A total of 40 8-10 week old wild-type female C57BL/6 mice weighing 16–22 g and 50 8–10 week female C3H mice weighing 16–22 g were purchased from the Animal Experimental Center of Chongqing Medical University (Chongqing, China). All mice were housed at a temperature of 23°C and humidity of 60% under a 12-h light/dark cycle. Food and water were supplied ad libitum. All protocols involving mice were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (16) and all experiments were approved by the Animal Care and Use Committee of Second Affiliated Hospital of Chongqing Medical University (Chongqing, China).

An improved Kamada's two-cuff method with minor modifications was used to perform OLT between female C57BL/6 donor mice and inbred female C3H recipient mice, as previously described (17). Donor and recipient mice received 1.9% ethyl ether anesthesia via nostril cannula. The use of ethyl ether was approved by the Animal Experimental Centre of Chongqing Medical University (Chongqing, China). Warm ischemia, cold ischemia and anhepatic times were set at 0 min, 1 h and 20 min respectively. Only C3H recipient mice underwent the following experiments. C3H mice that had undergone single LT from donor mice but had not received subsequent treatments (n=5) were sacrificed 24, 48 and 72 h following surgery to determine KC activity and TIM-4 expression in the liver. Recipient mice (n=30) were then randomly divided into 3 treatment groups: A sham group (n=10) that did not receive a transplant but still underwent abdominal cutting and vascular exposures around the liver; a control mouse monoclonal antibody (mAb) group (n=10; cat. no. GTX14149; 1:50 dilution; mAb received from GeneTex, Inc., Irvine, CA, USA) treated with 0.35 mg/mouse mAb via portal vein injection to establish an LT model; and a T cell immunoglobulin mucin protein 4 (TIM-4) mAb group (n=10) that received 0.35 mg/mouse portal vein injections. The indicated treatments and dosages in each group were administered intravenously for 2 days following surgery at 24 h intervals. On day 7 following surgery, all mice were anaesthetized via inhaled 1.9% ethyl ether and 0.5 ml/mouse blood samples were drawn from the abdominal aorta. Mice were then sacrificed in a sealed-container with an overdose of carbon dioxide (CO₂ replacement rate: 20% of the container volume/min). Murine liver tissues were subsequently removed and examined and KCs were then isolated.

Another mouse model was then established. Donor mice were injected with clodronate liposomes (CL; 10 mg/kg; cat. no. 40337ES05/10; Shanghai Yeasen Biotechnology Co., Ltd., Shanghai, China) via the caudal vein to destroy liver KCs 24 h prior to surgery. Following CL pretreatment, OLT or sham surgery was performed (C57BL/6→C3H) and recipient C3H mice were administered the aforementioned treatments. Recipient mice were randomly divided into CL+sham, CL+control mAb and CL+TIM-4 mAb groups (all n=5). All recipient mice were sacrificed and underwent tissue section examination 7 days post-surgery.

OLT or sham surgery was performed and KCs were isolated. A modified method of the in vitro type IV collagenase digestion was used to dissociate liver tissue. Mice were anaesthetized using inhaled 1.9% ethyl ether. Murine heartbeats and thoracic breathing were monitored to ensure that mice were anesthetized and not sacrificed. The liver was perfused in situ with 10 ml PBS (3 ml/min) at 37°C to remove red blood cells. Liver tissues were then dispersed in RPMI-1640 (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 0.5% type IV collagenase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C for 30 min. Liver homogenate was filtered through a 200-mesh sieve to remove undigested tissue and non-parenchymal cells of the liver were obtained using the gradient centrifugation method. The cell suspension was centrifuged at 300 × g on an 5810R machine (Eppendorf, Hamburg, Germany) for 5 min at 4°C. The top aqueous phase was discarded and the cell sediment was reserved. Cell sediments were then resuspended with 10 ml RPMI-1640 and centrifuged at 300 × g for 5 min at 4°C, the top aqueous phase was discarded and cell sediments were reserved. Cell sediments were then resuspended with 10 ml RPMI-1640 and centrifuged at 50 × g for 3 min at 4°C. The top aqueous phase (cleared cell suspension) was transferred into a new 10 ml centrifuge tube and centrifuged at 300 × g for 5 min at 4°C, the top aqueous phase was discarded and cell sediments were reserved. To further purify KCs, the selective adherence to plastic method was used following the protocol described by Li et al (18). Cells were seeded in a 6-well plate at a density of 1–3×10⁷/well in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum (both HyClone, GE Healthcare Life Sciences, Logan, UT, USA) and 100 U/ml penicillin/streptomycin (Sigma-Aldrich; Merck KGaA) and incubated for 2 h in a 5% CO₂ atmosphere at 37°C. Following washing with PBS, non-adherent cells were removed from the dish; adherent cells were KCs.

