BTLA–HVEM Checkpoint Axis Regulates Hepatic Homeostasis and Inflammation in a ConA-Induced Hepatitis Model in Zebrafish

Wei Shi, Tong Shao, Jiang-yuan Li, Dong-dong Fan, Ai-fu Lin, Li-xin Xiang and Jian-zhong Shao
J Immunol November 1, 2019, 203 (9) 2425-2442; DOI: https://doi.org/10.4049/jimmunol.1900458

Key Points

  • BTLA–HVEM checkpoint axis regulates hepatic homeostasis and inflammation.

  • Engagement of BTLA by HVEM triggers inhibitory signals for CD8+ T cell tolerization.

  • A potential BTLA–HVEM–LIGHT modulatory network is involved in hepatitis.

Abstract

The BTLA−HVEM checkpoint axis plays extensive roles in immunomodulation and diseases, including cancer and autoimmune disorders. However, the functions of this checkpoint axis in hepatitis remain limited. In this study, we explored the regulatory role of the Btla–Hvem axis in a ConA-induced hepatitis model in zebrafish. Results showed that Btla and Hvem were differentially expressed on intrahepatic Cd8+ T cells and hepatocytes. Knockdown of Btla or Hvem significantly promoted hepatic inflammation. Btla was highly expressed in Cd8+ T cells in healthy liver but was downregulated in inflamed liver, as evidenced by a disparate proportion of Cd8+Btla+ and Cd8+Btla– T cells in individuals without or with ConA stimulation. Cd8+Btla+ T cells showed minimal cytotoxicity to hepatocytes, whereas Cd8+Btla– T cells were strongly reactive. The depletion of Cd8+Btla– T cells reduced hepatitis, whereas their transfer enhanced hepatic inflammation. These observations indicate that Btla endowed Cd8+Btla+ T cells with self-tolerance, thereby preventing them from attacking hepatocytes. Btla downregulation deprived this tolerization. Mechanistically, Btla–Hvem interaction contributed to Cd8+Btla+ T cell tolerization, which was impaired by Hvem knockdown but rescued by soluble Hvem protein administration. Notably, Light was markedly upregulated on Cd8+Btla– T cells, accompanied by the transition of Cd8+Btla+Light– to Cd8+Btla–Light+ T cells during hepatitis, which could be modulated by Cd4+ T cells. Light blockade attenuated hepatitis, thereby suggesting the positive role of Light in hepatic inflammation. These findings provide insights into a previously unrecognized Btla–Hvem–Light regulatory network in hepatic homeostasis and inflammation, thus adding a new potential therapeutic intervention for hepatitis.

Introduction

T cell activation is strictly regulated by numerous costimulatory and coinhibitory molecules that comprise diverse immune checkpoint axes of adaptive immunity (1). These immune checkpoint axes are crucial for self-tolerance, which prevents the immune system from indiscriminately attacking cells to maintain homeostasis (2, 3). The dysregulation of immune checkpoint axes leads to various diseases, including cancer, inflammation, and autoimmune disorders (4, 5). Among the growing family of checkpoint inhibitors, the B and T lymphocyte attenuator (BTLA), which is associated with the herpesvirus entry mediator (HVEM), is a promising target for immunotherapy because of its great potential use in multiple types of cancer and other diseases (5–7).

BTLA is an Ig superfamily member that mainly serves as a coinhibitor similar to programmed cell death 1 (PD-1) and CTL-associated protein 4 (CTLA-4) in humans. This ligand is a type I membrane glycoprotein that contains a type V Ig domain in its extracellular region and three functional tyrosine residues (Tyrs) embedded in one growth factor receptor–bound protein 2 (Grb2) binding site and two immunoreceptor tyrosine-based motifs in its cytoplasmic tail, which are essential for the recruitment of Grb2 and Src homology phosphatase-1/-2 (6). HVEM is a member of the TNFR superfamily and is a type I membrane protein with an N terminus extracellular region and a cytoplasmic segment closely associated with TNFR-associated factors (TRAFs) and STAT3 signaling pathways (8–10). The ectodomain of HVEM is composed of four cysteine-rich domains (CRDs). CRD1 is combined with BTLA and CD160, whereas CRD2 and CRD3 are mainly responsible for recognizing lymphotoxin-α and LIGHT (lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed on T cells) (11–13). BTLA is predominantly expressed in B cells and various T cells, including Th1, Tfh, Th17, γδ T, and NKT cells (14–16). HVEM is extensively expressed in dendritic cells, epithelial cells, hematopoietic cells, B lymphocytes, and hepatoma cells (12, 17, 18). The engagement of BTLA by HVEM triggers the inhibitory activity of the former on T and B cell activation by decreasing the accumulation of phosphorylated TCR and BCR signals. This occurrence leads to the tolerance or anergy of CD4+ T and CD8+ T cells and inhibition of cytokine release of NKT cells to avoid excessive inflammatory reactions and tissue injury (19–21). The BTLA–HVEM interaction can be regulated by LIGHT, the latter of which is also known as TNF superfamily member 14 (TNFSF14) (22). LIGHT, a type II membrane protein with a C terminus extracellular TNF homology domain (THD), assembles in homotrimers and modulates immune responses by interacting with three TNFR superfamily members, namely, HVEM, lymphotoxin β receptor, and decoy receptor 3 (22–24). Two LIGHT isoforms, namely, membrane-bound LIGHT (mLIGHT) and soluble LIGHT (sLIGHT), have been identified in humans and mouse models. mLIGHT can break the interaction between BTLA and HVEM because its affinity to HVEM is higher than that of BTLA to HVEM. By contrast, sLIGHT tends to combine with the BTLA–HVEM complex and firms the link between the two components because of the opposite binding region of BTLA and LIGHT to HVEM (25–27). Thus, mLIGHT disrupts the inhibitory effect of BTLA on T cells by competitively binding to HVEM, whereas sLIGHT enhances the functional role of BTLA (25, 27).

BTLA, HVEM, and LIGHT are closely associated with various cancers, such as melanoma; hepatocellular carcinoma; diffuse large B-cell lymphoma; invasive breast and colon cancers (5, 28–30); autoimmune disorders, including systemic lupus erythematosus, experimental autoimmune encephalomyelitis, type 1 diabetes, spontaneous urticaria, lung fibroblasts, and dermatitis (6, 31, 32); and inflammatory diseases, such as intestinal inflammation and diet-induced obesity (4, 33). However, investigations on the functional roles of BTLA, HVEM, and LIGHT in hepatitis remain limited. Although the involvement of BTLA and HVEM in hepatitis has been preliminarily explored, the results have been elusive because of some controversial conclusions from different research groups. For example, BTLA-deficient (BTLA−/−) mice display autoimmune hepatitis-like features with CD4+ T and NKT cell infiltration, spotty necrosis in the liver, and elevated transaminase levels in serum (34, 35). In accordance with this observation, HVEM-deficient (HVEM−/−) mice exhibit increased morbidity and mortality, with high levels of multiple proinflammatory cytokines in a ConA-induced hepatitis model, depending on the presence of CD4+ T cells (36). However, HVEM−/− mice also show low serum transaminase and IFN-γ levels, high protective IL-22 serum levels, and attenuated liver histopathology in a liver invariant NKT cell–dependent manner (37). These observations suggest that intensive studies are warranted to further explore the complex regulatory mechanisms underlying BTLA, HVEM, and LIGHT interactions during hepatic inflammation.

In this study, the functional roles of Btla, Hvem, and Light in liver homeostasis and inflammation were explored in a ConA-induced hepatitis model in zebrafish (Danio rerio), which is attractive for the study of comparative immunology and diseases (38). The Btla–Hvem interaction between intrahepatic Cd8+ T cells and hepatocytes contributes to liver homeostasis. The engagement of Btla by Hvem triggers inhibitory signals for the tolerization of Cd8+ T cells. The breakdown of the Btla–Hvem interaction by upregulated Light on Cd8+ T cells activates the cytotoxicity of Cd8+ T cells, thereby leading to the disruption of liver homeostasis and occurrence of hepatitis. These findings suggest the importance of the Btla–Hvem axis and Btla–Hvem–Light network in the liver and reveal a previously unrecognized mechanism underlying hepatic homeostasis and inflammation.

Materials and Methods

Experimental fish

One-year-old wild-type AB zebrafish (D. rerio) with body weight of 0.5–1.0 g were raised and maintained at 28°C on a 12 h/12 h light/dark cycle in a standard circulating system, as previously described (39). All fish used in experiments were siblings generated after at least two generations of inbreeding. Only healthy fish, as determined by general appearance and activity level, were used. All the animal experiments were conducted in accordance with the guiding principles for the care and use of laboratory animals and were approved by a local ethics committee.

Molecular cloning

The genome databases maintained by the National Center for Biotechnology Information, the Genome Browser of the University of California Santa Cruz, and Ensembl were used to predict zebrafish btla, hvem, and light homologs. Total RNA was extracted from zebrafish livers by using an RNAiso Plus kit (Takara Bio). The btla, hvem, and light cDNAs were amplified by RT-PCR with primers shown in Supplemental Table I. The cDNA products were ligated into pGEM-T easy vectors (Promega) and sequenced on a 3730 XL sequencer (Applied Biosystems), as previously described (39).

Bioinformatics analysis

Genome locations of btla, hvem, and light genes were retrieved from the Genome Data Viewer in the National Center for Biotechnology Information database. Primers for gene cloning were predicted by the Primer-BLAST program. Gene organization was elucidated by comparing cDNAs of btla, hvem, and light with genome sequences by using BLAT and drawn by GeneMapper 2.5. Multiple alignments were analyzed using Clustal X (version 1.8) and GeneDoc. Putative extracellular, transmembrane, and cytoplasmic regions were predicted by TMHMM Server (version 2.0). Phylogenetic trees were generated by MEGA 7.0 with the neighbor-joining or maximum likelihood method. Potential tertiary structures and functional domains were predicted using PROSITE, SWISS-MODEL, and PyMOL software (40).

