Elevated Hepatic Cd1d Levels Coincide with Invariant NKT Cell Defects in Chronic Hepatitis B Virus Infection

Elevated Hepatic CD1d Levels Coincide with Invariant NKT Cell Defects in Chronic Hepatitis B Virus Infection

This information is current as Xiaosheng Tan, Yajie Ding, Peng Zhu, Rui Dou, Zhihui of September 24, 2021. Liang, Daofeng Yang, Zhiyong Huang, Wei Wang, Xiongwen Wu and Xiufang Weng J Immunol published online 11 April 2018 http://www.jimmunol.org/content/early/2018/04/10/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 11, 2018, doi:10.4049/jimmunol.1701801 The Journal of Immunology

Elevated Hepatic CD1d Levels Coincide with Invariant NKT Cell Defects in Chronic Hepatitis B Virus Infection

Xiaosheng Tan,* Yajie Ding,* Peng Zhu,† Rui Dou,* Zhihui Liang,* Daofeng Yang,‡ Zhiyong Huang,† Wei Wang,* Xiongwen Wu,* and Xiufang Weng*

Activation of invariant NKT (iNKT) cells manifests antiviral immune responses in vivo. However, clinical trials have failed to show consistent hepatitis B virus (HBV) DNA reduction postadministration of iNKT cell–speciﬁc agonist a-galactosylceramide (a-GalCer). In this study, we aimed to investigate HBV infection–related iNKT cell defects and explore iNKT cell–based therapeutic potential for chronic hepatitis B (CHB). Liver specimens from 30 HBV-infected hepatocellular carcinoma patients were collected for CD1d/hepatitis B surface Ag (HBsAg) staining and/or intrahepatic iNKT cell assay. Two hundred and six chronic HBV-infected patients (including 130 CHB patients) were enrolled in the study of circulating iNKT cell frequency and function.

We found that liver and hepatoma tissue that positively stained for HBsAg had higher CD1d expression as compared with HBsAg Downloaded from negatively stained counterparts. The elevated CD1d expression in infected tissue is supposed to facilitate the iNKT cell–based antiviral effects locally. However, iNKT cell defects that related with disease progression suggested iNKT cells attenuated their effects during chronic HBV infection. The residual iNKT cells in CHB patients showed aberrant activation and hyporesponsiveness to a-GalCer. Exogenous IL-2 fully rescued a-GalCer–induced expansion of iNKT cells from CHB patients, and synergistic effects of IL-2 and IL-15 helped to recover the CD1d-dependent IFN-g production. In conclusion, our results highlight the

increased CD1d expression in HBV-infected liver and differential iNKT cell defects associated with disease progression during http://www.jimmunol.org/ chronic HBV infection. The reversibility of iNKT cell defects suggests protective immune responses could be partially recovered in CHB. The Journal of Immunology, 2018, 200: 000–000.

hronic hepatitis B (CHB) caused by hepatitis B virus recover after viral clearance by the development of a vigorous (HBV) infection is associated with significant mortality immune response associated with acute inflammatory liver dis- C and morbidity (1). Approximately 15–40% of patients eases (3). However, 90% of neonates and 30–50% of children with CHB infection develop fibrotic reaction and cirrhosis, which under 5 y of age exposed to HBV become chronic carriers, are associated with over 50% of the total cases of hepatocellular resulting in over 240 million people being chronically infected by guest on September 24, 2021 carcinoma (HCC) worldwide (2). Most HBV-infected adults (4, 5). Besides aiding in transformation, HBV plays a crucial role in enhancing immune suppression (6). During chronic HBV infection, T cells lost effector function after exposure over decades *Department of Immunology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China; to high levels of the virus (7). The failure of the immune system to †Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong Univer- eradicate the virus and to halt progression of HCC is closely linked ‡ sity of Science and Technology, Wuhan, Hubei 430030, China; and Department of with pathogenesis and the survival of patients with HBV infection Infectious Disease, Institute of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, (8, 9). Therefore, reconstitution of an efficient anti-HBV immune re- China sponse is still a promising approach for treatment of hepatitis B (10). ORCIDs: 0000-0001-9920-1555 (X.T.); 0000-0003-3647-8201 (R.D.); 0000-0003- CD1d-restricted invariant NKT (iNKT) cells that recognize lipid 2625-2137 (X. Weng). Ags are characterized by expression of an invariant TCR a-chain Received for publication January 2, 2018. Accepted for publication March 15, 2018. (Va14Ja18 in mice; Va24Ja18 in human) paired with a limited This work was supported by the National Nature Science Foundation of China (Grant array of TCR b-chains (Vb8.2, Vb7, or Vb2 in mice; Vb11 in 31570913 to X. Weng and 31370885 to X. Wu) and the 973 Program from the human) (11). As one of the predominant lymphocyte populations Ministry of Science and Technology of China (2013 CB530505). in liver, iNKT cells are potent regulators of the local immune X.T. and X. Weng conceived and designed the experiments. X.T., Y.D., W.W., and R.D. performed the experiments. P.Z., D.Y., and Z.H. collected clinical samples. X.T., system (11). Upon activation, iNKT cells respond with robust Th1 P.Z., D.Y., Z.L., X. Wu, and X. Weng analyzed the data. X.T., X. Wu, and X. Weng and Th2 cytokine production, helping to modulate both innate and wrote the article. adaptive immune responses (12). Despite low frequency in hu- Address correspondence and reprint requests to Dr. Xiufang Weng and Dr. Xiongwen mans, iNKT cell activity is now recognized to play important roles Wu, Department of Immunology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, No. 13 Hangkong Road, Wuhan, in infectious disease (13). In humans with X-linked lymphopro- Hubei 430030, China. E-mail addresses: [email protected] (X. Weng) and liferative syndrome, a selective defect in iNKT cells is associated [email protected] (X. Wu) with lethal EBV infection and increased lymphoma (14, 15). Al- The online version of this article contains supplemental material. though studies on transgenic mouse models of HBV infection have Abbreviations used in this article: ALT, alanine aminotransferase; AST, aspartate suggested iNKT cell control of HBV replication (16), less is aminotransferase; CHB, chronic hepatitis B; ER, endoplasmic reticulum; FasL, Fas ligand; a-GalCer, a-galactosylceramide; HBeAb, hepatitis B e Ab; HBeAg, hepatitis known about the role of iNKT cells in chronic HBV infection in B e Ag; HBsAg, hepatitis B surface Ag; HBV, hepatitis B virus; HCC, hepatocellular humans. Current descriptions of frequency and functional status of carcinoma; HD, healthy donor; IC, inactive carrier; iNKT, invariant NKT; IT, im- iNKT cells are highly inconsistent in chronic HBV-infected mune tolerance; MNC, mononuclear cell. patients. Both increased circulating iNKT cell frequency with Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 elevated IFN-g production (17) and reduced circulating iNKT cell

