Breast Cancer Research and Treatment

, Volume 140, Issue 2, pp 273–284 | Cite as

Exosomal pMHC-I complex targets T cell-based vaccine to directly stimulate CTL responses leading to antitumor immunity in transgenic FVBneuN and HLA-A2/HER2 mice and eradicating trastuzumab-resistant tumor in athymic nude mice

  • Lu Wang
  • Yufeng Xie
  • Khawaja Ashfaque Ahmed
  • Shahid Ahmed
  • Amer Sami
  • Rajni Chibbar
  • Qingyong Xu
  • Susan E. Kane
  • Siguo Hao
  • Sean J. MulliganEmail author
  • Jim XiangEmail author
Preclinical study


One of the major obstacles in human epidermal growth factor receptor 2 (HER2)-specific trastuzumab antibody immunotherapy of HER2-positive breast cancer is the development of trastuzumab resistance, warranting the search for other therapeutic strategies. Using mouse models, we previously demonstrated that ovalbumin (OVA)-specific dendritic cell (DC)-released exosome (EXOOVA)-targeted CD4+ T cell-based (OVA-TEXO) vaccine stimulates efficient cytotoxic T lymphocyte (CTL) responses via exosomal peptide/major histocompatibility complex (pMHC)-I, exosomal CD80 and endogenous IL-2 signaling; and long-term CTL memory by means of via endogenous CD40L signaling. In this study, using two-photon microscopy, we provide the first visual evidence on targeting OVA-TEXO to cognate CD8+ T cells in vivo via exosomal pMHC-I complex. We prepared HER2/neu-specific Neu-TEXO and HER2-TEXO vaccines using adenoviral vector (AdVneu and AdVHER2)-transfected DC (DCneu and DCHER2)-released EXOs (EXOneu and EXOHER2), and assessed their stimulatory effects on HER2/neu-specific CTL responses and antitumor immunity. We demonstrate that Neu-TEXO vaccine is capable of stimulating efficient neu-specific CTL responses, leading to protective immunity against neu-expressing Tg1-1 breast cancer in all 6/6 transgenic (Tg) FVBneuN mice with neu-specific self-immune tolerance. We also demonstrate that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2+HER2+ BL6-10A2/HER2 B16 melanoma in 2/8 double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Furthermore, we demonstrate that HER2-TEXO vaccine stimulates responses of CD8+ T cells capable of not only inducing killing activity to HLA-A2+HER2+ BL6-10A2/HER2 melanoma and trastuzumab-resistant BT474A2 breast cancer cells in vitro but also eradicating 6-day palpable HER2+ BT474A2 breast cancer (3–4 mm in diameter) in athymic nude mice. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2+ breast cancer, especially for trastuzumab-resistant HER2+ breast cancer patients.


HER2 T cell-based vaccine Trastuzumab resistance Transgenic HLA-A2/HER2 mice 



This research work was supported by research grants from Canadian Institutes of Health Research (MOP 89713) and Saskatchewan Cancer Agency (413092). Lu Wang and Yufeng Xie were supported by Scholarship of China Scholarship Council and Postdoctoral Fellowship of Saskatchewan Health Research Foundation & Saskatchewan Cancer Agency, respectively. We appreciated Mark Boyd for help in flow cytometry.

