Anthony T. Tan

IL-7 Licenses Activation of Human Liver Intrasinusoidal Mucosal-Associated Invariant T Cells

Human mucosal-associated invariant T (MAIT) cells are a T cell population characterized by the expression of a semi-invariant TCR capable of recognizing bacterial products in the context of MR1. MAIT cells are enriched in the human liver, which is constantly exposed to bacterial products from the intestine. Whether this specific parenchymal localization influences their function remains unknown. We analyzed MAIT cells resident in the vascular bed of livers and showed that they represented the majority of T cells expressing NK markers and the dominant IL-17A+ T cell subset in the human liver sinusoids. In comparison with MAIT cells purified from peripheral blood, intrasinusoidal MAIT cells expressed markers of T cell activation; however, TCR-mediated cytokine production was equally suppressed in both circulating and intrasinusoidal MAIT cells. MAIT cells also expressed high levels of IL-7R, and we showed that IL-7, a cytokine produced by hepatocytes during inflammation, regulated TCR-mediated activation of MAIT cells, licensing them to dramatically increase Th1 cytokines and IL-17A production. Our quantitative and functional data indicate that MAIT cells are a specialized cell population highly adapted to exert their immune functions in the vascular network of the liver.

Preserved T-Cell Function in Children and Young Adults With Immune-Tolerant Chronic Hepatitis B

BACKGROUND & AIMS Chronic hepatitis B (CHB) infection acquired perinatally or in early childhood has been associated with a prolonged phase of immune tolerance from viral exposure into early adulthood. The immune-tolerant phase of the disease is characterized by high levels of hepatitis B virus (HBV) DNA and normal liver biochemistry, with minimal or no fibrosis. We investigated whether the age of patients with CHB affects their antiviral immunity and whether children and young adults have a veritable state of immunologic tolerance. METHODS We isolated T cells from different age groups of patients with CHB and used flow cytometric methods to measure production of effector and inflammatory cytokines (interferon, tumor necrosis factor, interleukin [IL]-17A, IL-22, and IL-8), T-helper (Th)2 cytokines (IL-10, IL-4), Th1 cytokines (IL-2 and IL-21), and the CC chemokine CCL3 (MIP-1). We also measured markers of T-cell exhaustion or inhibition (PD-1, LAG-3, TIM3, LAIR-1, and CTLA-4) and HBV-specific T cells. RESULTS Young patients with CHB have a Th1-cell cytokine profile and a partial profile of T-cell exhaustion. Direct quantification of the HBV-specific T-cell response showed that young patients with CHB have more HBV-specific T cells with the ability to proliferate and produce cytokines than adult patients with CHB. CONCLUSIONS HBV infection in younger patients is not associated with an immune profile of T-cell tolerance. On the contrary, children and young adults with chronic HBV infection have an HBV-specific immune profile that is less compromised than that observed in older patients.

A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B

BACKGROUND & AIMS The pathogenesis of hepatic flares (HF) in patients chronically infected with hepatitis B virus (HBV) is controversial. Therefore, we studied the kinetics of innate and adaptive immune activation during HF in chronic hepatitis B. METHODS Soluble (IFN-alpha, IL-1beta, TNF-alpha, IL-6, IL-8, IL-10, CCL-2, CCL-3, CXCL-9, CXCL-10) and cellular (HBV-specific T cells, NK, Treg) immunological parameters were measured longitudinally (10 month-4 week intervals) in patients (n=5) who developed HF after therapy withdrawal and cross-sectionally in chronic (n=29) and acute hepatitis B patients (n=5). Hepatic expression of different chemokines was studied by co-incubating cytokines (IFN-alpha, IFN-gamma, TNF-alpha) and activated T, NK and monocytes with hepatocytes and hepatocyte-like cells. RESULTS A progressive increase of HBV replication precedes HF but occurs without detection of innate immune activation, with the exception of increased serum CXCL-8. Despite the absence of increased circulatory HBV-specific T or activated NK cells, HF were temporally associated with high serum levels of IFN-gamma inducible chemokines CXCL-9 and CXCL-10 (but not CCL-2 or CCL-3). CXCL-9 and CXCL-10 displayed different in vitro requirements for activation and are differentially produced in liver injury present in acute or chronic patients. CONCLUSIONS CXCL-9 and CXCL-10 play a major role in the development of HF. Their differential expression in acute versus chronic patients suggests the presence of different mechanisms that govern liver injury during acute and chronic hepatitis B.

Toll-Like Receptor 8 Agonist and Bacteria Trigger Potent Activation of Innate Immune Cells in Human Liver

The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161Bright mucosal-associated invariant T (MAIT) and CD56Bright NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.

