key: cord-338165-mgrf1odm
authors: Wang, Xiaojing; Yan, Weiming; Lu, Yulei; Chen, Tao; Sun, Ying; Qin, Xiaomin; Zhang, Jiangguo; Han, Meifang; Guo, Wei; Wang, Hongwu; Wu, Di; Xi, Dong; Luo, Xiaoping; Ning, Qin
title: CD4–CD8-T cells contribute to the persistence of viral hepatitis by striking a delicate balance in immune modulation
date: 2012-11-30
journal: Cellular Immunology
DOI: 10.1016/j.cellimm.2012.11.010
sha: 
doc_id: 338165
cord_uid: mgrf1odm

Abstract Viral hepatitis remains the most common cause of liver disease and a major public health problem. Here, we focus on the role of CD4 CD8 double negative T (DN T) cells involved in the mechanisms of viral persistence in hepatitis. C3H/HeJ mice infected with murine hepatitis virus strain 3 (MHV-3) were used to display chronic viral hepatitis. DN T cells dramatically increased in MHV-3 infected mice. Adoptive transfer of DN T cells from MHV-3 infected mice led to a significant increase in mice survival. The DN T cells with production of IFN-γ and IL-2 are able to kill virus-specific CD8+ T cells via the Fas/FasL dependent pathway. The delicate balance of multiple effects of DN T cells may lead to viral persistence in MHV-3 induced hepatitis. In short, our study identified DN T cells contributing to viral persistence in MHV-3 induced hepatitis in C3H/HeJ mice, which provides a rationale for modulating DN T cells for the management of viral hepatitis.

Viral hepatitis remains a major public health problem in the world with considerable morbidity and mortality. It is estimated that there are over 350 million carriers of hepatitis B virus (HBV) and 250 million carriers of hepatitis C virus (HCV) worldwide [1, 2] . Persistent infection may progress to chronic liver disease, cirrhosis and primary liver cancer [3] .

The viral proteins expressed in hepatocytes may influence the severity and progression of liver disease. However, extensive studies suggest that the mechanisms of liver injury in viral hepatitis are due to the host immune responses, but not to the direct cytopathic effects of viruses. The direct killing of infected cells by virusspecific CD8 + cytotoxic T lymphocytes (CTLs) is considered as the central mechanism resulting in both liver damage and virus control. In addition, CD4 + T helper response is also found to have a strong association with viral clearance in the acute phase of hepatitis [4] .

Viral persistence during hepatitis infection may be the direct result of a weak antiviral immune response to the viral antigens, with corresponding inability to eradicate virus within the infected cells. Accumulating evidence has indicated that regulatory T (Treg) cells play an important role in the suppression of virus specific immune responses [5] [6] [7] . Many subsets of Treg cells have been studied including CD4 + CD25 + Tregs [8] [9] [10] [11] [12] [13] , CD4 + DX5 + T cells [14] , Agspecific CD4 + or CD8 + T cells that secrete the immuno-regulatory cytokines IL-10 (Tr1 cells) or TGF-b (Th3 cells), TCRcd + T cells [15, 16] , and TCRab + CD3 + CD4 À CD8 À double-negative (DN) T cells [17] [18] [19] .

Among Treg cells, CD4 + CD25 + Tregs are the most extensively studied. A transcription factor Foxp3 is considered as the optimal marker of classic natural Treg [20] . In patients with HBV or HCV infection, CD4 + CD25 + Foxp3 + Tregs have increased levels and impair the immune responses directed against hepatitis viruses, leading to persistent infections and chronic liver injury [21] [22] [23] . Lately, Shalev et al. reported that adoptive transfer of Tregs from fgl2 +/+ mice into fgl2 À/À mice resulted in increased mortality to MHV-3 infection, demonstrating the critical role for CD4 + CD25 + Tregs in the pathogenesis of MHV-3 induced fulminant hepatitis [24] .

