Tumor sample from a 63-year-old woman with metastasis ESCC from Peking University Cancer Hospital was obtained in our preclinical research with informed consent. The tumor sample was collected in tubes containing sterile saline solution and resected into several fragments for a) TIL culture; b) generation of patient-derived xenograft (PDX) model. This study was approved by the Institutional Review Board of the Peking University School of Oncology, China.
Cell lines and monoclonal antibodies
HEK 293-FT (Life Technologies), a packaging cell line used to produce high-titer lentivirus supernatants, were cultured in complete Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum(FBS, Gibco, USA) containing 0.1 mM MEM Non-Essential Amino Acids, 1 mM sodium pyruvate and 2 mM Glutamax (Life Technologies, USA) at 37 °C with 5% CO2.
Flow cytometry staining antibodies were shown as below: CD3-BUV395(Clone: UCHT1), CD4-PE-CF594(Clone: RPA-T4), CD8-PE-Cy7(Clone: RPA-T8), PD-1-BUV737(Clone: EH12.1), CCR7-PE(Clone: 150503), CD45RA-APC(Clone: HI100), CD137-APC(Clone: 4B4–1), CD107A-AF647(Clone: H4A3), CD107b-AF647(Clone: H4B3), Fixable Viability Stain 780 (FVS780). All antibodies were from BD Biosciences, except for anti-mouse TCR-β constant region (clone H5–597, eBioscience, USA). The blocking antibody against HLA class I were from eBioscience (clone: W6/32).
HLA typing for the patient and donors
DNA of autologous tumor cells or the patient or donors’ peripheral blood were exstracted with DNeasy Blood & Tissue Kit (QIAGEN, Germany) according to the manufacturer’s protocol. Genotyping of HLA alleles was performed using high-resolution, high-throughput HLA genotyping with deep sequencing (BGI Diagnosis, Shenzhen, China). The HLA types of the patient and donors were outlined in Additional file 2: Table S1.
Generation of TILs, PDX models and autologous tumor cells
TILs were generated as previous described [14, 15] with slightly modification. Briefly, tumor sample was minced into approximately 1–2 mm fragments and each fragment was placed into a well of a 24-well plate comprised of T cell media and 50 ng/mL OKT3 antibody (ACRO, USA). T cell media consisted of X-VIVO 15 (Lonza, USA); Glutamax (Life Technologies, USA); IL-2 (50 U/mL, Perprotech, USA), IL-7 (10 ng/mL, Perprotech, USA); IL-15(10 ng/mL, Perprotech, USA). T cells were incubated at 37 °C with 5% CO2, and passaged to maintain a density of 1 × 106 cells/mL until there were enough TILs used for screening of tumor-specific T cells.
PDX model was established by implanting tumor fragments mixed with matrigel subcutaneously into immunodeficient NOD-SCID mice to generate PDX model. ESCC PDX models could be promising for individual therapy, since we have verified that the molecular characteristics of ESCC PDXs were in accordance with primary patient tumors in our previous study .
Autologous tumor cells were generated from tumor specimens based on successfully established PDX model . Dissected tumor samples were dissociated into single-cell suspension, using the human Tumor Dissociation Kit (Miltenyi Biotech, Germany) with gentleMACS Octo Dissociator with Heaters (Miltenyi Biotech, Germany) according to the manufacturer’s instructions. Single-cell suspensions were harvested, washed with 1 × phosphate-buffered saline (PBS), and then resuspended in 6-well plate with complete media (DMEM, 20% FBS, 2 mM Glutamax) at 37 °C with 5% CO2.
TILs phenotypic characterization by flow cytometry
Single-cell pellets of TILs were stained with CD3-BUV395, CD4-PE-CF594, CD8-PE-Cy7, CCR7-PE, CD45RA-APC, PD-1-BUV737 antibody cocktails. Cells were washed with PBS prior to acquisition on BD FACS Aria III flow cytometer. Data files were analyzed using FlowJo vX software (FlowJo, Tree Star), gated on single cells. Dead cells and debris were excluded by staining with Fixable Viability Stain 780 (FVS780). Phenotypes of T cells were gated on total CD3+ T cells.
Initial screening of TILs for recognition of autologous tumor cells
Autologous tumor cells were pretreated with complete media containing DAC cocktails (DAC (10 μM, Sigma Aldrich, USA), IFNγ (100 U/mL, ACRO, USA) and TNF-α(10 ng/mL, ACRO,USA) for 48 h, which restored cell surface expression of HLA molecules through enhancing the mRNA expression of HLA-related molecular including TAP or LMP genes or inhibiting DNA methylation [18,19,20]. Both IFN-γ enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assays (ELISA) were used to screen tumor-reactive TILs cocultured with pretreated ATCs.
IFN-γ ELISPOT assay
Human IFN-γ ELISPOT Kit (with precoated plates, Abcam, USA) was performed as the protocol’s procedure. Briefly, 2 × 104 T cells, rested in cytokines-free media overnight, and 1 × 104 PBS-washed autologous tumor cells were incubated together for approximately 20 h in the absence of exogenous cytokines at 37 °C with 5% CO2. The number of colored spots was determined by ImmunoSpot plate reader and associated software (Celluar Technologies, USA).
IFN-γ ELISA assay
1 × 106 responder cells (T cells) and 1 × 105 target cells (ATCs) were incubated together in a 96-well plate in the absence of exogenous cytokines for 18-24 h. Coculture supernatant was transferred into a new 96-well plate, and IFN-γ concentration was measured by using commercially available human IFN-γ ELISA kit (ExCell Bio, China) as manufacturer’s protocols. In HLA blocking experiment, ATCs were pretreated with HLA-blocking antibody (clone W6/32, 50 μg/mL). Following a 3.5-h incubation, 5 × 104 target cells (ATCs) and 1 × 105 responder cells (T cells) were cocultured overnight for assessment of IFN-γ releasing level with the standard IFN-γ ELISA procedure .
