Peyer's patch-involved gut microbiota facilitates anti-HBV immunity in mice.

BACKGROUND: Gut microbiota is crucial for immune homeostasis and is associated with the prognosis of chronic hepatitis B infection. Peyer's patches (PPs), characterized by intestinal mucosa localization, are involved in the gut microbiota-mediated immune response. However, whether and how PPs orchestrate gut microbiota-modulated anti-hepatitis B virus (HBV) response remain elusive. This study aims to elucidate the role of PPs in gut microbiota-mediated anti-HBV adaptive immunity. METHODS: We investigated the effects of gut microbiota and PPs on adaptive immune responses by transcriptomic, phenotypic, and functional analyzes from an HBV mouse model with gut commensal microbiota and PP-depleting interventions. RESULTS: Depletion of gut microbiota impaired systemic adaptive immune responses, resulting in a delayed HBV antigen clearance. Differentially expressed genes analysis of PPs revealed that pathways related to adaptive immune responses were significantly downregulated in gut microbiota-deficient mice. Notably, the depletion of PPs could abolish gut microbiota-boosted intrahepatic HBV-specific T cell response, leading to a higher serum hepatitis B surface antigen level in mice. CONCLUSION: PPs orchestrate gut microbiota-mediated intrahepatic anti-HBV cellular immunity, underlining the significance of remote manipulating the "gut microbiota-PPs" axis for achieving optimum anti-HBV response.

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.

High L-Carnitine Levels Impede Viral Control in Chronic Hepatitis B Virus Infection.

Persistent antigen exposure during chronic hepatitis B infection leads to exhausted immune responses, thus impeding viral control. In recent years, immunometabolism opens new therapeutic possibilities for the modulation of immune responses. Herein, we investigated the immunomodulatory effect of L-carnitine (L-Cn) on immune cells in chronic HBV infection. In this study, 141 treatment-naïve patients with chronic HBV infection, 38 patients who achieved HBsAg loss following antiviral treatment, and 47 patients who suffered from HBV-related HCC from real-life clinical practice were recruited. The plasma L-Cn levels were measured by ELISA. RNA sequencing was conducted to define the transcriptional profiles of peripheral blood mononuclear cells after L-Cn stimulation. In vitro assays were performed to assess the effect of L-Cn on immune cells; the frequencies and function of immune cells were analyzed by flow cytometry. We found that compared with patients with HBsAg loss, patients with HBsAg positivity and patients who suffered from HBV-related HCC had higher levels of L-Cn, and the plasma levels of L-Cn in the HBeAg-positive chronic hepatitis patients who had elevated ALT were significantly higher than that of HBeAg-negative chronic infection and HBsAg loss groups. Moreover, a positive correlation between plasma levels of L-Cn and HBsAg levels was found. Additionally, RNA sequencing analysis demonstrated that L-Cn altered the transcriptional profiles related to immune response. In vitro assays revealed that L-Cn suppressed the proliferation of and IFN-γ production by CD4+ and CD8+ T cells. It also down-regulated the proliferation and IgG production of B cells. Notably, L-Cn enhanced IL-10 secretion from regulatory T cells and up-regulated the expression of inhibitory receptors on T cells. Moreover, a variant in CPT2 (rs1799821) was confirmed to be associated with L-Cn levels as well as complete response in CHB patients following Peg-IFNα antiviral therapy. Taken together, the immunosuppressive properties of L-Cn may hinder the control of HBV in chronic HBV infection, implicating that L-Cn manipulation might influence the prognosis of patients with 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.

Split chimeric antigen receptor-modified T cells targeting glypican-3 suppress hepatocellular carcinoma growth with reduced cytokine release.

