Cell lines and culture media
CaSki, SiHa, U87, T98G and T2 cell lines were purchased from ATCC. Cell lines were cultured in RPMI-1640 (CaSki, SiHa and T2), or DMEM (293GP, U87 and T98G) supplemented with 10% (v/v) FBS (Lonsera), GlutaMAX (Life Technologies), 100 U/ml penicillin (Life Technologies) and 100 µg/ml streptomycin (Life Technologies). U87-pp65-EGFP, and T98-pp65-EGFP are U87 or T98-based cell lines with stable expression of pp65-GFP. These cell lines were generated by transduction of retroviral vectors encoding target proteins, and a subsequent cell sorting of GFP+ cells with flow cytometry. All cell lines that used in the experiments tested negative for mycoplasma. The culture medium for T cell in vitro peptide sensitization was X-VIVO™ 15 (Lonza) with 5% (v/v) human AB serum and 50 U/ml IL-2 (Peprotech). The medium used for quick expansion of T cell was X-VIVO™ 15 with 10% (v/v) FBS serum and 300 U/ml IL-2 and 50 ng/mL IL-15 (Peprotech).
Generation of virus-specific T cell clones
PBMC were obtained by leukapheresis, prepared with density gradient centrifugation (human T lymphocyte separation medium from Dakewe Biotech), and cryopreserved. For in vitro sensitization, CD8+ T cells were first isolated from human PBMC using Dynabeads™ CD8 positive isolation kit (Life Technologies), and then 3 × 105 CD8+ T cells were stimulated with irradiated, viral peptide-loaded autologous PBMC (1 × 106, CD8+ T cell-depleted PBMC) in each well of a 24-well plate. Cultures were maintained individually for 7–10 days and replaced with fresh medium every 2 days. The reactivities of cells were tested by the IFN-γ release assay against T2 cells pulsed with cognate viral peptides or irrelevant peptides. T cell cultures with pronounced and specific recognition of the target epitope (OD450 ≥ 1 after subtracting control signal) were subjected to the limiting dilution cloning (3 cells/well). Specifically, T cells were stimulated with 2 × 106 irradiated allogenic PBMC from three healthy donors and 30 ng/mL anti-CD3 antibody (OKT3) in the quick expansion medium. After 12–14 days of culture, plates were screened by ELISA again to isolate virus-specific T cells with high positive signal. The quick expansion protocol was used to expand positive T cells in T25 flasks . After tetramer-guided selection, 1–2 × 106 T cells were used to isolate RNA (RNeasy plus universal mini kit, Qiagen) and were sent to GENEWIZ (Nanjing, China) for ImmunoSEQ.
Construction of TCR retroviral vector and T cell transduction
TCR nucleotide sequences were synthesized and cloned into the MSGV1-1D3-28Z.1–3 mut (addgene) retroviral vector. The human TCR constant regions were replaced with murine TCR constant regions, and a furin P2A linker was used to connect the TCR chains in the α-β order. Retroviral vectors encoding the target TCRs were first generated by transfecting 293GP packaging cells with pMSGV1-TCR and VSV-G plasmids. Viral vector-containing supernatant were harvested 48 and 72 h post-transfection. PG13-TCR producer cells were established by two consecutive transductions of PG13 cells with harvested viral supernatant. Human PBMC from healthy donors were isolated as mentioned above. CD3+ T cells were isolated from PBMCs with Dynabeads™ untouched™ human T cells kit (Life Technologies) and stimulated for 2 days with Dynabeads human T-activator CD3/CD28 (Life Technologies) at a bead to cell ratio of 1:1. Retroviral vectors that produced from PG13-TCR cells were preloaded into RetroNectin-coated 6-well plates (Takara), and activated T cells were transduced with two cycles of spinoculation.
The following fluorescently conjugated antibodies were purchased from BD Biosciences: CD3-BV605/APC/PE (SK7), CD8-APC-H7/FITC (HIT8a), CD4-PE/BV786/APC (SK3), murine TCRβ constant region (mTRBC)-PE-Cy7. Tetramers (HLA-A*02:01-HPV16-E711–19, HLA-A*02:01-EBV-LMP2356–364, HLA-A*02:01-EBV-LMP2426–434 and HLA-A*02:01-CMVpp65495–503) were purchased from MBL International. Data were acquired with a BD FACSAria™ II flow cytometer (BD Biosciences) and analyzed with FlowJo software (FlowJo).
T cell in vitro functional assays
For cytokine production assays, 1 × 105 T cells were cocultured with 5 × 104 target cells in 96-well U-bottom plates at 37 °C. For peptide titration experiments, T2 cells were pulsed with different concentrations of peptides overnight. After washing, 5 × 104 peptide-pulsed T2 cells were cocultured with 1 × 105 T cells as mentioned above. Coculture supernatants were harvested after 16–18 h, and IFN-γ concentration was measured by ELISA (Thermo Scientific). Cytotoxicity of T cells was determined by lactate dehydrogenase (LDH) release assays. T cells were cocultured with target cells at the indicated effector-to-target ratios, and LDH in coculture media were quantified according to the manufacturers’ instructions (Pierce LDH cytotoxicity assay kit, Thermo Scientific).
Treatment of cervical cancer in a xenograft murine model
Animal research protocols were approved by the Animal Experiment Ethics Committee of Shenzhen Second People's Hospital, Shenzhen, Guangdong Province, China. Female (4–6 weeks old) NCG mice (NOD-Prkdcem26Il2rgem26/Nju) were purchased from GemPharmatech (Jiangsu, China). Tumors were initiated by a subcutaneous injection of 2 × 106 CaSki-luc cancer cells on the flank of mice. Twenty-one-days following tumor initiation, T cells (E7 specific TCR-T cells or untransduced T cells were administered by tail vein injection. Mice received adjuvant IL-2 (Jiangsu Kingsley Pharmaceutical Co., Ltd.) 50,000 IU by intraperitoneal injection daily for 3 days beginning immediately after T cell infusion. Tumor load was measured by bioluminescence imaging. Isoflurane-anesthetized animals were imaged using the IVIS system (Xenogen Corp.) 10 min after intraperitoneal injection of 150 mg/kg VivoGlo™ luciferin (Promega). Mice were regularly examined for weight loss, or signs of stress, and euthanized by cervical dislocation according to the preset criteria. At the end of the experiment, Heparinized blood samples were collected by intracardiac puncture under general anesthesia (a single dose of intraperitoneal injection of 250 mg/kg ketamine and 10 mg/kg xylazine). After that, mice were immediately euthanized by cervical dislocation, and tumors and tissues were collected.
Statistical analyses were performed with Prism 5 (GraphPad Software). A two-tailed unpaired t test was used to compare the differences between G8 TCR-T cell group and control groups. A P value of less than 0.05 was considered significant.