Roles Of Tim-3in The Exhaustion Of CD8T Cells In Chronic Hepatitis B

Hepatitis B virus (HBV) contains a small (3.2kb), circulular, double stranded DNA genome. The chronic infection of hepatitis B is one of the leading causes of cirrhosis and hepatocellular carcinoma. Accumulated evidence confirmed that HBV is not directly cytopathic for the hepatocytes. The antiviral immune response is the main cause of liver injury during HBV infection. The resolusion of acute self-limited hepatitis B is associated with strong CD4T and CD8T cells responses and type Ⅰ cytokine expression. There is evidence that CD8T cells clear HBV by two mechanisms, directly killng infected hepatocye and producing antiviral cytokines IFN-γ and TNF-α. But there is also evidence that CD8T cells responses are exhausted, weak or anergy in chronically persistent infected people, and the exact mechanism is still unkown.T cell immunoglobulin domain and mucin domain protein (Tim) gene family is first discovered in2001and is thougt to be associated with immune regulation. It's located on human chromosome5q33.2, and consists of three members in human, encoding Tim-1, Tim-3and Tim-4. Tim-3was originally identified as a mouse Thl-specific cell surface marker that was expressed after several rounds of Th1differentiation in vitro. Human Tim-3consists of a181amino acids extracellular domain, a21aa transmembrane segment, and a78aa cytoplasmic tail. In humans, Tim-3is expressed on a subset of CD4T cells, on CD8T cells, on Th17cells. Tim-3is also expressed on cells of the innate immune system including natural killer cells, dendritic cells and monocytes/macrophages. Tim-3expressed on the surface of activated Th1cells interacting with galectin-9triggers the apoptosis pathway of Th1cells, mediates immune tolerance, negatively regulates Th1responses in EAE and NOD mouse models. Two recent reports have implicated Tim-3in mediating T-cell dysfunction involved in chronic viral infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Virus-specific CD8T cells in both chronic viral infections progressively develop a range of functional impairments in cytokine production, cytotoxic activity, and proliferation. Our group previously found that Tim-3suppressed NK cells responses in chronic hepatitis B. Whether Tim-3plays roles in CD8T cells responses is still unkown in the pathogenesis of chronic hepatitis B.In this study, we investigated the expression of Tim-3on peripheral CD8T cells, analyzed the correlation between Tim-3expression and clinical results, and the role and mechanism of Tim-3on CD8T cells in CHB by cell-cell coculture system and blocking antibobies in vitro.Part I The augmented expression of Tim-3mediates exhaustion of CD8T cells in patients with CHBTim-3is expressed on many kinds of immune cells and regulates both innate and adaptive immune responses by many different ways. CD8T cells is the main effective immune cells in antiviral immune responses, its quantitaty and quality play important roles in the resolution of viral infection. There is evidence that Tim-3inhibited the function of CD8T cells in HIV and HCV infections, but what roles Tim-3play on the exhaustion of CD8T cells in CHB is still unknown. Firstly, we detected the expression of Tim-3on CD8T cell in the peripheral blood in CHB patients and healthy controls.1. The augemented Tim-3expression on CD8T cells correlates with serum viral load in CHB patientsA total of56CHB patients and34healthy controls were included for the test of Tim-3expression on peripheral of CD8T cells. Flow cytometry analysis showed that both the percentage of Tim-3expression (p=0.0008) and MFI levels (p=0.0005) was obviously up-regulated on CD8T cells from CHB patients. Tim-3expression on CD8T cells from HBeAg positive patients was obviously higher than those form HBeAg negative patients. Further statistical analysis showed that Tim-3expression is positively correlated with serum HBV-DNA load but not ALT level.2. Tim-3positive CD8T cells from CHB patients are activated but functionally exhausted28chronic hepatitis B patients and12healthy controls were admitted to detect the expression of CD28and Tim-3and further analysed the coexpression of these two molecules. Flow cytometry analysis results showed that Tim-3expression on CD8+CD28+T cells was obviously up-regulated from CHB patients rather than healthy controls. But Tim-3expression on CD8+CD28-Ts cells showed no obvious difference from both groups. This result reminded that Tim-3might be associated with the activation of CD8T cells. To further confirm the role of Tim-3on CD8T cells, we distinguished CD8T cell into two groups according to the expression of Tim-3. Phenotype analysis showed that Tim-3positive CD8T cells expressed higher levels of activation marker CD69(p=0.0072). The expression of Granzyme B (p=0.8430) and