Plasmid constructions

The encoding sequences for Btla, Hvem, and Light proteins and their ectodomains were amplified by RT-PCR with primers shown in Supplemental Table I. After gel extraction and digestion, the sticky fragments were inserted into pET41a (Invitrogen), pcDNA6/myc-His (Invitrogen), pEGFPN1 (BD Biosciences), pEGFPC1 (BD Biosciences), and pAcGHLTc (AB Vector) vectors to construct prokaryotic or eukaryotic expression vectors with GST, Myc, enhanced GFP (EGFP), and His tags, respectively. The resulting constructs designated as pET41a-btla, pAcGHLTc-hvem, pEGFPN1-btla, pEGFPC1-btla, pEGFPC1-hvem, pEGFPC1-light, pcDNA6/myc-His-hvem, and pcDNA6/myc-His-light were used for recombinant protein expression and subcellular localization examination.

Preparation of recombinant proteins

For prokaryotic expression of soluble Btla (sBtla) protein with ectodomain, pET41a-btla was transformed into BL21 (DE3; TransGen Biotech) competent cells, cultured in Luria–Bertani medium containing kanamycin (50 mg/l; Sangon Biotech) at 37°C with 200 rpm shaking, and induced by isopropyl-b-d-thiogalactoside (IPTG, 0.5 mM; Sangon Biotech) at 20°C for 12 h. After ultrasonication, the supernatants were collected for purification. For eukaryotic expression, pAcGHLTc-hvem and baculovirus vector DNA (AB Vector) were cotransfected into Sf9 (Spodoptera frugiperda) cells under the assistance of polyethylenimine (branched PEI; Sigma-Aldrich) in a T25 flask with SIM HF medium (Sino Biological) containing penicillin–streptomycin (Thermo Fisher Scientific). The cells were cultured at 28°C for 5 d, harvested by centrifugation (2000 rpm), and dissolved in lysing buffer (200 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 1 mM PMSF). The recombinant Btla–GST and Hvem–His proteins were purified by nickel–nitrilotriacetic acid agarose affinity chromatography (Qiagen), following the manufacturer’s manual, and then detected by SDS-PAGE (41).

Preparation of polyclonal Abs

The recombinant sBtla and sHvem proteins and an epitope peptide with 16 aa (SLHPKIPRPSIENSFL) predicted from the ectodomain of the Light protein were used to prepare Abs against Btla, Hvem, and Light proteins. The epitope peptide was chemically synthesized and conjugated with OVA (BankPeptide). Four-week-old male Institute of Cancer Research mice (∼15 g) or 6-wk-old male New Zealand rabbits (∼1.5 kg) were immunized with the proteins or peptide (20 μg or 0.5 mg) each time in CFA (Sigma-Aldrich) initially and then in IFA (Sigma-Aldrich) for four times thereafter at biweekly intervals, as previously described (39). Seven days after the final immunization, serum samples were collected. Abs were affinity purified by using Protein A Agarose Columns (Thermo Fisher Scientific), and their titers were examined by ELISA. The validity and specificity of the Abs were determined by Western blot analysis. Rabbit anti-Cd8α Ab and mouse anti-Cd4 Ab were produced in our previous studies (39, 42), and rabbit anti–betaine homocysteine S-methyltransferase (anti-BHMT) Ab was purchased from Thermo Fisher Scientific.

Quantitative real-time PCR

The transcript abundance of the target genes was analyzed via quantitative real-time PCR (qRT-PCR) on a CFX Connect Real-Time PCR Detection System (Bio-Rad). Total RNA was extracted from the samples by using an RNAiso Plus kit (Takara Bio) and reverse transcribed into cDNAs. The PCR experiments were performed in a total volume of 10 μl by using an iTaq Universal SYBR Green Supermix (Bio-Rad). The reaction mixtures were incubated for 2 min at 95°C, followed by 40 cycles of 15 s at 95°C, 15 s at 60°C, and 20 s at 72°C. Relative expression levels were calculated using 2−∆ cycle threshold and 2−∆∆ cycle threshold methods with rps18 (40S ribosomal protein S18) for normalization (43). Each PCR trial was run in triplicate parallel reactions and repeated three times. The primers used were listed in Supplemental Table I and were checked to have well efficiency.

Subcellular localization

HEK293T cells were seeded into 12-well plates (Corning) with cover glass and cultured in high-glucose DMEM (Life Technologies) in which 10% (v/v) FBS (Life Technologies) was added at 37°C in 5% CO2 to allow growth until 50–60% confluence. The cells in each well were transfected with 0.8 μg of pEGFPC1-btla, pEGFPC1-hvem, or pEGFPC1-light plasmid DNA combined with PEI reagent (3.2 μg per well) in accordance with the manufacturer’s protocol. At 48 h after the transfection, the cells were fixed by 4% (mass/volume) paraformaldehyde (PFA; Sigma-Aldrich) and stained with CM-DiI (1 μM; Thermo Fisher Scientific) and DAPI (100 ng/ml; Sigma-Aldrich). Fluorescence images were captured using a two-photon laser confocal scanning microscope (LSM-710; Zeiss) with ×630 magnification.

Generation of short hairpin RNAs encoding lentivirus

Short hairpin RNAs (shRNAs) carrying the small interfering RNAs (siRNAs) targeting btla and hvem mRNAs and shRNAs encoding lentiviruses (LVs) were designed and produced, as previously described (39). shRNAs were constructed into a pSUPER plasmid (pSUPER.retro.puro; OligoEngine) downstream of the H1 promoter. The constructs were transfected into HEK293T cells with pEGFPC1-btla or pEGFPC1-hvem for efficiency evaluation. The U6 promoter cassette in a lentiviral plasmid (pLB) was replaced by an H1–shRNA cassette from the constructs harboring effective shRNAs to produce pLB-sibtla and pLB-sihvem vectors. The shRNAs encoding LVs were generated by cotransfecting HEK293T cells with pLB-sibtla or pLB-sihvem and packaging vectors (pCMV-VSVG and pCMV-dR8.2). The viral supernatant was concentrated by ultracentrifugation (25,000 rpm, 90 min, 4°C). Viral titers were detected through EGFP signature in HEK293T cells under the fluorescent microscope or by flow cytometry (FCM) analysis. The silencing activity of the resulting LVs was determined in zebrafish intrahepatic leukocytes and Cd8+ T cells and hepatocytes by qRT-PCR or Western blot analysis after fish were i.p. injected with the LVs (2 × 105 transducing units [TU] per fish) once every 24 h for three to five times with or without ConA stimulation.

Immunofluorescence staining

Colocalizations of Cd8α and Btla, Cd8α and Light, and Bhmt and Hvem were determined by immunofluorescence staining. Leukocytes were isolated from the liver of zebrafish by Ficoll–Hypaque (1.080 g/ml; Sangon Biotech) centrifugation at 2500 rpm at 25°C for 25 min. The hepatocytes were separated from the liver digested with type IV collagenase (1.0 U/ml, at room temperature for 30 min; Sigma-Aldrich) by filtration through a 40-μm strainer (Falcon; BD Biosciences) and centrifugation at 100 × g at 4°C for 5 min. The cells were fixed with 4% PFA at room temperature for 10 min, blocked with 2% BSA (BSA; Sigma-Aldrich), and incubated with primary Abs in combinations of rabbit anti-Cd8α and mouse anti-Btla, rabbit anti-Cd8α and mouse anti-Light, and rabbit anti-BHMT and mouse anti-Hvem Abs at 4°C for 2 h. After washing with PBS, the cells were combined with FITC-conjugated goat anti-rabbit IgG and PE-conjugated goat anti-mouse IgG secondary Abs (Thermo Fisher Scientific), following the manufacturer’s instructions. The cells were washed with PBS and stained with DAPI (100 ng/ml) at room temperature for 5 min. Fluorescence images were captured using a two-photon laser confocal scanning microscope (LSM-710; Zeiss) with ×630 magnification.

Coimmunoprecipitation and Western blot analysis

Coimmunoprecipitation (Co-IP) was performed to detect the interaction between Btla and Hvem. HEK293T cells were cotransfected with pcDNA6/myc-His-hvem (3 μg) and pEGFPN1-btla (3 μg) in a 10-cm dish under the assistance of PEI. At 48 h posttransfection, the cells were lysed with precooling cell lysis buffer (Beyotime). The lysates were centrifuged (10,000 rpm at 4°C for 10 min), and the supernatants were incubated with mouse anti-EGFP mAb (Abmart) at 4°C overnight. The mixture was incubated with 50 μl of protein A agarose beads (Thermo Fisher Scientific) for 4 h. The beads were washed three times with lysis buffer, mixed with loading buffer, and heated to 100°C for 10 min to denature the proteins. After centrifugation, the proteins were separated by SDS-PAGE and transferred onto a 0.22-μm polyvinylidene difluoride membrane (PVDF; EMD Millipore) for Western blot analysis. After blocking with 2% BSA at 4°C for 1 h, the membrane was incubated with primary Abs at 4°C for 2 h, washed with TBST, and incubated with HRP-conjugated goat anti-mouse/rabbit IgG mAb (Abmart) at 4°C for 1 h. Detection was performed on a gel imaging system (Tanon 4500).

