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01.12.2017 | Letter to the Editor | Ausgabe 1/2017 Open Access

Journal of Hematology & Oncology 1/2017

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

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2017
Autoren:
Sheng Chen, Feifei Gu, Kang Li, Kai Zhang, Yangyang Liu, Jinyan Liang, Wei Gao, Gang Wu, Li Liu
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13045-017-0429-8) contains supplementary material, which is available to authorized users.
Abbreviations
CCR7
C-C chemokine receptor type 7
CD45
Cluster of differentiation 45
CTLs
Cytotoxic T lymphocytes
DCs
Dendritic cells
Her-2/neu
Human epidermal growth factor receptor 2
HIV
Human immunodeficiency virus
HLA
Human leukocyte antigen
IL-2
Interleukin-2
MHC
Major histocompatibility complex
PBMC
Peripheral blood mononuclear cell
TCR
T cell receptor
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 [ 15]. 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 CEM R.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).
For the expansion, starting from 1 × 10 5 total CD8 + T cells that were less than 0.05% Her-2/neu-specific, nearly 10 7 HER-2/neu (369–377) or HER-2/neu (435–443)-specific CD8 + T cells were expanded out in 8 weeks with the purity of around 85% (Fig.  1a, b). The expanded specific CD8 + T cells got massive co-stimulatory molecules and activation makers (Fig.  1c). The three controls got negative results. That was expected due to extremely low frequency of naturally occurring HIV gag (77–85) and insulin B chain (34–42)-specific naive CD8 + T cells as indicated in Additional file 3: Figure S2B and low affinity HER-2/neu (39–47) peptides as showed in Additional file 1: Table S1. We found these expanded HER-2/neu-specific CD8 + T cells were mainly effector and effector memory cells (Additional file 4: Figure S3).
The expanded Her-2/neu-specific CTL populations mediated dose-dependent lysis to HLA-A2-positive and Her-2/neu-overexpressed breast cancer cell line SK.BR.3, but not to the three control targets (Fig.  2a). The lysis attributed to over 50% perforin producing CTLs and more than 80% granzyme B producing CTLs (Fig.  2b). The cytotoxic activity against SK.BR.3 could significantly be eliminated by W6/32, but not by IgG2 (shown in Fig.  2c).
A major finding of this study was that the Her-2/neu-loaded T2 cells could expand out nearly 10 7 Her-2/neu-specific CTLs in 8 weeks. The expanding efficiency is equivalent to DCs previously reported by Marzocchetti et al. [ 68]. 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 [ 9]. 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.

Acknowledgements

Not applicable.

Funding

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/​EpitopePredictio​n.​htm.

Authors’ contributions

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.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

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.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://​creativecommons.​org/​licenses/​by/​4.​0/​), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated.

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