J. Miquel

Bim-mediated apoptosis and PD-1/PD-L1 pathway impair reactivity of PD1+/CD127− HCV-specific CD8+ cells targeting the virus in chronic hepatitis C virus infection

PD-1 molecule promotes anergy and IL-7 receptor (CD127) induces an anti-apoptotic effect on T cells. Correlation between PD-1/CD127 phenotype and hepatitis C virus (HCV)-specific CD8(+) cell reactivity in resolved infection (RI) after treatment and persistent HCV-infection (PI) was analysed. Directly ex vivo, PD-1 and CD127 expression on HCV-specific CD8(+) cells displayed a positive and negative correlation, respectively with viraemia. Proliferation after stimulation on PD-1(-)/CD127(+) cells from RI cases was preserved, while it was impaired on PD-1(+)/CD127(-) cells from PI patients. PD1(+)/CD127(+) population was observed in PI, and these maintained expansion ability but they did not target the virus. Frequency of PI cases with HCV-specific CD8(+) cell proliferation increased after anti-PD-L1 and anti-apoptotic treatment. Bim expression on HCV-specific CD8(+) cells from PI patients was enhanced. In conclusion, during chronic HCV infection non-reactive HCV-specific CD8(+) cells targeting the virus are PD-1(+)/CD127(-)/Bim(+) and, blocking apoptosis and PD-1/PD-L1 pathway on them enhances in vitro reactivity.

Adaptive immune response during hepatitis C virus infection.

Hepatitis C virus (HCV) infection affects about 170 million people worldwide and it is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV is a hepatotropic non-cytopathic virus able to persist in a great percentage of infected hosts due to its ability to escape from the immune control. Liver damage and disease progression during HCV infection are driven by both viral and host factors. Specifically, adaptive immune response carries out an essential task in controlling non-cytopathic viruses because of its ability to recognize infected cells and to destroy them by cytopathic mechanisms and to eliminate the virus by non-cytolytic machinery. HCV is able to impair this response by several means such as developing escape mutations in neutralizing antibodies and in T cell receptor viral epitope recognition sites and inducing HCV-specific cytotoxic T cell anergy and deletion. To impair HCV-specific T cell reactivity, HCV affects effector T cell regulation by modulating T helper and Treg response and by impairing the balance between positive and negative co-stimulatory molecules and between pro- and anti-apoptotic proteins. In this review, the role of adaptive immune response in controlling HCV infection and the HCV mechanisms to evade this response are reviewed.

Persistent hepatitis C virus (HCV) infection impairs HCV-specific cytotoxic T cell reactivity through Mcl-1/Bim imbalance due to CD127 down-regulation.

Summary.  In persistent hepatitis C virus (HCV) infection, HCV‐specific cytotoxic T lymphocyte (CTL) reactivity is impaired and this affects HCV control. Interleukin‐7 receptor (CD127) expression on these cells could regulate CTL reactivity through Mcl‐1/Bim balance modulation. Bim is a pro‐apoptotic molecule blocked by the action of Mcl‐1. Mcl‐1/Bim expression and T cell reactivity on HCV‐specific CTLs were compared according to CD127 phenotype. Peripheral blood lymphocytes (PBL) from HLA‐A2+ HCV+ patients were obtained. HCV‐specific CTLs were visualized by staining PBL with anti‐CD8 and HLA‐A2/peptide pentameric complexes (pentamer). Mcl‐1/Bim/CD127 phenotype of HCV‐specific CTLs was tested by staining detectable CD8+/pentamer+ cells with anti‐Mcl‐1/Bim/CD127 antibodies. HCV‐specific CTL proliferation ability after specific in vitro challenge was tested in the presence and absence of pancaspase inhibitor z‐VAD‐fmk. All stained cells were analysed by flow cytometry. CD127low‐expressing HCV‐specific CTLs associated with high HCV viraemia, while CD127high correlated with undetectable viral loads (P < 0.001). Directly ex vivo, pentamer+ cell frequency was similar according to CD127 expression level. Nevertheless, CD127low pentamer+ cell proliferation after specific in vitro challenge was impaired (P < 0.05), although this was corrected by z‐VAD‐fmk treatment (P < 0.05). Mcl‐1 expression was low directly ex vivo (P < 0.01), and Bim was up‐regulated after antigen encounter (P < 0.05) of CD127low pentamer+ cells. The ex vivo difference between Mcl‐1 and Bim expression on pentamer+ cells correlated positively with CD127 expression level (P < 0.001) and with pentamer+ cell reactivity (P < 0.05). In summary, a low ex vivo Mcl‐1 expression and Bim up‐regulation after antigen encounter are involved in CD127low HCV‐specific CTL hyporeactivity during chronic infection, but it can be overcome by apoptosis blockade.