Naive CD4⁺CD25⁻ T cells were isolated using the CD4⁺ T cell isolation kit II and CD25 microbeads (both Miltenyi Biotec GmbH, Bergisch Galdbach, Germany) were utilized to select the CD25⁻ population.

Liver tissues were fixed with 10% formaldehyde at room temperature for 4 h and subsequently embedded in paraffin. Paraffinized sections were placed in an oven at 58°C for 2 h, dewaxed with xylene, placed in 100% ethanol and rehydrated using 90–70% ethanol for 3 min at each stage. Sections were washed with PBS three times (3 min/wash), placed in a 0.01 M sodium citrate antigen retrieval solution (pH 6.0, 95°C) for 15 min and then cooled for 15 min at room temperature. Sections were washed with PBS three times (3 min/wash), treated with 4.3% H₂O₂ to block endogenous peroxidase for 20 min at room temperature. Sections were washed with PBS three times (3 min/wash) and then blocked with 10% fetal calf serum (FCS; cat. no. 25-01860; Shanghai Biomart Technology, Co., Ltd, Shanghai, China) for 30 min at room temperature. Sections were washed with PBS three times (3 min/wash) and incubated with anti-CD163 (EPR19; cat. no. ab182422; 1:100 dilution; Abcam, Cambridge, MA, USA) overnight at 4°C. Sections were washed with PBS three times (3 min/wash) and incubated with biotin-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin G secondary antibodies (cat. nos., BA1003 and BA1001, respectively; Wuhan Boster Biological Technology, Ltd., Wuhan; China) at 37°C for 90 min. Sections were washed with PBS three times (3 min/wash), treated with 3,3′-diaminobenzidine for 5 min at room temperature, stained with hematoxylin for 30 sec at room temperature and treated with hydrochloric acid for 1 sec. Sections were then placed in 70% ethanol for 3 min and then treated with 80–100% ethanol for 3 min each. Following dehydration, sections were mounted, observed using a XSP-13C-LB microscope (Shanghai Precision & Scientific Instrument Co., Ltd., Shanghai, China) at a magnification of ×400 and analyzed using Image pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

KCs derived from recipient (sham, control mAb or TIM-4 mAb mice) were collected and were fixed in 4% paraformaldehyde for 1 h at room temperature. Slides were washed with PBS three times (3 min/wash) and blocked with 30 min in 10% FCS at room temperature. Slides were then washed with PBS three times (3 min/wash) and incubated with TIM-4 antibodies (RMT4-53, cat. no. GTX14149; 1:100 dilution; GeneTex Inc.) overnight at 4°C. Slides were washed with PBS three times (3 min/wash) and incubated with specific secondary antibodies labeled with tetramethylrhodamine (red; 1:100 dilution; cat. no. GMS40036; Bio Valley, Shanghai; China) or DAPI (blue) for 30 min at room temperature. Slides were then washed with PBS three times (3 min/wash) and sealed with FluorFluoromount-G™ Slide Mounting medium (Southern Biotech, Birmingham, AL, USA). Images were taken using a Zeiss LSM 510 confocal microscope (magnification ×800; Zeiss AG, Thornwood, NY, USA) and were analyzed using an Image Analysis system, version 11.0 (Chang Heng Rong Technology Co., Ltd., Beijing; China).