DN T cells are a novel subset of Treg cells which is first identified by Zhang and colleagues. In mice, DN Treg cells could kill activated CD8 + T cells with the same TCR specificity, and infusion of in vitro-activated DN Treg cells led to significant prolongation of donor-specific skin and heart graft survival [17, [25] [26] [27] . Recently, increasing attention has been focused on these novel Treg cells. Crispín et al. demonstrated that DN T cells produce IL-17/IFN-c and contribute to the pathogenesis of kidney damage in patients with SLE [28] . Another study showed that DN T splenic cells from young NOD mice could provide long-lasting protection against type 1 diabetes [29] . In SIV-induced CD4 + T cell depletion in sooty mangabeys which do not present immune dysfunction and clinical AIDS, DN T cells partially compensates for CD4 + T cell function in these animals [30] . In cutaneous leishmaniasis infection, TCRab + DNT cells present an increased bias in their capacity to induce inflammatory immune responses, and TCRcd + DNT cells show a regulatory profile [31] .

In the present study, we investigated the characteristics and contribution of splenic DN T cells in a MHV-3 induced chronic viral hepatitis murine model. And our study provides a rationale for modulating DN T cells for the management of viral hepatitis.

All animal studies were carried out according to the guidelines of the Chinese Council on Animal Care, and were approved by the Tongji Hospital of Tongji Medical School Committees on Animal Experimentation (No.2009619).

MHV-3 was obtained from the American Type Culture Collection (ATCC), plaque-purified on a monolayer of DBT cells, and titered on L2 cells using a standard plaque assay. C3H/HeJ mice were purchased from Shanghai Laboratory Animal Center of Chinese Academy of Science (Shanghai, ROC). MHV-3 (10 Pfu/200 lL) was individually injected into the peritoneal cavity of C3H/HeJ mice as previously described [32] . Peripheral blood and livers were obtained on different time points after MHV-3 infection. C3H/HeJ mice receiving 200 lL of 0.9% NaCl were used as controls. The survival, serum biochemistry parameters including ALT, AST, TP, ALB, and liver histology were observed. Virus titers were determined in the liver tissue of MHV-3 infected C3H/HeJ mice at various time points by standard plaque assay as described before [32] .

Blood, spleen, and liver were collected from MHV-3 infected C3H/HeJ mice on indicated days post infections. At each time point, triplicate samples were examined. C3H/HeJ mice treated with 200 lL NaCl were used as control.

Three-color cytofluorometric analysis was performed on the FACSAria Flow Cytometer (BD Biosciences, San Jose, CA, USA). In all experiments, approximately 106 cells/100 L (from the single cell suspensions of processed PBMC, spleen and liver) were stained by FITC-anti-CD3, PE-anti-CD4 and PerCP-anti-CD8 (eBioscience, San Diego, CA, USA) monoclonal antibodies. Corresponding isotype antibodies were used as controls.

Five days after MHV-3 infection, three groups (30 mice in each group) of mice were treated respectively with DN T cells, splenocytes or DN T-depleted splenocytes from MHV-3 infected mice via the tail vein. Another 30 C3H/HeJ mice treated with PBS were used as controls. Liver tissue samples of recipient mice from each group (3-5 mice in each group) were harvested and stained with H&E 12 days post the adoptive transfer.

The Knodell hepatitis activity index (HAI) was used to evaluate the severity of the necroinflammation. The Knodell HAI score consists of four separate scores, including periportal necrosis with or without bridging necrosis (0-10), intralobular degeneration and focal necrosis (0-4), portal inflammation (0-4) and fibrosis (0-4) [33] . Virus titers in the liver tissues of recipient mice from each group were tested by standard plaque assay at indicated time points.

Expression of DN T cell surface markers were determined by flow cytometry analysis. Cell suspensions of processed spleens from C3H/HeJ mice on days 4, 10, 15, 20, and 30 post MHV-3 infection were stained with PEcy5.5-anti-CD3, FITC-anti-CD4, FITC-anti-CD8, APC-anti-CD25, APC-anti-TCRb, PE-anti-TCRcd, PE-anti-CD28/PE-anti-CD30, PE-anti-CD44, PE-anti-CD95L, PEanti-CD95 monoclonal antibodies or isotype control Ab (eBioscience, San Diego, CA, USA).

