Abstract
Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) play a vital role in viral control and clearance. Recent studies have elucidated that Tapasin, an endoplasmic reticulum chaperone, is a well-known molecule that appears to be essential in peptide-loading process. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway plays an important role in immune response regulation and cytokines secretion. We have previously verified that fusion protein CTP-HBcAg18–27-Tapasin could facilitate the maturation of bone marrow derived dendritic cells and enhance specific CTLs responses in vitro, which might be associated with the activation of JAK/STAT signaling pathway. To further explore whether JAK/STAT signaling pathway participated in specific immune responses mediated by CTP-HBcAg18–27-Tapasin, we suppressed the JAK/STAT pathway with pharmacological inhibitor (AG490) in vivo. Our studies showed that the number of IFN-γ+-CD8+ T cells was decreased significantly compared with other groups after being blocked by AG490. The percentage of IFN-γ+-CD4+ T cells and IL-2-CD4+ T cells was also decreased. Moreover, lower expression levels of Jak2, Tyk2, STAT1, and STAT4 were detected in AG490 group. In addition, the secretion levels of Th1-like cytokines were decreased and a weaker specific T-cell response was observed in AG490 group. Furthermore, the levels of HBV DNA and HBsAg in serum and expression levels of HBsAg and HBcAg in liver tissues were elevated after this pathway was inhibited in HBV transgenic mice. These results demonstrate that the JAK/STAT signaling pathway participates in Th1-oriented immune response induced by CTP-HBcAg18–27-Tapasin and this might provide a theoretical basis for HBV immunotherapy.
Introduction
Hepatitis B virus (HBV) infection is a major public-health threat, with an estimated 350 million people worldwide chronically infected. More than one million deaths are caused by HBV-related liver diseases, cirrhosis and hepatocellular carcinoma (HCC) each year [1]. Although preventive hepatitis B vaccinations have existed for several decades, chronically infected individual remains a reservoir for HBV persistence.
The precise mechanism of the diversity of chronic hepatitis B (CHB) infections and host immune responses has not been fully elucidated. However, it has been speculated that the resolution of HBV infection was associated with host immune response against HBV antigens [2]. Therefore, an ineffective antiviral immune response towards the virus might lead to the chronicity of HBV infection. The susceptibility of infected patients to become chronic is due to the immunodeficiency status manifested by imbalanced cellular response and impaired responses to HBV peptides [3]. Moreover, it has been speculated that an imbalanced cellular immune response is responsible for the persistence of HBV infection [4].
HBV-specific CD8+ T cells (CTL) are a major component of cellular adaptive immunity and play a major role in viral clearance [5]. The evolution of chronic hepatitis B (CHB) is dependent on the quality and quantity of the immune response. A robust and sustained CD8+ T activity was generated in acute self-limited HBV individuals [6]. Hence, most of the acutely infected individuals could efficiently inhibit the virus replication. In contrast, with a weak and narrow specific T-cell response, the infected adults from newborns and infants were predisposed to develop chronic infection [7]. Therefore, strategies aiming at strengthening CTL activity could lead to immunotherapy for terminating persistent HBV infection. The initial activation of naive CD8+ T cells into effector CTLs requires two predominant signals: T-cell receptor engagement (signal 1) and costimulation/IL-2 (signal 2) [8]. Recent studies have indicated that Th1-oriented CD4+ T cells are essential in the activation of CD8+ T cells [9]. Those Th1-oriented CD4+ T cells could migrate to the liver, secrete IFN-γ and TNF-α, and effectively suppress virus infection. IL-2 appears to have overlapping functions for TH1 differentiation with IL-12 which is a crucial regulator of cell differentiation. Thus, IL-2 can direct Th1 differentiation in the absence of IL-12 and influence cell differentiation decisions and elicit secondary expansion of memory CTL [10].
CD8+ T lymphocytes recognize short peptides (8-12 amino acids in length) presented by MHC class I (MHC-I) molecules. Tapasin, as a peptide-loading co-factor, could facilitate assembly and stability of MHC class I molecules with immune-dominant peptides [11]. The endoplasmic reticulum (ER) resident protein Tapasin is a crucial component of the peptide-loading complex (PLC). MHC class I molecule in Tapasin-deficient conditions was predisposed to bind lower affinity peptides, resulting in decreased expression of cell surface molecules and impaired immune recognition processes [12]. HLA-A2-restricted peptide HBcAg18–27 is dominant in the augment of CD8+ T-cell response. The deliberately designed cytoplasmic transduction peptide (CTP) could efficiently delivery the CTP-fused biomolecules [13]. Our previous results have confirmed that the fusion protein CTP-HBcAg18–27-Tapasin could evoke HBV-specific CTL immune responses and antiviral immunity in vivo [14]. However, the molecular mechanism of enhanced HBV-specific CTL immune response induced by fusion protein is still not clear.
