Cancer Immunology, Immunotherapy

, Volume 63, Issue 4, pp 335–345 | Cite as

Apoptotic blebs from leukemic cells as a preferred source of tumor-associated antigen for dendritic cell-based vaccines

  • Jurjen M. Ruben
  • Willemijn van den Ancker
  • Hetty J. Bontkes
  • Theresia M. Westers
  • Erik Hooijberg
  • Gert J. Ossenkoppele
  • Tanja D. de Gruijl
  • Arjan A. van de Loosdrecht
Original Article


Since few leukemia-associated antigens (LAA) are characterized for acute myeloid leukemia (AML), apoptotic tumor cells constitute an attractive LAA source for DC-based vaccines, as they contain both characterized and unknown LAA. However, loading DC with apoptotic tumor cells may interfere with DC function. Previously, it was shown in mice that apoptotic blebs induce DC maturation, whereas apoptotic cell remnants (ACR) do not. Here, we analyzed human monocyte-derived DC (MoDC) functionality in vitro, after ingesting either allogeneic AML-derived ACR or blebs. We show that MoDC ingest blebs to a higher extent and are superior in migrating toward CCL19, as compared to ACR-loaded MoDC. Although MoDC cytokine production was unaffected, co-culturing bleb-loaded MoDC with T cells led to an increased T cell proliferation and IFNγ production. Moreover, antigen-specific CD8+ T cells frequencies increased to 0.63 % by priming with bleb-loaded MoDC, compared to 0.16 % when primed with ACR-loaded MoDC. Importantly, CD8+ T cells primed by bleb-loaded MoDC recognized their specific epitope at one to two orders of magnitude lower concentrations compared to ACR-loaded MoDC. In conclusion, superior ingestion efficiency and migration, combined with favorable T cell cytokine release and CD8+ T cell priming ability and avidity, point to blebs as the preferred component of apoptotic leukemic cells for LAA loading of DC for the immunotherapy of AML.


Dendritic cell vaccination Dendritic cell loading Apoptotic tumor cells Blebs T cell priming Anti-tumor immunity 



We would like to thank Professor Dr. Rob Beelen and Donna Fluitsma from the department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, the Netherlands, for facilitating the electron microscopic analysis and their technical assistance therein. Furthermore, we would like to thank Dr. Teun de Vries, Ton Schoenmaker, and Cor Semeins from the department of Periodontology and Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, the Netherlands, for facilitating the confocal microscope and providing technical assistance.

Conflict of interest

The authors have no conflicts of interest to disclose.