BACKGROUND: Human glypican-3 (hGPC3) is a protein highly expressed in hepatocellular carcinoma (HCC) but limited in normal tissues, making it an ideal target for immunotherapy. The adoptive transfer of hGPC3-specific chimeric antigen receptor T (CAR-T) cells for HCC treatment has been conducted in clinical trials. Due to the rigid construction, conventional CAR-T cells have some intrinsic limitations, like uncontrollable overactivation and inducing severe cytokine release syndrome. METHODS: We redesigned the hGPC3-specific CAR by splitting the traditional CAR into two parts. By using coculturing assays and a xenograft mouse model, the in vitro and in vivo cytotoxicity and cytokine release of the split anti-hGPC3 CAR-T cells were evaluated against various HCC cell lines and compared with conventional CAR-T cells. RESULTS: In vitro data demonstrated that split anti-hGPC3 CAR-T cells could recognize and lyse hGPC3+ HepG2 and Huh7 cells in a dose-dependent manner. Impressively, split anti-hGPC3 CAR-T cells produced and released a significantly lower amount of proinflammatory cytokines, including IFN-γ, TNF-α, IL-6, and GM-CSF, than conventional CAR-T cells. When injected into immunodeficient mice inoculated subcutaneously with HepG2 cells, our split anti-hGPC3 CAR-T cells could suppress HCC tumor growth, but released significantly lower levels of cytokines than conventional CAR-T cells. CONCLUSIONS: We describe here for the first time the use of split anti-hGPC3 CAR-T cells to treat HCC; split anti-hGPC3 CAR-T cells could suppress tumor growth and reduce cytokine release, and represent a more versatile and safer alternative to conventional CAR-T cells treatment.

CXCL13-mediated recruitment of intrahepatic CXCR5+CD8+ T cells favors viral control in chronic HBV infection.

BACKGROUND & AIMS: Although CD8+T cell exhaustion hampers viral control during chronic HBV infection, the pool of CD8+T cells is phenotypically and functionally heterogeneous. Therefore, a specific subpopulation of CD8+T cells should be further investigated. This study aims to dissect a subset of CD8+T cells expressing C-X-C motif chemokine receptor 5 (CXCR5) in chronic HBV infection. METHODS: The frequency of CXCR5+CD8+T cells and the levels of C-X-C motif chemokine ligand 13 (CXCL13), a chemokine of CXCR5, were measured in patients with chronic HBV infection. C57BL/6, interleukin (IL)-21 receptor- or B cell-deficient mice were hydrodynamically injected with pAAV-HBV1.2 plasmids. Phenotype and functions of peripheral and intrahepatic CXCR5+ and CXCR5-CD8+T cells were assessed. RESULTS: CXCR5+CD8+T cells were partially exhausted but possessed a stronger antiviral ability than the CXCR5- subset in patients with chronic HBV infection; moreover, CXCR5+CD8+T cells were associated with a favorable treatment response in patients with chronic hepatitis B (CHB). High levels of CXCL13 from patients with CHB facilitated the recruitment of intrahepatic CXCR5+CD8+T cells, and this subpopulation produced high levels of HBV-specific interferon (IFN)-γ and IL-21. Notably, PD1 (programmed death 1) blockade and exogenous IL-21 enhanced the production of IFN-γ. More strikingly, mice injected with CXCR5+CD8+T cells showed remarkably decreased expression of HBsAg. Additionally, an impaired production of HBV-specific IFN-γ from intrahepatic CXCR5+CD8+T cells was observed in IL-21 receptor- or B cell-deficient mice. CONCLUSION: CXCL13 promotes the recruitment of CXCR5+CD8+T cells to the liver, and this subpopulation improves viral control in chronic HBV infection. The identification of this unique subpopulation may contribute to a better understanding of CD8+T cell functions and provide a potential immunotherapeutic target in chronic HBV infection. LAY SUMMARY: Exhaustion of CD8+ T cells is an important factor in the development of chronic hepatitis B virus (HBV) infection. CD8+ T cells expressing the receptor CXCR5 are partially exhausted, but have potent antiviral activity, as they produce high levels of HBV-specific cytokines in chronic HBV infection. Increased expression of CXCL13 within the liver facilitates the recruitment of CXCR5+CD8+T cells and establishes effective immune control of HBV infection.

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.