All animal experiments were conducted in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People’s Republic of China and complied with internationally accepted guidelines for laboratory animal care and use. All procedures were designed to minimize animal suffering. Mice were humanely euthanized in accordance with the AVMA Guidelines for the Euthanasia of Animals (2020) using intraperitoneal injection of sodium pentobarbital. The study protocol was reviewed and approved by the Laboratory Animal Welfare & Ethics Committee (LAWEC) of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention (Chinese Center for Tropical Diseases Research; approval ID: IPD-2022-015).

Six-week-old female C57BL/6 mice were obtained from Ji Hui Laboratory Animal Co., Ltd. (Shanghai, China). Tigit^fl/fl-Ncr1^iCre/+ mice were purchased from Mouse Treasure Biotechnology Co., Ltd. (Wuhan, China). S. japonicum cercariae (Chinese mainland strain) were provided by the snail breeding facility of our institute. All animals were maintained under specific pathogen-free (SPF) conditions in accordance with institutional animal care guidelines.

Female C57BL/6 mice were infected with S. japonicum cercariae using the abdominal patch method (20 ± 1 cercariae per mouse). Six mice were sacrificed prior to infection, and at 4, 6 and 8 weeks post-infection. All mice were humanely euthanized in strict accordance with the AVMA Guidelines for the Euthanasia of Animals (2020) via intraperitoneal injection of sodium pentobarbital (200 mg/kg body weight). Livers were perfused through the hepatic portal vein with phosphate-buffered saline (PBS). Nonparenchymal hepatic cells were isolated by double-density Percoll gradient centrifugation (Cytiva, Uppsala, Sweden). The white, cloud-like interphase fraction containing nonparenchymal cells was collected, washed twice with PBS, and treated with red blood cell lysis buffer to remove erythrocytes. The cell concentration was adjusted to 1 × 10⁵ cells/mL in fluorescence-activated cell sorting (FACS) buffer.

For flow cytometric analysis, cells were stained with the following fluorochrome-conjugated antibodies: Annexin V–BV605, CD3e–BB700, NK1.1–AF700, TIGIT–PE, IFNγ–PE–Cy7, and GZMB–APC (all from BD Biosciences, San Diego, CA, USA). NK cells were defined as CD3⁻NK1.1⁺ populations. Staining was performed for 30 min at room temperature (24–26 °C) and cells were washed once with FACS buffer. Data acquisition was performed using a BD FACSVerse flow cytometer (BD Biosciences), and data were analyzed with FlowJo v10 software (BD Biosciences). The proportions of NK cells and their intracellular levels of IFNγ and GZMB were subsequently evaluated.

The human NK92 cell line was obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). Cells were maintained in MyeloCult™ H5100 medium (StemCell Technologies, Vancouver, Canada) supplemented with 12.6% horse serum (Gibco, Grand Island, NY, USA) and 100 U/mL recombinant human interleukin-2 (IL-2; Miltenyi Biotec, Bergisch Gladbach, Germany). Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

To establish TIGIT-knockdown NK92 cells, the lentiviral vector pLent-EF1α-H1-GFP-Puro carrying the TIGIT short hairpin RNA (shRNA) sequence was used. The control and TIGIT -targeting lentiviruses were packaged and synthesized by Angel Biotechnology Co., Ltd. (Suzhou, China). NK92 cells were transduced with either the control or TIGIT knockdown lentivirus at a multiplicity of infection (MOI) of 100. After 72 h, puromycin (2 µg/mL) was added to the culture medium for selection, and stable cell lines were obtained after two weeks of continuous screening. The resulting negative control (NC) and TIGIT-knockdown (KD) NK92 cell lines were maintained under identical conditions.

For cell cycle analysis, cells were stained using a Cell Cycle Detection Kit (Beyotime Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s protocol, followed by flow cytometric analysis. Data were acquired using a BD FACSVerse flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed with FlowJo v10 software (BD Biosciences) to determine the distribution of cells across different phases of the cell cycle.

The human hepatic stellate cell line LX-2 was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37 °C in a humidified atmosphere containing 5% CO₂. NK92 cells from the negative control (NC) and TIGIT-knockdown (KD) groups were co-cultured with LX-2 cells at a ratio of 5:1 for 12 h.

After co-culture, LX-2 cells were harvested, and apoptosis was evaluated using an Annexin V/PI Apoptosis Detection Kit (Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer’s instructions. Flow cytometric analysis was performed on a BD FACSVerse flow cytometer (BD Biosciences, San Diego, CA, USA), and data were analyzed with FlowJo v10 software (BD Biosciences) to determine the proportion of apoptotic cells.

In parallel, the expression levels of Gzmb and Prf1 in NK92 cells were analyzed by flow cytometry and quantitative reverse transcription PCR (RT-qPCR). For flow cytometry, cells were stained with GZMB–PE and PRF1–PE–Cy7 antibodies (Thermo Fisher Scientific, Waltham, MA, USA) following standard protocols.

