Noncanonical regulation of HOIL-1 on cancer stemness and sorafenib resistance identifies pixantrone as a novel therapeutic agent for HCC.

BACKGROUND AND AIMS: Cancer stem cells (CSCs) contribute to therapy resistance in HCC. Linear ubiquitin chain assembly complex (LUBAC) has been reported to accelerate the progression of cancers, yet its role in the sorafenib response of HCC is poorly defined. Herein, we investigated the impact of LUBAC on sorafenib resistance and the CSC properties of HCC, and explored the potential targeted drugs. APPROACH AND RESULTS: We found that HOIL-1, but not the other components of LUBAC, played a contributing role in LUBAC-mediated HCC sorafenib resistance, independent of its ubiquitin ligase activity. Both in vitro and in vivo assays revealed that the upregulated HOIL-1 expression enhanced the CSC properties of HCC. Mechanistically, HOIL-1 promoted sorafenib resistance and the CSC properties of HCC through Notch1 signaling. Mass spectrometry, co-immunoprecipitation, western blot, and immunofluorescence were used to determine that the A64/Q65 residues of HOIL-1 bound with the K78 residue of Numb, resulting in impaired Numb-mediated Notch1 lysosomal degradation. Notably, pixantrone was screened out by Autodock Vina, which was validated to disrupt HOIL-1/Numb interaction to inhibit Notch1 signaling and CSC properties by targeting the Q65 residue of HOIL-1. Moreover, pixantrone exerted synergistic effects with sorafenib for the treatment of HCC in different HCC mouse models. CONCLUSIONS: HOIL-1 is critical in promoting sorafenib resistance and CSC properties of HCC through Notch1 signaling. Pixantrone targeting HOIL-1 restrains the sorafenib resistance and provides a potential therapeutic intervention for HCC.

Differential response of HBV envelope-specific CD4 + T cells is related to HBsAg loss after stopping nucleos(t)ide analogue therapy.

BACKGROUND AND AIM: Long-term maintenance of viral control, even HBsAg loss, remains a challenge for chronic hepatitis B (CHB) patients undergoing nucleos(t)ide analogue (NA) discontinuation. This study aimed to investigate the relationship between HBV-specific T-cell responses targeting peptides spanning the whole proteome and clinical outcomes in CHB patients after NA discontinuation. APPROACH AND RESULTS: Eighty-eight CHB patients undergoing NA discontinuation were classified as responders (remained relapse-free up to 96 weeks) or relapsers (relapsed patients who underwent NA retreatment for up to 48 weeks and reachieved stable viral control). HBV-specific T-cell responses were detected at baseline and longitudinally throughout the follow-up. We found responders had a greater magnitude of HBV polymerase (Pol)-specific T-cell responses than relapsers at baseline. After long-term NA discontinuation, simultaneously enhanced HBV Core-induced and Pol-induced responses were observed in responders. Particularly, responders with HBsAg loss possessed enhanced HBV Envelope (Env)-induced responses after short-term and long-term follow-up. Notably, CD4 + T cells accounted for the predominance of HBV-specific T-cell responses. Correspondingly, CD4-deficient mice showed attenuated HBV-specific CD8 + T-cell responses, reduced HBsAb-producing B cells, and delayed HBsAg loss; in contrast, in vitro addition of CD4 + T cells promoted HBsAb production by B cells. Besides, IL-9, rather than PD-1 blockade, enhanced HBV Pol-specific CD4 + T-cell responses. CONCLUSION: HBV-specific CD4 + T-cell responses induced by the targeted peptide possess specificities for long-term viral control and HBsAg loss in CHB patients undergoing NA discontinuation, indicating that CD4 + T cells specific to distinct HBV antigens may endow with divergent antiviral potential.

HBV Core-specific CD4+ T cells correlate with sustained viral control upon off-treatment in HBeAg-positive chronic hepatitis B patients.

