based immunotherapy. Acta Neuropathol 135, 923–938 (2018). https://doi.org/10.1007/s00401-018-1836-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00401-018-1836-9

Keywords

  • Glioblastoma
  • Immunotherapy
  • HLA ligand
  • Glioblastoma stem-like cells
  • GSC
  • Tumor-associated antigen