Upon recognition of cognate peptide–MHC complexes on DCs, naive T cells are activated and driven to differentiation into the cytotoxic T cells (CTL) via inflammatory cytokines receptor at T-cell surface [15]. Cytokines are crucial mediators of various biological responses, including immune regulation and inflammation which use selective component of the JAK/STAT pathway to mediate its specific biological responses. Surprisingly, the JAK/STAT signaling pathway plays a pivotal role in the early regulation of Th1 initiation, and induces proliferation of naive CD4+ T cells [16]. Of note, CD4+ T cells play an important role in the activation and long-term maintenance of CD8+ T-cell responses [17, 18]. We have previously shown that the fusion protein could induce potent CTL response that might be associated with the JAK/STAT signaling pathway. In the present study, we further elucidate the mechanisms of the JAK/STAT signal pathway involved in CTP-HBcAg18–27-Tapasin mediated specific immune responses with the inhibitor AG490 in HLA-A2 transgenic mice and HBV transgenic mice. Furthermore, this study may provide a scientific and theoretical basis for HBV immunotherapy in the future.
Materials and Methods
Reagents
The fluorescent antibodies and isotype controls were purchased from eBioscience (San Diego, USA). Phorbol 12-myristate 13-acetate (PMA), ionomycin, monensin, concanavalin A (ConA), and AG490 were obtained from Sigma (St Louis, USA). The enzyme-linked immunosorbent assay (ELISA) kits used to measure the levels of interferon (IFN)-γ and interleukin (IL)-2 were obtained from R&D Co., Ltd (Minneapolis, USA). HBcAg18–27 (FLPSDFFPSV) was synthesized by Sangon Biotech Co., Ltd (Shanghai, China). Enzyme-linked immunospot assay plates used for detecting IFN-γ were obtained from Dakewe Biotech Co., Ltd (Shenzhen, China). Interferon-α was purchased from Anhui Anke Biotechnology (Group) Co., Ltd (Hefei, China). Cell Counting Kit-8 was obtained from Dojindo (Osaka, Japan). The antibodies used for immunohistochemistry staining were purchased from Boster (Wuhan, China).
Animals
HLA-A2 transgenic mice (H-2Kb), 6–8 weeks old, were purchased from Jackson Laboratory (Bar Harbor, USA). C57BL/6-HBV transgenic mice carrying 1.3 times over-length of the HBV genome (ayw) were purchased from the Key Liver Army Laboratory (The No. 458 Hospital, Guangzhou, China). The description of the mice has previously been reported in detail [14]. Mice were bred and kept under specific pathogen-free conditions at the animal experiment center of the Shanghai Sixth People’s Hospital (Shanghai, China). All animal experiments were performed in accordance with protocols approved by the laboratory animal ethical commission of Shanghai Jiao Tong University (Shanghai, China).
Mouse immunization
HLA-A2 transgenic mice were randomly divided into six groups: CTP-HBcAg18–27-Tapasin (50 μg), CTP-HBcAg18–27 (50 μg), HBcAg18–27-Tapasin (50 μg), CTP-HBcAg18–27-Tapasin+AG490 (50 μg), AG490, and PBS. Mice were subcutaneously (S.C.) immunized with the recombinant protein [14] at 1-week intervals. Meanwhile, the two groups were daily treated with AG490 (5 mg/kg) via intraperitoneal injection in a total volume of 100 μl. And then serum samples, splenocytes, and livers were collected 1 week after the final immunization (Fig. 1A).
Productions of the cytokines IFN-γ and IL-2 (A) Working model summarizing our work. (B,C) Secretion of IFN-γ and IL-2 in HLA-A2 transgenic mice immunized with CTP-HBcAg18–27-Tapasin is significantly higher than those in the CTP-HBcAg18–27 or HBcAg18–27-Tapasin or CTP-HBcAg18–27-Tapasin +AG490 or AG490 or PBS group. (D,E) Levels of IFN-γ and IL-2 in HBV transgenic mice. Supernatant levels of IFN-γ and IL-2 were significantly decreased in AG490 group. Data are from at least three independent experiments. Data are expressed as the mean ± SD (n = 6). **P < 0.01.
C57BL/6-HBV transgenic mice were randomly divided into seven groups: CTP-HBcAg18–27-Tapasin (50 μg), CTP-HBcAg18–27 (50 μg), HBcAg18–27-Tapasin (50 μg), CTP-HBcAg18–27-Tapasin+AG490 (50 μg), AG490, 20,000 IU IFN-α, and PBS. The protocol was the same as that mentioned above. Mice were sacrificed at appropriate time interval.