Profile of Tumor Antigen-Specific CD8 T Cells in Patients With Hepatitis B Virus-Related Hepatocellular Carcinoma

BACKGROUND & AIMS Tumor and viral antigens are expressed by hepatocellular carcinoma (HCC) in patients with chronic hepatitis B, but little is known about the immunodominance and function of tumor- and virus-specific CD8+ T cells in these patients. METHODS HLA-A2-restricted T-cell responses to 16 tumor antigens and hepatitis B virus (HBV) proteins were tested using 49 previously described epitopes. Cells from 30 HLA-A2+, HBV-infected patients (10 with HCC, 10 with HBV cirrhosis, and 10 HBV but no cirrhosis) were analyzed, after expansion, by enzyme-linked immunosorbent spot (ELISPOT). Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2 production, as well as expression of the degranulation marker CD107a on tumor-specific CD8+ T cells, were evaluated. RESULTS Cells from all groups had tumor-specific responses. The tumor antigens NY-ESO-1 and SSX-2 were most frequently targeted and were immunogenic in the HLA-A2 subtypes that are characteristic of Asian ethnicity. Tumor-specific T cells had low affinities; T cells from non-HCC patients were polyfunctional (IFN-gamma+, TNF-alpha+, CD107a+) and those from HCC patients displayed an exhausted phenotype (IFN-gamma+, CD107a+). Programmed Death 1 (PD-1) was expressed at higher levels on T cells from tumor and liver than peripheral blood from HCC patients and might contribute to T-cell exhaustion. Blocking PD-1/PD-L1 increased the frequency of tumor-specific T cells in HCC patients but did not restore T cell function. CONCLUSIONS Patients with or without HCC have a quantitative and functional hierarchy of tumor-specific T cells. HLA-A2-restricted T cells from HCC patients target NY-ESO-1, but exist in an exhausted state that might require additional activation to restore function.

Host Ethnicity and Virus Genotype Shape the Hepatitis B Virus-Specific T-Cell Repertoire

ABSTRACT Repertoire composition, quantity, and qualitative functional ability are the parameters that define virus-specific T-cell responses and are linked with their potential to control infection. We took advantage of the segregation of different hepatitis B virus (HBV) genotypes in geographically and genetically distinct host populations to directly analyze the impact that host and virus variables exert on these virus-specific T-cell parameters. T-cell responses against the entire HBV proteome were analyzed in a total of 109 HBV-infected subjects of distinct ethnicities (47 of Chinese origin and 62 of Caucasian origin). We demonstrate that HBV-specific T-cell quantity is determined by the virological and clinical profiles of the patients, which outweigh any influence of race or viral diversity. In contrast, HBV-specific T-cell repertoires are divergent in the two ethnic groups, with T-cell epitopes frequently found in Caucasian patients seldom detected in Chinese patients. In conclusion, we provide a direct biological evaluation of the impact that host and virus variables exert on virus-specific T-cell responses. The discordance between HBV-specific CD8 T-cell repertoires present in Caucasian and Chinese subjects shows the ability of HLA micropolymorphisms to diversify T-cell responses and has implications for the rational development of therapeutic and prophylactic vaccines for worldwide use.

New genetic associations detected in a host response study to hepatitis B vaccine

The immune response to hepatitis B vaccination differs greatly among individuals, with 5–10% of healthy people failing to produce protective levels of antibodies. Several factors have been implicated in determining this response, chiefly individual genetic variation and age. Aiming to identify genes involved in the response to hepatitis B vaccination, a two-stage investigation of 6091 single-nucleotide polymorphisms (SNPs) in 914 immune genes was performed in an Indonesian cohort of 981 individuals showing normal levels of anti-HBs versus 665 individuals displaying undetectable levels of anti-HBs 18 months after initial dose of the vaccine. Of 275 SNPs identified in the first stage (476 normal/372 nonresponders) with P<0.05, significant associations were replicated for 25 polymorphisms in 15 genes (503 normal/295 nonresponders). We validated previous findings (HLA-DRA, rs5000563, P-value combined=5.57 × 10−10; OR (95%CI)=0.61 (0.52–0.71)). In addition, we detected a new association outside of the human leukocyte antigen loci region that passed correction for multiple testing. This SNP is in the 3′ downstream region of FOXP1, a transcription factor involved in B-cell development (P-value combined=9.2 × 10−6; OR (95%CI)=1.38 (1.2–1.6)).These findings might help to understand the biological reasons behind vaccine failure and other aspects of variation in the immune responses of healthy individuals.