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10549_2013_2626_MOESM1_ESM.eps (2.4 mb)
sFigure 1. Schematic representation of adenovirus (AdV) vectors. The E1/E3-deleted replication-deficient AdV vectors are under the regulation of the cytomegalovirus (CMV) early/immediate promoter/enhancer. The AdV vectors include AdVnull without any transgene expression, AdVHER2 expressing HER2 transgene and AdVHLA-A2 expressing HLA-A2 alpha chain transgene. LITR, left inverted terminal repeat and RITR, right inverted terminal repeat
10549_2013_2626_MOESM2_ESM.eps (1.3 mb)
sFigure 2. Flow cytometry analysis. (A) DCOVA and EXOOVA were stained with a panel of specific Abs (solid lines) or isotype-matched irrelevant Abs (dotted lines), and analyzed by flow cytometry. (B) DCOVA, (Kb−/−)DCOVA, OVA-TEXO and (Kb−/−)TEXO were stained with anti-pMHC-I antibody (solid lines) or isotype-matched irrelevant antibody (dotted lines), and analyzed by flow cytometry. (C) ConA-T or OVA-TEXO were stained with a panel of specific Abs (solid lines) or isotype-matched irrelevant Abs (dotted lines), and analyzed by flow cytometry. (D) Supernatants from ConA-T cells or OVA-TEXO were measured for IL-2, IFN-γ and TNF-α secretion by using ELISA kits. All kits were purchased from BD biosciences (Mississauga, ON, Canada). One representative experiment of two is shown (1.4 mb)
sMovie 1. Pathways of naive OTI CD8+ T and OVA-TEXO cells. Naive OTI CD8+ T cells (red) move along behind OVA-TEXO (green). The pathways of an OVA-TEXO (green/grey line) and a CD8+ T cell (red/grey line) are remaining bound to each other during the time of imaging. Dimensions: 106 μm × 106 μm × 18 μm × 30 min

sMovie 2. Migration of naive OTI and polyclonal CD8+ T cells in lymph nodes with OVA-TEXO present. Unlabeled OVA-TEXO were injected 24 h before co-transfer of naive OTI (green) and polyclonal (red) CD8+ T cells to the same recipient C57BL/6 mouse. OTI CD8+ T cells (green tracks) show slower and much more confined movements than polyclonal CD8+ T cells (red tracks). Dimensions: 121 μm × 67 μm × 28 μm × 40 min (1.6 mb)
sMovie 3. Migration of naive OTI CD8+ T cells in lymph nodes with OVA-(Kb−/−)TEXO present. Interactions between OVA-(Kb−/−)TEXO cells (green) and naive OTI CD8+ T cells (red) are only intermittent. Dimensions: 106 μm × 106 μm × 27 μm × 30 min