Isolation of T lymphocytes from transgenic mice
Single cell suspension of splenocytes was derived from immunized HLA-A2 transgenic mice and HBV transgenic mice according to the manufacturer’s protocol (Beijing Combi Source Technology Co., Ltd, Beijing, China). T lymphocytes isolated from the above mixed lymphocytes as described previously [14] were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). The purity of the T cells was detected by flow cytometry after staining with anti-CD3-PE-Cy5 (eBioscience), and cells with the purity > 80% were used for this study.
ELISA
T lymphocytes (2 × 106 cells/ml) were cultured in 24-well plates at 37°C with 10 μg/ml HBcAg18–27. After 72 h incubation, titers of IFN-γ and IL-2 in the supernatant were assayed using commercial ELISA kit (R&D Co. Ltd). Absorbance was measured at 450 nm. Data are expressed as pg/ml.
Flow cytometry assay
To detect the proportion of IFN-γ secreting CD8+ T cells and IFN-γ or IL-2 secreting CD4+ T cells, the splenocytes prepared as above were stimulated for 6 h with HBcAg18–27 at a final concentration of 10 μg/ml. After stimulation for 3 h, 25 μg/ml of PMA, 1 μg/ml of ionomycin, and 1.7 μg/ml of Monessen were added and then incubated for another 3 h [19]. Cells were labeled with monoclonal anti-mouse CD8α-FITC antibody or anti-mouse CD4-FITC antibody followed by addition of the Fix and Perm reagent A and B (eBioscience). The intracellular cytokines were stained with anti-mouse IFN-γ-PE or anti-mouse IL-2-PE antibodies. Data were acquired by flow cytometry (Beckman, Pasadena, USA). Fluorescence analyses were performed on COULTER EPICS XL Flow Cytometer (Beckman) using Expo32-ADC software.
Enzyme-linked immunospot assay
The secretion of IFN-γ from the HBcAg18–27-specific effector cells was analyzed using enzyme-linked immunospot (ELISPOT) assay. Briefly, splenocytes prepared from transgenic mice (HLA-A2 and HBV transgenic mice) as described above were added into wells (5 × 105 cells/well) of pre-coated immunospot plates with anti-IFN-γ McAb (BD, San Diego, USA), and co-cultured with HBcAg18–27 peptide (10 μg/ml). Positive control with PHA (15 μg/ml) and negative controls without peptide were performed in each test. After overnight incubation at 37°C under 5% CO2 and extensive washing with the washing buffer 5–7 times for 30–60 s each time to remove free peptides, the production of IFN-γ was detected using biotinylated anti-IFN-γ antibody (Dakewe Biotech), followed by incubation with streptavidin-HRP solution and 3-amino-9-ethylcarbazole substrate at 37°C for 1 h. The number of IFN-γ-positive spot-forming cells (SFCs) was counted with Bioreader 4000 PRO-X (Bio-Sys GmbH, Karben, Germany). As the positive control, the lymphocyte mitogen, phytohemagglutinin (PHA) stimulation was used. As a background control, X-VIVO15 serum-free medium (Lonza, Basel, Switzerland) was used. Each condition was assessed in triplicate.
T lymphocyte proliferation assay
The collected T lymphocytes (2 × 106 cells/ml) were seeded into wells of 96-well plates and cultured in RPMI-1640 medium supplemented with 10% FBS. The proliferation was measured using Cell Counting Kit-8 (CCK8, Dojindo). After the cells were treated with Concanavalin A (ConA) for 48 h, the CCK8 reagents were added to each well at a volume ratio of 10:1. The plates were incubated for another 4 h at 37°C and the absorbance was measured at a wavelength of 450 nm.
Serological analysis
The levels of HBsAg and HBV DNA in serum samples were separately analyzed on Days 7 and 21. HBsAg was analyzed with the Axsym Abbott system (Abbott Diagnostics, Chicago, USA), and serum HBV DNA was determined by quantitative PCR (Terra PCR Direct Polymerase mix; Clontech, Mountain View, USA). The inhibitory effects of HBsAg were detected in each group. The HBV DNA levels were transformed logarithmically. Alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were detected with ARCHITECT Automatic Biochemistry Analyzer (Abbott Diagnostics).
Hematoxylin and eosin staining and immunohistochemistry assay
Liver tissues collected from HBV transgenic mice on Day 7 after the last immunization were fixed with 10% paraformaldehyde for 24 h and embedded in paraffin. Deparaffinized sections (3–5 μm) were stained with hematoxylin and eosin (H&E).