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701801 2 iNKT CELL DEFECTS IN CHRONIC HEPATITIS B frequency with preserved function (18) have been reported in Flow cytometry CHB patients. iNKT cells have also been implicated in liver injury PBS57/CD1d tetramer was kindly gifted by the National Institutes of Health and HCC through secreting IL-4 and IL-13 (19). Of note, char- Tetramer Core Facility. Cells were stained with PBS57/CD1d tetramer, acterization of iNKT cells and CD1d expression in HBV-infected corresponding Abs, and/or CFSE (Thermo Fisher). The IFN-g in-culture liver tissue is poorly elucidated. supernatant was determined by using Cytometric Bead Array Flex Sets Marine sponge–derived agent a-galactosylceramide (a-GalCer) (BD Biosciences). Data were collected using FACSVerse or LSR II cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star). is a specific agonist of iNKT cells and capable of expanding CFSE proliferation assay was analyzed by ModFit LT V3.2 (Verity Soft- iNKT cells in vivo and in vitro (20). Activation of iNKT cells by ware House). a-GalCer inhibits HBV replication and promotes the breakage of Immunofluorescence staining CD8+ T cell tolerance in HBV transgenic animals (21). Further- more, increased iNKT cell ratio is associated with enhanced an- Liver and hepatoma specimens were fixed in 4% paraformaldehyde, em- titumor effect against hepatitis B surface Ag (HBsAg)–expressing bedded in paraffin, and sectioned. Fluorescent double staining of CD1d and HBsAg in sections was performed with FITC-conjugated anti-HBsAg human hepatoma cells (22). Findings from these preclinical (Bioss) and anti-CD1d (NOR3.2; AbD Serotec). Whole sections were studies raise the rationale of exploring a-GalCer–mediated and scanned by Pannoramic MIDI (3D HISTECH). More than 60 images iNKT cell–based immunotherapies for the treatment of HBV in- without overlap were taken from each section, followed by integrated OD fection. Nonetheless, a recent finding from a randomized placebo- analysis for HBsAg and CD1d levels by ImageJ software. controlled phase I/II trial indicates that a-GalCer administration Real-time PCR failed to promote sustained antiviral activity in CHB patients (23).