BACKGROUND & AIMS: Treatment with nucleos(t)ide analogue (NA) efficiently suppresses viral replication in patients with chronic HBV infection, yet HBV relapses frequently upon NA withdrawal; the detailed immunomodulatory compounds for sustained viral control of HBV upon NA interruption have yet to be fully clarified. This study aimed to elucidate the role of T cells specific for distinct HBV peptides in sustained response upon discontinuation of antiviral treatment. METHODS: A total of 48 patients with HBeAg-positive chronic hepatitis B receiving NA treatment and withdrawal were included longitudinally in a retrospective and prospective cohort. Enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining (ICS) assays were performed to detect IFN-γ producing HBV-specific T cells following stimulation with overlapping peptides covering the whole HBV genome after 10 days of in vitro expansion. RESULTS: ICS assays revealed that T cells specific for HBV Core and Polymerase induced more robust IFN-γ responses compared to Envelope and HBx. Notably, at the time of NA discontinuation, the intensity and breadth of HBV Core peptides-induced responses, predominately targeted by CD4+ T cells but not CD8+ T cells, were associated with sustained viral control upon off-treatment. Further exploration of longitudinal features in patients with sustained viral control revealed that the breadth of HBV-specific T cell responses does not increase following treatment cessation. CONCLUSION: This report emphasizes the essential role of HBV Core-specific CD4+ T cells in sustained response after therapy withdrawal, indicating it is a potential candidate for immunotherapeutic approaches in chronic HBV patients.

CTLA4+CD4+CXCR5-FOXP3+ T cells associate with unfavorable outcome in patients with chronic HBV infection.

BACKGROUND: A major barrier to achieving a favorable outcome of chronic HBV infection is a dysregulated HBV-specific immune response resulting from immunosuppressive features of FOXP3+ T cells. A better definition of FOXP3+ T cells is essential for improving the prognosis of HBV infection. We aimed to investigate the role of CD4+CXCR5-FOXP3+ T cells with CTLA4 expression in patients with chronic HBV infection. METHODS: Treatment-naïve chronic HBV-infected patients, HBV-related hepatic failure, and a longitudinal cohort of chronic hepatitis B (CHB) patients with nucleos(t)ide analogue treatment were enrolled for analysis of CD4+CXCR5-FOXP3+ T cell responses by flow cytometry and single-cell RNA sequencing (scRNA-seq). RESULTS: ScRNA-seq revealed that circulating CD4+CXCR5-FOXP3+ T cells presented distinct inhibitory features compared to spleen tissue. Meanwhile, patients with treatment-naïve chronic HBV infection or with HBV-related hepatic failure showed an upregulation of immune-suppressive features (PD-1, CTLA4, GITR) on CD4+CXCR5-FOXP3+T cells; in vitro analysis found HBeAg and HBcAg stimulation induced elevated levels of inhibitory molecules. Notably, the frequency of CTLA4+CD4+CXCR5-FOXP3+ T cells was positively correlated with HBV DNA levels, and longitudinal analysis demonstrated a high frequency of this subset at 12 weeks of antiviral treatment predicted unfavorable outcome in CHB patients. CONCLUSIONS: CTLA4+CD4+CXCR5-FOXP3+ T cells are related to unfavorable outcomes in HBV-infected patients; these data indicated that alleviating CTLA4+CD4+CXCR5-FOXP3+ T cells may improve the prognosis of HBV infection.

Interferon α facilitates anti-HBV cellular immune response in a B cell-dependent manner.

OBJECTIVES: Dissecting the underlying mechanism of T cells remodeling mediated by interferon α (IFN-α) is indispensable for achieving an optimum therapeutic response in chronic hepatitis B (CHB) patients. However, little is known about B cells in this process. This study aims to elucidate the roles of B cells in IFN-α-mediated anti-hepatitis B virus (HBV) cellular immunity. METHOD: The effects of B cells on IFN-α-mediated T cell response were investigated in B cell-deficient mouse model with HBV and IFN-α plasmid hydrodynamic injection. Single-cell RNA sequencing was performed to dissect the crosstalk among B cell and T cell subsets and the underlying molecule and pathway signatures on longitudinal blood samples from IFN-α-treated CHB patients. RESULTS: B cell depletion impaired the functional T cell subsets, including HBV-specific CD8+ T cells, and engendered a delayed HBV clearance. IFN-α treatment boosted the response of HBV-specific CD8+ T cells, whereas such effects disappeared in B cell-deficient mice. The underlying mechanisms were associated with IFN-α-reinforced connections of B cells toward T cells as mediated by the antigen presentation and costimulatory functions in B cells. CONCLUSION: IFN-α orchestrates protective HBV-specific cellular immunity in a B cell-dependent manner.