For immunohistochemistry assay, deparaffinized 35 μm-thick tissue sections were incubated with 0.3% H2O2 to effectively inhibit the endogenous peroxidase activity. After being blocked with normal goat serum at room temperature for 30 min, the sections were labeled overnight at 4°C with goat anti-HBsAg polyclonal antibody or goat anti-HBcAg polyclonal antibody (Novus Biologicals, Littleton, USA). After being washed three times in PBS, sections were treated with biotinylated secondary antibody (Boster) and streptavidin –biotin–peroxidase complex, followed by visualizing by incubation with diaminobenzidine (DAB). Finally, sections were counterstained with hematoxylin.
Real-time quantitative PCR
Total RNA was extracted from T cells using TRIZOL reagent (Invitrogen, Carlsbad, USA). The first-strand cDNA was synthesized using the Superscript II Reverse Transcriptase (Invitrogen) with specific primer sets (Table 1) obtained from Takara Bio (Kusatsu, Japan). Real-time quantitative PCR was performed using SYBRsPremix Ex TaqTM reagents (Takara) on a Light-Cycler (Roche Diagnostic, Penzberg, Germany). Data analysis was performed by 2-△△Ct method. GAPDH were used for normalization.
The primer sequences used for quantitative real-time PCR (F): Forward primer; (R): reverse primer.
| mRNA | Primers |
|---|---|
| Jak1 | (F) 5′-CAGATGCCCACCATTACC-3′ |
| (R) 5′-CCCTCTTCACTCCCTTCC-3′ | |
| Jak2 | (F) 5′-GGCAGCAGCAGAACCTAC-3′ |
| (R) 5′-GTCTAACACCGCCATCCC-3′ | |
| Jak3 | (F) 5′-CCCATCCGCTGAGTTCCT-3′ |
| (R) 5′-GGCTGCTATCCGGGTCTT-3′ | |
| Tyk2 | (F) 5′-TTCCGTAGCAACCGTCTC-3′ |
| (R) 5′-CATCAAGCATCCTGTGGG-3′ | |
| STAT1 | (F) 5′-CTATGAGCCCGACCCTAT-3′ |
| (R) 5′-TTGAACTTCCGAAATCCT-3′ | |
| STAT4 | (F) 5′-CCTGCTGTTGGTTGGTGT-3′ |
| (R) 5′-CTTGAGGCTTTCCTGTGC-3′ | |
| STAT6 | (F) 5′-TCCTGGTCACAGTTCAATAA-3′ |
| (R) 5′-CGATCTCAGAGTCGCTAAA-3′ |
| mRNA | Primers |
|---|---|
| Jak1 | (F) 5′-CAGATGCCCACCATTACC-3′ |
| (R) 5′-CCCTCTTCACTCCCTTCC-3′ | |
| Jak2 | (F) 5′-GGCAGCAGCAGAACCTAC-3′ |
| (R) 5′-GTCTAACACCGCCATCCC-3′ | |
| Jak3 | (F) 5′-CCCATCCGCTGAGTTCCT-3′ |
| (R) 5′-GGCTGCTATCCGGGTCTT-3′ | |
| Tyk2 | (F) 5′-TTCCGTAGCAACCGTCTC-3′ |
| (R) 5′-CATCAAGCATCCTGTGGG-3′ | |
| STAT1 | (F) 5′-CTATGAGCCCGACCCTAT-3′ |
| (R) 5′-TTGAACTTCCGAAATCCT-3′ | |
| STAT4 | (F) 5′-CCTGCTGTTGGTTGGTGT-3′ |
| (R) 5′-CTTGAGGCTTTCCTGTGC-3′ | |
| STAT6 | (F) 5′-TCCTGGTCACAGTTCAATAA-3′ |
| (R) 5′-CGATCTCAGAGTCGCTAAA-3′ |
The primer sequences used for quantitative real-time PCR (F): Forward primer; (R): reverse primer.