  1. 1.
    Schechter AL, Hung MC, Vaidyanathan L, Weinberg RA, Yang-Feng TL, Francke U, Ullrich A, Coussens L (1985) The neu gene: an erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. Science 229(4717):976–978PubMedCrossRefGoogle Scholar
  2. 2.
    Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A et al (1989) Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244(4905):707–712PubMedCrossRefGoogle Scholar
  3. 3.
    Gonzalez-Angulo AM, Hortobagyi GN, Esteva FJ (2006) Adjuvant therapy with trastuzumab for HER-2/neu-positive breast cancer. Oncologist 11(8):857–867PubMedCrossRefGoogle Scholar
  4. 4.
    Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, Slamon DJ, Murphy M, Novotny WF, Burchmore M, Shak S, Stewart SJ, Press M (2002) Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20(3):719–726PubMedCrossRefGoogle Scholar
  5. 5.
    Berns K, Horlings HM, Hennessy BT, Madiredjo M, Hijmans EM, Beelen K, Linn SC, Gonzalez-Angulo AM, Stemke-Hale K, Hauptmann M, Beijersbergen RL, Mills GB, van de Vijver MJ, Bernards R (2007) A functional genetic approach identifies the PI3K pathway as a major determinant of trastuzumab resistance in breast cancer. Cancer Cell 12(4):395–402PubMedCrossRefGoogle Scholar
  6. 6.
    Nahta R, Esteva FJ (2006) Herceptin: mechanisms of action and resistance. Cancer Lett 232(2):123–138PubMedCrossRefGoogle Scholar
  7. 7.
    Nahta R, Esteva FJ (2006) HER2 therapy: molecular mechanisms of trastuzumab resistance. Breast Cancer Res 8(6):215PubMedCrossRefGoogle Scholar
  8. 8.
    Hao S, Yuan J, Xiang J (2007) Nonspecific CD4(+) T cells with uptake of antigen-specific dendritic cell-released exosomes stimulate antigen-specific CD8(+) CTL responses and long-term T cell memory. J Leukoc Biol 82(4):829–838PubMedCrossRefGoogle Scholar
  9. 9.
    Hao S, Liu Y, Yuan J, Zhang X, He T, Wu X, Wei Y, Sun D, Xiang J (2007) Novel exosome-targeted CD4+ T cell vaccine counteracting CD4+25+ regulatory T cell-mediated immune suppression and stimulating efficient central memory CD8+ CTL responses. J Immunol 179(5):2731–2740PubMedGoogle Scholar
  10. 10.
    Xie Y, Wang L, Freywald A, Qureshi M, Chen Y, Xiang J (2013) A novel T cell-based vaccine capable of stimulating long-term functional CTL memory against B16 melanoma via CD40L signaling. Cell Mol Immunol 10(1):72–77PubMedCrossRefGoogle Scholar
  11. 11.
    Ahmed KA, Wang L, Munegowda MA, Mulligan SJ, Gordon JR, Griebel P, Xiang J (2012) Direct in vivo evidence of CD4+ T cell requirement for CTL response and memory via pMHC-I targeting and CD40L signaling. J Leukoc Biol 92(2):289–300PubMedCrossRefGoogle Scholar
  12. 12.
    Sas S, Chan T, Sami A, El-Gayed A, Xiang J (2008) Vaccination of fiber-modified adenovirus-transfected dendritic cells to express HER-2/neu stimulates efficient HER-2/neu-specific humoral and CTL responses and reduces breast carcinogenesis in transgenic mice. Cancer Gene Ther 15(10):655–666PubMedCrossRefGoogle Scholar
  13. 13.
    Chan T, Sami A, El-Gayed A, Guo X, Xiang J (2006) HER-2/neu-gene engineered dendritic cell vaccine stimulates stronger HER-2/neu-specific immune responses compared to DNA vaccination. Gene Ther 13(19):1391–1402PubMedCrossRefGoogle Scholar
  14. 14.
    Chen Z, Huang H, Chang T, Carlsen S, Saxena A, Marr R, Xing Z, Xiang J (2002) Enhanced HER-2/neu-specific antitumor immunity by cotransduction of mouse dendritic cells with two genes encoding HER-2/neu and alpha tumor necrosis factor. Cancer Gene Ther 9(9):778–786PubMedCrossRefGoogle Scholar
  15. 15.
    Eddy SF, Kane SE, Sonenshein GE (2007) Trastuzumab-resistant HER2-driven breast cancer cells are sensitive to epigallocatechin-3 gallate. Cancer Res 67(19):9018–9023PubMedCrossRefGoogle Scholar
  16. 16.