Cells or liver homogenates were lysed in lysis buffer (Gibco; Thermo Fisher Scientific, Inc.) and the concentration of total protein was determined using a BCA Protein assay kit (Sangon Biotech Co., Ltd., Shanghai, China). A total of 40 μg protein was loaded per lane, separated using 12% SDS-PAGE gel and then electrotransferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories Inc., Hercules, CA, USA). Membranes were blocked using 5% non-fat milk in Tris-buffered-saline with Tween at room temperature for 1 h and incubated with primary antibodies overnight at 4°C. The primary antibodies used included: TIM-4 (RMT4-53, cat. no. GTX14149; 1:100 dilution; GeneTex Inc.), tumor necrosis factor-α (TNF-α; 52B38, cat. no. ab1793; 1:100 dilution; Abcam), interferon-γ (IFN-γ; #37895R, cat. no. MAB485R; 1:100 dilution; R&D Systems Inc., Minneapolis, MN, USA), chemokine ligand 2 (CCL2; 2D8, cat. no. GTX60582; 1:100 dilution; GeneTex Inc.), C-X-C motif chemokine ligand 2 (CXCL2; cat. no. GTX74085; 1:200 dilution; GeneTex Inc.), TGF-β (cat. no. ab92486; 1:100 dilution), phosphorylated (p)-signal transducer and activator of transcription 6 (STAT6; phospho Y641, cat. no. ab28829; 1:100 dilution), p-p65 (phospho S536, cat. no. ab86299; 1:100 dilution), p-p38 (phospho Y182, cat. no. ab47363; 1:100 dilution), forkhead box P3 (Foxp3; 236A/E7, cat. no. ab20034; 1:100 dilution), transcription factor gata3 (Gata3; cat. no. ab106625, 1:100 dilution) and β-actin (cat. no. ab8226; 1:100 dilution) (all from Abcam). Membranes were then washed with TBS-Tween-20 and incubated with peroxidase-conjugated secondary antibodies (cat. nos. A0545, A9044; 1:8,000 dilution; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. An enhanced chemiluminescence kit (GE Healthcare, Chicago, IL, USA) was used to perform chemiluminescence and protein bands were visualized using X-ray films. All images were analyzed using ImageJ software, version 14.8 (National Institutes of Health, Bethesda, MD, USA).

RNAs were extracted from cell lysates using TRIzol RNA-extraction reagent and reverse transcription was performed using PrimeScript™ 1st Strand cDNA Synthesis kit (cat. no. 6110A) (both from Takara Bio Inc., Otsu, Japan) following the manufacturer's protocol. qPCR assays were performed using SYBR Premix Ex Taq (Takara Bio Inc.) and a cDNA template on the Applied Biosystems 7500 Real-time PCR System (Applied Biosystems; Thermo Fisher Scientific Inc.). The primers used for qPCR amplification were as follows: TIM-4; forward, 5′-CAATCGAGGTGACAGTGGG-3′ and reverse, 5′-AAGGAGCCAGGTGTTGTTG-3′; β-actin; forward, 5′-GCTCTGGCTCCTAGCACCAT-3′ and reverse, 5′-GCCACCGATCCACACAGAGT-3′. qPCR was conducted in a 50 μl reaction system, including 1 μl forward and 1 μl reverse primers, 0.5 μl 20x SYBR-Green I, 25 μl 2x PCR buffer, 2 μl cDNA, and 20.5 μl diethyl pyrocarbonate. The thermocycling conditions used for qPCR were as follows: 94°C for 4 min, 30 cycles of 94°C for 20 sec, 60°C for 30 sec, 72°C for 30 sec, and 72°C for 5 min. Each individual sample was run in triplicate and the level of expression was quantified using the comparative cycle threshold (Cq) method. Results were normalized to β-actin expression and RNA enrichments were calculated using the 2^−ΔΔCq method (19).

Splenic CD4⁺ T cells were purified from recipient mice that had received single LT with no treatment (n=5). Cells were labeled with 5 μM CFSE (cat. no. 65-0850-84; eBioscience; Thermo Fisher Scientific, Inc.) for 20 min at 37°C in the dark. Cold RPMI-1640 medium was then added and cells were incubated on ice for 5 min. CD4⁺ T cells were then co-cultured with TIM-4⁺ KCs (1:1). The following 5 experimental groups were constructed: The control group of cells that were untreated; the control mAb group of cells that were treated with control mAb (0.5 μg/ml, 500 μl); the TGF-β group of cells that were treated with additional exogenous TGF-β (1 ng/ml, 100 μl) following co-culture; the TIM-4 mAb group of cells that were treated with TIM-4 mAb (0.5 μg/ml, 500 μl); and the TGF-β+TIM-4 mAb group of cells that were treated with exogenous TGF-β and TIM-4 mAb. All cells were co-cultured for 96 h at 37°C. Following washing, cells were re-suspended in RPMI-1640 medium with 10% fetal bovine serum and then blocked with 10% FCS for 30 min at room temperature. Cells were then stained with APC-CD4 antibodies (cat. no. GK1.5, 1:100 dilution; eBioscience; Thermo Fisher Scientific, Inc.) in the dark for 30 min at room temperature, centrifuged at 3,000 × g for 10 min at 4°C and resuspended twice in PBS prior to fluorescence-activated cell sorting (FACS) analysis. All experiments were performed in triplicate.