  1. 1.
    Banchereau J, Palucka K (2005) Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol 5:296–306. doi: 10.1038/nri1592 PubMedCrossRefGoogle Scholar
  2. 2.
    Steinman RM, Banchereau J (2007) Taking dendritic cells into medicine. Nature 449:419–426. doi: 10.1038/nature06175 PubMedCrossRefGoogle Scholar
  3. 3.
    Westers TM, Stam AGM, Scheper RJ et al (2003) Rapid generation of antigen-presenting cells from leukaemic blasts in acute myeloid leukaemia. Cancer Immunol Immunother 52:17–27. doi: 10.1007/s00262-002-0316-0 PubMedCrossRefGoogle Scholar
  4. 4.
    Westers TM, Van Den Ancker W, Bontkes HJ et al (2011) Chronic myeloid leukemia lysate-loaded dendritic cells induce T-cell responses towards leukemia progenitor cells. Immunotherapy 3:569–576PubMedCrossRefGoogle Scholar
  5. 5.
    Nestle FO, Alijagic S, Gilliet M et al (1998) Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 4:328–332PubMedCrossRefGoogle Scholar
  6. 6.
    Thurner B, Haendle I, Röder C et al (1999) Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med 190:1669–1678PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Van Tendeloo VF, Van de Velde A, Van Driessche A et al (2010) Induction of complete and molecular remissions in acute myeloid leukemia by Wilms’ tumor 1 antigen-targeted dendritic cell vaccination. Proc Natl Acad Sci USA 107:13824–13829. doi: 10.1073/pnas.1008051107 PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Boczkowski D, Nair SK, Snyder D, Gilboa E (1996) Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med 184:465–472PubMedCrossRefGoogle Scholar
  9. 9.
    Bontkes HJ, Kramer D, Ruizendaal JJ et al (2007) Dendritic cells transfected with interleukin-12 and tumor-associated antigen messenger RNA induce high avidity cytotoxic T cells. Gene Ther 14:366–375PubMedCrossRefGoogle Scholar
  10. 10.
    Willemijn VDA, Van Luijn MM, Westers TM et al (2010) Recent advances in antigen-loaded dendritic cell-based strategies for treatment of minimal residual disease in acute myeloid leukemia. Immunotherapy 2:69–83CrossRefGoogle Scholar
  11. 11.
    Van den Ancker W, van Luijn MM, Ruben JM et al (2011) Targeting Toll-like receptor 7/8 enhances uptake of apoptotic leukemic cells by monocyte-derived dendritic cells but interferes with subsequent cytokine-induced maturation. Cancer Immunol Immunother 60:37–47. doi: 10.1007/s00262-010-0917-y PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Kitawaki T, Kadowaki N, Fukunaga K et al (2011) Cross-priming of CD8(+) T cells in vivo by dendritic cells pulsed with autologous apoptotic leukemic cells in immunotherapy for elderly patients with acute myeloid leukemia. Exp Hematol 39(424–433):e2. doi: 10.1016/j.exphem.2011.01.001 PubMedGoogle Scholar
  13. 13.
    Liu K, Iyoda T, Saternus M et al (2002) Immune tolerance after delivery of dying cells to dendritic cells in situ. J Exp Med 196:1091–1097. doi: 10.1084/jem.20021215 PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Morelli AE (2006) The immune regulatory effect of apoptotic cells and exosomes on dendritic cells: its impact on transplantation. Am J Transplant 6:254–261. doi: 10.1111/j.1600-6143.2005.01197.x PubMedCrossRefGoogle Scholar
  15. 15.
    Municio C, Hugo E, Alvarez Y et al (2011) Apoptotic cells enhance IL-10 and reduce IL-23 production in human dendritic cells treated with zymosan. Mol Immunol 1–10. doi: 10.1016/j.molimm.2011.07.022
  16. 16.
    Ren G, Su J, Zhao X et al (2008) Apoptotic cells induce immunosuppression through dendritic cells: critical roles of IFN-gamma and nitric oxide. J Immunol 181:3277–3284PubMedGoogle Scholar
  17. 17.
    Sen P, Wallet MA, Yi Z et al (2007) Apoptotic cells induce Mer tyrosine kinase-dependent blockade of NF-κB activation in dendritic cells. Blood 109:653–660. doi: 10.1182/blood-2006-04-017368 PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Stuart LM, Lucas M, Simpson C et al (2002) Inhibitory effects of apoptotic cell ingestion upon endotoxin-driven myeloid dendritic cell maturation. J Immunol 168:1627–1635PubMedGoogle Scholar
  19. 19.
    Lomonosova E, Chinnadurai G (2008) BH3-only proteins in apoptosis and beyond: an overview. Oncogene 27(Suppl 1):S2–S19PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Franz S, Herrmann K, Fürnrohr BG et al (2007) After shrinkage apoptotic cells expose internal membrane-derived epitopes on their plasma membranes. Cell Death Differ 14:733–742. doi: 10.1038/sj.cdd.4402066 PubMedCrossRefGoogle Scholar
  21. 21.
    Lane JD, Allan VJ, Woodman PG (2005) Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells. J Cell Sci 118:4059–4071. doi: 10.1242/jcs.02529 PubMedCrossRefGoogle Scholar
  22. 22.
    Meesmann HM, Fehr E-M, Kierschke S et al (2010) Decrease of sialic acid residues as an eat-me signal on the surface of apoptotic lymphocytes. J Cell Sci 123:3347–3356PubMedCrossRefGoogle Scholar
  23. 23.
    Fransen JH, Hilbrands LB, Ruben J et al (2009) Mouse dendritic cells matured by ingestion of apoptotic blebs induce T cells to produce interleukin-17. Arthritis Rheum 60:2304–2313. doi: 10.1002/art.24719 PubMedCrossRefGoogle Scholar