    Piechocki MP, Ho YS, Pilon S, Wei WZ (2003) Human ErbB-2 (Her-2) transgenic mice: a model system for testing Her-2 based vaccines. J Immunol 171(11):5787–5794PubMedGoogle Scholar
  17. 17.
    Hao S, Bai O, Li F, Yuan J, Laferte S, Xiang J (2007) Mature dendritic cells pulsed with exosomes stimulate efficient cytotoxic T-lymphocyte responses and antitumour immunity. Immunology 120(1):90–102PubMedCrossRefGoogle Scholar
  18. 18.
    Xiang J, Huang H, Liu Y (2005) A new dynamic model of CD8+ T effector cell responses via CD4+ T helper-antigen-presenting cells. J Immunol 174(12):7497–7505PubMedGoogle Scholar
  19. 19.
    Wang JC, Livingstone AM (2003) Cutting edge: CD4+ T cell help can be essential for primary CD8+ T cell responses in vivo. J Immunol 171(12):6339–6343PubMedGoogle Scholar
  20. 20.
    Nanjundappa RH, Wang R, Xie Y, Umeshappa CS, Chibbar R, Wei Y, Liu Q, Xiang J (2011) GP120-specific exosome-targeted T cell-based vaccine capable of stimulating DC- and CD4(+) T-independent CTL responses. Vaccine 29(19):3538–3547PubMedCrossRefGoogle Scholar
  21. 21.
    Chen Y, Xie Y, Chan T, Sami A, Ahmed S, Liu Q, Xiang J (2011) Adjuvant effect of HER-2/neu-specific adenoviral vector stimulating CD8(+) T and natural killer cell responses on anti-HER-2/neu antibody therapy for well-established breast tumors in HER-2/neu transgenic mice. Cancer Gene Ther 18(7):489–499PubMedCrossRefGoogle Scholar
  22. 22.
    Press MF, Pike MC, Chazin VR, Hung G, Udove JA, Markowicz M, Danyluk J, Godolphin W, Sliwkowski M, Akita R et al (1993) Her-2/neu expression in node-negative breast cancer: direct tissue quantitation by computerized image analysis and association of overexpression with increased risk of recurrent disease. Cancer Res 53(20):4960–4970PubMedGoogle Scholar
  23. 23.
    Ross JS, Fletcher JA, Linette GP, Stec J, Clark E, Ayers M, Symmans WF, Pusztai L, Bloom KJ (2003) The Her-2/neu gene and protein in breast cancer 2003: biomarker and target of therapy. Oncologist 8(4):307–325PubMedCrossRefGoogle Scholar
  24. 24.
    Koeppen HK, Wright BD, Burt AD, Quirke P, McNicol AM, Dybdal NO, Sliwkowski MX, Hillan KJ (2001) Overexpression of HER2/neu in solid tumours: an immunohistochemical survey. Histopathology 38(2):96–104PubMedCrossRefGoogle Scholar
  25. 25.
    Press MF, Cordon-Cardo C, Slamon DJ (1990) Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 5(7):953–962PubMedGoogle Scholar
  26. 26.
    Ward RL, Hawkins NJ, Coomber D, Disis ML (1999) Antibody immunity to the HER-2/neu oncogenic protein in patients with colorectal cancer. Hum Immunol 60(6):510–515PubMedCrossRefGoogle Scholar
  27. 27.
    Kiessling R, Wei WZ, Herrmann F, Lindencrona JA, Choudhury A, Kono K, Seliger B (2002) Cellular immunity to the Her-2/neu protooncogene. Adv Cancer Res 85:101–144PubMedCrossRefGoogle Scholar
  28. 28.
    Baxevanis CN, Sotiropoulou PA, Sotiriadou NN, Papamichail M (2004) Immunobiology of HER-2/neu oncoprotein and its potential application in cancer immunotherapy. Cancer Immunol Immunother 53(3):166–175PubMedCrossRefGoogle Scholar
  29. 29.
    Baxevanis CN, Voutsas IF, Gritzapis AD, Perez SA, Papamichail M (2010) HER-2/neu as a target for cancer vaccines. Immunotherapy 2(2):213–226PubMedCrossRefGoogle Scholar
  30. 30.
    Baxevanis CN, Sotiriadou NN, Gritzapis AD, Sotiropoulou PA, Perez SA, Cacoullos NT, Papamichail M (2006) Immunogenic HER-2/neu peptides as tumor vaccines. Cancer Immunol Immunother 55(1):85–95PubMedCrossRefGoogle Scholar
  31. 31.
    Eck SC, Turka LA (2001) Adoptive transfer enables tumor rejection targeted against a self-antigen without the induction of autoimmunity. Cancer Res 61(7):3077–3083PubMedGoogle Scholar
  32. 32.
    Garcia-Hernandez Mde L, Gray A, Hubby B, Klinger OJ, Kast WM (2008) Prostate stem cell antigen vaccination induces a long-term protective immune response against prostate cancer in the absence of autoimmunity. Cancer Res 68(3):861–869PubMedCrossRefGoogle Scholar