Reduction of HBV replication prolongs the early immunological response to IFNα therapy

BACKGROUND & AIMS The interaction between HBV replication and immune modulatory effects mediated by IFNα therapy is not well understood. We characterized the impact of HBV DNA replication on the early IFNα-induced immunomodulatory mechanisms. METHODS We interrogated the transcriptional, serum cytokine/chemokine and cellular immune profiles of 28 patients with HBeAg+ chronic HBV infection (CHB) randomly assigned to one of 4 treatment cohorts (untreated n=5, weekly dosing of 360 μg Pegasys [PegIFNα] n=11, daily dose of 300 mg Viread [tenofovir disoproxil fumarate, TDF] n=6, or a combination of both n=6). Samples were characterized at multiple early time points through day 14 of therapy, after which all patients were given standard of care (180 μg Pegasys injected subcutaneously, weekly). RESULTS PegIFNα induced a distinct and rapid up-regulation of IFN signaling pathway that coincided with increase detection of distinct serum cytokines/chemokines (IL-15, IL-6, and CXCL-10) and the up-regulation of the frequency of proliferating NK and activated total CD8+ T cells. IFNα treatment alone did not result in rapid decay of HBV replication and was not able to restore the defective HBV-specific T cell response present in CHB patients. In addition, the IFNα immune-stimulatory effects diminished after the first dose, but this refractory effect was reduced in patients where HBV replication was simultaneously inhibited with TDF. CONCLUSIONS We present here the first comprehensive description of the early effects of IFNα treatment on immune and viral biomarkers in HBeAg+ CHB patients. Our results show that PegIFNα-induced innate immune activation directly benefits from the suppression of HBV replication.

Contribution of Herpesvirus Specific CD8 T Cells to Anti-Viral T Cell Response in Humans

Herpesviruses infect most humans. Their infections can be associated with pathological conditions and significant changes in T cell repertoire but evidences of symbiotic effects of herpesvirus latency have never been demonstrated. We tested the hypothesis that HCMV and EBV-specific CD8 T cells contribute to the heterologous anti-viral immune response. Volume of activated/proliferating virus-specific and total CD8 T cells was evaluated in 50 patients with acute viral infections: 20 with HBV, 12 with Dengue, 12 with Influenza, 3 with Adenovirus infection and 3 with fevers of unknown etiology. Virus-specific (EBV, HCMV, Influenza) pentamer+ and total CD8 T cells were analyzed for activation (CD38/HLA-DR), proliferation (Ki-67/Bcl-2low) and cytokine production. We observed that all acute viral infections trigger an expansion of activated/proliferating CD8 T cells, which differs in size depending on the infection but is invariably inflated by CD8 T cells specific for persistent herpesviruses (HCMV/EBV). CD8 T cells specific for other non-related non persistent viral infection (i.e. Influenza) were not activated. IL-15, which is produced during acute viral infections, is the likely contributing mechanism driving the selective activation of herpesvirus specific CD8 T cells. In addition we were able to show that herpesvirus specific CD8 T cells displayed an increased ability to produce the anti-viral cytokine interferon-γ during the acute phase of heterologous viral infection. Taken together, these data demonstrated that activated herpesvirus specific CD8 T cells inflate the activated/proliferating CD8 T cells population present during acute viral infections in human and can contribute to the heterologous anti-viral T cell response.

Increased Levels of Arginase in Patients With Acute Hepatitis B Suppress Antiviral T Cells

BACKGROUND & AIMS During viral infection, the activities of virus-specific CD8(+) T cells are carefully regulated to prevent severe damage of the infected organs. We investigated the mechanisms that control the functions of activated T cells. METHODS We measured the size of the population of activated and proliferating CD8(+) T cells and the functional pattern of CD8(+) T cells specific for the entire hepatitis B virus proteome and for selected heterologous virus (Epstein-Barr virus, human cytomegalovirus, and influenza virus) using blood samples from 18 patients with acute hepatitis B. We analyzed the effects of different modulatory mechanisms, such as inhibitory molecules, suppressive cytokines (interleukin-10), and arginase, on the activities of CD8(+) T cells. RESULTS In patients with acute hepatitis B, the expansion of activated and proliferating (HLA-DR/CD38(+), Ki-67(+)/Bcl-2(low)) CD8(+) T cells did not quantitatively match their specific functions ex vivo; virus-specific CD8(+) T cells had functional impairments that were temporally restricted to the acute phase of viral hepatitis. These impairments in function were not limited to HBV-specific CD8(+) T cells but were also observed in CD8(+) T cells with specificities for other viruses. We investigated possible causes of antigen-independent CD8(+) T cell inhibition and found that the increased levels of arginase observed in patients with acute hepatitis could suppress the function of activated, but not resting, CD8(+) T cells. CONCLUSIONS The increased level of arginase in patients with acute hepatitis B suppresses the functions of activated CD8(+) T cells. This mechanism might limit the amount of liver damage caused by activated CD8(+) T cells in patients with acute HBV infection.