Cells (KCs or T cells) were digested for 3 min in 0.25% trypsin at 37°C and collected following centrifugation at 50 × g for 5 min at room temperature. Cells were then rinsed in pre-cooled PBS (4°C) twice. Cells were then resuspended in 400 μl binding buffer and the cell concentration was diluted to 1×10⁶/ml. Fluorochrome-labeled mAbs, including CD14-phycoerythrin (PE; cat. no. Sa2-8; 12-0141, dilution, 1:100), CD163-APC (cat. no. GHI/61; 17-1639-42, dilution, 1:100), Foxp3-PE (cat. no. 150D/E4; 12-477, dilution, 1:100), CD25-fluorescein isothiocyanate (FITC; cat. no. CD25-4E3; 11-0257, dilution, 1:100), were purchased from eBioscience; Thermo Fisher Scientific, Inc. KCs were stained for CD14 and CD163 and T cells were stained for CD25 and Foxp3 for 1 h at 4°C in the dark. Flow cytometric cell sorting was performed using FACSAria; flow cytometric data were acquired using a FACSCalibur and analyzed using Cellquest version 5.1 software (BD Biosciences, Franklin Lakes, NJ, USA). Experiments were performed in triplicate.

Cardiac puncture blood samples were collected from mice following euthanasia 7 days post-operation and allowed to clot. Serum was obtained following centrifugation at 200 × g at 4°C for 10 min. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin in serum (TBIL) were determined using a Synchron CX7 analyzer (Beckman Coulter Inc., Brea, CA, USA) in the Clinical Biochemical Laboratory of Chongqing Medical University. Experiments were performed in triplicate.

Liver tissues were removed from mice 7 days following surgery. Then, liver tissues were sliced and washed repeatedly with saline and formed into homogenates using a tissue crusher. Liver homogenates were then centrifuged 3 times at 300 × g at 4°C for 15 min to obtain supernatants. TIM-4⁺ KCs were incubated with naive CD4⁺CD25^- T cells for 96 h at 37°C and were then centrifuged at 300 × g at 4°C for 10 min to obtain supernatants. Levels of inflammatory cytokines in liver homogenates or supernatants were assessed using the following ELISA kits: TNF-α (cat. no. MAT00B), IFN-γ (cat. no. MIF00), CCL2 (cat. no. MJE00), CXCL2 (cat. no. MM200), TGF-β (cat. no. MB100B), IL-4 (cat. no. M4000B), IL-6 (cat. no. M6000B) and IL-13 (cat. no. M1300CB; all R&D Systems, Inc.). All procedures were performed following the manufacturer's protocols. Absorbance was read at 450 nm and experiments were performed in triplicate.

To assess hepatic injury, liver tissues were fixed with 10% formaldehyde at room temperature for 4 h and then embedded in paraffin. Tissues were sliced into sections 5-μm thick prior to staining with hematoxylin for 10 min and eosin for 3 min at room temperature. AR was classified into four types using the Banff schema (20).

Apoptotic cells were detected using a commercial TUNEL kit (cat. no. 11684817910; Roche Inc., Switzerland) following the manufacturer's protocol. The TUNEL assay was performed for paraffin-embedded sections fixed with 4% paraformaldehyde for 1 h at room temperature and processed following the method described by Kitamoto et al (21). TUNEL + hepatocyte nuclei were quantified by calculating the mean number of TUNEL + hepatocytes in five random fields of view per animal with 100 nuclei counted per field. The mean percentage of apoptotic cells was calculated.

All values are expressed as the mean ± standard deviation. Data were analyzed using an unpaired two-tailed Student's t-test or one-way analysis of variance with Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant result. Graft survival was assessed using the Kaplan-Meier method and statistical differences were calculated using the log rank test.