Cells were harvested from the spleen of MHV-3 infected C3H/ HeJ mice. Flow cytometric detection of intracellular cytokines was performed as previously described. The cells were stimulated with 25 lmol/L ionomycin and 10 ng/mL PMA in the presence of monensin (10 lg/mL) for 4 h at 37°C in 5% CO 2 . Cells were washed and fixed in 1 mL 4% ice-cold paraformaldehyde for 20 min. Before the initial staining, 10 7 cells/tube were washed with saponin/PBS buffer to permeabilize the plasma and intracellular membranes. PE-anti-IL-2, PE-anti-IL-4, PE-anti-IFN-c, PE-anti-IL-10, PE-anti-TNFa, PE-Anti-perforin, PE-anti-Granzyme B or isotype-matched, irrelevant control Ab (eBioscience, San Diego, CA,USA) was respectively added to the suspension and incubated for 15 min. After two further washes in saponin/PBS buffer, cells were stained with PEcy5.5-anti-CD3, FITC-anti-CD4, FITC-anti-CD8 (eBioscience, San Diego, CA, USA) for 20 min. Then the cells were analyzed by flow cytometry.

The spleens from C3H/HeJ mice were made into suspensions and incubated with magnetic bead sorting buffer. Non-T cells, i.e. B cells, NK cells, dendritic cells, and macrophages were magnetically labeled by a cocktail of biotin-conjugated antibodies and anti-biotin microbeads (Miltenyi Biotec, Cologne, Germany). Isolation of highly pure T cells was achieved by depletion of magnetically labeled cells.

Using the CD4 + T Cell Isolation kit (Miltenyi Biotec, Cologne, Germany), CD4 + T cells were then isolated from T cells obtained previously by depletion of non-CD4 + T cells (negative selection). CD8 + T cells were isolated from the non-CD4 + T cells using the CD8 + T Cell Isolation kit (Miltenyi Biotec, Cologne, Germany). The unlabeled non-CD4 + , non-CD8 + T cells were DN T cells.

Cytotoxicity of DN T cells was measured using the CytoTox 96 Non-Radioactive Cytotoxicity assay kit (Promega, Madison, WI, USA) following the manufacturer's instruction. This cytotoxicity kit measures the lactate dehydrogenase (LDH) activity released into the culture medium by lysed cells. MHV-3 specific CD8 + T cells, normal CD8 + T cells, MHV-3 infected hepatocytes, and normal hepatocytes were used as target cells, DN T cells were used as effector T cells.

The experiment was repeated after FasL blockade with anti-mFasL antibodies (R&D Minneapolis, MN, USA) with a 5:1 ratio of DNT cells to CD8 + T cells.

Mice infected with the Smith strain of murine cytomegalovirus (MCMV) were taken as an unrelated control. CD8 + T cells were iso-lated from the spleens of C3H/HeJ mice post MCMV infection. The cytotoxic effect of DN T cells (from the spleens of MHV-3 infected mice) on CD8 + T cells (from the spleens of MCMV infected mice) was then examined.

Both DN T cells and CD8 + T cells were isolated from the spleen of MHV-3 infected C3H/HeJ mice. Transwell (Corning, Lowell, MA, USA) experiments were conducted in 24 wells in 0.8 mL of complete tissue culture medium. The semipermeable membrane separating the upper and lower chambers allows diffusion of soluble materials but not cells. To measure the effect of non-contact cytotoxicity of DN T cells on CD8 + T cells, CD8 + T cells (10 5 ) were cultured in the lower chamber and DN T cells (5 Â 10 5 or 2.5 Â 10 5 ) in the upper chamber. CD8 + T cells were cultured in the lower chamber alone as control. After an incubation period of 24 h, CD8 + T cells were stained with FITC-anti-CD8, washed, and then stained with APC-anti-annexin V (Bender Med system, Vienna, Austria) and PI (Jingmei Biotech, Shanghai,China). At least 5,000 CD8 + T events were collected for annexin V/PI analysis. In this approach, the percent of annexin V À /PI À events (viable cell population) were used to correct for spontaneous apoptosis with the following formula:

% Cytotoxicity ¼ ½% control À viable cells À ½% of coincubated À viable cells ½% control À viable cells

The result reflects the rate of cells undergoing apoptosis/death at the time of sampling.

