Liver-restricted Type I IFN Signature Precedes Liver Damage in Chronic Hepatitis B Patients Stopping Antiviral Therapy.

Immune-mediated liver damage is the driver of disease progression in patients with chronic hepatitis B virus (HBV) infection. Liver damage is an Ag-independent process caused by bystander activation of CD8 T cells and NK cells. How bystander lymphocyte activation is initiated in chronic hepatitis B patients remains unclear. Periods of liver damage, called hepatic flares, occur unpredictably, making early events difficult to capture. To address this obstacle, we longitudinally sampled the liver of chronic hepatitis B patients stopping antiviral therapy and analyzed immune composition and activation using flow cytometry and single-cell RNA sequencing. At 4 wk after stopping therapy, HBV replication rebounded but no liver damage was detectable. There were no changes in cell frequencies at viral rebound. Single-cell RNA sequencing revealed upregulation of IFN-stimulated genes (ISGs) and proinflammatory cytokine migration inhibitory factor (MIF) at viral rebound in patients that go on to develop hepatic flares 6-18 wk after stopping therapy. The type I IFN signature was only detectable within the liver, and neither IFN-α/ß or ISG induction could be detected in the peripheral blood. In vitro experiments confirmed the type I IFN-dependent ISG profile whereas MIF was induced primarily by IL-12. MIF exposure further amplified inflammatory cytokine production by myeloid cells. Our data show that innate immune activation is detectable in the liver before clinically significant liver damage is evident. The combination of type I IFN and enhanced cytokine production upon MIF exposure represent the earliest immunological triggers of lymphocyte bystander activation observed in hepatic flares associated with chronic HBV infection.

IL-2 produced by HBV-specific T cells as a biomarker of viral control and predictor of response to PD-1 therapy across clinical phases of chronic hepatitis B.

BACKGROUND: There are no immunological biomarkers that predict control of chronic hepatitis B (CHB). The lack of immune biomarkers raises concerns for therapies targeting PD-1/PD-L1 because they have the potential for immune-related adverse events. Defining specific immune functions associated with control of HBV replication could identify patients likely to respond to anti-PD-1/PD-L1 therapies and achieve a durable functional cure. METHODS: We enrolled immunotolerant, HBeAg+ immune-active (IA+), HBeAg- immune-active (IA-), inactive carriers, and functionally cured patients to test ex vivo PD-1 blockade on HBV-specific T cell functionality. Peripheral blood mononuclear cells were stimulated with overlapping peptides covering HBV proteins +/-α-PD-1 blockade. Functional T cells were measured using a 2-color FluoroSpot assay for interferon-γ and IL-2. Ex vivo functional restoration was compared to the interferon response capacity assay, which predicts overall survival in cancer patients receiving checkpoint inhibitors. RESULTS: Ex vivo interferon-γ+ responses did not differ across clinical phases. IL-2+ responses were significantly higher in patients with better viral control and preferentially restored with PD-1 blockade. Inactive carrier patients displayed the greatest increase in IL-2 production, which was dominated by CD4 T cell and response to the HBcAg. The interferon response capacity assay significantly correlated with the degree of HBV-specific T cell restoration. CONCLUSIONS: IL-2 production was associated with better HBV control and superior to interferon-γ as a marker of T cell restoration following ex vivo PD-1 blockade. Our study suggests that responsiveness to ex vivo PD-1 blockade, or the interferon response capacity assay, may support stratification for α-PD-1 therapies.

Congenic hematopoietic stem cell transplantation promotes survival of heart allografts in murine models of acute and chronic rejection.

The ability to induce tolerance would be a major advance in the field of solid organ transplantation. Here, we investigated whether autologous (congenic) hematopoietic stem cell transplantation (HSCT) could promote tolerance to heart allografts in mice. In an acute rejection model, fully MHC-mismatched BALB/c hearts were heterotopically transplanted into C57BL/6 (CD45.2) mice. One week later, recipient mice were lethally irradiated and reconstituted with congenic B6 CD45.1 Lin-Sca1+ckit+ cells. Recipient mice received a 14-day course of rapamycin both to prevent rejection and to expand regulatory T cells (Tregs). Heart allografts in both untreated and rapamycin-treated recipients that did not undergo HSCT were rejected within 33 days (median survival time = 8 days for untreated recipients, median survival time = 32 days for rapamycin-treated recipients), whereas allografts in HSCT-treated recipients had a median survival time of 55 days (P < 0.001 vs. both untreated and rapamycin-treated recipients). Enhanced allograft survival following HSCT was associated with increased intragraft Foxp3+ Tregs, reduced intragraft B cells, and reduced serum donor-specific antibodies. In a chronic rejection model, Bm12 hearts were transplanted into C57BL/6 (CD45.2) mice, and congenic HSCT was performed two weeks following heart transplantation. HSCT led to enhanced survival of allografts (median survival time = 70 days vs. median survival time = 28 days in untreated recipients, P < 0.01). Increased allograft survival post-HSCT was associated with prevention of autoantibody development and absence of vasculopathy. These data support the concept that autologous HSCT can promote immune tolerance in the setting of allotransplantation. Further studies to optimize HSCT protocols should be performed before this procedure is adopted clinically.

Optimized ex vivo stimulation identifies multi-functional HBV-specific T cells in a majority of chronic hepatitis B patients.

High antigen burden during chronic hepatitis B (CHB) results in a low frequency HBV-specific T cell response with restricted functionality. However, this observation is based on limited data because low T cell frequencies have hindered effective ex vivo analysis. We adapted the ELISpot assay to overcome this obstacle to measure ex vivo T cell responses in CHB patients. We modified the key variables of cell number and the peptide pulsing method to improve ex vivo detection of HBV-specific T cells. We detected IFN-γ responses in 10/15 vaccinated controls and 20/30 CHB patients, averaging 195 and 84 SFUs/2 × 106 PBMCs respectively. Multi-analyte FluoroSpots improved functional characterization of T cells. We detected IFN-γ responses in all tested vaccinated controls (n = 10) and CHB patients (n = 13). IL-2 responses were detectable in 9/10 controls and 10/13 patients. TNF-α displayed less sensitivity, detectable in only 7/10 controls and 7/13 patients. Antigen-specific analysis demonstrated that IFN-γ responses were dominated by polymerase and core, with weak responses to envelope and X. IL-2 responses were found in 3/5 patients and equally directed towards polymerase and core. While their ex vivo frequency is extremely low, a fraction of HBV-specific T cells are detectable and display multi-functionality ex vivo.

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.