Invariant NKT Cells In Chronic HBV Infection:the Roles And Mechanisms

Background and aim:Worldwide, more than300million people suffer from chromic hepatitis B virus (HBV) infection, leading to a wide spectrum of liver diseases including asymptomatic carrier (AsC), chronic hepatitis B (CHB), cirrhosis and hepatocellular carcinoma. In china, over7%of the population are infected with HBV, out of which30million people are developed to CHB. Moreover, there are about300thousand patients die from HBV infection related diseases in our country every year. Given that there is no ideal drug to eliminate HBV completely from human body currently, chronic HBV infection has become a heavy health burdern at present. The pathogenesis of CHB and cirrhosis is thought to be mediated by the immune response to the HBV rather than the HBV itself. Multiple types of immune cells and molecules are involed in HBV associated liver damage. However, the precise roles of these cells and molecules are still incompletely understood.Invariant NKT (invariant NKT) cells are a unique group innate T lymphocytes that express an identical T cell antigen receptor (TCR) a chain, Val4-Jα18in mice and Va24-Jα18in humans.invariant NKT cells differ from conventional T lymphocytes in that they recognize lipid or glycolipid antigens presented by the MHC class I-like molecule CD1d. When activated with CD1d tetramer or anti-CD3antibody, invariant NKT cells rapidly secrete a variety of Thl and Th2cytokines within a few hours. Although invariant NKT cells comprise a very small proportion of peripheral T cells, about1%in mice and0.2%in humans, they seem to play important roles in regulating a number of immune responses, including transplant rejection, cancer, autoimmunity, allergy, and infection.The liver contains a large number of invariant NKT cells relative to blood and other lymphoid organs. Increasing evidence suggests that invariant NKT cells contribute to a variety of liver disorders, including drug-induced liver injury, primary biliary cirrhosis, alcoholic liver injury, autoimmune hepatitis, hepatocellular carcinoma, non-alcoholic fatty liver disease, and viral hepatitis. In CHB, alpha-galactosylceramide (a-GalCer) activated invariant NKT cells are able to inhibit HBV replication in vivo and are implicated in the pathogenesis of cirrhosis by producing profibrotic cytokines. Activation of invariant NKT cells also promotes the loss of tolerance to HBV-specific CD8+T cell antigens. However, most of these reports are based on mouse models, and the role of invariant NKT cells in CHB patients is largely unknown. There are few reports about the changes in invariant NKT cell frequency or activity in CHB patients during antiviral therapy.In the present study, we compared the frequency of circulating invariant NKT cells in chronic HBV infection subjects and healthy individuals by flow cytometry. We also compared the expression of chemokine receptors on invariant NKT cells, the ability of invariant NKT cells to migrate toward different chemokines and the ability to secret cytokines between CHB patients and healthy controls. Afterward we analyzed the longitudinal changes of invariant NKT cells frequency in CHB patients who received antiviral therapy with telbivudine. Finally, we detected intrahepatic invariant NKT cells by immunohistochemistry and try to understand the mechanisms of the decrease of circulating invariant NKT cells in CHB patients and subsequent changes by expriments in vitro. Methods1. SubjectsThirty-five CHB patients,15immunetolerant carriers (IT),25inactive HBV carriers (IC) and36healthy controls (HC) were enrolled in the present study. CHB patients, IT and IC were diagnosed according to the AASLD criteria. The subjects with previous antiviral therapy, with co-infection by the HIV, other hepatitis virus, and with diabetes, severe systemic illness, regular alcohol consumption and hepatocellular carcinoma were excluded.2. Flow Cytometry AnalysisTen to thirty milliliter (ml) of heparinized blood was collected from all individuals at the time of recruitment. Of the CHB patients,19received anti-viral therapy with telbivudine and were followed up for at least52weeks, and additional blood samples were obtained at week12, week24and week52during the course of therapy. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll density gradient centrifugation. One million fresh isolated PBMC were washed and stained with fluorochrome-conjugated antibodies. The frequencies and phenotype of circulating invariant NKT cells were analysed.In some cases, the cytokine production within peripheral invariant NKT cells was determined by Intracellular cytokine staining.3. Migration Assay The ability of recombinant CC chemokine ligand5(CCL5) and CCL20to attract invariant NKT cells was examined using a transwell system with24-well and5μm pore size. One and half million fresh isolated PBMC were added to the upper chamber,500ng/ml CCL5or CCL20were added to the lower chamber. After10hours incubation at37℃, the cells in the lower chamber were harvested and analysed using flow cytometry. 