ConA-induced hepatitis model in zebrafish

ConA-induced hepatitis model was established by i.p. injection of zebrafish with various doses of ConA (50 μg to 300 μg/g body weight; Sigma-Aldrich) for different time periods (12–72 h) for optimization. Hepatitis was evaluated by the release levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in sera; histopathologic symptoms; expression of proinflammatory cytokines (Il-1β, Il-6, Tnf-α, and Ifn-γ); perforin; lysosomal-associated membrane protein 1 (Lamp-1); apoptotic regulators, including Fas ligand (Fasl) and B cell lymphoma 2 (Bcl-2) in livers; and apoptosis of hepatocytes through in situ detection and FCM analysis. Serum ALT and AST levels were detected using ALT and AST Assay Kits (Jiancheng Bioengineering Institute, Nanjing). The liver tissues were fixed in 4% PFA overnight. The paraffin sections were collected for H&E, immunohistochemical staining, and TUNEL assay for histopathology and in situ hepatocyte apoptosis analyses using a TUNEL Detection Kit (Beyotime). For immunohistochemical staining, the sections were blocked in 2% BSA at 25°C for 1 h and incubated with primary Abs (mouse anti-Btla/Hvem/Light and rabbit anti-Cd8α) or isotype control mouse/rabbit IgG (Sangon Biotech) at 4°C overnight after retrieving Ags and blocking endogenous peroxidase. Secondary Abs (HRP-conjugated goat anti-mouse/rabbit IgG Abs, Abmart) were incubated at 4°C for 1 h, and color was developed using a DAB mixture (Beyotime). Hematoxylin was stained to show the nuclei. Pictures were caught under a Zeiss microscope (Zeiss Axiostar Plus).

FCM analysis and sorting

Cells under detection or sorting were blocked with 1% goat serum for 1 h at 4°C and incubated with the defined primary Abs for 1 h at 4°C. Nonspecific rabbit or mouse IgG was served as the isotype control. After washing three times with D-Hank’s buffer, the cells were incubated with secondary Abs (PE-conjugated goat anti-mouse IgG mAb and FITC-conjugated goat anti-rabbit IgG mAb) for 1 h at 4°C. The cells were detected or sorted by the flow cytometer (FACSCalibur or FACSJazz; BD Biosciences). FCM analysis for lymphocytes was performed following previously described protocols (39, 42). At least 10,000 cells were acquired from the gate for analysis. FlowJo 7.6 software (BD Biosciences) was used for data processing. For hepatic apoptosis analysis, the hepatocytes were separated from the livers digested with type IV collagenase as described above, washed three times with PBS by centrifugation, resuspended in 1 × annexin-binding buffer, and labeled by Annexin V and propidium iodide following the protocol recommended by the manufacturer (Thermo Fisher Scientific). At least 10,000 cells were acquired from the gate for FCM analysis.

Deletion and adoptive transfer assays

Deletion and adoptive transfer assays were performed for functional evaluation of the cytotoxic activity of Cd8+ T cells and the regulatory role of the Btla–Hvem axis to the tolerization of Cd8+ T cells. For deletion assay, zebrafish was i.p. injected with anti-Cd8α Ab (20 μg/g body weight) three times in a 24-h interval, as described in our previous studies (39, 42). At the third administration, the fish were coinjected with Ab and ConA. One day after the last injection, the deletion efficiency of Cd8+ T cells was examined by FCM, and the level of hepatitis was assessed, as described above. For adoptive transfer assay, intrahepatic Cd8+, Cd8+Btla+, Cd8+Btla–, Cd8+Light+, and Cd8+Light– T cells were sorted from the donor zebrafish livers with or without ConA stimulation and i.p. transferred into the ConA stimulation–recipient fish at different cell dosages (104–106 cells per fish). In a specified blockade assay, Cd8+Btla+ and Cd8+Btla– or Cd8+Light+ and Cd8+Light– T cells were incubated with anti-Btla Ab or anti-Light Ab at 4°C for 1 h before transfer. In a knockdown assay, Cd8+Btla+ T cells were transferred into recipient fish receiving ConA (200 μg ConA/g body weight), sihvem-LV and ConA (2 × 105 TU per fish three times at a 24-h interval in combination with ConA at the last time of viral injection), and ConA, sihvem-LV, and sHvem (ConA and sihvem-LV in combination with sHvem at a dose of 2 μg/g body weight at the last time of viral injection). Cd8+, Cd8+Btla+, and Cd8+Btla– T cells were sorted from the liver by flow cytometer (FACSJazz; BD Biosciences), as mentioned above. Mock PBS was administered in the control groups. The effect of adoptive transfer was determined by the changes in serum ALT and AST levels and apoptosis of hepatocytes.

Cytotoxicity assay

The cytotoxicity of Cd8+Btla+ or Cd8+Btla– T cells to hepatocytes was examined by the release of lactate dehydrogenase by using a CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega). The sorted intrahepatic Cd8+Btla+ or Cd8+Btla– T effector cells were cocultured with the target hepatocytes in 96-well plate in different proportions (1:40, 1:20, 1:10, or 1:5) at 28°C for 4 h. In a specified blockade assay, Cd8+Btla+ T cells were pretreated with anti-Btla Ab (5 μg/ml) or nonspecific mouse IgG (isotype control, 5 μg/ml) at 4°C for 1 h. After incubation, the plate was centrifuged at 250 × g for 4 min, and the supernatant (50 μl) was collected from each well and mixed with the CytoTox 96 reagent (50 μl) for enzymatic assay. After the 30-min reaction in an opaque box, the stop solution (50 μl) was added to each sample. Absorbance (OD490) was detected by a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). The cytotoxicity (percentage) of the effector cells was calculated following a previously described protocol (44).

Functional evaluation of Cd4+ T cells

Coculture assay with Cd4+, Cd8+, and Cd8+Btla+ T cells was performed in a Transwell device (Corning) to evaluate the functional role of Cd4+ T cells in the transition of Cd8+Btla+ to Cd8+Btla– T cells and Cd8+Btla+Light– to Cd8+Btla–Light+ T cells. Cd4+, Cd8+, or Cd8+Btla+ T cells were isolated from the liver with or without ConA stimulation through FACS (FACSJazz; BD Biosciences). Cd4+ T cells (1 × 104) were added into each Transwell chamber with a 5.0-μm pore polycarbonate membrane. Cd8+ or Cd8+Btla+ T cells (2 × 104) were loaded per well in a 12-well plate. The cells were cultured in L-15 medium (Thermo Fisher Scientific) containing 10% FBS and penicillin–streptomycin. After 1 or 3 d of culture, the expression of Ifn-γ, Fasl, perforin, and Lamp-1 in Cd8+Btla+ T cells was examined by qRT-PCR. T-bet, Il-12 (p35), Il-12 (p40), Ifn-γ, Il-4, Cd154, and lymphocyte protein tyrosine kinase (Lck) in Cd4+ T cells with or without ConA stimulation were also examined by qRT-PCR. The expression level of Btla on Cd8+Btla+ T cells and the proportion changes in Cd8+Btla+Light– and Cd8+Btla–Light+ T cells were determined by FCM with primary (rabbit anti-Btla and mouse anti-Light) and secondary (FITC-conjugated goat anti-rabbit IgG and PE-conjugated goat anti-mouse IgG) Abs. Cd8+ T cells cultured alone without Cd4+ T cells were examined to show the initial expression levels of Btla and Light in Cd8+ T cells. Unlabeled Cd8+ T cells were adopted as negative control in the FCM analysis.

Statistical analysis

Statistical differences among means of experimental groups were evaluated by ANOVA and multiple Student tests. All data were presented as the mean ± SD of each group. Statistical significance was considered when p < 0.05 or p < 0.01. The sample number for each group was at least 20 fish of equal mean body weight. All data represented the means of at least three independent experiments.

Results

Identification of zebrafish btla, hvem, and light genes

With human (Homo sapiens) BTLA (HsBTLA), HVEM (HsHVEM), and LIGHT (HsLIGHT) gene sequences as queries, the corresponding zebrafish btla, hvem, and light homologous genes were predicted from the zebrafish genome database. The btla/hvem/light gene was located within a 2.98/4.20/12.68 kb genomic fragment on chromosome 1/8/3 and contained 7/8/4 exons and 6/7/3 introns, respectively. The genes adjacent to btla/hvem/light loci shared an overall conserved chromosome synteny to human genes, but some of the genes were in reverse order at the locus upstream or downstream of the btla/hvem/light gene relative to that of humans (Fig. 1A left; Fig. 1A right; Fig. 1D). Exon organization and encoding sequences for functional domains in the btla/hvem/light gene were also similar to those in the HsBTLA/HsHVEM/HsLIGHT gene or mouse (Mus musculus) BTLA/HVEM/LIGHT (MmBTLA/MmHVEM/MmLIGHT) gene (Fig. 1B, 1C, 1E). The cloned btla/hvem/light cDNA consisted of 1408/1604/3536 bp with an 80/433/327 bp 5′UTR, 927/813/708 bp open reading frame encoding 308/270/235 aa, and 401/358/2501 bp 3′UTR (Supplemental Fig. 1A, 1B), respectively. One alternatively spliced variant of Btla, which lacked 5 aa in the extracellular region, was cloned from zebrafish instead of two other variants lacking the extracellular Ig domain or transmembrane region in mammalian BTLAs (Fig. 1F).

FIGURE 1.
FIGURE 1.