KCs are important APCs that serve bimodal roles in the innate and adaptive immune response. It has been demonstrated that liver KCs engulf and degrade pathogenic microorganisms and remove endotoxins (22). To assess the expression of TIM-4 in KCs following hepatic AR, flow cytometry and immunohistochemistry were performed and the number of activated KCs in transplanted livers were assessed. The number of activated KCs in the LT group gradually increased with time following surgery compared with the sham group (Fig. 1A and B). KCs were then isolated from mice that had undergone LT and the expression of TIM-4 was measured using western blotting. Mice that had undergone LT exhibited significantly elevated TIM-4 expression 1, 3 and 7 days post-surgery (P<0.05 vs. sham group; Fig. 1C). The expression of TIM-4 on day 3 following surgery increased to a peak and remained at a relatively high level, indicating that the expression of TIM-4 is promoted by activated KCs following LT.

TIM-4 was demonstrated to be present in activated KCs at high levels; therefore, the role of TIM-4 signaling in the function of KCs was assessed. The fluorescent expression of KCs was markedly higher in the TIM-4 mAb group compared with the sham and control mAb groups (Fig. 2A). The effect of TIM-4 on KCs was determined using flow cytometric analysis. TIM-4 expression in the TIM-4 mAb group was almost entirely blocked compared with the control mAb group, indicating that the expression of TIM-4 in KCs was successfully disrupted (Fig. 2B).

Compared with the control mAb group, the blockade of KC TIM-4 expression significantly improved hepatic injury, as determined by the liver function indices of AST, ALT and TBIL (each P<0.05; Fig. 2C). Furthermore, it was revealed that TIM-4 disruption significantly reduced levels of the inflammatory cytokines TNF-α, IFN-γ, CCL2 and CXCL2 (each P<0.05 vs. control mAb group; Fig. 2D and F) and significantly enhanced the levels of TGF-β (P<0.05 vs. control mAb group; Fig. 2E) in the liver, thus reversing the effects of AR. Additionally, visible focal necrosis and vacuolization of liver parenchyma with lymphocyte infiltration was observed in the control mAb group and classified as severe AR according to the Banffe schema (20). However, tissues from mice in the TIM-4 mAb group exhibited reduced infiltration of lymphocytes surrounding the portal area. AR severity was determined following a previously described protocol (20) and the rejection activity index (RAI) was identified as being significantly lower than that of the control mAb group (P<0.05; RAI score: 4.2±0.7 vs. 8.5±0.5; Fig. 2G). These results suggest that the blockade of TIM-4 by mAb contributes to AR amelioration and inflammatory cytokine downregulation.

CL stimulates the depletion of macrophages in the liver (23). To determine the function of KCs in vivo, the livers of donor mice were treated with CL to destroy hepatic KCs. The RAI score of liver grafts treated with CL+TIM-4 mAb was associated with severe AR and there were no significant differences in RAI scores between the CL+TIM-4 mAb and CL+control mAb groups (Fig. 2H), suggesting that the disruption of TIM-4 expressed on KCs is critical to the improvement of AR, rather than the TIM-4 that is expressed on other hepatic APCs.

To analyze the possible pathways involved in this process, the expression of key phosphorylated proteins involved in the NF-κB and p38 MAPK signaling pathways, including p65 and p38 were assessed. A significant decrease in the expression of p-p65 and p-p38 was observed in mice following treatment with TIM-4 mAb (P<0.05 vs. control mAb group; Fig. 2I). Therefore, the blockade of KC TIM-4 may inhibit NF-κB and p38 MAPK signaling to improve liver transplant injury.

As aforementioned, it was determined that the blockade of TIM-4 expressed by KCs enhances the secretion of TGF-β, which is a protein that inhibits the polarization and facilitates the proliferation of Th1/Th2 and Tregs, respectively. Therefore, the effect of TIM-4 blockade (0.5 μg/ml) with exogenous TGF-β (1 ng/ml) on the differentiation of naive CD4⁺ T cells was assessed in vitro. TIM-4⁺ KCs were isolated from model mice and purified using FACS. There was no significant difference in the KC antigen-presenting ability between the control mAb and TIM-4 mAb groups. However, compared with the TIM-4 mAb group, the TGF-β+TIM-4 mAb group exhibited a significant decrease in KC antigen-presenting ability (P<0.05; Fig. 3A). These results indicate that the TIM-4 blockade does not decrease the antigen-presenting capacity of KCs, however additional TGF-β treatment does. TIM-4⁺ KCs were subsequently incubated with naive CD4⁺CD25⁻ T cells. It was revealed that the blockade of TIM-4 with added TGF-β caused a significantly greater reduction in CD4⁺CD25⁻ T cell proliferation compared with TGF-β and TIM-4 treatment alone (P<0.05; Fig. 3B). Furthermore, the measurement of supernatants co-cultured cohorts demonstrated that TGF-β treatment or TIM-4 blockade significantly decreased levels of the cytokines IL-4, IL-6 and IL-13 (each P<0.05 vs. control mAb; Fig. 3C). The combination of TGF-β and TIM-4 treatment further enhanced this effect (P<0.05; Fig. 3C), indicating that Th2 polarization was effectively inhibited. The proportion of Treg (CD4⁺CD25⁺Foxp3⁺) cells was significantly increased in the TGF-β and TIM-4 mAb groups compared with the control mAb group (P<0.05; Fig. 3D) and the proportion of Treg cells was significantly enhanced in the combined TGF-β+TIM-4 mAb group (P<0.05; Fig. 3D). These results suggest that the TIM-4 blockade serves a crucial role in directing the conversion of iTreg and that the addition of TGF-β treatment induces an even greater effect.