Given that iNKT cell roles during the development of CHB re- To determine the mRNA levels of CD1d and HBsAg, total RNA was Downloaded from extracted from liver tissue using RNAfast200 (Fastagen, Shanghai, China) main controversial, several concerns should be addressed regard- according to the manufacturer’s instructions; RNA was reverse-transcribed ing iNKT cell functional status and their therapeutic potential in with a PrimeScript RT Reagent Kit (RR037A; Takara) to generate cDNA. these patients. The PCR reaction mixture was prepared using a SYBR Green Premix Ex In this study, we evaluated hepatic CD1d levels as well as Taq II Kit (RR820A; Takara) with the primers as follows: CD1d (forward: 59-TCTCGTCCTTCGCCAATAGC-39; reverse: 59-GGGACCAAGGCTT- iNKT cell frequency and functional status in chronic HBV infection, CAGAGAG-39); HBsAg (forward: 59-ACAGGCGGGGTTTTTCTTGT-39; aiming to investigate the factors related to iNKT cell changes and reverse: 59-CGCAGACACATCCAGCGATA-39); and GAPDH (forward: http://www.jimmunol.org/ explore iNKT cell–based therapeutic potential for CHB. 59-CAGTCCATGCCATCACTGCCACCCAG-39;reverse:59-CAGTG- TAGCCCAGGATGCCCTTGAG-39). The relative mRNA levels of CD1d and HBsAg were normalized to GAPDH mRNA. Materials and Methods Statistics Patient populations Graphs were generated and analyzed by GraphPad Prism 5.0 (GraphPad Liver and tumor specimens were collected during hepatic resection from 30 Software). Data were analyzed by Student t test for comparisons of groups HBV-infected HCC, 3 HBV-negative HCC, and 10 HBV-negative hepatic with normal distribution and equal variance. For nonnormal distributed carcinoid patients in Tongji Hospital, Wuhan, China. Blood samples of 206 variables, Mann–Whitney U test or Wilcoxon matched-pairs signed rank patients with chronic HBV infection (HBsAg-positive .6 mo) and 279 test was performed. Correlations between HBsAg and CD1d levels were by guest on September 24, 2021 healthy donor (HD) samples were collected from Tongji Hospital and Wuhan assessed by Spearman rank correlation. The p values , 0.05 were con- Blood Center, respectively. The HBV-infected patients were classified into sidered statistically significant. four groups according to the disease stages as follows. 1) The immune tolerance (IT) group included patients with hepatitis B e Ag (HBeAg)–positive, Ethics statement evaluated viral load, and a normal level of aminotransferase. 2) Inactive carriers (IC) were defined by absence of HBeAg, presence of hepatitis B e The ethics committee of Tongji Medicine College, Huazhong University of Ab (HBeAb), persistently normal aminotransferase levels, and low levels of Science and Technology, granted approval for all aspects of this study. All HBV DNA (,1 3 104 copies/ml). 3) CHB patients included individuals in patients gave written informed consent. All human subjects were adults. either immune-active phase or reactivation phase, confirmed by elevated aminotransferase or active inflammation on liver biopsy. Cirrhosis was di- Results agnosed by imaging, biochemical, or histological examination. 4) HCC CD1d expression is upregulated in HBV-infected tissue patients were documented by magnetic resonance imaging or computerized tomography scan and, in surgical patients, by the histological analysis of the As CD1d-restricted self-lipids modified by HBV contribute to removed mass. Individuals with other concurrent types of viral hepatitis, activation of iNKT cells to control HBV infection (26), we first HIV, autoimmune liver disease, or alcoholic liver disease, were excluded. sought to evaluate whether HBV infection affected hepatocyte Patient characteristics are listed in Tables I and II. CD1d expression. Double staining of HBsAg and CD1d was Isolation and in vitro stimulation of intrahepatic mononuclear performed in liver tissue from HBV-infected HCC patients. Nor- cells and PBMCs mal liver sections from HBV-negative hepatic carcinoid patients served as controls (Table I). As shown in Fig. 1A and 1B, liver Intrahepatic mononuclear cells (MNCs) were first isolated by modified enzymatic dispersal protocol as previously described (24), followed by sections from HBV-negative individuals were weakly stained with Percoll density purification. Intrahepatic MNCs were collected from the CD1d on the cell surface (upper panel), whereas HBV-infected layer between 27% Percoll and 50% Percoll. PBMCs were isolated by tissue had significantly higher CD1d levels (lower panel). In ad- Ficoll density gradient centrifugation as described before (25). dition, CD1d-expressing lymphocytes were frequently found in 3 5 3 5 1 10 intrahepatic MNCs or 3 10 PBMCs were simulated with HBV-infected tissue (Fig. 1A, arrowhead). In magnified images, it 200 ng/ml a-GalCer (KRN7000; Avanti). Recombinant IL-2 and IL-15 (PeproTech) were used at 50 U/ml and 2.5 ng/ml, respectively. Purified was clear that hepatocytes in HBV-infected liver had elevated mouse anti-human CD1d (51.1; BioLegend) was used as a blocking Ab CD1d levels both on cell surface and in cytoplasm (Fig. 1B, at 5 mg/ml. Supernatants and cells were harvested on day 3 and day 7, asterisk). The higher CD1d staining was not likely due to non- respectively. specific staining, because the staining of CD1d isotype control Abs for flow cytometry in HBsAg-positive areas gave only trace background levels (Fig. 1C). Fluorescence-conjugated mAbs were purchased from BD Biosciences, eBioscience, and BioLegend: CD3 (UCHT1), CD14 (M5E2), CD19 After quantification by counting pixels, we found liver sections (HIB19), CD38 (HB-7), CD45 (HI30), CD69 (FN50), CD95 (DX2), CD95L from HBV-infected HCC patients had higher CD1d levels than (NOK-1), CD1d (51.1), PD-1 (MIH4), and HLA-DR (L243). those from HBV-negative individuals (Fig. 1D). Intrahepatic The Journal of Immunology 3