Molecular characterization of zebrafish (D. rerio) btla, hvem, and light genes. (A) Chromosomal localization analysis between btla and human (H. sapiens) BTLA genes and between hvem and human HVEM genes. The arrows indicate the transcriptional direction of the genes. (B and C) Gene organization of zebrafish btla, human, and mouse (M. musculus) BTLA genes (B) and zebrafish hvem, human, and mouse HVEM genes (C). The black squares, fold lines, and numbers represent exons, introns, and size of corresponding exons or introns, respectively. (D) Chromosomal localization analysis between zebrafish light and human LIGHT genes. The arrows indicate the transcriptional direction of the genes. (E) Gene organization of zebrafish light, human, and mouse LIGHT genes. The black squares, fold lines, and numbers represent exons, introns, and size of corresponding exons or introns, respectively. (F) Structural characterization of alternative spliced variants of Btla. The signs above the schematic show the amino acid residues or amino acid sequence encoded by the variants. The GenBank accession numbers of the sequences are as follows: H. sapiens BTLA, NM_181780.3; M. musculus BTLA, NM_001037719.2; D. rerio btla variant 1, MK112054; D. rerio btla variant 2, MK112055; H. sapiens HVEM, NM_001297605.1; M. musculus HVEM, NM_178931.2; D. rerio hvem, MK112056; H. sapiens LIGHT, NM_003807.4; M. musculus LIGHT, NM_019418.3; and D. rerio light, NM_001281995.1. CP, cytoplasmic region; SP, signal peptide; THD, TNF homology domain; TM, transmembrane region; YCR, tyrosine conserved region.

Structural characterization of btla, hvem, and light proteins

Btla was predicted as a type I transmembrane protein with the typical structural features of the Ig superfamily. The protein contained three major functional domains, including a single extracellular V-type Ig-like domain (40–136 aa), a transmembrane region (167–189 aa), and a cytoplasmic tail (190–308 aa). One ITIM and one immunoreceptor tyrosine-based switch motif were present in the distal cytoplasmic tail region, which also included two conserved Tyrs (Tyr277 and Tyr302) (Supplemental Fig. 2A). The functional domains of Btla exhibited overall conserved tertiary structures compared with those of HsBTLA and MmBTLA, but they shared moderate identities (30–32%) and minor divergence with their mammalian counterparts. For example, the N-terminal Ig-like domain of Btla was folded with two α helices and two β sheets with eight strands. One sheet was composed of B, E, and D strands, whereas the other consisted of A′, G, F, C, and C′ strands. The comparison indicated that the analogy model of HsBTLA displayed one α helix and two β sheets with a total of nine strands, including B, E, and D in one sheet and A′, C, C′, F, G0, and G in another sheet. Six cysteine residues were conserved between Btla and HsBTLA. These cysteine residues formed three disulfides between C33 and C64, C57 and C120, and C73 and C80. The C73–C80 disulfide connected strands C and C′, whereas the C57–C120 disulfide connected strands B and F, similar to the case in HsBTLA. Residues 34–42 (DVKLKVPRQ) in the Ig-like domain of Btla were similar to residues 35–43 (DVQLYIKRQ) in HsBTLA, the binding site to HVEM (Supplemental Fig. 1C). Some imperceptible differences still existed between Btla and HsBTLA/MmBTLA, despite their numerous similarities. For example, the Grb2 binding site (YDND-containing motif), which was present in the cytoplasmic tail of HsBTLA and MmBTLA, was absent in Btla and some other fish Btlas, such as carp Btla. This observation suggests a slight functional difference in intracellular signaling between Btla and mammalian BTLAs.

Hvem was also predicted as a type I transmembrane protein containing an extracellular region (1–195 aa) rich in cysteines (22 residues), a single transmembrane region (196–218 aa), and a cytoplasmic region (219–270 aa) (Supplemental Fig. 2B). The extracellular region of Hvem possessed four CRDs in different lengths, including CRD1 (24–59 aa), CRD2 (61–103 aa), CRD3 (111–146 aa), and CRD4 (149–187 aa). These structural features are typically observed in mammalian TNF superfamily members occupying one to six CRDs of ∼40 aa each. Hvem shared 41% amino acid sequence identity with that of HsHVEM. The domain architecture of CRDs was highly conserved between HsHVEM and Hvem. In HsHVEM, CRD1 was critical for the interaction of HsHVEM with HsBTLA through residues 73–77 (TVCEP), which was mainly located on CRD1. A loop structure formed by the disulfide bonds was also present within C57 and C75 as well as C54 and C67, a position named DARC (glycoprotein D and BTLA binding site on the TNFR HVEM in CRD1). A similar structure of DARC was also predicted in Hvem, in which a potential Btla binding site (residues 57–61, TTCVP) and a loop composed by the disulfide bonds within C41 and C59 as well as C38 and C51 were present (Supplemental Fig. 1D). The CRD1 and CRD2 of Hvem individually contained one sheet with two strands, resembling those of HsHVEM. A threonine phosphorylation site (Thr251) was predicted in the cytoplasmic region of Hvem, whose counterpart (Thr277) in HsHVEM is crucial for the recruitment of TRAFs (TRAFs 1, 2, 3, and 5) and activation of PKCθ downstream HsHVEM signaling pathway. Hence, Hvem may activate a signaling pathway (such as the TRAFs–PKC axis) similar to that of HsHVEM, thereby leading to NF-κB activation.

Light was predicted as a type II transmembrane protein consisting of a cytoplasmic region with N terminus (1–38 aa), a single transmembrane region (39–61 aa), and an extracellular region with C terminus (63–235 aa) (Supplemental Fig. 2C). Light shared 43% amino acid sequence identity to HsLIGHT. Light and HsLIGHT were composed of three protomers, with each protomer containing one α helix and two β sheets. The two β sheets contained a total of 10 β strands. Light showed the typical “jelly-roll” fold model similar to HsLIGHT, in which two β sheets were formed by strands A′, A, H, C′, and F (inner β sheet) and strands B′, B, G, D, and E (outer β sheet). A GH loop structure that linked the inner and outer β sheets and contributed to the interaction between HsLIGHT and HsHVEM in humans also existed in Light, thereby suggesting the potential interaction of Light with Hvem in zebrafish (Supplemental Fig. 1E). In the distal cytoplasmic region of Light, a potential serine phosphorylation site (Ser10) was predicted, whose counterpart in HsLIGHT (Ser10) is responsible for the activation of PKC-mediated ERK, PI3K, and NF-κB signaling pathways. This finding implies that Light plays similar functional roles by conserved signaling pathways. The phylogenetic analysis showed that Btla, Hvem, and Light were clustered with other BTLAs, HVEMs, and LIGHTs in different species with high bootstrap probability (Supplemental Fig. 1F, 1G).

Preparation of recombinant proteins, Abs, and siRNA-encoding LVs

Recombinant Btla, Hvem, and Light proteins with either the ectodomain or intact molecules were prepared from Escherichia coli, Sf9, and HEK293T cell lines with a GST or Myc tag (Supplemental Fig. 3A–C). Abs against Btla (anti-Btla), Hvem (anti-Hvem), and Light (anti-Light) were prepared from immunized mouse or rabbit sera into IgG isotypes by employing protein A affinity purification. The results showed that Abs exhibited high specificities to Btla, Hvem, and Light proteins from liver leukocytes or hepatocytes, with average titers above 1:10,000 based on ELISA and Western blot analyses (Supplemental Fig. 3A–C). Rabbit anti-BHMT Ab was also confirmed to be effective and specific to zebrafish Bhmt molecules, as determined by Western blot and FCM analyses (Supplemental Fig. 3D, 3E). To prepare siRNA-encoding LVs against btla and hvem, we predicted a total of six candidate siRNAs (sibtla-1 to sibtla-3 and sihvem-1 to sihvem-3) targeting different regions of btla and hvem mRNAs. sibtla-3 and sihvem-2 were siRNAs with the highest effectiveness in inducing the degradation of btla and hvem mRNAs. These siRNAs were selected for the development of siRNA-encoding LVs (sibtla-LV and sihvem-LV, Supplemental Fig. 3F–H). The titers of the two generated LVs were >1 × 106 TU/μl when assessed in HEK293T cells by EGFP-based FCM analysis (Supplemental Fig. 3I, 3J). The interference activities of the two LVs were determined by in vivo knockdown evaluation. Zebrafish under ConA stimulation were i.p. administered with sibtla-LV and sihvem-LV (2 × 105 TU per fish) or scrambled control siRNA-LV for three times within a 24-h interval. One day after the last injection, the expression levels of btla and hvem mRNAs and proteins in the liver were determined by qRT-PCR and Western blot analyses. The results showed that the btla and hvem mRNAs were significantly (p < 0.01) downregulated by 80–90% in the intrahepatic leukocytes and Cd8+ T cells as well as hepatocytes that received sibtla-LV and sihvem-LV compared with those administered with the scrambled control siRNA-LV (Supplemental Fig. 3K–M). The Btla and Hvem proteins were also dramatically downregulated in leukocytes and hepatocytes of the livers that received the sibtla-LV and sihvem-LV (Supplemental Fig. 3N).

Subcellular localization and hepatic distribution of btla, hvem, and light proteins

Subcellular localization analysis showed that the green fluorescence from the Btla and Light fusion proteins in HEK293T cells was predominantly colocalized with the red fluorescence from the membrane indicator CM-DiI, thereby suggesting that Btla and Light are typical membrane proteins. By contrast, the green signals from Hvem fusion protein was distributed on the cell membrane and in the cytoplasm (Fig. 2A–C). Interestingly, when the cells were stimulated with sBtla, the majority of the Hvem proteins displayed a distinct punctate cytoplasmic distribution on the lateral area of the nuclear membrane and a loss of their original membrane localization and random cytoplasmic distribution (Fig. 2D, 2E). Hence, Hvem acts as a trafficking protein under Btla stimulation. Immunohistochemical staining assay showed that considerable nonparenchymal cells in the liver sections were marked with anti-Btla and anti-Light (Fig. 3A, 3B). Most of the hepatocytes in the sample were positive for anti-Hvem (Fig. 3C). For further clarification, the hepatocytes and other nonparenchymal cells were separated from the liver. The distribution of Btla, Light, and Hvem were examined by an indirect immunofluorescence assay. Btla and Light clearly displayed dotlike signatures on the surface of cells coexpressed with Cd8α, suggesting the membrane localization of Btla and Light on Cd8+ T cells (Fig. 3D, 3E). Hvem was mainly detected on the surface of cells colocalized with Bhmt, which is a surface hallmark of hepatocytes in zebrafish. This finding suggests the distribution of Hvem in zebrafish hepatocytes (Fig. 3F).

