Breast cancer cell lines (MCF-7, MDAMB-231 and T47D) and the virus packaging cell line (293T) were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 U/ml streptomycin (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) in an incubator at 37°C and 5% CO₂. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by Ficoll gradient centrifugation with 700 × g for 20 min at 4°C, subsequent to obtaining written informed consent. The present study conformed to The Declaration of Helsinki and was approved by the Ethics Committee of Shenzhen Beike Cell Engineering Research Institute (Guangdong, China). Two male and two female patients participated in present study, with an age range from 25 to 40 years old and the mean age was 31.5 years old. The blood of healthy donors was collected at the Hengze Clinic (Shenzhen, Guangdong, China) on January 7, 2016. Subsequently, the cytotoxic (CD8+) T cells from PBMCs were selected using a negative selection procedure using a CD8+ T cell Isolation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's protocol. These isolated cells were cultured using the T cell Activation/Expansion kit (Miltenyi Biotec GmbH) and RPMI 1640 medium supplemented with 10% human serum from donors of AB blood group (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 400 U/ml interleukin (IL)-2 (Peprotech, Inc., Rocky Hill, NJ, USA) and 400 U/ml IL-15 (Peprotech, Inc., Rocky Hill, NJ, USA) at 37°C and 5% CO₂.

The lentiviral vector pCDH-EF1-MCS-Thosea asigna virus (T2A)-Puro and three packaging vectors, pLP1, pLP2 and pLP-VSVG, were obtained from Addgene, Inc. (Cambridge, MA, USA).

In order to generate cytotoxic T-lymphocytes (CTLs), the monocyte-derived dendritic cells (DC) of an HLA-A2+ donor were loaded with 5 µg/ml PLAC1_28–36-peptide (VLCSIDWFM) (GenScript, Jiangsu, China) for the stimulation of autologous CD8+ T cells. Following two stimulations, the HLA-A2+/PLAC1_28–36-multimer+ CD8+ T cells were sorted by flow cytometry (FACSAria; BD Biosciences, San Jose, CA, USA), and the data was analyzed using BD FACSDiva software (version 6.0; BD Biosciences). Subsequently, the sorted T cells were cloned using a limiting dilution as previously described (17,18) and after 2 weeks, screened for specific recognition of MCF-7 cells (PLAC1+, HLA-A2+) based on specific interferon γ (IFN-γ) secretion (data not shown), which was typically stimulated by antigen encounters, and was ascribed to the major histocompatibility complex (MHC)-dependent TCR activation (19). Then, the PLAC1-reactive clones were expanded using the T cell Activation/Expansion kit and RPMI 1640 supplemented with 10% human serum from donors of AB blood group, 400 U/ml IL-2, and 400 U/ml IL-15 at 37°C and 5% CO₂.

To identify the sequences of the TCR genes, a 5′-RACE-PCR GeneRacer kit (Invitrogen; Thermo Fisher Scientific, Inc.) amplifying the TCRα- and TCRβ-chains was performed using RNA isolated from the T cell clones using an RNA Isolation Kit (cat. no. R6731; Omega Bio-tek, Inc., Norcross, GA, USA) according to the manufacturer's protocol. The RACE-PCR products were sequenced, the TCRα- and TCRβ-chains were linked by a 2A peptide linker (TCRα-T2A-TCRβ) as previously described (17,18), and the whole constructs were codon-optimized and synthesized by GenScript. These synthesized fragments were cloned into the lentiviral vector pCDH-EF1-MCS-T2A-Puro via EcoRI and BamHI restriction sites.

A virus packaging cell line, 293T, was seeded at a density of 1×10⁷ cells/150-mm dish and then incubated at 37°C with 5% CO₂ for 24 h. After 24 h, the cells were subjected to transfection at 37°C for 4 h with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and 20.0 µg pCDH-TCR construct (or pCDH-EF1-MCS-T2A-Puro empty vector) with the pLP1, pLP2, and pLP-VSVG packaging vectors. After culturing the cells for 48 h, the filtered culture supernatants were used for infection. The CD8+ T cells from the PBMCs of healthy donors were transduced using the lentiviral vectors carrying TCR or control empty vector [negative control (NC)] in the presence of 5 µg/ml polybrene (Sigma-Aldrich; Merck KGaA) for 12 h, followed by incubation at 37°C for 48 h in RPMI 1640 supplemented with 10% human serum from donors of AB blood group, 400 U/ml IL-2 and 400 U/ml IL-15. The TCR-transduced CD8⁺ T cells were evaluated for the expression of appropriate TCR by multimer staining at 4°C for 20 min and flow cytometry.

