br Materials and methods br Results br Discussion
Materials and methods
Discussion It was previously shown that heterologous DNA vaccines composed of fused cDNA fragments encoding chimeric NH2-terminal human HER2 and COOH-terminal rat neu sequences stimulated stronger antibody responses and protective antitumor immunity than either HER2 or neu DNA vaccine in transgenic mice with HER2-specific self-immune tolerance , . In this study, we constructed heterologous human/rat HER2-specific T cell vaccine HuRt-Texo by using ConA-stimulated polyclonal CD4+ T Cetylpyridinium Chloride with the uptake of EXO expressing HER2- and neu-specific pMHC complexes (EXOHuRt) released by DCHuRt expressing heterologous NH2-terminus HER2 and COOH-terminus neu fusion protein. For the first time, we demonstrate that heterologous HuRt-Texo vaccine, in comparison to homologous HER2-Texo vaccine, stimulates not only potent HER-2-specific antibody responses, which is consistent to previous reports , , but also more potent HER2-specific effector CTL responses, leading to enhanced therapeutic immunity against early stage-established HER2-expressing 4T1HER2 breast cancer in its lung metastasis or s.c. tumor form in wild-type BALB/c mice. The Tg HER2 mice with HER2-specific self-immune tolerance  represent a useful animal model for assessing HER2-specific immunotherapies , . We recently established a double Tg HLA-A2/HER2 mouse model by cross-breeding of Tg HER2 male with Tg HLA-A2 female mice. Thus, the double Tg HLA-A2/HER2 mice mimic HLA-A2+HER-2+ breast cancer patients with HER2-specific self immune tolerance. We demonstrated that HER2-TEXO vaccine stimulated HER2-specific CTL responses and partially induced protective immunity against transgene HLA-A2+HER2+ BL6-10A2/HER2 melanoma in double Tg HLA-A2/HER2 mice . To assess whether heterologous human/rat HER2-specific T cell vaccine better circumvents HER2-specific immune tolerance, we immunized double Tg HLA-A2/HER2 mice with HuRt-Texo, followed by s.c. challenge of immunized mice with HLA-A2- and HER2-expressing BL6-10A2/HER2 tumor cells. We demonstrate that the HuRt-Texo vaccine triggers a strong protective immunity against BL6-10A2/HER2 tumors in all (8/8) tested double Tg HLA-A2/HER2 mice with HER2-specific self-immune tolerance, which is significantly more efficient, in comparison to the protective immunity induced in only three from eight (3/8) mice by the HER2-Texo vaccination . To assess the cytolytic effect of HuRt-TEXO-stimulated CTLs to trastuzumab-resistant breast cancer cells, we performed in vitro cytotoxicity assay, where HuRt-TEXO-stimulated CTLs and transgene HLA-A2-engineered trastuzumab-resistant breast cancer cell line BT474/A2 cells were used as effectors and targets, respectively. To examine the therapeutic effect of HuRt-TEXO-stimulated CTLs against trastuzumab-resistant breast cancer, we performed in vivo immunotherapeutic assay, in which BT474/A2 tumor-bearing athymic nude mice were adoptively transferred with HuRt-TEXO-stimulated CTLs. We found that HER2-specific CTLs derived from HuRt-TEXO-immunized HLA-A2/HER2 mice are not only cytolytic against BT474/A2 tumor cells in vitro, but also eradicate pre-established BT474/A2 tumors in athymic nude mice in vivo, revealing that HuRt-TEXO-stimulated CTLs are therapeutic against trastuzumab-resistant breast cancer. CD8+ CTLs detecting HER2-specific pMHC-I complexes on HER2+ breast cancer cells are critical in protecting high-risk HER2+ breast cancer patients from cancer recurrence . CD4+ helper T (Th) cells recognizing HER2-specific pMHC-II complexes provide important help for CD8+ CTL expansion and memory formation , . HER2-specific MHC-II-restricted Th-epitopes would strengthen CTL response and memory derived from the MHC-I-restrictive HER2 peptide vaccine in HER2+ breast cancer patients . Accumulating evidence also demonstrate that tumor-specific Th cells can also exhibit potent cytolytic effects against pMHC-II+ or pMHC-II- tumor cells , , , . The P30 (FNNFTVSFWLRVPKVSASHLE) sequence of tetanus toxin is a strong heterologous CD4+ T helper epitope  capable of enhancing CTL responses via breaking self-immune tolerance in Tg mouse mammary tumor virus (MMTV)-c-neu and rat insulin promoter-mOVA mice, respectively , . Vaccination with neu/P30 fusion protein completely inhibited neu+ breast cancer development in Tg FVNneuN mice . We previously reported that vaccination of DCs engineered with neu transgene encoding P30 enhanced neu-specific protective immunity against neu-expressing mouse breast cancer Tg1-1 in Tg FVBNeuN mice with neu-specific self-immune tolerance . In this study, we demonstrate that our heterologous human/rat HER2-specific T cell vaccine HuRt-TEXO stimulates stronger CD4+ T cell responses than the HER2-TEXO vaccine. We rationize that the potent CD4+ T cell responses in HuRt-TEXO-vaccinated mice are derived from the presence of heterologous Th epitopes within the COOH-neu481-590 portion of the HuRt-TEXO vaccine. It has been reported that eight HLA-DR-restricted HER2 Th epitopes were identified and clinically applied as a peptide vaccination to patients . Among them, two peptides (HER262-76 and HER2605-619) are within the HER2 extracellular domain, while another six peptides located in the HER2 intracellular domain are mostly deleted in our heterologous human/rat HER2-specific T cell vaccine. We found that only one Th epitope neu605-619 (KPDLSYMPIWKYPDE) is within COOH-neu481-590 region of the HuRt fusion protein, but it contains a single amino acid F616Y mutation compared to HER2605-619 (KPDLSYMPIWKFPDE). Therefore, we suspect that the F616Y mutation in Th epitope may be critical in HuRt-TEXO’s enhanced circumvention of the HER2 tolerance. To test our assumption, construction of new HuRt-TEXO vaccine with F616Y mutation by recombinant DNA technology  followed by experiments designed to examine whether the F616Y mutation contributes to HuRt-TEXO vaccine’s effective circumvention of HER2 tolerance in double Tg HLA-A2/HER2 mice is underway in our laboratory.