| mRNA | Primers |
|---|---|
| Jak1 | (F) 5′-CAGATGCCCACCATTACC-3′ |
| (R) 5′-CCCTCTTCACTCCCTTCC-3′ | |
| Jak2 | (F) 5′-GGCAGCAGCAGAACCTAC-3′ |
| (R) 5′-GTCTAACACCGCCATCCC-3′ | |
| Jak3 | (F) 5′-CCCATCCGCTGAGTTCCT-3′ |
| (R) 5′-GGCTGCTATCCGGGTCTT-3′ | |
| Tyk2 | (F) 5′-TTCCGTAGCAACCGTCTC-3′ |
| (R) 5′-CATCAAGCATCCTGTGGG-3′ | |
| STAT1 | (F) 5′-CTATGAGCCCGACCCTAT-3′ |
| (R) 5′-TTGAACTTCCGAAATCCT-3′ | |
| STAT4 | (F) 5′-CCTGCTGTTGGTTGGTGT-3′ |
| (R) 5′-CTTGAGGCTTTCCTGTGC-3′ | |
| STAT6 | (F) 5′-TCCTGGTCACAGTTCAATAA-3′ |
| (R) 5′-CGATCTCAGAGTCGCTAAA-3′ |
| mRNA | Primers |
|---|---|
| Jak1 | (F) 5′-CAGATGCCCACCATTACC-3′ |
| (R) 5′-CCCTCTTCACTCCCTTCC-3′ | |
| Jak2 | (F) 5′-GGCAGCAGCAGAACCTAC-3′ |
| (R) 5′-GTCTAACACCGCCATCCC-3′ | |
| Jak3 | (F) 5′-CCCATCCGCTGAGTTCCT-3′ |
| (R) 5′-GGCTGCTATCCGGGTCTT-3′ | |
| Tyk2 | (F) 5′-TTCCGTAGCAACCGTCTC-3′ |
| (R) 5′-CATCAAGCATCCTGTGGG-3′ | |
| STAT1 | (F) 5′-CTATGAGCCCGACCCTAT-3′ |
| (R) 5′-TTGAACTTCCGAAATCCT-3′ | |
| STAT4 | (F) 5′-CCTGCTGTTGGTTGGTGT-3′ |
| (R) 5′-CTTGAGGCTTTCCTGTGC-3′ | |
| STAT6 | (F) 5′-TCCTGGTCACAGTTCAATAA-3′ |
| (R) 5′-CGATCTCAGAGTCGCTAAA-3′ |
Western blot analysis
Total proteins from different groups were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Protein concentrations were measured with Pierce BCA protein assay reagent kit (Rockford, USA). The extracted protein were loaded on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in Tris-buffered saline-Tween (TBST) solution for 1 h at room temperature and then incubated with antibodies specific to Jak1 (1:2000; Abcam, Cambridge, USA), Jak2 (1:2000; Abcam), Jak3 (1:1000; CST, Beverly, USA), Tyk2 (1:1000; Abcam), STAT1 (1:1000; CST), STAT4 (1:1000; CST), STAT6 (1:1500; Abcam), and GAPDH (1:2000; Abcam), followed by incubation with horseradish peroxidase-conjugated secondary antibody. After the final wash with TBST, the protein bands was detected using an enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK). The signals were exposed on X-Ray film and the density of the bands was analyzed with an Xomat machine (Amersham Pharmacia, Cambridge, UK).
Statistical analysis
Data were expressed as the mean ± SD. SPSS16.0 software package was used to analyze all parameters. The HBV DNA levels were logarithmically transformed for analysis. The difference between two groups was determined by one-way ANOVA and post-hoc least significant difference (LSD) test. P < 0.05 was considered as significant.
Results
CTP-HBcAg18–27-Tapasin stimulates the secretion of cytokines via JAK/STAT pathway
Accumulating evidence implies that JAK/STAT pathway plays a crucial role in mediating cytokine family [20]. Incompetent CD8+ CTLs is associated with a weak Th1 immunity. Thus, we detected the levels of the cytokines IFN-γ, IL-2 (Th-1 like) in the splenocytes supernatants in the presence of HBcAg18–27 for 72 h. As shown in Fig. 1B,C, splenocytes from immunized mice with CTP-HBcAg18–27-Tapasin produced higher levels of IFN-γ (303.30 ± 9.93 pg/ml) and IL-2 (483.10 ± 18.52 pg/ml) compared with other groups. On the contrary, the administration with AG490 significantly reduced the levels of IFN-γ and IL-2. As shown in Fig. 1D,E, high levels of IFN-γ (172.33 ± 7.51 pg/ml) and IL-2 (350.67 ± 8.50 pg/ml) in the CTP-HBcAg18–27-Tapasin group was also observed in immunized HBV transgenic mice. In contrast, in inhibitor group the levels were obviously decreased. However, there were no significant differences in the levels of IFN-γ and IL-2 between other groups (P > 0.05). These results indicated that Th1-type immune response elicited by fusion protein could be attenuated with the inhibition of JAK/STAT pathway.