A total of 1×10⁶ T cells were collected by centrifugation with 650 × g for 10 min at room temperature, fixed with paraformaldehyde for 30 min at room temperature, washed twice with PBS, and then blocked with fluorescence-activated cell sorter (FACS) solution (PBS containing 2% FBS and 0.1% NaN₃) for 30 min at 4°C. Then cells were stained using fluorescein isothiocyanate (FITC)-labeled monoclonal antibody (mAb) against human CD8 (cat. no. 555366; dilution, 1:10; BD Biosciences, San Jose, CA, USA), PerCP-Cy5.5-labeled mAb against human CD3 (cat no. 560835; dilution, 1:10; BD Biosciences), and phycoerythrin (PE)-labeled PLAC1-multimer (code, F2A-G; dilution, 1:10; Proimmune, Ltd., Oxford, UK) diluted with FACS solution for 20 min at 4°C. Cancer cells were digested with 0.25% trypsin for 3 min at 37°C, collected by centrifugation at 300 × g for 10 min at room temperature, fixed with paraformaldehyde for 30 min at room temperature and washed twice with PBS. Then, cells were blocked with FACS solution for 30 min at 4°C. HLA-A2 expression in the cultured cancer cells was evaluated by flow cytometry using the PE-labeled HLA-A2 antibody (cat no. 558570; dilution, 1:10; BD Biosciences) and PE-labeled isotype control (cat no. 555743; dilution, 1:10; BD Biosciences) diluted with FACS solution for 20 min at 4°C. Isotype control antibody was used in order to eliminate nonspecific combinations in a previous test (data not shown). The cell was analyzed using flow cytometry (FACS Calibur; BD Biosciences) and the data was analyzed using CellQuest Pro.app software (version 6.0; BD Biosciences).

TCR-, NC-transduced or untransduced CD8+ T cells were serially diluted with RPMI 1640 medium supplemented with 10% FBS and co-cultured with 1×10⁴ target cells including MCF-7, MDA-MB-231 and T47D in 100 µl RPMI 1640 medium supplemented with 10% FBS, resulting in a gradient effector cell to target cell (E:T) ratio (20:1, 10:1 and 5:1, respectively). After 4 h, the culture supernatants were collected, and the level of IFN-γ and tumor necrosis factor α (TNF-α) determined using a commercial enzyme-linked immunosorbent assay kit (cat. no. DIF50 and DTA00C; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol.

For evaluating the efficacy of TCR- or NC-transduced CD8+ T cell-mediated lysis, the cells were serially diluted with RPMI 1640 medium supplemented with 10% FBS and co-cultured with 1×10⁴ target cells including MCF-7, MDA-MB-231 and T47D, resulting in a gradient E:T ratio as described above. After 4 h of co-culture at 37°C, the cytolysis was determined using a lactate dehydrogenase (LDH) activity assay (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's protocol, as previously described (20). The absorbance was measured on a microplate reader at 490 nm (BioTek Instruments, Inc., Winooski, VT, USA). Following background subtraction, the percentage lysis was calculated using the following equation: 100% × [(experimental release-effector spontaneous release-target spontaneous release)/(target maximum release-target spontaneous release)].

Immunofluorescence was performed as described previously (21,22). Briefly, MCF-7, MDA-MB-231 and T47D cells were fixed with 4% paraformaldehyde for 30 min at room temperature and permeabilized using 0.2% Triton X-100. Following blocking with 2% bovine serum albumin for 1 h at 4°C, the cells were incubated with a rabbit anti-PLAC1 antibody (cat. no. ab105395; dilution, 1:100; Abcam, Cambridge, UK) or a rabbit IgG (cat. no. A7016; dilution, 1:100; Beyotime Institute of Biotechnology, Haimen, China) overnight at 4°C. Subsequently, the cells were washed, followed by the addition of PE-conjugated goat anti-rabbit IgG (cat. no. HS121-01; dilution, 1:100; TransGen, Beijing, China) for 1 h at 37°C in the dark, and visualized using an inverted fluorescence microscope (magnification, ×100) (Olympus IX71; Olympus Corporation, Tokyo, Japan).

One pair of siRNA oligonucleotides corresponding to the target sequence for human PLAC1 (CACCTACCGTGTTACTGAA) were designed and synthesized by RiboBio (Guangzhou RiboBio Co., Ltd., Guangzhou, China). A negative-control siRNA (NC siRNA) (Guangzhou RiboBio Co., Ltd.) was used as the control. Cells were plated to achieve 40–60% confluency in RPMI 1640 containing 10% FBS without antibiotics. The siRNA was transfected into MCF-7 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, the cells were analyzed 48 h after transfection, and PLAC1 protein expression was determined using western blot analysis.