Table I. Clinical characteristics of patients enrolled in this study (for liver and tumor samples)

HCC

HBV-Positive HBV-Negative HBV-Negative Hepatic Carcinoida No. 30 3 10 Gender (male/female) 24/6 2/1 4/6 Age (y)b 51 (25–68) 69 (56–76) 49.5 (21–60) HBV-DNAb 5.36 ND ND (log10 copies/ml) (, 2.70–7.32), 11 ND ND ND HBeAg (positive/negative) 4/16, 10 ND 0/3 0/10 HBeAb (positive/negative) 15/5, 10 ND 0/3 0/10 ALTb 34.5 (15–145), 10 ND 12 (11–13), 1 ND 14 (7–40) ASTb 39.5 (14–110), 10 ND 17 (13–21), 1 ND 22 (15–30) aNine hepatic hemangioma patients and one hepatic cyst patient are enrolled. bData are shown as median (range). ND, not determined.

accumulation of HBsAg was positively correlated with CD1d The individual differences revealed by the asymmetric distri- Downloaded from staining, as indicated by correlation analysis of double-stained bution suggested that a large sample size would be beneﬁcial to nonmalignant liver sections from three HBV-infected HCC pa- characterize the frequency changes of iNKT cells in chronic tients (Fig. 1E). Meanwhile, HBsAg-positive hepatoma sections HBV infection. In this study, a reduction of iNKT cell proportion had signiﬁcantly higher CD1d levels than their negative counter- inPBMCswasobservedinHBV-infected patients (Fig. 3B). parts (Fig. 1F, 1G), further supporting the positive relationship T cell ratios in PBMCs from the patients also decreased as