Role of T cell death in maintaining immune tolerance during persistent viral hepatitis

Virus-specific T cells play an important role in the resolution of hepatic infection. However, during chronic hepatitis infection these cells lack their effector functions and fail to control the virus. Hepatitis B virus and hepatitis C virus have developed several mechanisms to generate immune tolerance. One of these strategies is the depletion of virus-specific T cells by apoptosis. The immunotolerogenic liver has unique property to retain and activate naïve T cell to avoid the over reactivation of immune response against antigens which is exploited by hepatotropic viruses to persist. The deletion of the virus-specific T cells occurs by intrinsic (passive) apoptotic mechanism. The pro-apoptotic molecule Bcl-2 interacting mediator (Bim) has attracted increasing attention as a pivotal involvement in apoptosis, as a regulator of tissue homeostasis and an enhancer for the viral persistence. Here, we reviewed our current knowledge on the evidence showing critical role of Bim in viral-specific T cell death by apoptotic pathways and helps in the immune tolerance.

HCV-specific CD8+ cell detection at week 12 of chronic hepatitis C treatment with PEG-interferon-α2b/ribavirin correlates with infection resolution.

Lower than 2-log viral-load (VL) decrease at week 12 (w12) of chronic hepatitis C (CHC) treatment with Peg-interferon/ribavirin has 100% negative predictive value (PV) of sustained virologic response (SVR), and this could be related with absence of HCV-specific cytotoxic T lymphocyte (CTL) response. In this study, percentage of cases with SVR, according to peripheral HCV-specific cytotoxic response at w12, was analysed (Group-1: detection(+), Group-2: detection(-)). SVR was higher in group-1 (93%) than in group-2 (47%) (p=0.003). An increase on HCV-specific CTL frequency between baseline and w12 and higher specific reactivity were observed in group-1 (p=0.011 and p=0.025). HCV-specific CTL detection at w12 correlated with level of VL decrease (p=0.016, r=0.389), and among HCV genotype-1 patients with either early or delayed virologic response (EDVR), 100% positive PV of SVR was observed. In summary, HCV-specific CTL detection at w12 of Peg-interferon/ribavirin treatment correlates with SVR and in EDVR genotype-1 cases predicts SVR.

According to Hepatitis C Virus (HCV) Infection Stage, Interleukin-7 Plus 4-1BB Triggering Alone or Combined with PD-1 Blockade Increases TRAF1low HCV-Specific CD8+ Cell Reactivity

