Department of Medicine II, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany.
Department of Medicine II, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Germany; Faculty of Chemistry and Pharmacy, University of Freiburg, Germany.
J Hepatol. 2019 Jun;70(6):1103-1113. doi: 10.1016/j.jhep.2019.02.016. Epub 2019 Feb 28.
BACKGROUND & AIMS: Current antiviral therapies lack the potential to eliminate persistent hepatitis B virus (HBV) infection. HBV-specific T cells are crucial for HBV control and have recently been shown to be protective in patients following discontinuation of antiviral therapy. Thus, T cell-based approaches may greatly improve the therapeutic landscape of HBV infection. We aimed to augment HBV-specific CD4 T cells from chronically infected patients by targeting different immunological pathways.
Expression of various co-stimulatory and inhibitory receptors on HBV- and influenza-specific CD4 T cells was analyzed directly ex vivo by MHC class II-tetramers. Patients infected with HBV genotype D were screened for CD4 T cell responses by IFN-γ ELISpot and intracellular cytokine staining following stimulation with overlapping peptides (OLPs) spanning the HBV-polyprotein. Stimulation with recombinant IL-7, an agonistic OX40-antibody or blockade of PD-L1 was performed in antigen-specific in vitro cultures. Cytokine secretion and expression of transcription factors were analyzed by flow cytometry. Responses targeting influenza, Epstein-Barr virus and tetanus toxoid served as controls.
Tetramer-staining revealed that the IL-7 receptor-alpha (CD127), OX40 and PD-1 constitute possible therapeutic targets as they were all strongly expressed on HBV-specific CD4 T cells ex vivo. The HBV-specific CD4 T cell responses identified by OLP screening targeted predominantly the HBV-polymerase and core proteins. Combined OX40 stimulation and PD-L1 blockade significantly augmented IFN-γ and IL-21 producing HBV-specific CD4 T cells in vitro, suggesting active T helper type 1 cell and follicular T helper cell programs. Indeed, transcription factors T-bet and Bcl6 were strongly expressed in cytokine-producing cells.
Combined OX40 stimulation and PD-L1 blockade augmented secretion of the helper T cell signature cytokines IFN-γ and IL-21, suggesting that immunotherapeutic approaches can improve HBV-specific CD4 T cell responses.
CD4 T cells are important in controlling viral infections but are impaired in the context of chronic hepatitis B virus (HBV) infection. Therapeutic approaches to cure chronic HBV infection are highly likely to require an immune-stimulatory component. This study demonstrates that HBV-specific CD4 T cells can be functionally augmented by combined stimulation of the co-stimulatory molecule OX40 and blockade of the inhibitory PD-1 pathway.
目前的抗病毒疗法缺乏消除持续性乙型肝炎病毒（HBV）感染的潜力。HBV 特异性 T 细胞对于 HBV 的控制至关重要，最近已证明在停止抗病毒治疗后，它们对患者具有保护作用。因此，基于 T 细胞的方法可能会极大地改善 HBV 感染的治疗前景。我们旨在通过靶向不同的免疫途径来增强慢性感染患者的 HBV 特异性 CD4 T 细胞。
通过 MHC Ⅱ四聚体直接在体外分析 HBV 和流感特异性 CD4 T 细胞上各种共刺激和抑制受体的表达。通过 IFN-γ ELISpot 和细胞内细胞因子染色对感染 HBV 基因型 D 的患者进行筛选，以检测与 HBV 多聚蛋白重叠肽（OLPs）刺激后的 CD4 T 细胞反应。在抗原特异性体外培养物中进行重组 IL-7、激动性 OX40 抗体或 PD-L1 阻断刺激。通过流式细胞术分析细胞因子分泌和转录因子的表达。针对流感、EB 病毒和破伤风类毒素的反应作为对照。
四聚体染色显示，IL-7 受体-α（CD127）、OX40 和 PD-1 可能是治疗靶点，因为它们在体外均强烈表达于 HBV 特异性 CD4 T 细胞上。通过 OLP 筛选鉴定的 HBV 特异性 CD4 T 细胞反应主要针对 HBV 聚合酶和核心蛋白。联合 OX40 刺激和 PD-L1 阻断可显著增强体外 HBV 特异性 CD4 T 细胞产生 IFN-γ 和 IL-21，提示存在活跃的辅助性 T 细胞 1 型细胞和滤泡辅助性 T 细胞程序。事实上，转录因子 T-bet 和 Bcl6 在产生细胞因子的细胞中强烈表达。
联合 OX40 刺激和 PD-L1 阻断增强了辅助性 T 细胞特征细胞因子 IFN-γ 和 IL-21 的分泌，表明免疫治疗方法可改善 HBV 特异性 CD4 T 细胞反应。
CD4 T 细胞在控制病毒感染中很重要，但在慢性乙型肝炎病毒（HBV）感染的情况下受到损害。治愈慢性 HBV 感染的治疗方法很可能需要免疫刺激成分。本研究表明，通过联合刺激共刺激分子 OX40 和阻断抑制性 PD-1 途径，可以增强 HBV 特异性 CD4 T 细胞的功能。