CTP-HBcAg18–27-Tapasin enhances specific CTL response
The intracellular secretion of IFN-γ in CD8+ and CD4+ T cells and intracellular secretion of IL-2 in CD4+ T cells were detected by flow cytometry. As shown in Fig. 2, more IFN-γ+CD8+ T cells were observed in HLA-A2 transgenic mice and HBV transgenic mice immunized with CTP-HBcAg18–27-Tapasin than other groups. Results demonstrated that delivery of Tapasin and HBcAg18–27 via CTP enhanced the generation of specific CTLs in vivo. As shown in Fig. 3, higher percentages of specific IFN-γ+CD4+ T cells and IL-2+CD4+ T cells were observed in HLA-A2 transgenic mice and HBV transgenic mice immunized with CTP-HBcAg18–27-Tapasin (2.80% ± 0.14%) than those in other groups. In contrast, the percentages of IFN-γ+CD4+ T cells and IL-2+CD4+ T cells were significantly lower in AG490 group compared with those of other groups. These results suggested that the frequency of Th1-oriented CD4+ T cells induced by CTP-HBcAg18–27-Tapasin could be suppressed by blocking JAK/STAT signaling pathway.
Intracellular IFN-γ production in CD8+T lymphocytes of transgenic mice (A,B) The level of intracellular staining of IFN-γ in CD8+ T cells from HLA-A2 transgenic mice. (C,D) The secretion levels of IFN-γ in CD8+ T lymphocytes from immunized HBV transgenic mice. Cells were labeled with FITC-CD8α and PE-IFN-γ antibodies. Data are expessed as the mean ± SD from six mice per group. **P < 0.01.
Intracellular IFN-γ and IL-2 production in CD8+T lymphocytes and CD4+T lymphocytes of transgenic mice The whole cell population was labeled with fluorescent material using FITC-CD4 and PE-IFN-γ and analyzed by flow cytometry. (A,B) The stained intracellular cytokine IFN-production in CD4+ T lymphocytes of HLA-A2 transgenic mice. (C,D) The intracellular cytokine IFN-γ production in CD4+ T lymphocytes of HBV transgenic mice. (E,F) The stained intracellular cytokine IL-2 production in CD4+ T lymphocytes of HLA-A2 transgenic mice. (G,H) The intracellular cytokine IFN-γ production in CD4+ T lymphocytes of HLA-A2 transgenic mice. Data are expressed as the mean ± SD from six mice per group. **P < 0.01.
CTP-HBcAg18–27-Tapasin-enhanced specific CTL response and T-cell proliferation is weakened with the blocking of the JAK/STAT pathway
Furthermore, the ability of T lymphocyte proliferation was analyzed in different groups. CTP-HBcAg18–27-Tapasin significantly enhanced T lymphocyte proliferation activity when compared with other groups (Fig. 4A,C). Then ELISPOT assay was used to confirm the elevated specific T-cell response. As shown in (Fig. 4B,D), the number of IFN-producing T cells in AG490 group was dramatically reduced compared with CTP-HBcAg18–27-Tapasin group (**P < 0.01). Those observations suggested that stronger CTL response induced by CTP-HBcAg18–27-Tapasin was correlated with JAK/STAT pathway.
T lymphocyte proliferation activity and ELISPOT IFN-γ detection and changes in serum biochemical marker in HBV transgenic mice (A) The proliferation activity of T lymphocytes was determined using CCK8. (B) The number of IFN-γ spots from HLA-A2 transgenic mice. CTP-HBcAg18–27-Tapasin was found to generate stronger specific T-cell responses compared with control group. (C) The proliferation activity of T lymphocytes from HBV transgenic mice was determined using CCK8. (D) The number of IFN-γ SFCs per 106 splenocytes from HBV transgenic mice. CTP-HBcAg18–27-Tapasin induced higher secretion of IFN-γ compared with other groups, but after treatment with AG490, the level was decreased. (E,F) The serum levels of HBV DNA and HBsAg inhibitory rates in each group. (G,H) ALT and AST levels in HBV transgenic mice. Data are presented as the mean ± SD (n = 6). *P < 0.05, **P < 0.01.
Serum levels of HBsAg, HBV DNA, ALT, and AST
Subsequently, to explore whether the JAK/STAT signaling pathway is involved in the therapeutic effects of the fusion protein, we analyzed the serological changes in HBV transgenic mice. The titers of HBV DNA were decreased in CTP-HBcAg18–27-Tapasin group (Fig. 4E), which was consistent with the higher inhibition rate of serum HBsAg compared with other groups (Fig. 4F). However, these results were in contrast to those of pharmacological inhibitor AG490 group. The serum levels of ALT and AST in CTP-HBcAg18–27-Tapasin were higher than other groups (Fig. 4G,H). These results indicated that the effects of fusion protein were due to the enhanced immune responses via JAK/STAT pathway.
