A hybrid of B and T lymphoblastic cell line could potentially substitute dendritic cells to efficiently expand out Her-2/neu-specific cytotoxic T lymphocytes from advanced breast cancer patients in vitro
- Sheng Chen†1,
- Feifei Gu†1,
- Kang Li1,
- Kai Zhang1,
- Yangyang Liu1,
- Jinyan Liang1,
- Wei Gao2,
- Gang Wu1Email author and
- Li Liu1Email author
© The Author(s). 2017
Received: 7 January 2017
Accepted: 23 February 2017
Published: 28 February 2017
Adoptive transfer of cytotoxic T lymphocytes (CTLs) holds promises to cure cancer. However, this treatment is hindered by lacking a robust way to specifically expand out CTLs. Here, we developed a hybrid of B lymphoblastic cell line and T lymphoblastic cell line (T2 cells) as a substitute of dendritic cells, together with irradiated autologous peripheral blood mononuclear cell (PBMC) as feeder cells and rhIL-2, to activate and expand Her-2/neu-specific CD8+ T cells from human epidermal growth factor receptor 2 (Her-2/neu) and human leukocyte antigen (HLA)-A2 double positive advanced breast cancer patients in vitro. These Her-2/neu-loaded T2 cells reproducibly activated and expanded out Her-2/neu-specific CD8+ T cells to 107 in 8 weeks. Furthermore, these Her-2/neu-specific CD8+ T cells had good sensitivity of recognition and killing Her-2/neu-overexpressed breast cancer cell line SK.BR.3. This technique gives us another insight on how to rapidly obtain sufficient CTLs for adoptive cancer immunotherapy.
KeywordsHybrid of B and T lymphoblastic cell line Adoptive cancer immunotherapy Cytotoxic T lymphocytes Breast cancer
To the editor
The current standard way to expand specific cytotoxic T lymphocytes (CTLs) replies obtaining sufficient dendritic cells (DCs) from patients (Additional file 1). This method has several defects such as invasive, time-consuming, expansive, and unstable according to patients’ physical conditions [1–5]. Our group found that the T2 cells, which are a cloned hybrid between the 721.174 (variant of the B lymphoblastic cell line LCL 721) and CEMR.3 (variant of T lymphoblastic cell line CEM-C7), potentially conform to the demands. The cells are TAP and MHC class II deficient, but they do express HLA-A2 and massive co-stimulatory molecules (Additional file 2: Figure S1). These characters make T2 cells potentially useful to study CD8+ T cell recognition of MHC class I antigens, meanwhile, convenient to exclude from MHC class II antigen intervention. Subsequently, HER-2/neu(369–377) and HER-2/neu(435–443) which scores more than 20 by SYFPEITHI prediction was selected to load to T2 cells to expand Her-2/neu-specific CD8+ T cells. HIV gag(77–85), insulin B chain(34–42), and HER-2/neu(39–47) which scores minus 3 were performed as a control (see Additional file 1: Table S1). All peptides except low-affinity peptide HER-2/neu(39–47) could stabilize HLA-A2 molecules on the cell membrane obviously (Additional file 3: Figure S2A).
After co-culture with CD8+ T cells from Her-2/neu and HLA-A2 double positive advanced breast cancer patients, HER-2/neu(369–377)- and HER-2/neu(435–443)-loaded T2 cells lead to a large secretion of IFN-γ, but not the three controls (Additional file 3: Figure S2B).
A major finding of this study was that the Her-2/neu-loaded T2 cells could expand out nearly 107 Her-2/neu-specific CTLs in 8 weeks. The expanding efficiency is equivalent to DCs previously reported by Marzocchetti et al. [6–8]. And because the expansion was started from CD8+ T cells which were only from 5 ml blood, with an initial frequency of Her-2/neu-specific CD8+ T cells at 0.05%, it would be possible to amplify the expanding quantity if we isolate the Her-2/neu-specific CD8+ T cells firstly from more blood, 50 ml or more for example.
We found the expanded HER-2/neu-specific CTLs could recognize endogenous antigen on allogeneic breast cancer cell line SK.BR.3. This is critically important because previously, many expanding techniques lead to CTLs that could only kill targets pulsed with related peptides but not targets that endogenously processed the antigen of interest . Yee et al. [10, 11] found low affinity of induced CTLs leads to failure of recognition of endogenous antigen.
Thus, T2 cells have shown promise as a convenient tool to rapidly expand out Her-2/neu-specific CTLs in vitro. But so far, we are not able to transfer the expanded Her-2/neu-specific CTLs to breast cancer patients directly as they are mixed with T2 cells, and for the same reason, we cannot compare its anti-tumor effect with trastuzumab in breast cancer patients either. This technique might accelerate to study expanding CTLs in vitro and promote the development of safe and effective adoptive cancer immunotherapy in the future.