4. Elispot AssayIFN-γ Elispot assay was performed to detect the effect of recombination human IL-21on the IFN-y production of invariant NKT cells stimulated with a-GalCer. Multiscreen-iP96-well plates were coated with capture mouse antihuman IFN-y monoclonal antibody, then blocked with RPMI1640-10%FCS. PBMC were seeded in the wells and a-GalCer was added to stimulate invariant NKT cells. Plates were incubated overnight in the presence or absence of IL-21. After washing, biotinylated secondary mouse antihuman IFN-y monoclonal antibody was added. After3h of incubation, goat alkaline phosphatase antibiotin antibody was added and the plates were incubated for a further2h. Substrate was added and the colorimetric reaction was stopped by washing with distilled water. Plates were air dried, and spots were photographed and counted. In some cases, plates were coated with recombinate HBeAg or HBcAg to evaluate the function of invariant NKT cells on the production of HBV specific antibody.5. ImmunohistochemistryParaffin-embedded, formalin-fixed liver tissue from patients with liver surgery was cut into5μm sections and placed on polylysine-coated slides. Antigen retrieval was achieved via pressure cooking for10minutes in citrate buffer. Mouse anti-human invariant NKT cells monoclonal was added and incubated for45minutes. EnVisionTM Detection Kit was used to stain invariant NKT cells followed by counterstaining with hematoxylin.6. Statistical AnalysisComparisons between different groups were performed using the Mann-Whitney U test. Within-subject data were compared with the repeated measures ANOVA followed by Tukey’s or Bonferroni post-tests. The Spearman rank order correlation coefficient was used for correlation analyses. Categorical variables were compared by Fisher’s exact test. The area under receiver operating characteristics (ROC) curves were calculated to assess the use of peripheral invariant NKT cells frequency at baseline to predict HBeAg seroconversion. For all tests, a two-sided P<0.05was considered significant.Results1. We compared the frequencies of peripheral invariant NKT cells in subjects from the HC, IT, CHB and IC groups.invariant NKT cells were detected by flow cytometry using a monoclonal antibody6B11, which specifically reacts with the complementarity determining region3(CDR3) of the Va24-Ja18T cell receptor of human invariant NKT cells. We found that the frequency (medium, range) of invariant NKT cells in CHB patients (0.13%,0.027%-1.071%) was significantly lower than that in HC (0.24%,0.064%-0.81%, P=0.0143) and IC (0.19%,0.06%-0.38%, P=0.0235). By contrast, there was no significant difference between HC and IC (P=0.703). Invariant NKT cells are heterogeneous and can be categorized into two subsets according to the expression of CD4, namely CD4+and CD4-invariant NKT cells. A comparison of the frequency of CD4+and CD4-invariant NKT cells was performed in the three groups. We found that the frequency of CD4-invariant NKT cells in CHB patients (0.071%,0.013%-0.98%) was decreased compared to HC (0.175%,0.027%-0.7%, P<0.01), and the frequency of CD4-invariant NKT cells in IC (0.072%,0.019%-0.73%) was also significantly lower compared to HC (P=0.023). There was no significant difference (P=0.458) between CHB and IC in the frequency of CD4-invariant NKT cells. By contrast, the frequency of CD4+invariant NKT cells in CHB patients (0.053%,0.007%-0.83%) was significantly lower than IC (0.1%,0.027%-0.65%, P=0.019), but was similar to HC (0.055%,0.01%-0.2%,P=0.7).2. We next compared the ratio of CD4-/CD4+invariant NKT cells in the different groups, and found that the ratio was decreased in CHB patients (1.21,0.11-10.77) relative to the HC (2.61,0.53-9.55, P<0.01), and even further decreased in the IC (0.78,0.11-8.5) relative to CHB (P=0.018). We also analyzed the correlation between peripheral invariant NKT cells and plasma HBV DNA load and serum alanine aminotransferase (ALT) level in the CHB patients (n=30). The results indicated that there was no correlation between the frequency of total invariant NKT cells and plasma HBV DNA load or serum ALT level. By contrast, CD4-invariant NKT cells and the ratio of CD4-/CD4+invariant NKT cells were negatively correlated with ALT levels.3. We detected the expression of CCR5and CCR6on peripheral invariant NKT cells in CHB patients (n=6) by flow cytometry and found that the proportion (mean±SD) of invariant NKT cells expressing CCR5(82.83%±9.87%) or CCR6(67.67%±16.83%) was dramatically higher than that of other T cells (CCR5,30.5%±5.65%, CCR6,14.02%±5.92%, both P<0.001). Further analysis revealed that the percentage of CD4-invariant NKT cells expressing CCR5or CCR6was also significantly higher than that of CD4+invariant