between HBV infection and upregulation of CD1d. What’s more, compared with HDs (Supplemental Fig. 1C). Further analyzing http://www.jimmunol.org/ tissue from HBV-infected patients expressed higher mRNA levels the changes of circulating iNKT cells in different disease stages of CD1d than those from HBV-uninfected individuals (Fig. 1H). A during chronic HBV infection, we found iNKT cell proportion positive correlation was found between hepatic CD1d and HBsAg among peripheral T cells significantly decreased in IC, CHB, and transcripts in HBV-infected patients (Fig. 1I). Collectively, our HCC patients, but not in IT individuals (Fig. 3C). results unveiled elevated CD1d expression in HBV-infected liver The role of iNKT cells in infection remains controversial, as both and hepatoma tissue. protective and pathogenic effects of iNKT cells have been proposed (19, 26, 27). To evaluate the relationship between the number or iNKT cell defects are observed in the liver from HBV-infected functional status of iNKT cell and liver injury, CHB patients were HCC patients selected for further analysis because this group of patients showed by guest on September 24, 2021 The elevated CD1d in HBV-infected liver and hepatoma tissue is a wide range of alanine aminotransferase (ALT)/aspartate ami- expected to facilitate an iNKT cell–based immunosurveillance notransferase (AST) levels. Notably, the CHB patients had an even effect during chronic HBV infection. However, we found a re- lower circulating iNKT cell proportion as compared with IC markable reduction of iNKT cell proportion in the liver and hepa- groups (Fig. 3C). The number of circulating iNKT cells in CHB toma tissue from HBV-infected HCC patients (Fig. 2A–C). The patients significantly reduced (Fig. 3D). Moreover, the frequency percentage of CD3 and PBS57/CD1d tetramer double-positive of iNKT cells reduced to a greater extent in CHB patients with (CD3+CD1d Tet+) iNKT cells ranged from 0.26 to 1.04% in he- cirrhosis or high HBV DNA levels (Fig. 4A, 4B). The iNKT cell patic T cells from HBV-uninfected patients. This ratio decreased frequencies were negatively associated with the degree of liver significantly in liver and hepatoma tissue from HBV-infected HCC injury because patients with higher serum AST/ALT tended to patients (Fig. 2B, 2C). Furthermore, intrahepatic MNCs in hepa- have fewer iNKT cells (Fig. 4C). toma and adjacent liver tissue from HBV-infected HCC lost To assess the functional status of residual iNKT cells in CHB a-GalCer–induced IFN-g production (Fig. 2D, middle and right patients, we stimulated PBMCs with a-GalCer to check iNKT cell panel), whereas those from HBV-negative liver had significant expansion and IFN-g production. As expected, culturing purified IFN-g secretion (Fig. 2D, left panel). The impaired responsiveness PBMCs from HDs with a-GalCer led to CD1d-dependent IFN-g to a-GalCer was most likely due to reduced number and impaired production and significant expansion of iNKT cells (Fig. 3E–G, function of iNKT cells in the HBV-infected HCC patients. HDs). However, in PBMCs from CHB patients, impaired IFN-g production as well as reduced expansion rate of iNKT cells were Circulating iNKT cells are reduced and hyporesponsive in observed (Fig. 3E–G, CHB). The iNKT cell expansion generated CHB patients by a-GalCer stimulation among HD and CHB patients was ac- To assess the reciprocal effects between iNKT cells and HBV tually the result of proliferation, as indicated by CFSE staining infection, peripheral blood samples from 268 HDs and 191 HBV- (Supplemental Fig. 2). Although the lower iNKT cell ratio in infected patients in different disease stages were analyzed for patients could partially account for lower IFN-g production, it is changes of iNKT cell proportion (Table II). Prevalence of circu- worthy to note that the increased folds of ratio and number were lating iNKT cells was considerably low, ranging from 0.0002 to much less in iNKT cells from CHB patients than those from HDs 1.08% among all detected subjects (Fig. 3A, 3B). Unlike the (Fig. 3G). What’s more, lower expansion rates were found among Gaussian distribution of conventional T cell frequency in the iNKT cells from CHB patients with high AST levels (Fig. 4D), and peripheral blood, the ratios of circulating iNKT cells from both there was no CD1d-dependent IFN-g production by iNKT cells HDs and patients exhibited skewed distribution with a long right from those with more severe liver injury (Fig. 4E). The in vitro tail (Supplemental Fig. 1A), which could fit a normal distribution hyporesponsiveness of iNKT cells was unlikely to be the result of only after logarithmic transformation (Supplemental Fig. 1B). impairment of CD1d presentation because there was no decrease in 4 iNKT CELL DEFECTS IN CHRONIC HEPATITIS B Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. Intrahepatic CD1d expression elevates in HBV-infected liver tissue. (A and B) HBsAg and CD1d expression in liver sections from HBV- negative hepatic carcinoid patients (HBV2) and HBV-infected patients (HBV+). White asterisk, HBsAg-positive hepatocytes; white arrowhead, CD1d- expressing lymphocytes. (C) Double staining of HBsAg (upper panel) and CD1d isotype control (lower panel) in HBV-positive liver sections. (D) Integrated OD per 104 pixels for CD1d and HBsAg of entire liver section were quantiﬁed in HBV2 (n = 4) and HBV+ (n = 9) patients. Data are presented as box-and- whisker plots. Data are from at least four independent experiments. (E) Spearman rank correlation analysis of CD1d and HBsAg level of liver sections from three HBV-infected patients. (F) CD1d and HBsAg staining in HBsAg-negative hepatoma tissue (HBsAg2 HCC) and HBsAg positively stained hepatoma tissue (HBsAg+ HCC). (G) CD1d levels in HBsAg2 HCC and HBsAg+ HCC. (H) Relative mRNA levels of hepatic CD1d to GAPDH in HBV-uninfected and -infected patients. (I) Spearman rank correlation analysis of CD1d and HBsAg mRNA levels from HBV-infected patients. Data are shown by mean 6 SEM from at least three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, Mann–Whitney U test. The Journal of Immunology 5

FIGURE 2. Hepatic iNKT cells are reduced and lost a-GalCer–induced IFN-g production in HBV-infected liver and hepatoma tissue. Intrahepatic MNCs from HBV-negative and HBV-positive individuals were isolated. (A) Representative plots for the intrahepatic iNKT cells were shown. (B and C) iNKT cell frequencies in normal liver tissue from HBV- negative (HBV2 liver) patients, and hepatoma and adjacent liver tissue from HBV-infected HCC patients (HBV+ HCC) were analyzed. Representative dot plots (B) and summary scatter graph with geometric mean 6 95% CI (C) are shown. **p , 0.01, Mann–Whitney U test. (D) Intrahepatic lymphocytes from HBV2 Downloaded from liver (n = 7), HBV+ liver (n = 13), and hepatoma (n = 14) were cultured with vehicle or a-GalCer for 3 d. The concentrations of IFN-g released are shown in bar graph. Data are from at least six separate experiments. *p , 0.05, Wilcoxon matched-pairs signed rank test. http://www.jimmunol.org/