FIGURE 2.
FIGURE 2.

Subcellular localization analysis of Btla, Light, and Hvem proteins in HEK293T cells. (A–C) Detection of subcellular localization of Btla, Light, and Hvem proteins in HEK293T cells transfected with pEGFPC1-btla/-light/-hvem for 48 h. The green, blue, and red fluorescence shows Btla–/Light–/Hvem–EGFP fusion proteins, DAPI-labeled nuclei, and CM-DiI–labeled cell membranes. (D and E) Perinuclear distribution of Hvem proteins in HEK293T cells enhanced by sBtla protein stimulation (E) compared with that in the control group without sBtla stimulation (D). Scale bar(s), 10 μm.

FIGURE 3.
FIGURE 3.

Cellular distribution of Btla, Light, and Hvem proteins in liver. (A–C) Immunohistochemical staining with anti-Btla (A), anti-Light (B), and anti-Hvem (C) Abs in zebrafish liver paraffin sections. Tan spots marked by red arrowheads show the positive cells. Scale bar(s), 50 μm. (D–F) Cellular distribution of Btla, Light, and Hvem proteins in cells isolated from liver. Double immunofluorescence staining shows the colocalization of Cd8α and Btla, Cd8α and Light, and Bhmt and Hvem on the surfaces of intrahepatic lymphocytes and hepatocytes. The nuclei of cells were labeled by DAPI. Scale bars are present in the first diagram of each line.

ConA-induced hepatic inflammation in zebrafish

ConA-induced hepatic inflammation, one of the most widely used hepatitis models, was developed in zebrafish to investigate the functional roles of Btla, Hvem, and Light in liver inflammatory reactions. ConA induced hepatic inflammation in a dose-dependent manner, with an optimal concentration of ∼200 μg/g of body weight. The induction of hepatic inflammation was determined by the increased levels of ALT and AST in sera; upregulation of Il-1β, Il-6, Tnf-α, and Ifn-γ in the liver; and enhanced apoptosis of hepatocytes accompanied by the upregulation of Fasl and downregulation of Bcl-2. Hepatic inflammation peaked at 24 h under ConA stimulation at a dose of 200 μg/g of body weight. The most strikingly upregulated proinflammatory cytokines, such as Il-1β and Tnf-α, were increased by more than 50-fold (Fig. 4A–F, 4J). Histopathological observation through H&E staining showed evident hepatic plate structure disorder, punctate/lytic necrosis, and edema of hepatocytes in the liver after ConA treatment. Lymphocyte infiltration markedly occurred in the liver (Fig. 4G). Given that ConA-induced hepatitis in mouse models is a T cell–dependent disorder, this characteristic was also evaluated in a zebrafish model by using the T cell inhibitor cyclosporin A (CsA). Expectedly, hepatic inflammatory indicators, including serum ALT and AST levels; Il-1β, Il-6, Tnf-α, Ifn-γ, perforin, Fasl, and mRNA levels; and percentage of apoptotic hepatocytes in ConA-induced livers, were significantly diminished by CsA treatment in a dose-dependent manner. In accordance with the decreased hepatocyte apoptosis, Bcl-2 mRNA was upregulated (Fig. 4H–K).

FIGURE 4.
FIGURE 4.

ConA-induced hepatic inflammation in zebrafish. (A–F) Changes in ALT and AST release levels in sera and apoptosis of hepatocytes after ConA treatment (200 μg/g body weight) at different doses and time periods. Mock PBS–injected groups served as controls. (G) H&E staining of zebrafish liver paraffin sections of PBS-treated control group (left) and ConA- (200 μg/g body weight) stimulated group (middle). The area in the red, dashed rectangle frame is enlarged for a detailed view (right). Red arrow indicates the area of punctate necrosis of hepatocytes accompanying lymphocyte infiltration. Scale bar(s), 50 μm. (H–K) Changes in ALT and AST release levels in sera; transcription levels of proinflammatory factors, perforin, Lamp-1, and apoptosis-related factors in the livers; and apoptosis of hepatocytes induced by ConA (200 μg ConA/g body weight) or ConA plus CsA (2 or 10 μg CsA/g body weight) administration. Mock PBS treatment served as the control. Broken y-axis with disparate scales was adopted to display the disproportionate data. Error bars indicate SD. *p < 0.05, **p < 0.01.

Btla and hvem contribute to liver homeostasis and hepatitis suppression

Given the immune tolerance nature of the liver, this organ satisfies the requirements for studying the negative regulatory role of coinhibitors (such as BTLA) in the maintenance of immune homeostasis, the disruption of which boosts inflammatory reactions and leads to various diseases, including hepatitis. Thus, the potential roles of Btla in the maintenance of liver homeostasis were examined by siRNA-based knockdown assays. Transaminases (ALT and AST), inflammatory cytokines (Il-1β, Il-6, Tnf-α, and Ifn-γ), perforin, Lamp-1, and Fasl were significantly induced (p < 0.05 or p < 0.01) in the sera and livers of zebrafish in which sibtla-LV had been administered compared with those injected with the scrambled control siRNA-LV (Fig. 5A–C). Hepatocyte apoptosis was significantly increased, accompanied by Lamp-1 and Fasl upregulation (Fig. 5D). The livers also showed evident hepatic plate structure disorder, punctate/lytic necrosis, lymphocyte infiltration, and edema of hepatocytes after Btla knockdown for 3 d (Fig. 5E). These observations indicate that Btla greatly contributes to the immune homeostasis of the liver. Furthermore, fish with Btla or Hvem knockdown markedly exacerbated ConA-induced hepatic inflammation, as shown by the significantly higher levels of ALT and AST in sera and Il-1β, Il-6, Tnf-α, and Ifn-γ in the livers of fish in which sibtla-LV and/or sihvem-LV had been administered than those in scrambled siRNA-LV and ConA-treated fish (Fig. 5F–H). The TUNEL assay showed a remarkably increased number of apoptotic hepatocytes in the livers of fish in which sibtla-LV or sihvem-LV had been administered under ConA stimulation (Fig. 5I). The FCM analysis results indicated that the apoptotic cell rate was 30.16 ± 0.15% or 31.13 ± 0.49% in ConA-induced livers or ConA- and scramble siRNA–treated livers, which significantly increased to 43.63 ± 0.67% and 44.60 ± 0.50% with the administration of sibtla-LV and sihvem-LV, respectively (Fig. 5J). These results suggest the inhibitory role of Btla and Hvem in hepatic inflammation, whose disruption would augment hepatic inflammatory responses.

FIGURE 5.
FIGURE 5.

Evaluation of the effect of Btla or Hvem on liver homeostasis and ConA-induced hepatitis. (A–D) Changes in ALT and AST release levels in sera, transcription levels of proinflammatory factors, perforin, Lamp-1, and apoptosis-related factors in the livers and apoptosis of hepatocytes after knockdown of Btla by administration of sibtla-LV for 3 and 5 d. (E) H&E staining shows the histopathological changes in zebrafish livers that received sibtla-LV injection for 3 d. Red arrow indicates the area of punctate necrosis of hepatocytes accompanying lymphocyte infiltration. Scale bar(s), 50 μm. (F–H) Changes in ALT and AST release levels in sera and transcription levels of proinflammatory factors, perforin, Lamp-1, and apoptosis-related factors in the livers of ConA- (200 μg/g body weight) stimulated zebrafish after the knockdown of Btla and Hvem by sibtla-LV and sihvem-LV administration, respectively, for 3 d. (I and J) TUNEL assay and FCM analysis show the increased apoptotic hepatocytes in sibtla-LV– and sihvem-LV–treated groups. The tan spots indicate the apoptotic hepatocytes. Mock PBS-injected group served as the control. Broken y-axis with disparate scales was adopted to display the disproportionate data. Error bars represent SD. Scale bar(s), 50 μm. *p < 0.05, **p < 0.01.

Involvement of Cd8+ T cells in ConA-induced hepatic inflammation

ConA-induced hepatitis in zebrafish is T cell dependent, and immunohistochemical results showed that a considerable percentage of Cd8+ T cells infiltrated into the ConA-induced liver (Fig. 6A). This finding suggests that Cd8+ T cells are largely involved in the ConA-induced hepatitis model. To provide support for this hypothesis, we performed a Cd8+ T cell deletion assay by using anti-Cd8α Ab. The FCM analysis showed that the number of Cd8+ T cells was significantly decreased by ∼91% in the ConA-induced liver after anti-Cd8α Ab was administered three times within a 24-h interval (Supplemental Fig. 3O). The Cd8+ T cell–deleted fish displayed lower ALT and AST release, downregulation of inflammatory cytokines (Il-1β, Tnf-α, and Ifn-γ) and perforin, and decreased apoptotic hepatocytes from 30.17 ± 0.25% to 16.17 ± 0.50% compared with the fish that received ConA treatment alone (Fig. 6B–E). The TUNEL assay showed a remarkably decreased number of apoptotic hepatocytes in the livers of fish that received anti-Cd8α Ab under ConA stimulation (Fig. 6F). To support this finding, we performed an adoptive transfer assay by sorting Cd8+ T cells from the ConA-induced livers and transferring them into Cd8+ T cell–depleted fish at different doses (104, 105, and 106 cells per fish) under ConA stimulation. As expected, the adopted fish showed significant restoration of inflammation, as determined by the upregulated serum ALT and AST levels, along with the increased dosage of Cd8+ T cells (Fig. 6G).