Hepatocyte-Derived L-Carnitine Restricts Hepatitis B Surface Antigen Loss Through an Immunosuppressive Effect on Germinal Center-Related Immune Cells.

BACKGROUND: The outcome of hepatitis B virus (HBV) infection is significantly affected by host immune response; herein, we aim to dissect the effect of L-carnitine (L-Cn) on germinal center (GC)-related immune cells and the influence on the prognosis of HBV infection. METHODS: In vitro and in vivo experiments were performed in patients with chronic HBV infection and a hydrodynamic injection mouse model. RESULTS: In vitro assays revealed that L-Cn significantly reduced GC-related immune responses and enhanced immunosuppressive profiles. Intriguingly, L-Cn released from lysed hepatocytes was associated with the degree of liver damage. Besides, the administration of L-Cn in an HBV mouse model resulted in delayed clearance of hepatitis B surface antigen (HBsAg) in serum and decreased GC formation in the spleen. Notably, patients with HBsAg loss showed decreased plasma L-Cn levels, and longitudinal observations found that low baseline levels of L-Cn were associated with a favorable treatment response in patients with chronic hepatitis B. CONCLUSIONS: The suppressive effect of hepatocyte-derived L-Cn on GC-related immune cells may contribute to the inability of HBsAg clearance in chronic HBV infection, indicating that L-Cn might serve as a potential therapeutic target for the treatment of HBV infection.

Identification and Mapping of HBsAg Loss-Related B-Cell Linear Epitopes in Chronic HBV Patients by Peptide Array.

Identification of immunogenic targets against hepatitis B virus (HBV)-encoded proteins will provide crucial advances in developing potential antibody therapies. In this study, 63 treatment-naïve patients with chronic HBV infection and 46 patients who achieved hepatitis B surface antigen loss (sAg loss) following antiviral treatment were recruited. Moreover, six patients who transitioned from the hepatitis B e antigen-positive chronic infection phase (eAg+CInf) to the hepatitis phase (eAg+CHep) were enrolled from real-life clinical practice. Additionally, telbivudine-treated eAg+CHep patients and relapsers or responders from an off-treatment cohort were longitudinally studied. The frequencies and function of B cells were assessed by flow cytometry. We devised a peptide array composed of 15-mer overlapping peptides of HBV-encoded surface (S), core (C), and polymerase (P) proteins and performed a screening on B-cell linear epitopes with sera. Naïve B cells and plasmablasts were increased, whereas total memory, activated memory (AM), and atypical memory (AtM) B cells were reduced in sAg- patients compared with sAg+ patients. Importantly, longitudinal observations found that AtM B cells were associated with successful treatment withdrawal. Interestingly, we identified six S-specific dominant epitopes (S33, S34, S45, S76, S78, and S89) and one C-specific dominant epitope (C37) that reacted with the majority of sera from sAg- patients. Of note, more B-cell linear epitopes were detected in CHep patients with alanine aminotransferase (ALT) flares than in nonflare CInf patients, and five B-cell linear epitopes (S4, S5, S10, S11, and S68) were overwhelmingly recognized by ALT flare patients. The recognition rates of epitopes on C and P proteins were significantly increased in CHep patients relative to CInf patients. Strikingly, a statistically significant elevation in the number of positive epitopes was observed when ALT nonflare patients shifted into the flare phase. Moreover, S76 identified at baseline was confirmed to be associated with a complete response after 48 weeks of telbivudine therapy. Taken together, we identified several functional cure-related B-cell linear epitopes of chronic HBV infection, and these epitopes may serve as vaccine candidates to elicit neutralizing antibodies to treat HBV infection.

Identification of the association between HBcAg-specific T cell and viral control in chronic HBV infection using a cultured ELISPOT assay.