ABSTRACT Hepatitis C virus (HCV)-specific CD8+ T cells suffer a progressive exhaustion during persistent infection (PI) with HCV. This process could involve the positive immune checkpoint 4-1BB/4-1BBL through the loss of its signal transducer, TRAF1. To address this issue, peripheral HCV-specific CD8+ T cells (pentamer-positive [pentamer+]/CD8+ T cells) from patients with PI and resolved infection (RI) after treatment were studied. The duration of HCV infection and the liver fibrosis progression rate inversely correlated with the likelihood of detection of peripheral pentamer+/CD8+ cells. In PI, pentamer+/CD8+ cells had impaired antigen-specific reactivity that worsened when these cells were not detectable ex vivo. Short/midduration PI was characterized by detectable peripheral PD-1+ CD127low TRAF1low cells. After triggering of T cell receptors (TCR), the TRAF1 level positively correlated with the levels of CD127, Mcl-1, and CD107a expression and proliferation intensity but negatively with PD-1 expression, linking TRAF1low to exhaustion. In vitro treatment with interleukin-7 (IL-7) upregulated TRAF1 expression, while treatment with transforming growth factor-β1 (TGF-β1) did the opposite, suggesting that the IL-7/TGF-β1 balance, besides TCR stimulation, could be involved in TRAF1 regulation. In fact, the serum TGF-β1 concentration was higher in patients with PI than in patients with RI, and it negatively correlated with TRAF1 expression. In line with IL-7 increasing the level of TRAF1 expression, IL-7 plus 4-1BBL treatment in vitro enhanced T cell reactivity in patients with short/midduration infection. However, in patients with long-lasting PI, anti-PD-L1, in addition to the combination of IL-7 and 4-1BBL, was necessary to reestablish T cell proliferation in individuals with slowly progressing liver fibrosis (slow fibrosers) but had no effect in rapid fibrosers. In conclusion, a peripheral hyporeactive TRAF1low HCV-specific CD8+ T cell response, restorable by IL-7 plus 4-1BBL treatment, characterizes short/midduration PI. In long-lasting disease, HCV-specific CD8+ T cells are rarely detectable ex vivo, but treatment with IL-7, 4-1BBL, and anti-PD-L1 recovers their reactivity in vitro in slow fibrosers. IMPORTANCE Hepatitis C virus (HCV) infects 71 million people worldwide. Two-thirds develop a chronic disease that can lead to cirrhosis and hepatocellular carcinoma. Direct-acting antivirals clear the infection, but there are still patients who relapse. In these cases, additional immunotherapy could play a vital role. A successful anti-HCV immune response depends on virus-specific CD8+ T cells. During chronic infection, these cells are functionally impaired, which could be due to the failure of costimulation. This study describes exhausted specific T cells, characterized by low levels of expression of the signal transducer TRAF1 of the positive costimulatory pathway 4-1BB/4-1BBL. IL-7 upregulated TRAF1 expression and improved T cell reactivity in patients with short/midduration disease, while in patients with long-lasting infection, it was also necessary to block the negative PD-1/PD-L1 checkpoint. When the results are taken together, this work supports novel ways of restoring the specific CD8+ T cell response, shedding light on the importance of TRAF1 signaling. This could be a promising target for future immunotherapy.

Is it possible to stop nucleos(t)ide analogue treatment in chronic hepatitis B patients

Chronic hepatitis B (CHB) remains a challenging global health problem, with nearly one million related deaths per year. Nucleos(t)ide analogue (NA) treatment suppresses viral replication but does not provide complete cure of the hepatitis B virus (HBV) infection. The accepted endpoint for therapy is the loss of hepatitis B surface antigen (HBsAg), but this is hardly ever achieved. Therefore, indefinite treatment is usually required. Many different studies have evaluated NA therapy discontinuation after several years of NA treatment and before HBsAg loss. The results have indicated that the majority of patients can remain off therapy, with some even reaching HBsAg seroconversion. Fortunately, this strategy has proved to be safe, but it is essential to consider the risk of liver damage and other comorbidities and to ensure a close follow-up of the candidates before considering this strategy. Unanswered questions remain, namely in which patients could this strategy be effective and what is the optimal time point at which to perform it. To solve this enigma, we should keep in mind that the outcome will ultimately depend on the equilibrium between HBV and the host’s immune system. Viral parameters that have been described as good predictors of response in HBeAg(+) cases, have proven useless in HBeAg(-) ones. Since antiviral immunity plays an essential role in the control of HBV infection, we sought to review and explain potential immunological biomarkers to predict safe NA discontinuation in both groups.