FIGURE 6.
FIGURE 6.

Determination of the involvement of Cd8+ T cells in ConA-induced hepatic inflammation. (A) Immunohistochemical staining with anti-Cd8α Ab shows the increased infiltration of Cd8+ T cells (red arrowheads) in ConA- (200 μg/g body weight) treated group (right) or mock PBS–injected control group (left). Scale bars, 50 μm. (B–E) Changes in ALT and AST release levels in sera; transcription levels of proinflammatory factors, perforin, Lamp-1, apoptosis-related factors in the livers; and apoptotic hepatocytes after deletion of Cd8+ T cells by anti-Cd8α Ab administration. (F) TUNEL analysis was performed for apoptosis determination. The tan spots indicate the apoptotic hepatocytes. Scale bar(s), 50 μm. (G) Adoptive transfer of Cd8+ T cells from the inflamed livers shows restoration of hepatic inflammation, as determined by the upregulation of serum ALT and AST levels. Broken y-axis with disparate scales was adopted to display the disproportionate data. Error bars represent SD. **p < 0.01.

Downregulation of btla on Cd8+ cells enhances hepatic inflammation

Immunohistochemical staining and FCM analyses showed two subsets of Cd8+ T cells (i.e., Cd8+Btla+ and Cd8+Btla–) in the livers with or without ConA stimulation. The proportion of Cd8+Btla+ T cells to the total Cd8+ T cells decreased from 15.13 ± 0.96% to 2.46 ± 0.26% in the ConA-induced liver compared with that of the untreated liver. The ratio of Cd8+Btla– T cells increased from 83.13 ± 0.75% to 97.03 ± 0.15%, accompanied with the decline of Cd8+Btla+ T cells (Fig. 7A). These observations suggest that Cd8+Btla– T cells are cytotoxic in hepatic inflammation, whereas Cd8+Btla+ T cells are anergic because of the expression of the inhibitory Btla protein. To provide evidence for this hypothesis, we isolated Cd8+Btla+ and Cd8+Btla– T cells and hepatocytes from the ConA-induced livers for in vitro cytotoxicity assay. As predicted, Cd8+Btla+ T cells showed no significant cytotoxicity to hepatocytes at a wide range of effector/target ratios (1:5 to 1:40), whereas Cd8+Btla– T cells displayed high cytotoxicity with an optimal effector/target ratio of 1:20. After treatment with anti-Btla Ab, Cd8+Btla+ T cells exhibited considerable cytotoxic activity to target cells (Fig. 7B). To indicate that the Cd8+Btla+ T cells are anergic rather than just naive, we examined the expression levels of Il-2, Lck, Cxcr3, and Ccr7, a set of markers for Cd8+ T cell activation in Cd8+Btla+ T cells. Intrahepatic Cd8+Btla+ T cells exhibited high expression levels of Il-2, Lck, and Cxcr3 but low Ccr7 expression. These values were comparable with those of the activated Cd8+Btla− T cells. This expression pattern was opposite of that of the inactivated naive Cd8+ T cells from the head kidney (a hematopoietic tissue) of fish without any stimulation (Fig. 7C). In vivo adoptive transfer assay was performed for further evaluation. Cd8+Btla+ and Cd8+Btla– T donor cells sorted from the inflamed livers were transferred (1 × 105 cells per fish) into the recipient fish that received the optimized ConA treatment (200 μg/g body weight for 1 d). Hepatitis was significantly enhanced by the transfer of Cd8+Btla– T cells, as determined by the increased serum levels of ALT and AST and the occurrence of hepatic apoptosis. By contrast, hepatic inflammation stopped developing in the ongoing ConA-induced hepatitis in the Cd8+Btla+ T cell–transferred liver, as evident by the maintained basal levels of serum levels of ALT and AST and the apoptosis of hepatocytes in comparison with those in the control groups. However, after the transfer of Cd8+Btla+ T cells that were pretreated with anti-Btla Ab for the blockade of Btla, hepatic inflammation resumed (Fig. 7D–F). The treatment of Cd8+Btla– T cells with anti-Btla Ab did not influence the cytotoxic activity of Cd8+Btla– T cells, suggesting that Btla is the direct target of anti-Btla Ab. In this case, the ratio of hepatic apoptosis in fish that received a transfer of Cd8+Btla– T cells that were pretreated with anti-Btla Ab was 39.03 ± 0.46%, similar to the 39.23 ± 0.31% ratio in the control group without anti-Btla Ab treatment. These results indicate that Cd8+Btla– T cells contributed to cytotoxicity during hepatic inflammation and Cd8+Btla+ T cells were anergic because of Btla expression. Moreover, the cytotoxic effector molecules perforin, Lamp-1, and Fasl were significantly upregulated in Cd8+Btla– T cells in the liver that suffered from inflammation (Fig. 7G).

FIGURE 7.
FIGURE 7.

Evaluation on the contribution of Btla and Btla–Hvem association to the tolerization of Cd8+ T cells in the liver. (A) The ratio of Cd8+Btla+ and Cd8+Btla− T cells in the control liver without stimulation and inflamed liver under ConA (200 μg/g body weight) stimulation suggests that downregulation of Btla on Cd8+ T cells enhanced hepatic inflammation. (B) In vitro cytotoxicity assay showing that Cd8+Btla+ T cells are anergic, whereas Cd8+Btla− T cells are cytotoxic. After treatment with anti-Btla Ab (5 μg/ml), the Cd8+Btla+ T cells displayed cytotoxicity to the target cells. (C) Different expression levels of Il-2, Lck, Cxcr3, and Ccr7 in Cd8+Btla+ and Cd8+Btla− T cells sorted from ConA-stimulated liver. The inactivated Cd8+ T cells sorted from the head kidney of fish without stimulation were selected as a control. (D–F) In vivo adoptive transfer assay showing Cd8+Btla– T cells, but not Cd8+Btla+ T cells, promoted hepatic inflammation, as determined by the release levels of ALT and AST and changes in hepatocyte apoptosis. (G) Different transcription levels of perforin, Ifn-γ, Lamp-1, and Fasl in Cd8+Btla+ and Cd8+Btla− T cells. (H) Co-IP assay showing the association of Btla with Hvem. Btla–EGFP and Hvem–Myc fusion proteins were coexpressed on HEK293T cells. Mouse anti-EGFP and anti-Myc Abs and nonrelevant control IgG isotype were used for the examination. (I and J) Functional evaluation on the negative regulatory role of Btla–Hvem association in ConA-induced (200 μg/g body weight) hepatic inflammation by the adoptive transfer of Cd8+Btla+ T cells into fish that had or had not been administered sihvem-LV and/or soluble Hvem protein. The changes in ALT and AST release levels and apoptotic hepatocyte ratios were determined in the transfer assays to evaluate whether the tolerization of Cd8+Btla+ T cells is dependent on Hvem receptor proteins. Broken y-axis with disparate scales was adopted to display the disproportionate data. Error bars indicate SD. *p < 0.05, **p < 0.01.

Btla–hvem association contributes to the tolerization of Cd8+Btla+ T cells

The association of Btla with Hvem was initially examined by Co-IP assay to evaluate whether they act as reciprocal molecules in a manner similar to that in mammals. The result showed that Btla–EGFP interacted with Hvem, whereas EGFP–tag protein did not combine with Hvem (Fig. 7H). The contribution of Btla–Hvem association to the tolerization of Cd8+Btla+ T cells to hepatocytes was functionally evaluated by adoptive transfer assays. The Cd8+Btla+ T cells from ConA-induced donor fish were transferred into Hvem-knockdown recipient fish that received sihvem-LV and ConA stimulation in accordance with the above-mentioned method. The tolerization of Cd8+Btla+ T cells in hepatitis was significantly impaired, along with the downregulation of Hvem in the liver. This finding suggests that the cytotoxic anergy of Cd8+Btla+ T cells on hepatocytes was abolished when the degree of association between Btla and Hvem decreased. The attenuation of Cd8+Btla+ T cell tolerization was determined by the increased ALT and AST release and the occurrence of hepatic apoptosis from 31.90 ± 1.08% in the untreated liver to 50.80 ± 0.40% in Hvem-knockdown liver. When the Hvem-knockdown recipient fish were coadministered Cd8+Btla+ T cells and a soluble recombinant Hvem protein (sHvem, 2 μg/g body weight), the cytotoxic anergy of Cd8+Btla+ T cells was significantly restored, as shown by the decline in ALT and AST release and apoptotic hepatocytes from 50.80 ± 0.40% to 34.20 ± 2.26% (Fig. 7I, 7J). By contrast, when the Hvem-knockdown recipient fish were coadministered Cd8+Btla− T cells and sHvem (2 μg/g body weight), the cytotoxic activity of Cd8+Btla− T cells remained unchanged. In this case, the ratio of hepatic apoptosis in the recipients was 49.00 ± 0.66%, similar to the 50.70 ± 2.01% ratio in the control group in which Cd8+Btla– T cells were transferred but did not receive sHvem administration. This finding suggests that the suppressive axis of Btla−Hvem in the Hvem-knockdown recipient fish with Cd8+Btla− T cell transfer could not be restored, even when sHvem was replenished. In addition, the maintenance of the cytotoxic anergy of Cd8+Btla+ T cells depends on the association of Btla with Hvem largely provided by hepatocytes. The soluble Hvem protein plays a regulatory role in hepatic inflammation, which is of potential importance for clinical therapeutic purposes.