NK92 cells were seeded in six-well plates at a density of 1 × 10⁶ cells/mL and treated with 100 μmol IFNγ antagonist 1 (MedChemExpress, Monmouth Junction, NJ, USA) for 48 h. Cells cultured under identical conditions without antagonist treatment served as the control group.

Total RNA was extracted from mouse liver tissue and NK92 cells using a Cell and Tissue RNA Extraction Kit (Sikejie Biotechnology Co., Ltd., Shandong, China) according to the manufacturer’s instructions. The concentration and purity of RNA were determined spectrophotometrically, and 1 μg of total RNA was reverse-transcribed into complementary DNA (cDNA) using a Reverse Transcription Kit (Sikejie Biotechnology). Quantitative real-time PCR (RT-qPCR) was then performed to evaluate the expression of target genes associated with liver fibrosis (Acta2, Col1a1, Fn) and NK cell function (Tigit, Ifnγ, Gzmb, Ifi30). Primer sequences used in this study are listed in Table 1.

NK92 cells from the NC and KD groups (three biological replicates per group) were collected and washed three times with PBS to remove residual contaminants. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and quantified spectrophotometrically to assess purity and concentration. High-quality RNA samples were submitted to Biomarker Technologies Co., Ltd. (Beijing, China) for library preparation and sequencing. Differentially expressed genes (DEGs) were identified between the NC and KD groups. Functional enrichment and signaling pathway analyses of DEGs were performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to elucidate the biological processes and molecular pathways affected by TIGIT knockdown.

Liver tissues were fixed in 4% paraformaldehyde, routinely dehydrated, and paraffin-embedded. Tissue sections (4 µm thick) were stained using Hematoxylin and Eosin (HE) and Masson Staining Kits (Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s protocols. For each group, three ± two representative sections containing single egg granulomas were imaged under a microscope using CellSens imaging software (Olympus, Tokyo, Japan). The proportion of positively stained areas was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The hepatic egg burden was quantified using an alkaline digestion method to assess the intensity of S. japonicum infection. Mice were sacrificed at 6 weeks post-infection and humanely euthanized in strict accordance with the AVMA Guidelines for the Euthanasia of Animals (2020) via intraperitoneal injection of sodium pentobarbital (200 mg/kg body weight). The liver was immediately harvested, and approximately 0.1 g of tissue from each mouse was accurately weighed and transferred to a centrifuge tube. Liver samples were digested in 8 mL of 4% potassium hydroxide (KOH) solution at 37 °C for 2 h with constant shaking at 150 rpm to completely dissolve the tissue and release schistosome eggs. Following digestion, the suspension was thoroughly vortexed, and a 10 μL aliquot of the homogenized solution was placed onto a glass slide for egg counting under a light microscope. Egg counts were performed independently in triplicate for each sample, and the mean value was calculated. The total egg number in the entire digest was extrapolated based on the aliquot-to-total volume ratio. Hepatic egg burden was ultimately expressed as the number of eggs per gram of liver tissue.

Total protein was extracted from liver tissue or cultured cells using 1 mL of RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Solarbio Science & Technology Co., Ltd., Beijing, China). Lysates were centrifuged at 12,000 rpm (12,830 × g) for 5 min at 4 °C, and the protein concentration of the supernatant was determined using the bicinchoninic acid (BCA) assay (Solarbio Science & Technology). Equal amounts of protein (30–50 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes using a semi-dry transfer apparatus (Bio-Rad, Hercules, CA, USA).

Membranes were blocked and then incubated overnight at 4 °C with the following primary antibodies: Mouse Anti-GAPDH (Cat. No. 5174; CST, Danvers, MA, USA), Rabbit Anti-Bcl-2 (Cat. No. 4223; CST), Rabbit Anti-Bax (Cat. No. 50599-2-Ig; ProteinTech, Wuhan, China), Rabbit Anti-Collagen I (Cat. No. 72026; CST), Rabbit Anti-α-Smooth Muscle Actin (α-SMA; Cat. No. 19245; CST), Rabbit Anti-TIGIT (Cat. No. 99568; CST), and Rabbit Anti-IFI30 (Cat. No. 15112-1-AP; ProteinTech). After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, sheep anti-rabbit IgG or sheep anti-mouse IgG (1:4000; Jackson ImmunoResearch, West Grove, PA, USA), for 1 h at room temperature. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL) reagents (Solarbio Science & Technology).

Three to four biological replicates were included for each experimental group. Band intensities were quantified using ImageJ software (NIH, Bethesda, MD, USA), and protein expression levels were normalized to GAPDH.

All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) and Microsoft Excel 2010 (Microsoft, Redmond, WA, USA). Differences between two groups were evaluated using a two-tailed paired Student’s t-test or a two-tailed Mann–Whitney U test, as appropriate. Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA). Data are presented as mean ± standard deviation (SD), and a P value of less than 0.05 was considered statistically significant.