CD1d-expressing APCs in CHB patients (Supplemental Fig. 3). HCC patients showed no increase in PD-1 level either, although Altogether, these results unveiled distinct iNKT cell defects in the hepatic CD8+ T cells from hepatoma tissue had elevated expression

patients at different disease statuses. The defects associated with of PD-1 (Fig. 5D). Altogether, our results suggested aberrant acti- by guest on September 24, 2021 disease progression suggested iNKT cells attenuated their effects in vation, but not PD-1 upregulation, was associated with iNKT cell CHB patients with cirrhosis, high virus load, and severe liver injury. defects in CHB patients. iNKT cells in CHB patients exhibit overactivated phenotype Exogenous IL-2 and/or IL-15 partially overcome the It is reported that iNKT cells are rapidly eliminated by apoptosis hyporeponsiveness of iNKT cells from CHB patients mediated by Fas/Fas ligand (FasL) interaction after activation (28). It is widely accepted that IL-2 signiﬁcantly promotes iNKT cell It could therefore be speculated that this mechanism was also proliferation and overcomes the hyporesponsive phenotype of responsible for the reduction of iNKT cells in CHB patients. a-GalCer–pretreated iNKT cells (30). Moreover, IL-15 is reported Consistent with this notion, we observed upregulation of both Fas to play a critical role in iNKT cell homeostasis and expansion via (CD95) and FasL (CD95L) in iNKT cells from CHB patients as promoting production of IL-12 by monocytes (31). As lower ex- compared with those from HDs (Fig. 5A, 5B). Further, CD69, pansion rate and less CD1d-dependent IFN-g production were CD38, and HLA-DR levels also increased on iNKT cells from the found among CHB patients with severe liver injury (AST .33 patients (Fig. 5A, 5B). Despite the PD-1:PD-L pathway playing a upper limit of normal), we tested the capacity of IL-2 and IL-15 to critical role in iNKT cell anergy after antigenic stimulation (29), rescue proliferation and cytokine production of the iNKT cells. As there was no upregulated PD-1 expression on circulating iNKT cells expected, IL-2 and IL-15 signiﬁcantly increased iNKT cell ex- of CHB patients (Fig. 5C). Furthermore, hepatic iNKT cells from pansion and CD1d-dependent IFN-g production of PBMCs from

Table II. Clinical characteristics of patients enrolled in this study (for HBV+ blood samples)

IT IC CHB HCC Undeﬁned No. 8 23 130 25 20 Gender (male/female) 4/4 18/5 118/12 21/4 13/7 Age (y)a 29 (17–49) 43 (18–64) 43 (18–66) 51 (22–75) 31 (22–47) HBV-DNAa 7.23 , 2.70 4.10 , 2.70 , 2.70 ( log10 copies/ml) (4.51–8.09) (, 2.70–3.63) (, 2.70–9.16), 9 ND (, 2.70–6.97), 3 ND (, 2.70–6.21) HBeAg (positive/negative) 8/0 0/23 39/81, 10 ND 3/22, 3 ND 6/14 HBeAb (positive/negative) 0/8 23/0 54/66, 10 ND 14/8, 3 ND 0/20 ALTa 14.5 (11–25) 20 (10–30) 354.1 (11–3369) 31 (11–500) 15.5 (7–35) ASTa 17.5 (15–28) 21 (15–28) 229 (20–3365) 43 (17–1285) 21.5 (16–32) aData are shown as median (range). ND, not determined. 6 iNKT CELL DEFECTS IN CHRONIC HEPATITIS B Downloaded from

FIGURE 3. Reduction and hyporesponsiveness of circulating iNKT cells in CHB patients. PBMCs were separated from 268 HDs and 191 chronic HBV-infected patients. (A) Representative dot plots for circulating iNKT cell and conventional T cell frequencies. (B) Circulating iNKT cell ratio from 268 HDs and 191 chronic HBV- infected patients. Results are shown by geometric mean 6 95% CI. ***p , 0.001, Mann–Whitney U test. (C) Circulating iNKT cell ratio among HDs and patients in http://www.jimmunol.org/ different disease phases including IT (n = 8), IC (n =23),CHB(n = 115), and HCC (n = 25). Scatter plots are shown with geometric mean 6 95% CI. **p , 0.01, ***p , 0.001, Mann–Whitney U test. (D) Absolute count of circulating iNKT cells among HD and CHB patients. ***p , 0.001, Mann–Whitney U test. (E)Bargraph depicts mean 6 SEM of IFN-g production by a-GalCer–stimulated PBMCs with or without anti-CD1d Ab. *p , 0.05, ***p , 0.001, Wilcoxon matched-pairs signed rank test. (F) Representative iNKT cell frequencies at day 0 and day 7 post–a-GalCer stimulation. (G) Increase folds of iNKT cell ratio and number after a-GalCer stimulation (relative to control). Results are shown by mean 6 SEM. Data are from at least 10 independent experiments. ***p , 0.001, Mann–Whitney U test.