  24. 24.
    Suber T, Rosen A (2009) Apoptotic cell blebs: repositories of autoantigens and contributors to immune context. Arthritis Rheum 60:2216–2219. doi: 10.1002/art.24715 PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Wlodkowic D, Skommer J, Pelkonen J (2007) Towards an understanding of apoptosis detection by SYTO dyes. Cytom Part A J Int Soc Anal Cytol 71:61–72. doi:  10.1002/cyto.a.20366 CrossRefGoogle Scholar
  26. 26.
    Liu Y, Qureshi M, Xiang J (2002) Antitumor immune responses derived from transgenic expression of CD40 ligand in myeloma cells. Cancer Biother Radiopharm 17:11–18. doi: 10.1089/10849780252824028 PubMedCrossRefGoogle Scholar
  27. 27.
    Schreurs MWJ, Scholten KBJ, Kueter EWM et al (2003) In vitro generation and life span extension of human papillomavirus type 16-specific, healthy donor-derived CTL clones. J Immunol 171:2912–2921PubMedGoogle Scholar
  28. 28.
    Hooijberg E, Ruizendaal JJ, Snijders PJ et al (2000) Immortalization of human CD8+ T cell clones by ectopic expression of telomerase reverse transcriptase. J Immunol 165:4239–4245PubMedGoogle Scholar
  29. 29.
    Mathivanan S, Ji H, Simpson RJ (2010) Exosomes: extracellular organelles important in intercellular communication. J Proteomics 73:1907–1920. doi: 10.1016/j.jprot.2010.06.006 PubMedCrossRefGoogle Scholar
  30. 30.
    Fischer E, Kobold S, Kleber S et al (2010) Cryptic epitopes induce high-titer humoral immune response in patients with cancer. J Immunol 185:3095–3102PubMedCrossRefGoogle Scholar
  31. 31.
    Fransen JH, Hilbrands LB, Jacobs CW et al (2009) Both early and late apoptotic blebs are taken up by DC and induce IL-6 production. Autoimmunity 42:325–327PubMedCrossRefGoogle Scholar
  32. 32.
    Verdijk P, Aarntzen EHJG, Lesterhuis WJ et al (2009) Limited amounts of dendritic cells migrate into the T-cell area of lymph nodes but have high immune activating potential in melanoma patients. Clin Cancer Res 15:2531–2540. doi: 10.1158/1078-0432.CCR-08-2729 PubMedCrossRefGoogle Scholar
  33. 33.
    Wickman GR, Julian L, Mardilovich K et al (2013) Blebs produced by actin-myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs. Cell Death Differ. doi: 10.1038/cdd.2013.69 PubMedCentralPubMedGoogle Scholar
  34. 34.
    Gallucci S, Lolkema M, Matzinger P (1999) Natural adjuvants: endogenous activators of dendritic cells. Nat Med 5:1249–1255. doi: 10.1038/15200 PubMedCrossRefGoogle Scholar
  35. 35.
    Herr W, Ranieri E, Olson W et al (2000) Mature dendritic cells pulsed with freeze-thaw cell lysates define an effective in vitro vaccine designed to elicit EBV-specific CD4(+) and CD8(+) T lymphocyte responses. Blood 96:1857–1864PubMedGoogle Scholar
  36. 36.
    Schnurr M, Galambos P, Scholz C et al (2001) Tumor cell lysate-pulsed human dendritic cells induce a T-cell response against pancreatic carcinoma cells: an in vitro model for the assessment of tumor vaccines. Cancer Res 61(17):6445–6450PubMedGoogle Scholar
  37. 37.
    Kokhaei P, Rezvany MR, Virving L et al (2003) Dendritic cells loaded with apoptotic tumour cells induce a stronger T-cell response than dendritic cell-tumour hybrids in B-CLL. Leukemia 17:894–899. doi: 10.1038/sj.leu.2402913 PubMedCrossRefGoogle Scholar
  38. 38.
    Wieckowski E, Chatta GS, Mailliard RM et al (2011) Type-1 polarized dendritic cells loaded with apoptotic prostate cancer cells are potent inducers of CD8(+) T cells against prostate cancer cells and defined prostate cancer-specific epitopes. Prostate 71:125–133. doi: 10.1002/pros.21228 PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Dolan BP, Li L, Takeda K et al (2010) Defective ribosomal products are the major source of antigenic peptides endogenously generated from influenza A virus neuraminidase. J Immunol 184:1419–1424PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Yewdell JW, Nicchitta CV (2006) The DRiP hypothesis decennial: support, controversy, refinement and extension. Trends Immunol 27:368–373. doi: 10.1016/ PubMedCrossRefGoogle Scholar
  41. 41.
    Li Y, Wang L-X, Pang P et al (2011) Tumor-derived autophagosome vaccine: mechanism of cross-presentation and therapeutic efficacy. Clin Cancer Res 17:7047–7057. doi: 10.1158/1078-0432.CCR-11-0951 PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Jurjen M. Ruben
    • 1
  • Willemijn van den Ancker
    • 1
  • Hetty J. Bontkes
    • 1
  • Theresia M. Westers
    • 1
  • Erik Hooijberg
    • 2
  • Gert J. Ossenkoppele
    • 1
  • Tanja D. de Gruijl
    • 3
  • Arjan A. van de Loosdrecht
    • 1
  1. 1.Department of Hematology, Cancer Center AmsterdamVU University Medical CenterAmsterdamThe Netherlands
  2. 2.Department of Pathology, Cancer Center AmsterdamVU University Medical CenterAmsterdamThe Netherlands
  3. 3.Department of Medical Oncology, Cancer Center AmsterdamVU University Medical CenterAmsterdamThe Netherlands

Personalised recommendations