Histopathological changes in HBV transgenic mice
Changes in H&E and immunohistology were analyzed in liver tissues from the various groups. As shown in Fig. 5A, tremendous numbers of lymphocytes infiltration were detected in the liver of mice treated with CTP-HBcAg18–27-Tapasin, compared with the other groups. Furthermore, the immunohistological changes were analyzed in each group. Higher levels of the cytoplasmic HBsAg and nuclear HBcAg were found in AG490 group (Fig. 5B,C) when compared with the positive control group. These observations implied that JAK/STAT signal pathway might be effective in the potent antiviral immune responses induced by CTP-HBcAg18–27-Tapasin-enhanced immune response.
Histopathologic changes and immunohistological detection of HBsAg and HBcAg in liver biopsies of HBV transgenic mice (A) Hematoxylin and eosin staining of liver sections examined by light microscopy. (B,C) Immunohistochemical staining with HBsAg and HBcAg antibody, respectively. Representative photographs are presented.
CTP-HBcAg18–27-Tapasin enhances the mRNA and protein expression levels of target molecules in JAK/STAT pathway
To further explore the molecular mechanisms, the JAK/STAT pathway was blocked with the pharmacological inhibitor AG490 and then the expressions of Jak1, Jak2, Jak3, Tyk2, STAT4, STAT1, and STAT6 were further analyzed at protein and mRNA levels in HBV transgenic mice. As shown in Fig. 6. The expression levels of Jak2, Tyk2, STAT1, and STAT4 were significantly up-regulated in transgenic mice immunized with CTP-HBcAg18–27-Tapasin compared with other groups. However, the expression levels of Jak1, Jak3, and STAT6 were not significantly different between CTP-HBcAg18–27-Tapasin+AG490-treated group and vehicle one (P > 0.05; Fig. 6). These results implied that molecules in JAK/STAT pathway might play a crucial role in robust CTLs induced by CTP-HBcAg18–27-Tapasin.
mRNA and protein expressions of related molecules of JAK/STAT pathway in HBV transgenic mice (A) The expression levels of Jak1, Jak2, Jak3, Tyk2, STAT1, STAT4, and STAT6 protein. Lane 1, AG490; Lane 2, CTP-HBcAg18–27-Tapasin+AG490; Lane 3, PBS; Lane 4, HBcAg18–27-Tapasin; Lane 5, CTP-HBcAg18–27; Lane 6, CTP-HBcAg18–27-Tapasin. (B) The relative expression of the above proteins. The expressions of Jak2, Tyk2, STAT1, and STAT4 were significantly up-regulated in CTP-HBcAg18–27-Tapasin group compared with other groups. (C) The mRNA expression levels of molecules of the JAK/STAT pathway. Data are presented as the mean ± SD (n = 6). *P < 0.05, **P < 0.01.
Discussion
HBV infection is highly prevalent in developing countries, despite the current availability of efficient vaccine and antiviral drugs [21]. The persistent infection of HBV leads to the development of end-stage liver disease and HCC. HBV acts as a stealth virus to escape the recognition by host innate immune response sensor molecules [22]. Therefore, the clearance of HBV and disease pathogenesis was largely mediated by the adaptive immune response whose major component was HBV-specific CD8+ T cells. The ER resident protein tapasin could facilitate the loading of HBV antigen peptide. It has been demonstrated that the expression level of tapasin was significantly downregulated in chronically infected patient (CHB) individuals compared with healthy controls [23].
The virus-specific CD8+ T-cell responses are vigorous and multi-specific in acute HBV individuals who successfully clear the virus. However, cytotoxic T lymphocyte (CTL) responses are relatively undetectable in CHB individuals, suggesting the HBV-specific CTL response in the clearance of HBV [2]. In CHB individuals, HBV-specific T cells are dysfunctional or exhausted owing to the prolonged exposure to HBV antigens. Therefore, the restoration of HBV antigen-specific immune response and the induction of Th1 immunity might inhibit HBV infection.
It has been demonstrated that the activation and differentiation of naive CD8+ T cells into effector CTLs requires help from CD4+ Th cells which are activated and provide help via production of cytokines such as IL-2 [24, 25]. Preferential production of Th1-type immune response is detected in the course of acute self-limited hepatitis B that is manifested by predominant production of IFN-γ and IL-2, which induces T-cell proliferation and differentiation [26]. Our results showed that the secretion levels of Th-1 like cytokines (IFN-γ and IL-2) were significantly increased in CTP-HBcAg18–27-Tapasin group, compared with AG490 treatment group in HBV transgenic and HLA-A2 transgenic mice. Moreover, there is no significant difference between the CTP-HBcAg18–27-Tapasin+AG490 group and the PBS group. The results of flow cytometry analysis and ELISPOT assay showed that the robust immune response induced by fusion protein was diminished after the blocking of JAK/STAT pathway.
