Study animals

Female C57BL/6 mice (6–8 weeks old) were purchased from the Laboratory Animal Center of the Chinese Academy of Medical Sciences and housed in well-ventilated cabinets under standard environmental conditions (temperature 21 ± 2 °C, 50-60% relative humidity, and a conventional 12/12 h light/dark cycle). DEREG mice (C57BL/6 background, MMRRC Stock No: 32050-JAX) were purchased from The Jackson Laboratories (USA). The use of animals was ethically approved by the Tianjin Medical University ethics committee. Mice (8–10 mice/group) received intraperitoneal injections of 1 × 106 ID8 cells 10 days before initiating treatments. Ovarian cancer mice underwent three intraperitoneal injections at 4-day intervals with 100 µg of control (Ultra-LEAF™ Purified Mouse IgG1, κ Isotype Ctrl Antibody, Catalog#401414, BioLegend) or anti-TIGIT monoclonal antibody (Clone 1B4, Catalog#Ab01258, Absolute Antibody Ltd.) [20] following the same method as in our previous research [18]. Seven days later, spleens and ascites from treated mice were harvested, and mononuclear cells were isolated and analyzed by flow cytometry.

For survival experiments, mice (20 mice/group) received intraperitoneal injections of 1 × 106 ID8 cells 10 days before treatment. In the treatment groups, mice were administered three doses of 100 µg of control or anti-TIGIT monoclonal antibody at 4-day intervals. Mice were weighed bi-weekly, and daily observations were made for signs of swollen bellies indicative of ascites formation. Additionally, evidence of toxicity, such as weight loss, hunched posture, mobility, diarrhea, failure to eat, and respiratory distress, was assessed daily. Mice were euthanized upon the development of ascites and a weight increase exceeding 30%, following institutional guidelines. The mean survival time of mice was calculated using the Kaplan-Meier survival curve and log-rank test.

Tregs depletion

To deplete Tregs, we utilized DEREG mice, which express DTR under the control of the Foxp3 promoter (Foxp3-DTR mice). As previously described in detail [21], these mice were administered an intraperitoneal injection of 1 µg DT (Catalog #322326, Merck) or PBS 24 h before 1 × 106 ID8 cells injection. Subsequently, 1 µg DT was administered on day 7 following tumor implantation. Control mice were treated with PBS injection. Flow cytometry in blood were used to confirm Tregs depletion.

Tumor volume measurement

To remove bias, a blind tumor volume measurement was done. Tumor volume was calculated using the following formula [22]: Tumor volume (mm3) = (L × W2) / 2. “L” and “W” represented the longest and shortest diameter of the tumor, respectively.

Cell lines

ID8, a well-established clone of the MOSEC ovarian cancer derived from C57BL/6 mice (Catalog #SCC145), was generously provided by the University of Pennsylvania. Prior to cell suspension preparation and subsequent administration to mice, ID8 cells underwent cultivation under controlled conditions at 37 °C in an atmosphere of 5% CO2. This cultivation process occurred in a complete DMEM medium, enriched with essential components, including 10% Fetal Bovine Serum (FBS) (Catalog #10099, Gibco), 100 U/mL of penicillin, and 100 µg/mL of streptomycin.

Antibodies and flow cytometry analysis

Flow cytometry analyses were conducted on multiple occasions, up to three times, to comprehensively characterize the phenotypes of immune cells present in both the spleen and ascites. A panel of monoclonal antibodies, including CD3 PerCP (Clone SK7, Catalog #145-2C11, BD Biosciences), CD4 Pacific Orange (Clone RM4-5, Catalog #MCD0430, Invitrogen), CD8 Pacific Blue (Clone SK1, Catalog #344718, BioLegend), CD25 PE-Cy7 (Clone PC61, Catalog #552880, BD Biosciences), PD-1 FITC (Clone 29 F.1A12, Catalog #135214, BioLegend), FoxP3 PE (Clone MF23, Catalog #560408, BD Biosciences), and TIGIT BV605 (Clone 1G9, Catalog #744212, BD Biosciences), were thoughtfully selected for immune cell staining.

To perform these analyses, mononuclear cells (1.5 × 106 cells) isolated from both the spleen and ascites were incubated with prepared antibody mixtures using FACS buffer, composed of PBS with 2% BSA and 0.05% sodium azide, at a dilution of 1:100. This incubation occurred for 15 min at a controlled temperature of 4 °C, followed by two thorough washes. Subsequently, for intracellular staining of FoxP3 following the manufacturer’s instructions, the cells were fixed and permeabilized using a BD Cytofix/Cytoperm kit (Catalog #554714, BD Biosciences).

