Materials
Reagents and antibodies used in the present study are listed in Supplementary Table 1.
Cell culture
The 4T1 cells, a murine TNBC cell line, which is derived from a spontaneous mammary carcinoma in a BALB/c mouse, were obtained from the American type culture collection (ATCC) and cultured according to the recommended protocols. The cells were cultured in RPMI 1640 medium, supplemented with 10% foetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37℃ in the presence of 5% CO2.
Mice
The study was conducted in 6–8-week-old female BALB/c mice purchased from Vital River Laboratory Animal Technology Co. Ltd. Beijing, China. All animal experiments were performed under specific pathogen-free conditions. The animal experiments were approved by the Ethics Committee of the Affiliated Hospital of Qingdao University, Qingdao, China.
Tumour-bearing mouse model
To prepare TILs, 1 × 106 4T1 cells were injected into the mammary fat pad of female BALB/c mice. For the TIL-ACT experiment, 1 × 105 4T1 cells were injected directly into the mammary fat pad. The mice were monitored daily, and the tumour volume was measured every 2–3 days by using a caliper and determined using the following formula: ℼ/6 × length × width2, where length is the longest diameter and width is the shortest diameter.
Preparation of TILs and conventional T cells
Tumours could be palpated subcutaneously 7–9 days after inoculation of 4T1 cells. The fresh tumours were isolated, broken down into smaller fragments, and digested using the tumour dissociation Kit for the enrichment of single tumour cell suspensions. TILs were sorted using the CD45 (TIL) MicroBeads on a clean platform. TILs were incubated in complete RPMI 1640 medium supplemented with 10% inactivated FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 1 × non-essential amino acid, and 55 µM 2-mercaptoethanol at 37℃ in the presence of 5% CO2. Approximately 30 ng/mL OKT3 antibody was added to the fresh complete RPMI medium containing 6000 U/mL of IL-2 and 20 ng/mL of IL-7. Depending on TIL growth, OKT3 antibody, IL-2, and IL-7 were supplemented twice for re-stimulation. TILs were harvested on days 12–14, resuspended to a final concentration of 5 × 107 cells/mL. The conventional T cells were isolated from the fresh spleen of tumour-bearing mice by using the Pan T Cell Isolation Kit II and were prepared using the same method.
IFN-γ and TNF-α release assays
The TIL antitumour reactivity was determined using the IFN-γ and TNF-α release assay in vitro. TILs or conventional T cells were co-cultured overnight with the 4T1 cells in different ratios, as indicated in a capture antibody-coated 96-well plate. Then, the cells were removed, supernatant was collected, and secreted IFN-γ and TNF-α levels were determined through ELISA.
Flow cytometry
The anti-mouse monoclonal antibodies used for T cells were: CD3-APC, CD4-BV421, CD8-FITC, and CD279-PerCP-eFluor 710 (PD-1). CD45-FITC, CD11b-Percpcy5.5, and F4/80-PE were used for the surface staining of tumour-associated macrophages (TAMs). The T-cell or tumour cell pellet was washed with the FACS buffer and then incubated with the anti-mouse CD16/32 for 10 min at room temperature to block Fc receptors. The surface antibodies were stained in the dark for approximately 30 min.
To determine the intracellular IFN-γ levels of T cells, 1–2 × 106 TILs/T cells were stimulated using the cell activation cocktail for 2 h. After stimulation, Fixable Viability Stain 780 or 520 (APC-CY7 or FITC) dye was used to exclude dead cells before Fc receptor blocking and cell surface staining. Then, the cells were fixed and permeabilised using a Fixation/Permeabilisation kit, followed by staining with IFN-γ (FITC) antibody. The cells were washed twice with the FACS staining buffer or Perm/Wash™ buffer prior to acquisition on an Arial II-Optics flow cytometer. All data were gated on live and single cells. The data were analysed using the FlowJo software.
Antitumour effects in vivo
A total of 1 × 105 4T1 cells were subcutaneously injected into the mammary fat pad of female BALB/c mice. When the average tumour volume reached approximately 50 mm3, all the mice were pretreated with cyclophosphamide (CTX, 100 mg/kg) for lymphodepletion (day 1). A total of 1 × 107 TILs or conventional T cells were injected into the tumour-bearing mice through the tail vein (day 0), and rhIL-2 (100000 units) was injected intraperitoneally for 3 consecutive days (days 1–3). The anti-PD-1 (10 mg/kg) or an equal volume of PBS was injected intraperitoneally 4 times (days 5, 8, 11, and 14). The tumour growth was monitored through caliper measurements. The tumour-bearing mice were sacrificed on day 27. The tumour tissue, lung, liver, kidney, and intestine of each mice were harvested for pathological analysis.
Immunofluorescence analysis and histopathological evaluation
Tumour tissues were perfused with 0.1 M PBS, embedded into an optimal cutting temperature compound, and frozen for cryostat section. Cryostat sections were fixed with 4% paraformaldehyde for 15 min at and cryostat sections were incubated in the blocking solution for 30 min at room temperature. CD3 and CD137 expressions in tumour tissues were assessed immunohistochemically by using anti-mouse CD3 and anti-mouse CD137 antibodies as the primary antibodies, whereas Cy3-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-rabbit IgG were used as the secondary antibodies. The nuclei were stained with 4′,6-Diamidino-2-phenylindole (DAPI). ECLIPSE C1 orthofluorescent microscopy (Nikon, Japan) was used to view and acquire immunofluorescent microscopic images.
For histopathological examination, the samples were fixed with neutrally buffered 3.5% formaldehyde and subjected to haematoxylin and eosin (H&E) staining. The microscopic evaluation of H&E-stained images was performed using DS-U3 (Nikon, Japan).
Statistical analysis
A completely randomised, balanced design was used for all experiments. Statistical analyses were performed using the paired Student’s t-test in Prism software (GraphPad). Data are represented as mean ± standard error of mean. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered statistically significant.