All data are presented as mean ± SEM. Comparisons of data were performed by using the Student's two-tailed t-test or oneway analysis of variance (ANOVA), followed by the Student-Newman Keuls test. Statistical package for the social science (SPSS) was used for data analysis. In all cases, significance was determined at P < 0.05.

In order to investigate the role of DN T cells in antiviral responses, we developed a chronic hepatitis model by infecting C3H mice with MHV-3 as formerly reported. For the first time we extensively characterized the disease chronicity, liver function, and pathology of viral hepatitis in this model.

The infected C3H/HeJ mice began to have anorexia and acratia 4-6 days post infection. Between Day 6 and Day 15 post MHV-3 infection, approximately 76.7% mice died, and the survivors became persistently infected afterwards (Fig. 1A) . The virus in the liver tissues of infected mice could be detected during the whole course of observation. The virus replication peaked on day 12, and then decreased and maintained at a low level. (Fig. 1B) Various degrees of hydropic degeneration, hepatocellular necrosis and inflammatory infiltration were observed in the liver of C3H/HeJ mice after 4 days of infection. The most severe liver injury was observed between day 10 and day12, with dramatically impaired liver function, including increased ALT and AST as well as decreased total protein and albumin (Table. 1 ). After that, the hepatocellular injury in the survived mice was alleviated, accompanied with less infiltration and improved liver function. But all the inflammatory changes could still be observed in the livers of survivors during the period of observation (Fig. 1C) .

The proportion of DN T cell in the blood, liver, and spleen rose significantly after MHV-3 infection in C3H/HeJ mice ( Fig. 2A, C) , peaked between Day 12 and 15 and then decreased gradually. The absolute count of DN T cells in either liver or spleen presented a similar change as the proportion did (Fig. 2B ).

3.3. Adoptive transfer of DN T cells from MHV-3 infected C3H/HeJ mice increased the survival rate and improved liver histology of recipient mice infected by the same virus strain but had little impact on the virus titer of liver tissue Fig. 3A showed that no significant difference in virus titer of liver tissue was observed among DN T cells group, splenocytes group, DNT-depleted splenocytes group and PBS control group.

After adoptive transfer of either DN T cells or splenocytes from MHV-3 infected C3H/HeJ mice, the survival rate of the recipient mice raised notably to 46.67% and 43.33%, respectively, while only 23.33% of mice receiving PBS or 20% of mice receiving DN T-depleted splenocytes survived (Fig. 3B) .

As shown in Fig. 3C , mice receiving DN T cells or splenocytes had alleviated hydropic degeneration, inflammatory cells infiltration, as well as bridge necrosis in their livers compared with mice receiving PBS or DN T-depleted splenocytes. Compared with splenocytes group, the hepatocytes swelling in the recipient mice in DN T cells group were not as much severe. Comparison of Knodell score (HAI) among the four groups on day 12 after the adoptive transfer was shown in Fig. 3D . Significant difference was found when DN T cells group vs. DNT-depleted splenocytes group (2.0 ± 0 versus 7.3 ± 1.1, ⁄ P < 0.01), and DN T cells group vs. PBS control group (2.0 ± 0 versus 5.7 ± 0.6, ⁄ P < 0.01).

To further characterize the DN T cells, we detected the cell surface markers of DN T cells from spleens of C3H/HeJ mice post MHV-3 infection (Fig. 4A) . The majority of DN T cells expressed TCRab + while only a small part expressed TCRcd + . Adhesion molecule CD44 was highly expressed on DN T cells, while activation markers CD25, CD28 or CD30 were rarely detected. Moreover, these DN T cells did not recognize aÀGalcer (which NKT cells do in an Ag-specific fashion), demonstrating that DN T cells are different from NKT cells (data not shown). Intracellular cytokines of DN T cells were detected on Day 0, 6, 10, 15, 20, and 30 after MHV-3 infection. The infected DN T cells either producing IL-2 or IFN-c have increased since day 7 after MHV-3 infection, peaked at day 15 and maintained a stable level hence. Only marginal levels of IL-4, IL-10, TNF-a, perforin and granzyme B were detected, and there was no significant difference between infected and noninfected mice in the expression of these intracellular cytokines at any time points (Fig. 4B, C, D) .