Following the staining and preparation steps, the cells were promptly analyzed using a BD LSR II multicolor flow cytometer (BD Biosciences). The acquired data underwent in-depth analysis using FlowJo software version 10, developed by Tree Star.

Isolation of splenic CD4+CD25+ Tregs and CD4+CD25− T effector cells

Spleens were harvested and processed into a single-cell suspension by passing through 40 μm filters twice. Mononuclear cells were then isolated using Ficoll-Paque density gradient centrifugation. CD4+CD25+ Tregs and CD4+CD25− T effector cells were isolated utilizing a mouse CD4+CD25+ Tregs isolation kit (Catalog #130-091-041, Miltenyi Biotec) [23]. The purity of T cells was estimated to be > 90% by FACS. The isolated cells were cultured in RPMI 1640 supplemented with 10% FBS.

Coculture

Splenic CD4+ Tregs were isolated from both untreated normal mice and ovarian cancer mice, including those in the isotype control and anti-TIGIT treatment groups. Subsequently, they were co-cultured with normal splenic CD4+CD25− T effector cells from untreated normal mice for a period of 24 h, maintaining a ratio of 1:1 (2 × 105 cells/well). The co-cultures were then subjected to treatment with anti-CD3 (5µg/ml) and anti-CD28 (2 µg/ml) for the polyclonal activation of T cells following the same method as in our previous research [18]. The proliferation and apoptotic rate of CD4+CD25− T effector cells were assessed using CCK-8 and Annexin-V Staining, while the secretory capacity (IFN-γ and IL-4) was determined via ELISA.

CCK-8 measurement

Splenic CD4+CD25− T effector cells from untreated normal mice and splenic CD4+ Tregs, obtained from untreated normal mice, as well as from ovarian cancer mice in the isotype control and anti-TIGIT treatment groups, were co-cultured at a 1:1 ratio. Following a 24-hour co-culture, cells in the supernatant were gathered and distributed to 96-well plates in triplicate, with a seeding density of 1 × 105 cells per well. Subsequently, 10 µl of CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was introduced to each well, and the cells were incubated for 4 h at 37 °C in the absence of light exposure. Absorbance was measured using a microplate reader (Spectra MR, Dynex, Richfield, MN) at OD450nm.

Annexin-V staining

Cell apoptosis was assessed using the Annexin V-fluorescein isothiocyanate apoptosis kit (Nanjing Keygen Biotech, Nanjing, China) in accordance with the manufacturer’s instructions. In brief, cells were resuspended in 100 µl of binding buffer containing 5 µl of Annexin-V and 5 µl of 7-AAD, followed by a 15-minute incubation at room temperature. Afterward, the cells were washed twice with cold PBS. Subsequently, cells were diluted by adding 300 µl of binding buffer and promptly analyzed using flow cytometry within 1 h. The data are presented as the percentage of Annexin-V-positive cells.

ELISA

The supernatants obtained from the co-culture were collected for the quantification of IFN-γ and IL-4 levels using ELISA kits (Catalog #BMS606, #BMS613, Invitrogen). The procedure strictly adhered to the protocols provided by the manufacturer. In summary, the supernatants were diluted 1:1 with sample dilution buffer, and 100 µl of each sample was added to the respective wells. For the calibration curve, a dilution series of the standard was prepared on the same plate. The plate was incubated at 37 °C for 1 h. Following this, the samples were removed, and the wells were washed five times with the washing solution. An enzyme-labeled secondary antibody, diluted with sample dilution buffer, was added (100 µl per well) and incubated at 37 °C for 1 h. After the reaction, the secondary antibody was removed, and the wells were washed five times with the washing solution. A substrate solution was added, and the color developed during incubation. Upon sufficient color development, a stop solution was added, and the absorption at 450 nm was measured using a plate reader (Spectra MR, Dynex).

Statistics

Statistical analyses were performed using GraphPad Prism V9.0 (GraphPad Software, La Jolla, California, USA). The data shown are representative of three independent experiments. Continuous variables in figures are expressed as the mean ± SEM. The Mann-Whitney test, univariate analysis of variance (ANOVA, Least Significant Difference test), and Student t-test were two-tailed and employed to calculate P values. A significance level of P < 0.05 was considered statistically significant.