Functional role of Cd4+ T cells for the downregulation of btla on Cd8+ T cells

CD4+ T cells play an important role in hepatitis in humans and ConA-induced mouse models. Thus, we explored whether the downregulation of Btla on Cd8+ T cells during hepatic inflammation was modulated by Cd4+ T cells. Cd4+, Cd8+Btla+, and Cd8+Btla– T cells were sorted from ConA-induced livers by FACS. Cd8+Btla+ and Cd8+Btla– T cells were cocultured with Cd4+ T cells through a Transwell chamber. The transcription level of btla mRNA in Cd8+Btla+ T cells significantly declined. In contrast, Ifn-γ, Fasl, and Lamp-1 were upregulated after 24 or 72 h of coculture (Fig. 8A, 8B). The transcripts of T-bet, Il-12, and Ifn-γ, which are three representative Th1-typic factors, were remarkably increased in Cd4+ T cells from the ConA-induced livers compared with those of Cd4+ T cells from the unstimulated livers (Fig. 8C). These observations suggest that Cd4+ T cells underwent Th1 cell differentiation under hepatic inflammation and secreted Th1-typic cytokines to suppress Btla expression in Cd8+Btla+ T cells, which then differentiated into cytotoxic Cd8+Btla– T cells. Supporting this hypothesis, the mean fluorescence intensity of Btla in Cd8+Btla+ T cells was significantly (p < 0.01) declined after 24 or 72 h of coculture with Cd4+ T cells, as determined by the FCM analysis (Fig. 8D).

FIGURE 8.
FIGURE 8.

Determination of the functional role of Cd4+ T cells for the downregulation of Btla on Cd8+ T cells and involvement of Light in Btla–Hvem–mediated hepatic inflammation. (A and B) Changes in the transcriptional levels of btla and some other proinflammatory and apoptosis-related factors, including Ifn-γ, Fasl, perforin, and Lamp-1, which are expressed in Cd8+Btla+ T cells after coculture with Cd4+ T cells for 24 or 72 h. (C) Changes in the transcriptional levels of Th1- or Th2-associated factors in Cd4+ T cells from the livers with or without ConA stimulation (200 μg/g body weight). (D) Mean fluorescence intensity (MFI) of Btla in Cd8+Btla+ T cells after coculture with Cd4+ T cells for 24 or 72 h. (E) The light mRNA is differentially expressed in Cd8+Btla+ and Cd8+Btla− T cells. (F) The ratio of Cd8+Light+ and Cd8+Light− T cells in the livers without (mock PBS–injected control) or with ConA stimulation (200 μg/g body weight). (G) The ratio of Cd8+Btla+Light− and Cd8+Btla−Light+ T cells in Cd8+ T subsets sorted from the livers without (mock PBS–injected control, Cd8+ TCtrl cells) or with ConA stimulation (200 μg/g body weight, Cd8+ TConA cells). (H–J) Evaluation on the functional role of Light by blockade of Light on Cd8+Btla–Light+ T cells through anti-Light Ab. Changes in ALT and AST levels in sera and transcription levels of proinflammatory factors, perforin, Lamp-1, and apoptosis-related factors in the livers were examined in the blockade assays. Mock PBS and nonrelated IgG were used in the control groups. Broken y-axis with disparate scales was adopted to display the disproportionate data. (K) Adoptive transfer assay of Cd8+Light+ and Cd8+Light– T cells or Cd8+Light+ T cells that were pretreated with anti-Light Ab. Changes in serum ALT and AST levels were examined. (L) Ratios of intrahepatic Cd8+Btla+Light– and Cd8+Btla–Light+ T cells after coculture with Cd4+ T cells for 24 h. Unlabeled Cd8+ T cells served as negative control group. Error bars represent SD. *p < 0.05, **p < 0.01.

Involvement of light in btla–hvem–mediated hepatic inflammation

LIGHT, another ligand of HVEM, serves as a costimulatory molecule during T cell activation in humans and other mammalian models. Finally, we explored whether Light plays a regulatory role in Btla–Hvem–mediated hepatic inflammation in the zebrafish model. The qRT-PCR results showed that light mRNA was expressed in low quantities in Cd8+Btla+ T cells but was significantly (p < 0.01) upregulated in Cd8+Btla– T cells (Fig. 8E). The FCM analysis showed that the ratio of Cd8+Light+ to total Cd8+ T cells was significantly (p < 0.01) enhanced by ∼2.4-fold in the ConA-stimulated livers compared with that in the control group treated with mock PBS (20.03 ± 1.17% versus 8.23 ± 1.00%, Fig. 8F). Accordingly, a Cd8+Btla–Light+ and a Cd8+Btla+Light– T subset population were detected in the sorted intrahepatic Cd8+ T cells. The number of Cd8+Btla–Light+ T cells significantly increased (p < 0.01) from 2.15 ± 0.45% in Cd8+ T cells sorted from healthy livers without ConA stimulation (Cd8+ TCtrl cells) to 38.35 ± 2.90% in those from the inflamed livers with ConA stimulation (Cd8+ TConA cells). The number of Cd8+Btla+Light– T cells significantly decreased (p < 0.01), accompanied by the increase in the percentage of Cd8+Btla–Light+ T cells, from 12.37 ± 2.82% in healthy livers to 1.22 ± 0.67% in the ConA-stimulated livers (Fig. 8G). The disparate ratio of Cd8+Btla–Light+ and Cd8+Btla+Light– T cells in the healthy and hepatitis livers suggests the different roles between Light and Btla. The upregulation of Light on Cd8+Btla–Light+ T cells may contribute to the hepatic inflammation in response to ConA stimulation. To provide support for this hypothesis, we abolished the functional role of Light on Cd8+Btla–Light+ T cells by a blockade assay via the administration of anti-Light Ab (20 μg/g body weight) into fish. As predicted, the blockade of Light markedly impaired ConA-induced hepatic inflammation, as shown by the significantly decreased levels of ALT and AST in the sera and Il-1β, Il-6, Tnf-α, Ifn-γ, perforin, Lamp-1, and Fasl in the livers, accompanied by the upregulation of Bcl-2 (Fig. 8H–J). The adoptive transfer of Cd8+Light+ T cells (1 × 105 cells per fish) from the inflamed livers into the ConA-stimulated recipient fish significantly aggravated hepatitis and increased the levels of ALT and AST released. However, this aggravation was impaired when the transferred Cd8+Light+ T cells were pretreated with anti-Light Ab (Fig. 8K). No significant enhancement of hepatitis was observed after transferring Cd8+Light– T cells with or without the treatment of anti-Light Ab. These observations support the notion that the upregulation of Light on Cd8+ T cells exacerbates hepatitis. When Cd8+ T cells were cocultured with Cd4+ T cells through a Transwell chamber for 24 h, the percentage of CD8+Btla+Light– T subsets decreased from 41.33 ± 0.95% to 0.51 ± 0.05%, which was accompanied with an increase in the number of Cd8+Btla–Light+ T cells from 1.41 ± 0.41% to 67.27 ± 0.42% (Fig. 8L). This result suggests that Cd4+ T cells also play a modulatory role in the upregulation of Light on Cd8+ T cells, in addition to their function in the downregulation of Btla in Cd8+ T cells.

Discussion

The BTLA, HVEM, and LIGHT immune checkpoint regulators have become increasingly explored because of their important modulatory roles in immunity and extensive involvement in various diseases of humans and other mammals (28, 32). However, the occurrence and functions of these checkpoint molecules in nonmammalian species remain poorly understood (45). In the current study, we identified Btla, Hvem, and Light homologs from zebrafish. These homologs were characterized to share similar structural features with their mammalian counterparts, thereby suggesting their functional conservation in vertebrate evolutionary history. For functional study, a ConA-induced hepatitis model was established in zebrafish. The regulatory functions of Btla, Hvem, and Light homologs were then extensively investigated in this inflammatory disease model.

A number of experimental lines substantially supported the conclusion that the Btla–Hvem–Light checkpoint network regulates liver homeostasis and hepatic inflammation in the ConA-induced hepatitis model. For example, the knockdown of Btla and Hvem in healthy or ConA-stimulated livers significantly induced hepatic inflammatory reactions. These results provide initial insights into the negative regulation of the Btla–Hvem axis in liver homeostasis and inflammation, given that the disruption of this axis augmented the occurrence of hepatitis. Immunohistochemical staining showed the presence of a considerable number of Cd8+ T cells in ConA-induced liver. The depletion of these Cd8+ T cells decreased hepatic inflammation and tissue injury, which could be restored by the adoptive transfer of Cd8+ T cells from the livers that suffered from hepatitis. This result suggests the involvement of Cd8+ T cells in ConA-induced hepatitis. The FCM analysis indicated that Btla was differentially expressed on Cd8+ T cells in the livers with or without ConA stimulation. Btla was highly expressed in Cd8+ T cells in healthy liver without stimulation. By contrast, Btla expression was dramatically decreased in Cd8+ T cells in liver with hepatitis. This observation was determined by the high proportion of Cd8+Btla+ T cells in healthy liver, which decreased in the ConA-induced liver, accompanied with the increase in the number of Cd8+Btla– T cells. In the in vitro cytotoxicity assay, Cd8+Btla+ T cells showed no significant cytotoxicity to hepatocytes, whereas Cd8+Btla– T cells exhibited strong cytotoxicity. Similarly, the in vivo transfer of Cd8+Btla– T cells into ConA-induced liver enhanced hepatic inflammation and liver injury, whereas the transfer of Cd8+Btla+ T cells did not. These results indicate that Btla endowed Cd8+ T cells with self-tolerance, which prevented Cd8+ T cells from attacking hepatocytes, thereby maintaining liver homeostasis. However, Btla downregulation in Cd8+ T cells deprived the tolerization of Cd8+ T cells, leading to the disruption of liver homeostasis and the occurrence of hepatitis. Cd8+Btla+ T cells showed high expression levels of Il-2, Lck, and Cxcr3 but a low level of Ccr7. These values were similar to those of the activated Cd8+Btla− T cells. Given the upregulation of Il-2, Lck and Cxcr3 combined with the downregulation of Ccr7 are typical markers for Cd8+ T cell activation (39, 46–48), indicating that Cd8+Btla+ T cells are anergic rather than just naive, and their activity is largely suppressed by Btla expression. The Co-IP assay showed that Btla clearly associated with Hvem in a manner similar to that in mammals. The tolerization of Cd8+Btla+ T cells in hepatitis was dramatically impaired with Hvem knockdown in the liver but could be rescued by the administration of a recombinant soluble Hvem protein. These observations suggest that the maintenance of Cd8+ T cell tolerization depends on the association of Btla with Hvem. The transition of Cd8+Btla+ T cells to Cd8+Btla– T cells in the ConA-induced liver may largely contribute to hepatic inflammation.