The TIM-4 blockade of KCs reduced the expression of IL-4 and triggered the conversion of iTregs. The addition of exogenous low-dose TGF-β amplified this effect. It has been demonstrated that STAT6 signaling inhibits iTreg by binding to the Foxp3 promoter, which directly interferes with the transcription of Foxp3 (24). To determine the role of the IL-4/STAT6/Gata3 pathway in the generation of iTregs, fluorescent cell sorting was performed to attain TIM-4⁺ and TIM-4⁻ KCs in the LT group. These were then co-cultured with naive CD4⁺ T cells and the expression of p-STAT6 in T cells was assessed. The results demonstrated that levels of p-STAT6 in CD4⁺ T cells co-cultured with TIM-4⁺ KCs were significantly higher than those in CD4⁺ T cells co-cultured with TIM-4⁻ KCs (P<0.05; Fig. 4A). TIM-4⁺/TIM-4⁻ KCs were then co-cultured with naive CD4⁺ T cells (which received either no pretreatment or pretreatment with TIM-4 mAb) to detect levels of p-STAT6. It was demonstrated that the addition of TIM-4 mAb significantly blocked the expression of p-STAT6 in CD4⁺ T cells co-cultured with TIM-4⁺ KCs (P<0.05), but did not affect p-STAT6 expression in CD4⁺ T cells co-cultured with TIM-4⁻ KCs (Fig. 4B). The exogenous addition of IL-4 to TIM-4⁺ KCs co-cultured with naive CD4⁺ T cells demonstrated that the inhibition of p-STAT6 expression induced by TIM-4 mAb was significantly reversed by IL-4 (P<0.05; Fig. 4C). Furthermore, the blockade of TIM-4 with additional TGF-β in the co-cultured model significantly promoted the expression of Foxp3 (P<0.05) and had a greater suppressive effect on Gata3 compared with TIM-4 blockade alone (P<0.05; Fig. 4D). Collectively, these data reveal that the generation of the iTreg phenotype by TIM-4 blockade is dependent on the inhibition of the IL-4/STAT6/Gata3 signaling pathway.

The effect of TIM-4 blockade in combination with TGF-β on AR was assessed in vivo. Recipient mice were injected with either anti-TIM-4 mAb (0.35 mg/mouse), 0.5 ml TGF-β (1 ng/ml) or the two in combination via murine portal veins to establish a model of LT. Treatment was administered continuously at the same dose for a total of 2 days following surgery. The results of hepatic function and apoptotic tests indicated that TIM-4 mAb combined with TGF-β induced a more significant improvement of liver injury compared with the TGF-β or TIM-4 mAb groups (P<0.05; Fig. 5A) and evidently significantly ameliorates hepatocyte apoptosis in LT models on day 7 following surgery (P<0.05; Fig. 5B). As the present study demonstrated, TIM-4 mAb combined with TGF-β induced the generation of significantly more CD4⁺CD25⁺Foxp3⁺ T cells than in mice treated with TGF-β or TIM-4 mAb alone (P<0.05; Fig. 5C). The mean survival time of mice treated with TGF-β and TIM-4 mAb was markedly extended (>100 days) and was significantly longer than those that received TGF-β or TIM-4 mAb alone (P<0.05; Fig. 5D). These results indicate that TIM-4 blockade combined with additional TGF-β may ameliorate AR more effectively and potentially induce the formation of immunosuppressive cells.