perforin (p=0.3984) on both Tim-3positive group and Tim-3negative group showed no difference. All these results showed that Tim-3positive CD8T cells exhibited an activated phenotype and functionally exausted.3. Tim-3suppresses the IFN-γ expression and proliferation of CD8T cells in PBMC from CHB patients3.1Compared to Tim-3-CD8+T cells, peripheral Tim-3+CD8+T cells from CHB patients has decreased proliferation and IFN-γ production.PBMCs were prepared from CHB patients and stimulated by HBs peptide for6h. BFA was added in the last2hours. PBMCs were collected for flow cytometry analysis. The results showed that Tim-3positive CD8T cells expressed less IFN-γ than Tim-3negative CD8T cells but not statistically significant.(p=0.1823) PBMCs prepared from CHB patients were incubated with CFSE for15min. The CFSE labeled PBMCs were stimulated by HBs peptide for4days. Cells were collected and stained with PE-anti human Tim-3, PEcy5-anti human CD8. Flowcytometry analysis showed that the proliferation of Tim-3positive CD8T cells decreased compared to Tim-3negative group.All these results reminded that the up-regulation of Tim-3might be related to CD8T cells dysfunction.3.2Blocking Tim-3pathway increases the activity of CD8T cells in PBMC of CHB patientsTo further confirm the effect of Tim-3on CD8T cells, Tim-3-Fc was used to blocking Tim-3pathway (IgG as control), and then the IFN-γ production of CD8T cells were analysed. Flowcytometry analysis showed that blocking Tim-3pathway obviously up-regulated the IFN-γ expression in CD8T cells compared to IgG control group.(p=0.0071). PBMCs prepared from CHB patients were incubated with CFSE for15min. Then cells were pre-incubated with Tim-3-Fc or IgG for40min to blocking Tim-3pathway. Both groups were stimulated by HBs peptide for4days. Cells were collected and stained with and PEcy5-anti human CD8. Flowcytometry analysis showed that blocking Tim-3pathway obviously up-regulated the proliferation of CD8T cells compared to IgG control group.(p=0.0206)4. Tim-3increases the apoptosis sensitivity of CD8T cells in PBMC from CHB patients4.1The apoptosis sensitivity was obviously up-regulated on CD8T cells especially Tim-3positive CD8T cells in PBMC from CHB patientsWe next investigated whether Tim-3levels affect the apoptosis sensitivity in CHB patients. Annexin Ⅴ labeling experiments showed that the apoptosis sensitivity of CD8T cells was up-regulated in PBMCs from CHB patients than healthy controls.(p=0.0097) Further analysis showed that Tim-3positive CD8T cells are always more sensitive to apoptosis than Tim-3negative CD8T cells in PBMCs form CHB patients.(p=0.0002) All these results reminded that Tim-3might increase the apoptosis sensitivity in CHB patients.4.2The sensitivity to AICD was increased on Tim-3positive CD8T cells and positively correlated Tim-3expression on CD8T cells in PBMC of CHB patientsTo further investigate the effect of Tim-3in apoptosis of CD8T cells, we induced CD8T cell activated induce cell apoptosis (AICD) by PMA, ionomycin and anti-human CD3pathway. Annexin Ⅴ labeling experiments also showed that CD8T cells in PBMCs from CHB patients were more sensitive to apoptosis than those from healthy controls.(p<0.0001) Further analysis showed that Tim-3positive CD8T cells were always more sensitive to AICD than Tim-3negative group.(p=0.0018) Correlation analysis showed that Tim-3expression was positively correlated with Annexin Ⅴ expression.(r=0.5965, p=0.0001)4.3Blocking Tim-3pathway decreases the apoptosis of CD8T cells in PBMCs from CHB patiensPBMCs were prepared from CHB patients and were pre-incubated with Tim-3-Fc or IgG for40min to blocking Tim-3pathway. The Cells were stimulated by PMA, ionomycin and anti-human CD3to induce AICD. Annexin Ⅴ labeling experiments also showed that blocking Tim-3pathway obviously down regulated AICD of CD8T cells in PBMCs from CHB patients.(p=0.0153) In conclusion, Tim-3expression was augmented on CD8T cells in CHB patients. The up-regulated Tim-3inhibited the antiviral cytokine IFN-y expression and proliferation of CD8T cells and promoted the apoptosis sensitivity of CD8T cells in CHB patients.Part II NK cells participates Tim-3-meidated CD8T cells suppression in CHBIt is reported that Tim-3interacting with its ligand galectin-9inhibited Thl responses and mediated immune tolerance. But the role of Tim-3on CD8T cells responses is still unkown. In this study, we will further explore the mechanism how Tim-3mediated the exhaustion of CD8T cells in CHB patients.1. Tim-3-meidated CD8T cells suppression in CHB needs the presence of other preipheral blood cellsIn the first part, we have confirmed that Tim-3indeed regulated the activity of CD8T cells in CHB patients. To further confirm the regulatory functions of Tim-3on CD8T cells, we collected purified CD8T cells from CHB patients