The cytotoxic effects of DN T cells from the spleen of C3H/HeJ mice 0, 4, 15, 30, 40 days after MHV-3 infection were examined. DNT cells had significant cytotoxic effects on MHV-3 infected CD8 + T cells, but no apparent effect on infected hepatocytes or non-infected CD8 + T cells and hepatocytes (Fig. 5A) .

To further investigate the cytotoxic specificity of DN T cells, a murine cytomegalovirus (MCMV) was used as an unrelated control. After peritoneal MCMV infection, the livers of C3H/HeJ mice were evidently infected by MCMV, and virus replication was observed in the liver (as revealed by a standard plaque assay; data not shown). DN T cells from the spleens of MHV-3 infected mice showed no obvious cytotoxic effect on CD8 + T cells from the spleens of MCMV-infected mice, strongly suggesting that DN T cells only specifically kill CD8 + T cells with same virus specificity, and that this effect is not due to bystander cytotoxicity (Fig. 5B) .

A transwell experiment was performed to determine whether the cytotoxic effect is contact-dependent or is mediated by cytokines. Results showed that DN T cells cytotoxicity was remarkably weaker when DN T cells and CD8 + T cells were in different chambers as opposed to being in the same chamber (Fig. 5C) . To identify the precise mechanism involved in DN T cells cytotoxicity, the expression of FasL, perforin, and granzyme B in DN T cells was studied. Perforin and granzyme B were rarely detected (Fig. 4B) , while (47.53 ± 12.83) % of DN T cells express FasL. Moreover, DNT cells cytotoxicity dramatically decreased when FasL was blocked (effector:target = 5:1) (Fig. 5D ).

Lacking of adequate animal model has long being a major obstacle in studying viral hepatitis. Only chimpanzee and a few other primates are susceptible to hepatitis viruses, but the endangered status and financial considerations limit their widespread use. Researchers have also used surrogate animal models, such as ground squirrel, duck, tamarins and woodchuck. In addition to the wild-type animal models, inbred strains of animals are valuable to investigate the pathogenesis of viral infection. There are transgenic mouse models as well as immunodeficient mice or tolerized mice or rats, transplanted with human hepatocytes or hepatoma cells. Animal models mentioned above enable us to better understand the pathogenesis of viral hepatitis, but all have limitations [34, 35] .

Using MHV-3, which produces a strain-dependent viral hepatitis in inbred strains of mice, has brought insights into the pathogenesis of viral hepatitis from our group and others [32, [36] [37] [38] [39] [40] . MHV-3 belongs to the coronavirus family and has a singlestranded positive-sense RNA genome. As reported, susceptible inbred mouse strains such as Balb/cJ or C57BL/6 develop fulminant hepatitis and die within 3-5 days following peritoneal inoculation of the virus. In contrast, A/J mice are resistant and develop no clinical signs of hepatitis, and clear the virus within 10 days of infection [41] . C3H/HeJ mouse, a semisusceptible mouse strain develops acute hepatitis which progresses to mild chronic hepatitis [42] . In our study, the mice have presented acute inflammation from 6 to 15 days after infection, and a proportion of the infected mice died. However, after this phase, the liver injury in the survived mice was alleviated, accompanied with the recovery of the liver function. Only mild inflammation has been observed in the livers of the survivors, with persistent low-level virus replication, suggesting the persistence of viral infection. Since the disease course, histopathology, and serum biochemical changes observed in the mouse model are similar to those observed in patients with chronic viral hepatitis, this mouse model could be useful to understand the pathogenesis of chronic viral hepatitis in humans.

Host immune response exercises a great influence on the pathogenesis and outcome of viral hepatitis. Some viruses can be eliminated by the host immune system in the acute phase of infection, but certain viruses, like HBV and HCV, can evade the host immune responses, resulting in viral persistence [43] . In order to better understand the mechanisms leading to virus persistence in the model of MHV-3 infected C3H/HeJ mice, we studied the variations of different subgroups of T cells in C3H/HeJ mice after MHV-3 infection. The results showed that the frequency of DN T cells rose significantly in blood, liver, and spleen after MHV-3 infection. We also observed an increase of CD4 + CD25 + Tregs (data not shown) in the liver, but the increase of DN T cells was more pronounced than the increase of CD4 + CD25 + Tregs, indicating that DN T cells might play a critical role in controlling the pathogenesis of the disease.