Western blot analysis was performed as described previously (23). Briefly, the TCR-transduced or NC-transduced CD8+ T cells, MCF-7 cells and PLAC1 gene silencing- and NC-silencing-MCF-7 cells were lysed using radioimmunoprecipitation assay buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and the protein concentrations measured using a BCA Assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). Equivalent amounts of protein (20 µg) were separated using a 10% gel and SDS-PAGE and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were probed with primary antibodies against phosphoinositide 3-kinase γ (PI3Kγ; cat. no. 5405S; 1:1,000), protein kinase B (AKT; cat. no. 9272S; 1:1,000), phosphorylated (p)-AKT (cat. no. 4060S; 1:1,000), extracellular signal-regulated kinase (ERK; cat. no. 4695S; 1:1,000), p-ERK (cat. no. 4377S; 1:1,000), p38 (cat. no. 8690S; 1:1,000), p-p38 (cat. no. 4631S; 1:1,000), c-Jun N-terminal kinases (JNK; cat. no, 9252S; 1:1,000), p-JNK (cat. no. 4668S; 1:1,000), nuclear factor-κB (NF-κB; cat. no. 4764S; 1:1,000), β-actin (cat. no. 4970L; 1:1,000) and GAPDH (cat. no. 5174S; 1:1,000) (all from Cell Signaling Technology, Inc., Danvers, MA, USA), and PLAC1 (cat. no. ab105395; 1:1,000; Abcam, Cambridge, UK) in TBS-Tween-20 (TBST) containing 5% non-fat milk (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 4°C overnight, followed by three washes in TBST for 10 min per wash. Subsequently, the membranes were incubated with the IRDye 800CW goat anti-rabbit secondary antibody (1:5,000 dilution in TBST containing 5% non-fat milk; cat. no. 926-32211; LI-COR Bioscience; Lincoln, Nebraska, USA) for 2 h at room temperature. The protein bands were visualized using an Infrared Imaging System Odyssey (LI-COR Bioscience; Lincoln, Nebraska, USA). Quantity One software (version 4.62; Bio-Rad Laboratories, Inc., Hercules, California, USA) was used to quantify of the western blots.

A total of 15 subcutaneous transplant breast cancer model female nude mice (BALB/c-nu) (4–5-week-old) weighing between 18 and 20 g, were purchased directly from the Medical Laboratory Animal Center of Guangdong Province (Guangdong, China). The mice were housed under controlled 12 h light-dark cycles, with constant temperature (22–24°C) and humidity (55–60%), and were given sterilized food and tap water ad libitum. Briefly, the mice were pre-treated with 200 µg/kg estradiol valerate (Sigma-Aldrich; Merck KGaA) by gastric perfusion, starting 1 week prior to cell implantation as previously described (24). Subsequently, each of the mice were administered ~5×10⁶ MCF-7 cells (in 100 µl serum-free media) mixed with 100 µl Matrigel (BD Biosciences) into the left flank subcutaneously. Protocols for the treatment of nude mice were approved by the Laboratory Animal Ethics Committee of the Medical Laboratory Animal Center of Guangdong Province. When large tumors reached a mean volume of 150 mm³, the mice were randomly divided into 3 groups (5 animals/group) and subjected to intravenous tail vein transplantation with normal saline, 1×10⁷ TCR-, or NC-transduced CD8+ T cells twice with a 1 week interval. The tumors were measured once a week using electronic calipers. The longest length and width were recorded and the tumor volume was calculated according to the formula π/6 [(length × (width)²]. The mice were anesthetized prior to cervical dislocation using 45 mg/kg pentobarbital sodium at the culmination of the experiment.

Data were expressed as the mean ± standard deviation. SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used for analysis. All experiments (excluding the nude mice subcutaneous transplant tumor model experiment) were repeated at least 3 times. A one-way analysis of variance with Bonferroni correction was used for analysis. P<0.05 was considered to indicate a statistically significant difference.

cDNAs encoding TCRα- and β-chains specific for HLA-A2-restricted PLAC1 were cloned from a CTL generated by the in vitro stimulation of CD8+ T cells using a HLA-A2+ DC loaded with PLAC1_28–36-peptide (VLCSIDWFM). The TCRα- and TCRβ-chains were linked by a 2A peptide linker (TCRα-T2A-TCRβ), and the whole constructs were codon-optimized and synthesized. These synthesized fragments were then cloned into the lentiviral vector pCDH-EF1-MCS-T2A-Puro via EcoRI and BamHI restriction sites (Fig. 1A). To further characterize the TCR, the lentiviral vectors carrying TCR or NC were used for the transduction of the CD8+ T cells, isolated from PBMCs (99.1% purity), with a negative selection procedure using magnetic beads (Fig. 1B). The results revealed that the accurately expressing and matching efficiency of TCRα- and TCRβ-chains in CD8+ T cells was up to 25.16%, as detected by FITC-labeled CD8 mAb and PE-labeled PLAC1-multimers (Fig. 1C). These HLA-A*0201-restricted and PLAC1-specific TCR-engineered CD8+ T cells were then used for subsequent experiments.