A schistosomiasis infection mouse model was established by percutaneous exposure of mice to S. japonicum cercariae. HE and Masson staining confirmed the successful establishment of liver fibrosis at 6 weeks post-infection, as indicated by marked deposition of schistosome eggs within the liver (Figure 1A). Pronounced infiltration of inflammatory cells forming ring-shaped granulomas around the eggs and a substantial increase in collagen fiber accumulation were evident in these regions (Figures 1B, C). Consistently, the liver-to-body weight ratio was significantly elevated at 6 weeks post-infection, further validating the successful construction of the fibrosis model (Figure 1D). RT-qPCR analysis revealed that the expression levels of Acta2, Col1a1, and Fn were markedly upregulated in the liver at 6 weeks post-infection, accompanied by a significant increase in Tigit expression (Figure 1E).

Flow cytometric analysis of hepatic immune cells demonstrated a time-dependent dysregulation of NK cell populations during S. japonicum infection. Although the overall proportion of NK cells among hepatic nonparenchymal cells increased at 6 weeks post-infection (Figures 1F, G), there was a concomitant and progressive expansion of NK cells expressing the inhibitory immune checkpoint receptor TIGIT, which peaked at the same time point (Figure 1H). Importantly, these TIGIT⁺ NK cells displayed profound functional impairment, as indicated by significantly reduced production of IFNγ and granzyme B (Figures 1I, J). Our findings indicate that the progression of schistosomiasis-induced liver fibrosis is associated with the accumulation of TIGIT⁺ hepatic NK cells that exhibit functional impairment, defining an exhausted phenotype.

To investigate the effect of TIGIT on NK cell function, a TIGIT-knockdown NK92 cell line was established. As shown in Figure 2A, the transduced cells exhibited strong green fluorescence, confirming successful lentiviral transfection. RT-qPCR analysis further verified efficient TIGIT knockdown in NK92 cells (Figure 2B). Flow cytometric analysis demonstrated that TIGIT knockdown significantly increased IFNγ expression compared with control NK92 cells (Figures 2C, E), whereas GZMB expression remained unchanged (Figures 2D, F). Moreover, when co-cultured with LX-2 cells for 6 h, TIGIT-knockdown NK92 cells induced markedly higher levels of both early and late apoptosis in LX-2 cells compared with the control group (Figures 2G–I). These findings indicate that loss of TIGIT enhances IFNγ secretion and partially restores the cytotoxic activity of NK92 cells against hepatic stellate cells.

RNA-seq analysis was performed to compare gene expression profiles between TIGIT-knockdown and control NK92 cells, with transcriptome validation and GO enrichment results shown in Supplementary Figures 2A, B. The resulting volcano plot of differentially expressed genes (DEGs) identified 122 upregulated and 84 downregulated genes following TIGIT knockdown (Figure 3A). KEGG pathway enrichment analysis revealed that the DEGs were primarily associated with immune cytokine signaling and apoptotic pathways (Figure 3B). Validation by RT-qPCR and WB confirmed that TIGIT downregulation led to a marked increase in IFI30 expression (Figures 3C–E). Flow cytometric analysis further showed that the proportion of NK92 cells in the proliferative (S) phase was significantly higher after TIGIT knockdown, indicating enhanced proliferative activity (Figures 3F, G). Treatment of NK92 cells with an IFNγ antagonist for 48 h resulted in downregulation of Ifi30 expression, accompanied by decreased expression of the anti-apoptotic protein BCL2 and increased expression of the pro-apoptotic protein BAX (Figures 3H–J). These findings suggest that TIGIT modulates NK cell proliferation and survival through the IFNγ–IFI30 signaling axis.

To determine the in vivo role of TIGIT in schistosomiasis-induced liver fibrosis, NK cell–specific Tigit-knockout mice were generated using gene-editing technology and subsequently infected with S. japonicum. Hepatic egg burden was first quantified to assess infection intensity. No significant difference in liver egg counts was observed between wild-type (WT) and Tigit knockout mice, indicating that the differences observed in subsequent pathological and immunological outcomes were not attributable to variations in parasite burden (Supplementary Figure 2C). Six weeks after S. japonicum infection, liver fibrosis was evaluated in both WT and Tigit-knockout mice by HE and Masson staining (Figure 4A). Compared with WT mice, Tigit-knockout mice exhibited markedly reduced inflammatory cell infiltration on HE staining (Figure 4B) and a notable decrease in collagen fiber deposition on Masson staining (Figure 4C). Consistently, the liver-to-body weight ratio was significantly lower in Tigit-knockout mice than in WT controls at 6 weeks post-infection (Figure 4D).

Molecular analyses supported these histological findings. Both RT-qPCR (Figure 4E) and WB (Figures 4F, G) demonstrated that the hepatic expression levels of Acta2, Col1a1, Fn, and Tigit were significantly decreased in Tigit-knockout mice compared with WT counterparts. Flow cytometric analysis of hepatic nonparenchymal cells isolated 6 weeks after infection revealed an increased proportion of NK cells (Figures 4H, I) and a significant reduction in TIGIT⁺ NK cells (Figures 4J, K) in Tigit-knockout mice. Moreover, IFNγ secretion by NK cells was markedly enhanced following Tigit deletion (Figures 4L, M). These results indicate that NK cell–specific Tigit knockout effectively alleviates schistosomiasis-induced liver fibrosis by restoring NK cell abundance and functional activity.