The JAK/STAT signaling pathway is crucial in mediating cell differentiation and cytokine production [27]. The relative expression of specific transcription factors could modulate the differentiation of Th cells. JAK phosphorylates the downstream protein STATs to activate the related genes and exert its corresponding response. Unphosphorylated STATs have initially been thought to be functionless. Selective combination with members of the JAK and STAT families by a given cytokine receptor is responsible for the specificity of a cytokine action. However, recent studies have suggested that the pathway is far more complicated than previously thought to be, and unphosphorylated STATs are also able to enter the nuclear and bind to DNA as either a dimer or a monomer and regulate the expressions of related genes [28–30]. Nevertheless, further study is needed to confirm the role of unphosphorylated STATs. In our previous work, CTP-HBcAg18–27-Tapasin was found to induce the HBV-specific CTLs, and intracellular signaling pathway might be involved in the above immune response. In the present study, we further explored the participation of JAK/STAT signaling pathway in robust CTL response mediated by CTP-HBcAg18–27-Tapasin to inhibit HBV replication.
Several studies have implied that STAT1 and STAT4 are critical in Th1 cell differentiation [31, 32]. Our results revealed that the expression levels of Jak2, Tyk2, STAT1, and STAT4 were significantly up-regulated in HLA-A2 transgenic mice and HBV transgenic mice immunized with CTP-HBcAg18–27-Tapasin, when compared with other groups. In contrast, the amounts of Jak2, Tyk2, STAT1, and STAT4 in inhibitor group were markedly decreased in line with the reduced levels of Th-1 cytokines (IL-2 and IFN-γ). We further measured the levels of HBV DNA and HBsAg in serum and the expression levels of HBsAg and HBcAg in liver tissues in HBV transgenic mice treated with CTP-HBcAg18–27-Tapasin+AG490. However, the changes of the inflammatory reaction in AG490 group were no significant. Therefore, we hypothesize that the fusion protein may promote Th1 cell differentiation and induce potent CTLs via JAK/STAT pathway.
Several studies have showed that the boosted efficiency of CD8+ T-cell response is closely associated with early CD4+ T-cell priming. The deficient early CD4+ T-cell priming might result in the inefficient CD8+T-cell cytotoxicity [27]. Strong and persistent CD8+ and CD4+ T-cell responses are critical in HBV clearance and cytokine-induced factors that can directly inhibit virus replication. The chronicity of HBV infection is marked by a dysfunctional CD8+ CTLs and dramatic reduction of CD4+ Th cells in the liver [33]. IL-2 is a pleotropic cytokine that plays a crucial role in effective Th1 development [34]. We further measured the intracellular cytokine levels of IL-2 in T cells by flow cytometry. Results demonstrated that CTP-HBcAg18–27-Tapasin up-regulated the frequencies of IL-2-secreting CD4+ T cells in HLA-A2 transgenic mice and HBV transgenic mice. Studies have also shown that IL-2 could stimulate the transfer of activated CD8+ T cells into effective memory CD8+ T cells. Furthermore, CTP-HBcAg18–27-Tapasin significantly enhanced T lymphocyte proliferation and differentiation, which was higher than the control groups. Our observations demonstrated that the percentages of IFN-γ producing CD8+ T cells and CD4+ T cells (Th1) were enhanced in HLA-A2 transgenic mice and HBV transgenic mice immunized with CTP-HBcAg18–27-Tapasin. In contrast, there is no significant difference in AG490 groups. These results indicated that CTP-HBcAg18–27-Tapasin could enhance CTL and CD4+ T (Th1) cell response via JAK/STAT pathway to inhibit HBV replication.
In summary, our results demonstrated that fusion protein CTP-HBcAg18–27-Tapasin could increase the percentages of CTLs and elicit Th1 immune response with robust HBV-specific CTL activity by targeting Jak2, Tyk2, STAT1 and STAT4 molecules in the JAK/STAT signaling pathway. Further research is needed to explore the mechanisms underlying DNA-binding and regulation of gene expression by these unphosphorylated proteins.
Funding
This work was supported by the grants from Scientific Research Innovation project of Shanghai Education Committee (No. 15ZZ013) and the Shanghai Natural Science Foundation (No. 17ZR1421500).
References
Author notes
Shanshan Wu and Xiaohua Chen contributed equally to this work.