and did next experiments.1.1Blocking Tim-3pathway didn't change HBs-stimulated IFN-y expression of purified CD8T cells from CHB patientsTo further confirm the effect of Tim-3on CD8T cells in CHB patients, we isolated CD14+monocytes and CD8T cells from PBMCs of CHB patients. Purified CD14+monocytes were stimulated by GM-CSF and IL-4for7days to get immature DCs. Immature DCs were stimulated by HBs peptide for another24h. The mature DCs were cocultured with purified CD8T cells with Tim-3pathway blocking (isotype control IgG for control) for24h in the presence of HBs peptide. We next investigated whether blocking Tim-3pathway might influence the IFN-y expression of purified CD8T cells by flowcytometry. Our result showed that blocking Tim-3pathway did not increase the IFN-y expression of purified CD8T cells.(p=0.5552)1.2Blocking Tim-3pathway didn't upregulate HBs-stimulated proliferation of purified CD8T cells from CHB patientsThe mature DCs were cocultured with CFSE dyed CD8T cells with Tim-3pathway blocking (isotype control IgG for control) for7days in the presence of HBs peptide. Tim-3 blockade also did not increase the accumulation of CFSE low HBV-specific CD8T cells.(p=0.8857)1.3Blocking Tim-3pathway didn't down-regulate AICD of purified CD8T cells from CHB patientsWe isolated CD8T cell from PBMCs of CHB patients. Purified CD8T cells were incubated with Tim-3-Fc or IgG for40min and then were stimulated by PMA, ionomycin and anti human CD3for14h. Our results showed that blocking Tim-3did not down regulated AICD of purified CD8T cells.(p=0.4033)In conclusion, the negative regulation of Tim-3on the activity of CD8T cells was dependent on the help of other cells. There must be other immune cells providing Tim-3ligand to interact with Tim-3and further mediated the suppression of CD8T cells.2. NK cells is needed in Tim-3-mediated suppression of CD8T cells in CHB patientsNK cells are important innate immune cells in anti-virual immunity. It is reported that NK cells not only directly killed virus infected cells but also have regulatory role in adaptive immunity. Our previous results showed that Tim-3expression up-regulated on NK cells in CHB patients and further inhibited the function of NK cells. In this study, NK cells are isolated from PBMCs of CHB patients according to manufacture's instructions. PBMC deleted NK cells were collected and stimulated by HBs peptide for5days with Tim-3pathway blocked by Tim-3-Fc or IgG. Intracellular staining assay revealed that IFN-y expression in HBs-specific CD8T cells showed no difference in both groups (p=0.9160). Flowcytometry analysis showed that the percentage of CFSE low of HBV-specific CD8T cells was also not up-regulated in PBMC deleted NK cells (p=0.5102). All these results showed that NK played a regulating role in CD8T cells in PBMCs from CHB patients.3. NK cells take part in Tim-3-mediated CD8T cells suppressionMany studies confirmed that NK cells was not only an important innate immune cells and also played an important roles in immune regulatory effects. NK cells can regulate adaptive immune cells by direct cell contact and also by expressing cytokine to regulate adaptive immune cells indirectly. Our previous results showed that NK cells were necessary in Tim-3mediated CD8T cells suppression. To further confirm the molecular mechanism, we prepared to do some studies on two aspects.1) Whether NK cells provided Tim-3ligand to suppress the activity of CD8T cells by Tim-3/galectin-9interaction in CHB patients.2) Our previous experiments have confirmed that Tim-3suppressed the function of NK cells especially IFN-y expression which played important roles in immune regulation and increased CD8T cells response. So Whether Tim-3suppressed NK cells expressing IFN-y and further suppressed the activity of CD8T cells is still unkown.3.1NK cells provide Tim-3ligand to suppress the activity of CD8T cells in CHB patients3.1.1Tim-3ligand galectin-9is expressed on purified NK cells from CHB patientsWhether the up-regulated Tim-3expression on CD8Tcells interacting with its ligand galectin-9mediated the suppression of IFN-y expression and proliferation of CD8T cells was the main point in this part. PBMC, purified CD8T cells, purified NK cells from CHB patients and NK92cell line were prepared. Galectin-9mRNA expression was detected by RT-PCR. The result showed that galectin-9expressed on all these cells and much more higher in NK cells than CD8T cells. It reminded us that NK cells might provide galectin-9to interact with Tim-3on CD8T cells and further inhibited the function of CD8T cells.3.1.2Galectin-9inhibition increases the activity of CD8T cells from CHB patientsPurified NK cells were suspended in30mM lactose to inhibit galectin-9and sucrose as control. PBMC deleted NK cells were incubated with Tim-3-Fc for40min and washed with PBS, and then cocultured with purifed NK cells and stimulated by HBs peptide. IFN-y expression in CD8T cells was greatly increased in lactose group.