Hepatitis B virus (HBV)-specific T cells play a critical role in determining the outcome of HBV infection. However, T cell response induced by predominant Ag in chronic infection is hardly detectable owing to the lack of a suitable assay. We herein established an optimized method to enumerate HBV-specific T cells and assessed the association between HBV surface Ag (HBsAg) and HBV DNA. Sixty chronic HBV infection patients were enrolled. HBV-specific T cells were expanded by using overlapping peptide pools covering the entire sequence of HBV genotypes B and C. IFN-γ-producing HBV-specific T cells were detected by a cultured enzyme-linked immunospot (ELISPOT) assay, ex vivo ELISPOT assay, or flow cytometry staining. The association between HBV-specific T cells and serum levels of HBsAg and HBV DNA were analyzed. Cultured ELISPOT assay had a higher sensitivity than ex vivo ELISPOT in the detection of HBV-specific T cells. Moreover, consistent results were acquired by flow cytometry analysis and cultured ELISPOT assay, but the latter required only a limited number of cells for detection. Interestingly, HBV core peptide pool induced a robust HBV-specific T cell response in patients with lower levels of HBV DNA and HBsAg. Specifically, the frequency of HBV core Ag-specific IFN-γ+ spot-forming cells was inversely correlated with serum levels of HBV DNA and HBsAg. An optimized cultured ELISPOT assay reveals the association between HBV core Ag-induced T cell response and HBV control; this method may favor the investigation of HBV-specific T cell in chronic HBV infection.

Expanded circulating follicular dendritic cells facilitate immune responses in chronic HBV infection.

BACKGROUND: The restoration of host hepatitis B virus (HBV)-specific antiviral immunity is an effective strategy for hepatitis B recovery. Follicular dendritic cells (FDCs) play a crucial role in immune regulation. The goal of the present study was to investigate the characteristics and functions of FDCs in chronic HBV infection. METHODS: The frequencies of FDCs in peripheral blood, liver, and spleen were measured in patients with chronic HBV infection. Isolated FDCs from splenic tissues of HBV-related liver cirrhosis-induced hypersplenism patients were cultured with autologous intrasplenic CD4+ T cells and CD19+ B cells. RESULTS: We observed that patients with chronic HBV infection had a significantly increased frequency of circulating FDCs compared to that of healthy controls. Additionally, the frequency of circulating FDCs was positively correlated with that of intrahepatic and intrasplenic counterparts. Moreover, positive correlations were observed between the frequencies of circulating FDCs and plasmablast and memory B cells, as well as C-X-C motif chemokine receptor type 5 (CXCR5)+CD4+ T cells and CXCR5+CD8+ T cells. Notably, in vitro experimental results demonstrated that FDCs derived from splenic tissues of chronic HBV patients facilitated interferon-γ and interleukin-21 production from autologous intrasplenic CD4+ T cells and promoted the proliferation of autologous intrasplenic CD19+ B cells. CONCLUSIONS: Expanded FDCs in patients with chronic HBV infection may favor host immune responses against HBV. The identification of this unique population of cell may contribute to a better understanding of the immune regulatory mechanisms associated with chronic HBV infection and provide a potential immunotherapeutic target for this disease.

IL-21 receptor signaling is essential for control of hepatocellular carcinoma growth and immunological memory for tumor challenge.

Hepatocellular carcinoma (HCC) is a typical inflammation-associated cancer. IL-21 regulates both innate and adaptive immune responses and has key roles in antitumor and antiviral responses. However, the role of IL-21 in HCC development is poorly defined. In the current study, we explored the role of IL-21R signaling in HCC growth by using IL-21R knockout mice and HCC mouse models. We discovered that IL-21R signaling deficiency promoted HCC growth in tumor-bearing mice. We showed that IL-21R deletion reduced T cells infiltration and activation as well as their function but increased the accumulation of myeloid-derived suppressor cells in tumor tissues to enhance HCC growth. Furthermore, loss of IL-21R signaling in tumor-bearing mice resulted in an imbalance of the systemic immune system characterized by decreased antitumor immune cells and increased immunosuppressive cells in the spleen and lymph nodes. In addition, we revealed that IL-21R signaling is critical for the expansion of antitumor immune cells in the memory immune response to tumor rechallenge. Finally, we showed that the transcriptional levels of IL-21 in the peritumoral region and IL-21R within the tumor are associated with survival and recurrence of HCC patients. In conclusion, our study demonstrates that IL-21R signaling is essential for controlling the development of HCC and immunological memory response to tumor challenge.