HDs in response to a-GalCer (Fig. 6). For the patients, culture of with our ﬁndings above (Fig. 3). IL-2 fully rescued the a-GalCer– PBMCs with a-GalCer in the absence of the cytokines resulted in low induced expansion of iNKT cells from the CHB patients to reach the expansion rate of iNKT cells (Fig. 6A, 6B), which was consistent comparable expansion rate as those from HDs (Fig. 6B). by guest on September 24, 2021

FIGURE 4. The degree of iNKT cell defects is associated with liver injury and HBV DNA load. (A) Circulating iNKT cell ratio in CD3+ T cells among CHB patients with (n = 54) or without (n = 61) cirrhosis. (B) Circulating iNKT cell ratio among CHB patients with different serum HBV DNA levels (n $ 13 per group). (C) Circulating iNKT cell ratio among CHB patients with different AST and ALT levels (n $ 19 per group). Scatter plots are shown with geometric mean 6 95% CI. (D and E) PBMCs from CHB patients with different levels of AST were stimulated with a-GalCer and analyzed for CD1d-dependent IFN-g production (day 3) as well as iNKT cell ratios and numbers (day 7) (n $ 8 per group). Fold changes in iNKT cell ratio and number (D)andIFN-g levels in the presence or absence of anti-CD1d (E) are shown by bar graphs with mean 6 SEM. Results are pooled from at least three separate experiments. Mann–Whitney U test was used for unpaired groups and Wilcoxon matched-pairs signed rank test was performed for paired samples. *p , 0.05, **p , 0.01, ***p , 0.001. ns, no signiﬁcance; ULN, upper limit of normal. The Journal of Immunology 7

FIGURE 5. Overactivated status of iNKT cells from CHB patients. (A–C) PBMCs were separated from HD and CHB patients. Flow cytometry was performed to analyze CD95, CD95L, CD69, CD38, HLA-DR, and PD-1 levels. Representative histogram (A) and summary bar graph (B) display CD95, CD95L, CD69 CD38, and HLA- DR levels on circulating iNKT cells (n $ 7 per group). (C) PD-1 levels on circulating iNKT, CD4+ T, and CD8+ T cells are compared between HD and CHB patients (n = 7 per group). (D) Intrahepatic MNCs were isolated from HBV– livers of hepatic carcinoid patients and hepatoma and adjacent liver tissue from HBV+ HCC patients. Bar graph represents PD-1 levels on hepatic Downloaded from iNKT, CD4+ T, and CD8+ T cells. Bar graphs are shown with mean 6 SEM. Data are pooled from four separate experiments. *p , 0.05, **p , 0.01, ***p , 0.001, Mann–Whitney U test. http://www.jimmunol.org/

Unexpectedly, IL-2 alone was not sufficient to recover the CD1d- administration of a-GalCer with IL-2 and IL-15 might help to dependent cytokine production of PBMCs from patients (Fig. 6C). restore the Th1 response of iNKT cells in CHB patients. Although IL-15 was less efficient in promoting a-GalCer–induced expansion of iNKT cells than IL-2, the synergistic effect of IL-2 Discussion and IL-15 recovered the CD1d-dependent IFN-g production of Previous studies display conflicting results regarding changes in iNKT cells from the CHB patients (Fig. 6C). This suggested that circulating iNKT cells in chronic HBV-infected patients (17, 18, 27). by guest on September 24, 2021

FIGURE 6. IL-2 and/or IL-15 partially overcome the hyporesponsiveness of iNKT cells from CHB patients. PBMCs from HDs (n = 12) and CHB patients (n =8)werestim- ulated with a-GalCer in the presence of IL-2, IL-15, and/or anti-CD1d Ab. PBMCs cultured with vehicle served as control. At day 3, supernatants were collected for IFN-g detection by cytometric bead array. At day 7, cells were harvested and iNKT cell ratio was detected by ﬂow cytometry. Representative dot plots for iNKT cell ratio (A) and fold changes of iNKT cell ratio and number (B)areshown.(C)Bar graphs show IFN-g concentration in different groups. Bar graphs are shown as mean 6 SEM. Data are from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, Wil- coxon matched-pairs signed rank test. 8 iNKT CELL DEFECTS IN CHRONIC HEPATITIS B

Given the highly diverse iNKT cell frequencies in the human response toward HBV, and exert direct cytotoxicity against in- population, we enlarged the sample size by enrolling 268 healthy sub- fected and transformed cells in the liver (21). Given the elevated jects and 191 chronic HBV-infected patients in this study. Changes of CD1d level on HBV-infected liver and hepatoma tissue, a-GalCer iNKT cell frequency and their responsiveness to a-GalCer were ana- administration combined with IL-2 and IL-15 that restored lyzed in patients in different phases of HBV infection. We found sig- iNKT cell ratio and function in CHB patients might harness the nificant reduction in iNKT cell ratio and expansion rate in CHB patients, antiviral and immunosurveillance effects of iNKT cells. whereas there was no reduction of iNKT cells in IT individuals (Fig. 3). Because of the overall defects in iNKT cells from progressive CHB Acknowledgments patients, administration of a-GalCer alone was unlikely to have a potent We thank the National Institutes of Health Tetramer Facility for CD1d antiviral effect. This could partially explain the negative result of the tetramers. We also thank Sreya Bagchi for critical reading of the article. phase I/II trail of a-GalCer treatment for chronic HBV infection. In this study, we highlighted the elevated CD1d expression in Disclosures HBV-infected hepatocytes. It is reported that HBV infection in- The authors have no financial conflicts of interest. duces endoplasmic reticulum (ER)–associated lipid alterations on HBV-expressing hepatocytes, thus helping activation of NKT cells (26). Events in the ER normally regulate CD1d trafficking to References plasma membranes (32). Therefore, the alteration of lipids in ER 1. Kane, M. 1995. Global programme for control of hepatitis B infection. Vaccine probably affects the CD1d expression on HBV-infected cells. 13(Suppl. 1): S47–S49.