Human non-metastatic breast cancer cells (MCF-7) and TNBC cells (MDAMB-231) were examined for the baseline expression of PLAC1 by immunofluorescence. As presented in Fig. 2A, the two cell lines expressed PLAC1. Next, these two different cancer cell lines were serotyped with an HLA-A2 antibody by flow cytometry and were revealed to be HLA-A2 positive. The HLA-A2 expression efficiency in MCF-7 and MDAMB-231 cell lines reached 92.07 and 98.63%, respectively (Fig. 2B), thereby resulting in the selection of these 2 cell lines for further studies.

In order to evaluate the recognition of PLAC1 TCRs, transduced CD8+ T cells were subjected to a co-culture assay with MCF-7 (PLAC1+, HLA-A2+) and MDAMB-231 (PLAC1+, HLA-A2+) cells. A significantly higher release of IFN-γ and TNF-α was observed in TCR-engineered CD8+ T cells co-cultured with HLA-A2+/PLAC1+ MCF-7 and MDAMB-231 cell lines compared with NC-transduced and untransduced CD8+ T cells co-cultured with these two cell lines (P<0.01; Fig. 3A and B). As NC-transduced and untransduced CD8+ T cells could not mediate the release of effector cytokines when co-cultured with HLA-A2+/PLAC1+ MCF-7 and MDAMB-231 cell lines, only the NC-transduced CD8+T cells were selected as the control in subsequent studies.

Subsequently, the specific lysis of breast cancer cell lines by the engineered CD8+ T cells was measured using an LDH assay. PLAC1 TCR-transduced CD8+ T cells demonstrated an significantly increased lytic function against HLA-A2+/PLAC1+ MCF-7 and MDAMB-231 cells compared with NC-transduced CD8+ T cells, which demonstrated little reactivity against any of the target cells (P<0.05; Fig. 3C). PLAC1 TCR-transduced CD8+ T cells were not capable of lysing T47D cells, which were PLAC1-positive, however, this cell line was identified to be HLA-A2 negative in the immunofluorescence and flow cytometry assays (Fig. 4A-C). As PLAC1 is generally expressed in the breast cancer cells, it is difficult to identify a PLAC1-negative cell line. Therefore, the PLAC1 gene was silenced using siRNA in MCF-7 cells, to further detect the function of PLAC1 TCR-engineered CD8+ T cells. The results revealed that the significant PLAC1 gene silencing in MCF-7 cells compared with NC MCF-7 cells (P<0.01; Fig. 4D) resulted in the significant decrease in the specific lysis of breast cancer cells by the engineered CD8+ T cells compared with NC cells (P<0.05; Fig. 4E). These results indicated that the PLAC1 TCR-engineered CD8+ T cells may efficiently and specifically recognize and kill breast cancer cells.

A previous study demonstrated that PI3Kγ kinase activity is essential for optimal T cell activation and differentiation, in addition to an efficient T cell-mediated immune response (25). Upon TCR engagement, multiple signaling mechanisms, including mitogen-activated protein kinase (MAPK) and NF-κB, modulated by the PI3K/AKT signaling pathway, are activated during the process of T cell activation, which results in gene induction and cell cycle progression (26–29). In order to investigate the mechanism of the enhanced activation of TCR-transduced CD8+ T cells, western blot analysis was performed and demonstrated that the expression of PI3Kγ, and the phosphorylation of AKT (p-AKT) and ERK1/2 (p-ERK) in addition to the expression of NF-κB, but not the expression of JNK, p-JNK, P-38 and p-p38 in TCR-transduced CD8+ T cells were enhanced as compared with the NC-transduced CD8+ T cells (Fig. 5). These results suggested that the PI3K pathway possessed the capacity to develop the T cells in order to respond to the engineered TCR stimulation.

To examine the in vivo efficacy of PLAC1 TCR-transgenic CD8+ T cells, 5×10⁶ MCF-7 cells were inoculated into BALB/c-nu mice to establish a subcutaneous transplant tumor model. Upon achieving a mean tumor volume of 150 mm³, the mice were randomly divided into 3 groups (5 animals/group) and subjected to intravenous tail vein transplantation of either normal saline, 1×10⁷ PLAC1 TCR-transduced CD8+ T cells or NC-transduced CD8+ T cells, twice with a 1 week interval. Slowing of tumor growth was observed 14 days post-injection. At day 28, the tumors in the PLAC1 TCR-transduced cells group were significantly smaller, ~50% smaller compared with the normal saline or NC-transduced cells group (P<0.05; Fig. 6). These results indicated that TCR-transduced CD8+ T cells inhibited the tumor growth in vivo.