C-C chemokine receptor type 7
Cluster of differentiation 45
Cytotoxic T lymphocytes
Human epidermal growth factor receptor 2
Human immunodeficiency virus
Human leukocyte antigen
Major histocompatibility complex
Peripheral blood mononuclear cell
T cell receptor
This work was supported by the National Natural Sciences Foundation of China (NO: 81000911, 81372260, 81300586).
Availability of data and materials
The dataset supporting the conclusions of this article is available in the SYFPEITHI repository, http://www.syfpeithi.de/bin/MHCServer.dll/EpitopePrediction.htm.
SC, LL, and GW conceived and designed the study. SC wrote the manuscript. KL and FG performed the experiments. FG collected and assembled the data. WG performed the statistical analysis. LL reviewed the manuscript. All authors approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The study was reviewed and approved in 2010 by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. All subjects were performed in accordance with the medical experiment guidelines of Huazhong University of Science and Technology, which abides by the Helsinki Declaration on ethical principles for medical research involving human subjects. Written informed consent to participate under the ethics, consent, and permissions was obtained from all ten patients.
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- Teague RM, Sather BD, Sacks JA, Huang MZ, Dossett ML, Morimoto J, Tan X, Sutton SE, Cooke MP, Ohlén C, Greenberg PD. Interleukin-15 rescues tolerant CD8+ T cells for use in adoptive immunotherapy of established tumors. Nat Med. 2006;12(3):335–41.View ArticlePubMedGoogle Scholar
- Disis ML, Bernhard H, Jaffee EM. Use of tumour-responsive T cells as cancer treatment. Lancet. 2009;373(9664):673–83.View ArticlePubMedPubMed CentralGoogle Scholar
- Vignard V, Lemercier B, Lim A, Pandolfino MC, Guilloux Y, Khammari A, Rabu C, Echasserieau K, Lang F, Gougeon ML, Dreno B, Jotereau F, Labarriere N. Adoptive transfer of tumor-reactive Melan-A-specific CTL clones in melanoma patients is followed by increased frequencies of additional Melan-A-specific T cells. J Immunol. 2005;175(7):4797–805.View ArticlePubMedGoogle Scholar
- Yee C, Thompson JA, Byrd D, Riddell SR, Roche P, Celis E, Greenberg PD. Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci U S A. 2002;99(25):16168–73.View ArticlePubMedPubMed CentralGoogle Scholar
- Almand B, Resser JR, Lindman B, Nadaf S, Clark JI, Kwon ED, Carbone DP, Gabrilovich DI. Clinical significance of defective dendritic cell differentiation in cancer. Clin Cancer Res. 2000;6(5):1755–66.PubMedGoogle Scholar
- Marzocchetti A, Lima M, Tompkins T, Kavanagh DG, Gandhi RT, O'Neill DW, Bhardwaj N, Koralnik IJ. Efficient in vitro expansion of JC virus-specific CD8(+) T-cell responses by JCV peptide-stimulated dendritic cells from patients with progressive multifocal leukoencephalopathy. Virology. 2009;383(2):173–7.View ArticlePubMedGoogle Scholar
- Tuettenberg A, Jonuleit H, Tüting T, Brück J, Knop J, Enk AH. Priming of T cells with Ad-transduced DC followed by expansion with peptide-pulsed DC significantly enhances the induction of tumor-specific CD8+ T cells: implications for an efficient vaccination strategy. Gene Ther. 2003;10(3):243–50.View ArticlePubMedGoogle Scholar
- Wolfraim LA, Takahara M, Viley AM, Shivakumar R, Nieda M, Maekawa R, Liu LN, Peshwa MV. Clinical scale electroloading of mature dendritic cells with melanoma whole tumor cell lysate is superior to conventional lysate co-incubation in triggering robust in vitro expansion of functional antigen-specific CTL. Int Immunopharmacol. 2013;15(3):488–97.View ArticlePubMedGoogle Scholar
- Heiser A, Coleman D, Dannull J, Yancey D, Maurice MA, Lallas CD, Dahm P, Niedzwiecki D, Gilboa E, Vieweg J. Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest. 2002;109(3):409–17.View ArticlePubMedPubMed CentralGoogle Scholar
- Yee C, Savage PA, Lee PP, Davis MM, Greenberg PD. Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers. J Immunol. 1999;162(4):2227–34.PubMedGoogle Scholar
- Gritzapis AD, Mahaira LG, Perez SA, Cacoullos NT, Papamichail M, Baxevanis CN. Vaccination with human HER-2/neu (435-443) CTL peptide induces effective antitumor immunity against HER-2/neu-expressing tumor cells in vivo. Cancer Res. 2006;66(10):5452–60.View ArticlePubMedGoogle Scholar