  33. 33.
    Lane C, Leitch J, Tan X, Hadjati J, Bramson JL, Wan Y (2004) Vaccination-induced autoimmune vitiligo is a consequence of secondary trauma to the skin. Cancer Res 64(4):1509–1514PubMedCrossRefGoogle Scholar
  34. 34.
    Palmer DC, Chan CC, Gattinoni L, Wrzesinski C, Paulos CM, Hinrichs CS, Powell DJ Jr, Klebanoff CA, Finkelstein SE, Fariss RN, Yu Z, Nussenblatt RB, Rosenberg SA, Restifo NP (2008) Effective tumor treatment targeting a melanoma/melanocyte-associated antigen triggers severe ocular autoimmunity. Proc Natl Acad Sci USA 105(23):8061–8066PubMedCrossRefGoogle Scholar
  35. 35.
    Bos R, van Duikeren S, Morreau H, Franken K, Schumacher TN, Haanen JB, van der Burg SH, Melief CJ, Offringa R (2008) Balancing between antitumor efficacy and autoimmune pathology in T-cell-mediated targeting of carcinoembryonic antigen. Cancer Res 68(20):8446–8455PubMedCrossRefGoogle Scholar
  36. 36.
    Baxevanis CN, Perez SA, Papamichail M (2009) Cancer immunotherapy. Crit Rev Clin Lab Sci 46(4):167–189PubMedCrossRefGoogle Scholar
  37. 37.
    Sumoza-Toledo A, Eaton AD, Sarukhan A (2006) Regulatory T cells inhibit protein kinase C theta recruitment to the immune synapse of naive T cells with the same antigen specificity. J Immunol 176(10):5779–5787PubMedGoogle Scholar
  38. 38.
    Whittington PJ, Piechocki MP, Heng HH, Jacob JB, Jones RF, Back JB, Wei WZ (2008) DNA vaccination controls Her-2+ tumors that are refractory to targeted therapies. Cancer Res 68(18):7502–7511PubMedCrossRefGoogle Scholar
  39. 39.
    Radkevich-Brown O, Jacob J, Kershaw M, Wei WZ (2009) Genetic regulation of the response to Her-2 DNA vaccination in human Her-2 transgenic mice. Cancer Res 69(1):212–218PubMedCrossRefGoogle Scholar
  40. 40.
    Dai S, Wan T, Wang B, Zhou X, Xiu F, Chen T, Wu Y, Cao X (2005) More efficient induction of HLA-A*0201-restricted and carcinoembryonic antigen (CEA)-specific CTL response by immunization with exosomes prepared from heat-stressed CEA-positive tumor cells. Clin Cancer Res 11(20):7554–7563PubMedCrossRefGoogle Scholar
  41. 41.
    Saha A, Chatterjee SK, Foon KA, Celis E, Bhattacharya-Chatterjee M (2007) Therapy of established tumors in a novel murine model transgenic for human carcinoembryonic antigen and HLA-A2 with a combination of anti-idiotype vaccine and CTL peptides of carcinoembryonic antigen. Cancer Res 67(6):2881–2892PubMedCrossRefGoogle Scholar
  42. 42.
    Saha A, Bhattacharya-Chatterjee M, Foon KA, Celis E, Chatterjee SK (2009) Stimulatory effects of CpG oligodeoxynucleotide on dendritic cell-based immunotherapy of colon cancer in CEA/HLA-A2 transgenic mice. Int J Cancer 124(4):877–888PubMedCrossRefGoogle Scholar
  43. 43.
    Hao S, Ye Z, Li F, Meng Q, Qureshi M, Yang J, Xiang J (2006) Epigenetic transfer of metastatic activity by uptake of highly metastatic B16 melanoma cell-released exosomes. Exp Oncol 28(2):126–131PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Lu Wang
    • 1
  • Yufeng Xie
    • 1
  • Khawaja Ashfaque Ahmed
    • 1
  • Shahid Ahmed
    • 2
  • Amer Sami
    • 2
  • Rajni Chibbar
    • 3
  • Qingyong Xu
    • 3
  • Susan E. Kane
    • 5
  • Siguo Hao
    • 6
  • Sean J. Mulligan
    • 4
    Email author
  • Jim Xiang
    • 1
    • 7
    Email author
  1. 1.Cancer Research UnitSaskatchewan Cancer AgencySaskatoonCanada
  2. 2.Department of OncologyUniversity of SaskatchewanSaskatoonCanada
  3. 3.Department of PathologyUniversity of SaskatchewanSaskatoonCanada
  4. 4.Department of Physiology, College of MedicineUniversity of SaskatchewanSaskatoonCanada
  5. 5.Beckman Research Institute of the City of HopeDuarteUSA
  6. 6.Xinhua HospitalJiao-Tong UniversityShanghaiChina
  7. 7.Saskatoon Cancer CenterSaskatoonCanada

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