J.R. Larrubia

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.

Specific CD8+ T cell response immunotherapy for hepatocellular carcinoma and viral hepatitis

Hepatocellular carcinoma (HCC), chronic hepatitis B (CHB) and chronic hepatitis C (CHC) are characterized by exhaustion of the specific CD8(+) T cell response. This process involves enhancement of negative co-stimulatory molecules, such as programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), 2B4, Tim-3, CD160 and LAG-3, which is linked to intrahepatic overexpression of some of the cognate ligands, such as PD-L1, on antigen presenting cells and thereby favouring a tolerogenic environment. Therapies that disrupt these negative signalling mechanisms represent promising therapeutic tools with the potential to restore reactivity of the specific CD8(+) T cell response. In this review we discuss the impressive in vitro and in vivo results that have been recently achieved in HCC, CHB and CHC by blocking these negative receptors with monoclonal antibodies against these immune checkpoint modulators. The article mainly focuses on the role of CTLA-4 and PD-1 blocking monoclonal antibodies, the first ones to have reached clinical practice. The humanized monoclonal antibodies against CTLA-4 (tremelimumab and ipilimumab) and PD-1 (nivolumab and pembrolizumab) have yielded good results in testing of HCC and chronic viral hepatitis patients. Trelimumab, in particular, has shown a significant increase in the time to progression in HCC, while nivolumab has shown a remarkable effect on hepatitis C viral load reduction. The research on the role of ipilimumab, nivolumab and pembrolizumab on HCC is currently underway.

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.

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.