298 SUSTAINED VIROLOGIC RESPONSE IN HIGH VIRAEMIC GENOTYPE-1 HCV INFECTION CORRELATES WITH PERIPHERAL HCV-SPECIFIC CYTOTOXIC RESPONSE RESTORATION

Background and Aims: Programmed cell death-1 receptor (PD-1) plays pivotal roles in regulating host immune responses. This study aimed to characterize PD-1 expression in patients with HBVrelated acute-on-chronic liver failure (ACLF) and further evaluate its prognostic significance and functions associated with the disease progression. Methods: Sixty-four patients with HBV-related ACLF (Early, n = 23; Intermediate, n = 21; Late, n = 20) and twenty healthy controls (HC) were enrolled in this study. Flow cytometric, immunohistochemical and functional analyses were employed in the study. Results: Our study found that the expression of PD-1 on circulating CD8T cells was significantly upregulated in ACLF patients from early, intermediate to late stage. Both immunohistochemical staining and flow cytometric analyses revealed a further enhanced PD-1-expression in the liver compared with its counterparts in the peripheral blood in ALCF patients. Moreover, we found the expression of PD-L1, the ligand for PD-1, was also greatly enhanced on the hepatocytes. Correlation analysis with the clinical parameters showed that the expression of PD-1 on CD8T cells positively correlated with MELD Score (model for endstage liver disease) and aspartate aminotransferase (AST), and negatively correlated with prothrombin activity (PTA). Notably, longitudinal study showed that there were gradually decreased PD1-expressions on CD8T cells in patients with improved prognosis, compared with sustained or even increased expressions in those with poor prognosis. Furthermore, the expression of several common g-chain cytokines, including IL-2, IL-7 and IL-15, were greatly upregulated in the liver of ACLF patients, which would contribute to the upregulation of PD-1 on CD8T cell. To evaluate the function of PD-1 on CD8T cells, the in-vitro assays showed that blocking PD-L1 could revitalize the abilities of CD8T cells in cytokine-secretion and proliferation. Conclusions: PD-1 upregulation could mitigate pathogenic CD8Tcell responses, whereas sustained overexpression of PD-1 on CD8T cells is associated with poor prognosis, indicating an uncontrollable immune injury in the liver of ACLF patients. Therefore, PD-1 expression on CD8-T cells could serve as a potential prognostic marker for patients with HBV-related ACLF, which would be valuable for better clinical management and new therapy development.

679 CD127/PD-1 PHENOTYPE DEFINES THE HCV-SPECIFIC CYTOTOXIC T CELL REACTIVITY AFTER ANTIGEN ENCOUNTER DURING CHRONIC HEPATITIS C VIRUS INFECTION

Methods: 20 HCV-seronegative HLA-A2+ individuals without risk for HCV-acquisition were studied. HCV-NS3(1073)-specific cell lines were generated by short culturing of CD8 T-cells with APCs pulsed with HCV-NS3(1073) peptide. After one-week in-vitro stimulation proliferation by CFSE, after three weeks cytokine responses and tetramer binding were analyzed. Individuals were categorized into non-responder and responder based on proliferation and tetramer frequency. TCR repertoire-analysis of tetramer-sorted cells was performed by RT-PCR with Vb-specific primer and subcloning and sequencing of the CDR3-region. TCR repertoire of HCV-seronegative responder was compared with the TCR repertoire of patients with acute HCV-infection. Results: In 8/20 individuals, HCV-NS3(1073)-specific cells proliferated and variable frequencies (0.4–76%) of tetramer positive cells were documented. TCR reper-analysis of the highly responding individual showed usage of only TCR repertore-Vb6.2. Subcloning of Vb6.2 revealed an oligoclonal TCR repertoire. Interestingly, the CDR3-region of the dominant clone (Vb6.2-QEAGAP-Jb2.2) revealed the presence of similar amino acids found in patients with acute HCV-infection (Acute HCV motifs: Vb6.2-LxGxP-Jb 2.7 and Vb6.2ExAxG-Jb 2.3). Conclusions: Our study confirmed that HCV-NS3(1073)-specific CD8 T-cells exist in HCV-seronegative individuals. The variability between individuals is consistent with the concept of private specificity. The identified TCR repertoire was oligoclonal reminiscent to previously identified cross-reactive responses in mouse models and similar to clones in patients with acute HCVinfection. We suggest that this clone may cross-react during acute HCV-infection leading either to protection, immune pathology or immune-escape due to its oligoclonality. The polyspecificity of memory T-cells needs to be considered when developing peptide based vaccines.