Meanwhile, inflammation caused by infection, cytokines, and 2. El-Serag, H. B. 2012. Epidemiology of viral hepatitis and hepatocellular carci- Downloaded from noma. Gastroenterology 142: 1264–1273.e1261. TLR signals also increases CD1d levels, which promotes 3. Hyams, K. C. 1995. Risks of chronicity following acute hepatitis B virus in- NKT cell activation (33, 34). Viral danger signals such as type I fection: a review. Clin. Infect. Dis. 20: 992–1000. 4. European Association for the Study of the Liver. 2017. EASL 2017 Clinical IFN and viral TLR ligands are reported to enhance CD1d de novo Practice Guidelines on the management of hepatitis B virus infection. J. Hepatol. synthesis through increasing the number of CD1d transcripts (34). 67: 370–398. In this study, it remained a question whether HBV infection itself 5. Franco, E., B. Bagnato, M. G. Marino, C. Meleleo, L. Serino, and L. Zaratti. 2012. Hepatitis B: epidemiology and prevention in developing countries. World or the subsequent inflammatory response contributed to the CD1d J. Hepatol. 4: 74–80. http://www.jimmunol.org/ upregulation in HBsAg-positive livers. However, aberrant CD1d 6. Zhang, H. H., M. H. Mei, R. Fei, F. Liu, J. H. Wang, W. J. Liao, L. L. Qin, expression on nonprofessional APCs has been shown to induce L. Wei, and H. S. Chen. 2010. Regulatory T cells in chronic hepatitis B patients affect the immunopathogenesis of hepatocellular carcinoma by suppressing the overactivation and hyporesponsiveness of NKT cells (35, 36). anti-tumour immune responses. J. Viral Hepat. 17(Suppl. 1): 34–43. Considering the enhanced CD69 and Fas/FasL expression in 7. Maini, M. K., and A. Schurich. 2010. The molecular basis of the failed immune response in chronic HBV: therapeutic implications. J. Hepatol. 52: 616–619. iNKT cells from CHB patients (Fig. 5), it is reasonable to spec- 8. Hato, T., L. Goyal, T. F. Greten, D. G. Duda, and A. X. Zhu. 2014. 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80 100 *** 100 60 100 40 Counts Counts

50 50 Counts 20 80 0 0 0 (%) (%) (log%) -8.5 -4.5 -0.5 60 T in PBMC T 60 HBV con-T 200 HBV iNKT 60 HBV iNKT + 40

150 40

40 % CD3 100 20 Counts Counts 20 Counts 20 50 0 CHB 0 0 0 HD (%) (%) (log%) -8.5 -4.5 -0.5

Supplemental Figure 1. The distribution of iNKT cells and conventional T (con-T) cells in HBV-infected patients. (A) The percentages of circulating con-T cells in healthy donors (HD) and HBV-infected patients (HBV) show Gaussian distribution, while circulating iNKT cell ratios show skewness distribution. (B) Logarithmic transformation of iNKT cell ratios to fit the Gaussian distribution. (C) Circulating T cell ratios among HD and chronic Hepatitis B patients (CHB). Scatter plots are shown with geometric mean ± 95% CI. Data are pooled from at least 10 separate experiments. Mann-Whitney U test, ***P < 0.001.

iNKT con-T control +αGC control +αGC

Parents Generation1 HD Generation2 Generation3

Generation4 Count

CHB

CFSE

Supplemental Figure 2. iNKT cells proliferate upon α-GalCer stimulation. CFSE staining of iNKT and conventional T cell (con-T) expansion by α-GalCer (+αGC) stimulation among HD and CHB patients at day7. Cells cultured in mediums served as controls. The populations of parents and proliferated generations are shown in different colors. A B Ratio of APCs CD1d level 1500

25 20 *

% 20

+ 1000 15

% 10 10 500 CD1d inPBMCs 5 0 CD1dMFI 0 0 HD CHB HD CHB HD CHB HD CHB HD CHB B Mo B Mo

Supplemental Figure 3. No change in CD1d-expressing cells in peripheral blood from CHB patients. (A) Proportion of CD1d positive cells among 10 HD and 17 CHB patients. (B) Percentages and CD1d levels of circulating B cell and monocyte. Bar graphs are shown as mean ± SEM. Data are pooled from at least 3 independent experiments. Mann-Whitney U test, *P < 0.05.