(p=0.0033)Purified NK cells were suspended in30mM lactose to inhibit galectin-9and sucrose as control. CFSE labeled PBMC deleted NK cells were incubated with Tim-3-Fc for40min and washed with PBS, and then cocultured with purifed NK cells and stimulated by HBs peptide. The proliferation of CD8T cells was greatly increased in lactose group.(p=0.0464)In conclusion, the up-regulated Tim-3expression on CD8Tcells interacted with its ligand galectin-9and further mediated the suppression of IFN-y expression and proliferation of CD8T cells.3.2Tim-3inhibites NK cells producing IFN-y and further suppressed the activity of CD8T cells from CHB patientsIt is reported that activated NK cells produce immune regulatory cytokine IFN-y and IFN-y can regulate the response of CD8T cells in chronic virus infection. While in chronic hepatitis B whether NK cells can also regulate the response of CD8T cells by producing IFN-y is still unknown.3.2.1NK cells depletion increases the IFN-y expression and proliferation of CD8T cells from CHB patientsWe first detected whether NK cells had a regulatory effect on CD8T cells. NK cells were depleted by using magnetic cell separation from PBMCs of CHB patients. The rest cells named PBMC-NK and were stimulated with HBs peptide, PBMCs were used as control. The results showed that IFN-y expression of CD8T cells were obviously up-regulated in PBMC-NK group.(p=0.0284)NK cells were depleted by using magnetic cell separation from PBMCs of CHB patients. The rest cells named PBMC-NK were labeled with CFSE and stimulated with HBs peptide, PBMCs were used as control. The results showed that the proliferation of CD8T cells were obviously up-regulated in PBMC-NK cells.(p=0.002)All these results showed that NK cells suppressed the activity of CD8T cells in CHB patients. This might be the main cause of exhaustion of CD8T cells in CHB patients.3.2.2Blocking Tim-3pathway on NK cells enhance HBs-specific CD8T cells functions in CHB patientsOur previous results showed that Tim-3suppressed NK cells function in CHB patients. To further confirm whether Tim-3involved in the regulatory effects of NK cells, purified NK cells blocked Tim-3pathway by using anti-human Tim-3, were cocultured with PBMC-NK, and stimulated with HBs peptide. Flowcytometry results showed that IFN-y expression was greatly increased in Tim-3blocking group.(p=0.0042)NK cells were purified by using magnetic cell separation from PBMCs of CHB patients. The rest cells named PBMC-NK were labeled with CFSE. Purified NK cells blocked Tim-3pathway by using anti-human Tim-3, were cocultured with PBMC-NK, and stimulated with HBs peptide. The results showed that was greatly increased in Tim-3blocking group.(p=0.0218)These results indicated that Tim-3played roles in NK cells regulatory effects on the exhaustion of CD8T cells. 3.2.3Anti-IFN-yR treatment promotes the NK-mediated suppression on HBs-specific CD8Tcells in CHB patientsIt is reported that NK cells IFN-y expression play roles in immune regulation of CD8T cells in LCMV infection. Our previous studies showed that blocking Tim-3pathway up-regulated IFN-y expression on NK cells in CHB patients. In this part we will explore whether Tim-3inhibited NK cells expressing IFN-y and further suppressed the acitivity of CD8T cells in CHB patients.NK cells were purified from PBMCs in CHB patients and incubated with anti-human Tim-3for40min. PBMC-NK cells were blocked with anti-human IFN-rR or isotype IgG, then cocultured with purified NK cells blocking Tim-3pathway and then stimulated with HBs peptide. The results showed that IFN-y expression is down-regulated on CD8T cells in Anti-IFN-yR treatment group (p=0.0466).CFSE labeled PBMC-NK cells were blocked with anti-human IFN-rR or isotype IgG, then cocultured with purified NK cells blocking Tim-3pathway and then stimulated with HBs peptide. The results showed that IFN-y expression is down-regulated on CD8T cells in Anti-IFN-γR treatment group (p=0.0020).All these results showed that Chronic HBV infection increased the Tim-3expression on NK cells, the up-regulated Tim-3expression inhibited NK cells to produce IFN-y, in this environment, the activity of CD8T cells was suppressed. In this study, for the first time, we confirmed that Tim-3expression on NK cells indirectly suppress the immune response of CD8T cells dependent on IFN-y pathway.In conclusion, Chronic HBV infection increased the Tim-3expression on CD8T cells, the up-regulated Tim-3expression inhibited CD8T cells to produce IFN-y, proliferate and promoted apoptosis by different ways. These results provided new explanations on CD8T cells exhaustion in the pathogenesis of CHB and new targets on the therapy of CHB.