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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

Abstract

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.

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Acknowledgments

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.

Author information

Correspondence to Sean J. Mulligan or Jim Xiang.

Electronic supplementary material

Below is the link to the electronic supplementary material.

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

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

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

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

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

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Wang, L., Xie, Y., Ahmed, K.A. et al. 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. Breast Cancer Res Treat 140, 273–284 (2013). https://doi.org/10.1007/s10549-013-2626-7

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Keywords

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