The costimulatory signal for the cytotoxic activity of Cd8+Btla– T cells was further explored. In mammals, LIGHT is another ligand of HVEM, which plays a costimulatory role in the activation of several T cell models. Thus, the involvement of zebrafish Light in the Btla–Hvem axis and Cd8+Btla– T cell–elicited hepatitis was investigated. light mRNA was weakly detected in Cd8+Btla+ T cells but was highly expressed in Cd8+Btla– T cells. This observation was supported by the detection of Cd8+Light+ T cells, which were markedly increased in liver with hepatitis. For clarification, Cd8+Btla–Light+ and Cd8+Btla+Light– T subsets were further distinguished from the sorted total liver Cd8+ T cells. The number of Cd8+Btla–Light+ T cells significantly increased in the ConA-stimulated liver, accompanied with the decrease in the percentage of Cd8+Btla+Light– T cells and the occurrence of hepatitis. The disparate expression of Btla and Light on Cd8+ T cells during hepatitis suggests that the latter plays a costimulatory role in the initiation of the cytotoxic activity of Cd8+ T cells, whose performance was opposite to the inhibitory effect of Btla on Cd8+ T cells. Thus, the transition of Cd8+Btla+Light– to Cd8+Btla–Light+ T cells under ConA stimulation may largely contribute to hepatic inflammation. Cd8+Btla–Light+ T cells were abolished by blockade of Light to provide functional support. As predicted, the blockade of Light attenuated the ConA-induced hepatitis. By contrast, the adoptive transfer of Cd8+Light+ T cells exacerbated the hepatitis.

Given that CD4+ T cells play crucial roles in ConA-induced hepatitis in mouse models, the modulatory function of Cd4+ T cells for the downregulation of Btla in Cd8+Btla+Light– T cells and the upregulation of Light in Cd8+Btla–Light+ T cells were explored. As predicted, Btla expression was significantly declined at the mRNA and protein levels when Cd8+Btla+Light– T cells were cocultured with Cd4+ T cells prestimulated to express Th1-typic cytokines, such as T-bet, Il-12, and Ifn-γ, in the hepatitis-burdened liver. This process was accompanied by the upregulation of Light in Cd8+Btla–Light+ T cells, as determined by the increased percentage of Cd8+Btla–Light+ T subset and the decreased proportion of Cd8+Btla+Light– T subset. These results suggest that Cd4+ Th1 cells modulate the polarization of Cd8+ T cells from Cd8+Btla+Light– to Cd8+Btla–Light+ T subsets, which is a key event in hepatitis. To our knowledge, this work is the first to report the existence of Cd8+Btla+Light– and Cd8+Btla–Light+ T subsets in the liver. The Cd8+Btla+Light– T subset acts as an anti-inflammatory population and contributes to liver homeostasis and inhibition of hepatitis. The Cd8+Btla–Light+ T subset serves as a proinflammatory population and promotes hepatitis. However, the precise mechanisms underlying Cd4+ T cell regulation for Cd8+ T subset cell polarization remain to be further clarified.

Liver is a metabolic and an immune organ with numerous metabolites and exogenous toxins (49, 50). Hence, the liver is in an immunotolerant state to prevent the improper activation of immune reactions in healthy individuals. Liver self-tolerance is sustained through numerous approaches, including cellular and molecular mechanisms (51). For example, T regulatory/Th17 cells maintain the balance of the liver, in which T regulatory cells expanded by Kupffer cells or primed by liver sinusoidal endothelial cells maintain the tolerant state by inhibiting Th17 and Th1 cell activation (52). In addition, PD-1 and CTLA-4 coinhibitory molecules expressed on exhausted T cells help sustain homeostasis (53). In the current study, we provided new (to our knowledge) insights into the contribution of the Btla–Hvem checkpoint axis to liver homeostasis by maintaining the tolerization of Cd8+ T cells, whose disruption leads to the occurrence of hepatitis.

Hepatitis is the inflammation of liver tissue and a severe clinical disease worldwide. The common causes of hepatitis include viruses, heavy alcohol use, nonalcoholic steatohepatitis, toxins, certain medications, and autoimmune diseases (50, 54–56). To date, the main research animals for hepatitis are mammals, such as mouse and tree shrew (Tupaia belangeri), which are used as viral, alcoholic, and drug-induced hepatitis models (55–57). ConA-induced hepatitis is one of the most widely used drug-induced hepatitis models that mimic T cell–dependent acute or chronic hepatitis in humans (58). In the current study, we established a ConA-induced hepatitis model in zebrafish. The model was substantially verified by various pathological characteristics similar to those in mouse models. Such characteristics include lymphocyte infiltration; elevation of serum ALT and AST; upregulation of proinflammatory cytokines, such as IL-1β, TNF-α, and IFN-γ; and increased apoptosis of hepatocytes and tissue injury in the liver. Together with other liver disease models, such as alcoholic hepatitis, which was effectively established in zebrafish, we conclude that zebrafish is a suitable nonmammalian model organism for studying the mechanisms of liver diseases.

Various cells, such as liver sinusoidal endothelial cells, Kupffer cells, NK, NKT, and CD4+ T cells, are involved in ConA-induced hepatitis. CD4+ T cells are considered the predominant population for the occurrence of hepatitis because they initiate humoral autoimmune reactions through association with liver sinusoidal endothelial cells and Kupffer cells (59–61). However, the functional performance of CD8+ T cells in ConA-induced hepatitis remains elusive because controversial observations have been obtained by different research groups. For example, a mouse administered with anti-CD4 mAb can be effectively protected from ConA-induced liver injury, whereas anti-CD8 mAb treatment fails in the protection (58). The activated CD8+ T cells under ConA stimulation induce the activation of ST2+ type 2 innate lymphoid cells, which can promote hepatic inflammation (62). Consistent with our results, CD8+ T cells contribute to hepatic inflammation provoked by ConA. Nevertheless, knowledge on the mechanisms underlying CD8+ T cell regulation is still incomplete and requires extensive investigations. In the current study, we described the differential functional roles of Cd8+Btla+Light− and Cd8+Btla−Light+ T subsets in the pathogenesis of hepatitis. We added two new (to our knowledge) Cd8+ T cell populations into the intrahepatic Cd8+ T cell family. This finding uncovered a previously unknown (to our knowledge) cellular mechanism underlying hepatic inflammation, which will benefit the development of therapeutic interventions for hepatic inflammatory disorders.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Ying-ying Huang, Xing-hui Song, and Jing-yao Chen for technical support for FACS and immunohistochemical staining and She-long Zhang and Xin-hang Jiang for two-photon laser confocal scanning microscope capture and ultracentrifugation.

Footnotes

  • This work was supported by grants from the National Natural Science Foundation of China (31630083, 31572641), the National Key Research and Development Program of China (2018YFD0900503, 2018YFD0900505, 2016YFA0101001), the Open Fund of the Laboratory for Marine Biology and Biotechnology, the Qingdao National Laboratory for Marine Science and Technology, Qingdao, China (OF2017NO02), the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, and the Zhejiang Major Special Program of Breeding (2016C02055-4).

  • The sequences presented in this article have been submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers MK112054, MK112055, and MK112056.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ALT
    alanine aminotransferase
    AST
    aspartate aminotransferase
    Bcl-2
    B cell lymphoma 2
    BHMT
    Bhmt, betaine homocysteine S-methyltransferase
    BTLA
    Btla, B and T lymphocyte attenuator
    Co-IP
    coimmunoprecipitation
    CRD
    cysteine-rich domain
    CsA
    cyclosporin A
    EGFP
    enhanced GFP
    Fasl
    Fas ligand
    FCM
    flow cytometry
    Grb2
    growth factor receptor–bound protein 2
    HVEM
    Hvem, herpesvirus entry mediator
    Lamp-1
    lysosomal-associated membrane protein 1
    Lck
    lymphocyte protein tyrosine kinase
    LIGHT
    Light, lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator
    LV
    lentivirus
    mLIGHT
    membrane-bound LIGHT
    PFA
    paraformaldehyde
    qRT-PCR
    quantitative real-time PCR
    sBtla
    soluble Btla
    shRNA
    short hairpin RNA
    siRNA
    small interfering RNA
    sLIGHT
    soluble LIGHT
    TRAF
    TNFR-associated factor
    TU
    transducing unit.

  • Received April 24, 2019.
  • Accepted August 29, 2019.

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