Based on our observation, the liver injury of infected mice began around 4-5 days after infection. We assumed that the adoptive transfer of DN T cells at this time point might interfere the development of liver injury. Therefore, we chose to transfer DN T cells to recipient mice at 5 days post infection. The results demonstrated that DN T cells could lead to both the dramatic increase in the survival rate of recipient mice and amelioration in liver histology. However, differences in the virus titers of liver tissue had barely been observed between DN T group and control groups. We presume that DN T cells may play a role in inhibiting the host immune responses which cause liver injury. Nevertheless, the virus replication and clearance appear to reach a dynamic balance in the process.

The following cytotoxic assays revealed that after MHV-3 infection, DN T cells showed significant cytotoxic effects on virusspecific CD8 + T cells. Since the virus-specific CD8 + T cells play a key role in causing liver damage, this result made a sensible explanation to the survival increase and improvement of liver histology in recipient mice in adoptive transfer experiment. Transwell experiments indicated that cell-cell contact is necessary to the cytotoxicity mediated by DN T cells. Moreover, the Fas/FasL pathway instead of perforin/granzyme pathway plays a vital part in DN T cell killing. Thus we presume that, in MHV-3 infected C3H mice, DN T cells rapidly proliferated following the development of liver inflammation, and contributed to the control of liver injury by killing the virus-specific CD8 + T cells. The phenomenon observed here is in agreement with what has been described for TCRab + CD25 + CD30 + DN T cells, which are able to prevent the rejection of skin and heart allografts by specifically inhibiting CD8 + T cells through Fas-FasL interaction [17] . However, Zhang et al. reported that B220 + CD25 + DN T cells can control B and T cell responses in perforin/granzyme-dependent mechanisms [25] . Voelkl also reported that human DN Tregs do not eliminate effector T cells by Fas/FasL-mediated apoptosis, but to suppress by an active cell contact-dependent mechanism [44] . Thus, it seems that the functions of DN Tregs change in accordance with the different phenotypes in specific settings.

We found that the splenic DN T cells of the infected mice bear a distinctive array of phenotypes (TCRab + CD4 -CD8 -CD25 -CD28 -CD30 -CD44 + ) which were completely different from previously described T cells [45, 46] . Meanwhile, these DN T cells showed a relatively increased production of IFN-c and IL-2, but low IL-4, IL-10, TNF-a. It is reported that, in other disease models, DN T cells with different phenotypes present different cytokine profiles. In autoimmune diseases like SLE, TCRab + TCRVa24 À DN T cells are the major producers of IL-17, one of the key inflammatory cytokine involved in SLE. And the results suggested that these DN T cells contribute to the pathogenesis of kidney damage in patients with SLE [28] . In CD4-low mangabeys, DN T cells have increased expression of IFN-c(Th1), IL-4(Th2) and IL-17(Th17), suggesting these cells may be capable of performing multiple functions, including CD4 helper-like function and regulatory function. And the latter could potentially contribute to controlling immune activation during nonpathogenic SIV infection [30] . In our study, we assume that the DN T cells may, to some degree, exert the role of Th1 cells, which promote viral elimination. Nevertheless, the limited quantity of these cytokines constrained their power for viral clearance and allowed the low-level viral persistence. We postulate that it was the delicate balance of multiple effects of DN T cells contributing to the viral persistence in this model.

In conclusion, we report herein for the first time that splenic DN T cells with production of IFN-c/IL-2 have profound immunomodulatory effects by killing viral-specific CD8 + T cells via Fas/FasL pathway in MHV-3 induced viral hepatitis, suggesting the contribution of these DN T cells to viral persistence. Our findings may provide a rationale for modulating DN T cells for the management of viral hepatitis.

The authors have no financial conflict of interest.

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The authors thank Prof. Li Zhang, for her inspiring advices and critical reading for the manuscript. The authors also thank Dr. Guanxin Shen and Gary Levy for their comments in the process of this project, and Dr. Feng Fang for providing MCMV.