CFSE-based cytotoxity assay was performed as previously described [22, 23] with slightly alteration. Target cells were labeled with 5 μM CFSE (BD Biosciences) for 10 min and then cocultured with TCR-Ts at 37 °C for 4 h, at E: T ratio of 1:1 and 4:1. After the coculture, 1 μg/mL propidium iodide (PI, BD Biosciences) was added for assigning the ratio of cell death, and the samples were analyzed by flow cytometry.
Enrichment and isolation of tumor-reactive TILs after repeated stimulation with autologous tumor cells
For enrichment of tumor-reactive TILs, 2 × 106 TIL-F1 and TIL-F4 were stimulated with 2 × 105 autologous tumor cells pretreated with DAC cocktails in T-cell media for 1 week, respectively, after which they were stimulated with autologous tumor cells one more time. Moreover, both pre- and post-stimulated TILs were cocultured with 1 × 105 autologous tumor cells (E: T = 5:1) to evaluate their ability of specifically identifying and killing autologous tumor cells using flow cytometry. Data were analyzed using FlowJo vX software (Treestar Inc) after gating on live cells (FVS780 negative). Meanwhile, CD8+CD137+ T cells of pre- and post-stimulated TIL-F1 were sorted into 96-well PCR plates by single-cell sorting (Additional file 1: Figure S3). And then 96-well PCR plates were immediately put into liquid nitrogen and conserved in minus 80°C prior to running single-cell RT-PCR.
T cell receptor sequencing and analysis
For single-cell PCR, all the primer sequences were listed in Additional file 2: Table S2 as previously described  except that the primers of TRBC were optimized, the final concentration of each Vα and Vβ region primers was 5 μM, and final concentration of TRAC and TRBC primers was 20 μM. The single-cell RT-PCR reaction condition for first-step RT-PCR was as follows: 30 min at 50 °C for RT reaction; 95°Cfor 15 min and 30 cycles of 94 °C for 30s, 52 °C for 30s, 72°C for 1 min; 72°C for 10 min. For the second cycle, 2 μL of cDNA product was used as template for TCRα/β separately in total 20 μL 2nd-PCR TRA/TRB mix (Additional file 2: Table S3), containing multiple internal primer sequences (INT) of Vα/Vβ and one primer for Cα/Cβ, with PrimeSTAR® HS DNA Polymerase (Takara Bio, Japan). The cycling program was 98 °C for 1 min; 98 °C for 10s,52°C for 10s,72°C for 45 s × 43;72 °C for 10 min.The second PCR products were analyzed with TRA sequencing primer for TCRα or TRB sequencing primer for TCRβ (Additional file 2: Table S3). PCR products were purified and sequenced by Sanger sequencing method. The TCR sequences were analyzed using IMGT/V-Quest tool (http://www.imgt.org/).
Construction of lentivirus vectors and transduction of PBLs
TCRα/β chains were synthesized (GenScript) and cloned into the lentivirus vector. TCR was constructed in a β/α chain order and its constant regions were replaced by mouse counterparts modified with interchain disulfide bond and hydrophobic substitution as previously described, which not only was convenient for detection of TCR-T, but also improved TCR pairing and TCR/CD3 stability [17, 25]. However, since murine constant region of TCR-T could potentially be immunogenic in clinical application, human constant region could be essential for construction of TCR in order to improve the longevity of TCR-T persistence and enhance their therapeutic efficacy in patients.
Transduction of PBLs was conducted as previously delineated [17, 21, 26] with slightly modification. In brief, human peripheral blood mononuclear cells (PBMCs) were separated by centrifugation on a Ficoll-Paque Plus (GE Healthcare, USA) and then stimulated in T-cell media with 50 ng/ml OKT3 and 1 μg/ml anti-CD28 for 2 days before transduction. TCR lentivirus were generated by cotransfection of 293-FT cells with lentivector and packaging plasmids (ratio of pLP1: pLP2:pVSV-G is 2:2:1) using PEI MAX 40000 (Polysciences Inc. USA) . The lentiviral supernatants were harvested at 48 and 72 h after transfection and concentrated using optimized ultracentrifugation approaches with 20,000 g, 90 min at 4 °C . Activated T cells were transduced by concentrated lentivirus at the presence of 8 μg/mL polybrene (Sigma-Aldrich, USA). The transduction efficiency was assessed by flow cytometry using mouse TCR-β chain constant region staining.
Treatment of PDX models by TCR-Ts
NOD/SCID mice were used to establish patient-derived xenograft approved by the Institutional Review Board of the Peking University School of Oncology, China. Tumor treatment was on days 5 and 12 following tumor inoculation and consisted of two intravenous injections of 4 × 106 T cells as well as a single intraperitoneal injection of 5 mg/kg DAC on day 5. Tumor size was determined by caliper measurement of perpendicular diameters of each tumor and was calculated using the following formula: tumor volume (mm3) = [(length) × (width) × (width)]/2.
Statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software, CA) and SPSS software (version 24; IBM SPSS, Armonk, NY, USA). Statistical comparison was conducted with Student’s t test, and two-way repeated measures ANOVA. All tests were two-sided and p value < 0.05 was considered statistically significant. All in vitro experiments were performed more than three independent experiments.