NKT cells (P<0.05and P<0.01respectively). Interestingly, the expression of CCR5and CCR6on invariant NKT cells was comparable between CHB patients and HC subjects.4. Our data above indicated that invariant NKT cells expressed higher levels of CCR5and CCR6than other T cells. Therefore, we performed a chemotaxis assay to test the hypothesis that CCL5and CCL20, the ligands of CCR5and CCR6respectively, could induce the migration of invariant NKT cells in vitro. We found that the frequency of invariant NKT cells in the lower chamber in response to CCL5(0.87%±0.24%) was significantly higher compared to medium alone (0.65%±0.23%, P<0.001). Surprisingly, the frequency of invariant NKT cells in the lower chamber in response to CCL20was similar to medium alone. 5. We also assessed the ability of invariant NKT cells to produce IFN-y and IL-4in response to the specific antigen a-GalCer and non-specific mitogen PMA by flow cytometry. The IFN-γ production of circulating invariant NKT cells in response to a-GalCer was dramatically higher compared to IL-4, irrespective of whether they were obtained from CHB patients or HC (both P<0.001). Interestingly, there were no differences between the two groups in both IFN-γ(P=0.673) and IL-4expression (P=0.8884). When stimulated with PMA, there were also no significant differences between CHB patients and HC in IFN-γ and IL-4production (P=0.12, P=0.75respectively). Surprisingly, the IFN-γ response to PMA was lower compared with the response to a-GalCer, regardless of CHB patients or HC (both P<0.001).6. We analyzed the changes in peripheral invariant NKT cell frequency in CHB patients on antiviral therapy. PBMC were obtained at baseline and at week12,24and52after initiating treatment with telbivudine (n=19). We found that the frequency of peripheral invariant NKT cells was significantly increased at week52(0.21%,0.05%-1.22%) compared with baseline (0.18%,0.03%-1.07%, P=0.0176). At week12and week24, the frequency of circulating invariant NKT cells was also increased compared to the baseline, but the difference did not reach statistical significance. Further analysis indicated that the frequency of CD4-but not CD4+invariant NKT cells was dramatically increased by week52(P<0.01, P=0.2898respectively).7. Of the19CHB patients treated with telbivudine,7(37%) achieved HBeAg seroconversion by week52. The plasma HBV DNA load in patients with seroconversion decreased more rapidly than patients not achieving seroconversion during the course of therapy, especially after week12. By contrast, both the seroconversion and non-seroconversion patients had the similar variation mode in serum ALT levels and circulating invariant NKT cells. The precise mechanisms contributing to HBeAg seroconversion during antiviral therapy are not fully understood. The patients who achieved HBeAg seroconversion tended to have a slightly higher ratio of CD4-/CD4+invariant NKT cells (1.47,1-3.56) compared to those who failed to achieve HBeAg seroconversion (0.915,0.11-3.4, P=0.0826). We also analyzed this data by dividing the patients into high (≥1) and low (<1) ratio groups and found that the patients with a high ratio of CD4-/CD4+invariant NKT cells at baseline were more likely to achieve HBeAg serocon-version than those with a low ratio (P=0.02). Next, a receiver operating characteristics (ROC) curve was constructed to determine whether the ratio of CD4-/CD4+invariant NKT cells could be used as a predictor of HBeAg seroconversion within52weeks of starting telbivudine therapy. The area under the ROC curve (AUC) indicated the existence of a significant association between the CD4-/CD4+invariant NKT ratio and HBeAg seroconversion (AUC=0.75,95%confidence interval0.507-0.993, P=0.04). A CD4-/CD4+invariant NKT cell ratio cutoff at baseline of0.94gave the maximum combination of sensitivity (100%) and specificity (58.33%) to predict HBeAg seroconversion.Conclusions1. Circulating invariant NKT cells are decreased in CHB patients and recovered to normal level after viral control. There is no correlation between the frequency of total invariant NKT cells and plasma HBV DNA load or serum ALT level. By contrast, CD4-invariant NKT cells and the ratio of CD4-/CD4+invariant NKT cells were negatively correlated with ALT levels.2. Invariant NKT cells express high levels of CCR5and CCR6, and migrate more likely toward CCL5than other conventional T cells, which may contribute to the decreasing of circulating invariant NKT cells. 3. The ability of peripheral invariant NKT cells to produce IFN-γ and IL-4is not impaired with chronic HBV infection and IFN-y is the dominant cytokine secreted by circulating invariant NKT cells in response to a-GalCer.4. The ratio of CD4-/CD4+invariant NKT cells in CHB patients at baseline is associated with HBeAg seroconversion by week52after telbivudine therapy, and has the potential to serve as a predictor for HBeAg seroconversion.