CD4+ T cells license Kupffer cells to reverse the CD8+ T cell dysfunction induced by hepatocellular priming

CD4⁺ T cells are believed to be essential for effective CD8⁺ T cell responses against hepatotropic viruses like hepatitis B virus (HBV). However, the specific locations and the cellular and molecular mechanisms through which CD4⁺ T cells help CD8⁺ T cells remain unclear. In this study, we generated HBV-specific CD4⁺ TCR transgenic mice to demonstrate that effector CD4⁺ T cells can prevent or reverse the dysfunction of CD8⁺ T cells caused by hepatocellular priming. This, in turn, enhances CD8⁺ T cell effector function and suppresses viral replication. Notably, CD4⁺ T cell help to CD8+ T cells occurs directly in the liver, independent of secondary lymphoid organs, and requires local antigen recognition but not epitope linkage. While dendritic cells were found to be dispensable, Kupffer cells (KCs) emerged as the critical cellular platform for this effect. Mechanistically, CD4⁺ T cells engage KCs via CD40-CD40L interactions, leading to the production of IL-12 and IL-27. IL-12 promotes further CD4⁺ T cell expansion, whereas IL-27 serves as the key cytokine that restores CD8⁺ T cell functionality. Furthermore, we show that exogenous IL-27 administration can reverse HBV-specific CD8⁺ T cell dysfunction in mice and in chronically infected patients. These findings reveal a novel mechanism of CD4⁺ T cell help to CD8⁺ T cells that occurs outside secondary lymphoid organs, specifically in the liver, and involves antigen-presenting cells other than conventional dendritic cells, offering new insights for immunotherapeutic strategies against chronic HBV infection. We generated an HBV-specific CD4+ TCR transgenic (Tg) mouse model. Wild-type (WT) mice were immunized with the I-Ab-restricted immunodominant Env126-138 peptide from the preS2 region of the HBV genome. After immunization, we isolated splenocytes and restimulated them ex vivo with the Env126-138 peptide. Single-cell sorting and TCR sequencing of IFN- producing CD4+ T cells identified two distinct variable TCR region genes for the alpha and beta chains. These genes were cloned into TCR expression vectors known for efficient TCR gene expression. The vectors were co-injected into fertilized C57BL/6 eggs, generating two transgenic mouse lines expressing the alpha and beta chains of the Env126-138 TCR. These lines were crossbred to produce HBV-specific CD4+ TCR transgenic mice (referred to as Env126 mice).

CD4⁺ T细胞（CD4⁺ T cells）被认为对针对嗜肝病毒（hepatotropic viruses）如乙型肝炎病毒（hepatitis B virus, HBV）的有效CD8⁺ T细胞（CD8⁺ T cells）应答至关重要。然而，CD4⁺ T细胞辅助CD8⁺ T细胞的具体位点、细胞及分子机制迄今仍未阐明。本研究构建了HBV特异性T细胞受体（T cell receptor, TCR）转基因小鼠模型，证实效应性CD4⁺ T细胞（effector CD4⁺ T cells）可预防或逆转肝细胞致敏（hepatocellular priming）引发的CD8⁺ T细胞功能障碍，进而增强CD8⁺ T细胞的效应功能并抑制病毒复制。值得注意的是，CD4⁺ T细胞对CD8⁺ T细胞的辅助作用直接发生于肝脏内，不依赖次级淋巴器官（secondary lymphoid organs），且仅需局部抗原识别而非表位连锁。尽管树突状细胞（dendritic cells, DCs）被证实并非该过程必需，但库普弗细胞（Kupffer cells, KCs）被确定为该效应的关键细胞平台。机制层面，CD4⁺ T细胞通过CD40-CD40L相互作用（CD40-CD40L interactions）与库普弗细胞结合，诱导白细胞介素-12（interleukin-12, IL-12）与白细胞介素-27（interleukin-27, IL-27）的产生：IL-12可促进CD4⁺ T细胞进一步扩增，而IL-27则是恢复CD8⁺ T细胞功能的关键细胞因子。此外，本研究证实外源性IL-27给药可逆转小鼠及慢性HBV感染患者体内的HBV特异性CD8⁺ T细胞功能障碍。本研究揭示了一种全新的CD4⁺ T细胞辅助CD8⁺ T细胞的机制：该过程不依赖次级淋巴器官，特异性发生于肝脏，且涉及传统树突状细胞（conventional dendritic cells）之外的抗原呈递细胞（antigen-presenting cells），为慢性HBV感染的免疫治疗策略提供了全新的研究思路。
本研究构建了HBV特异性T细胞受体（TCR）转基因（transgenic, Tg）小鼠模型：将HBV基因组preS2区域的I-Ab限制性Env126-138免疫显性肽段（immunodominant peptide）免疫野生型（wild-type, WT）小鼠。免疫完成后，分离小鼠脾细胞（splenocytes）并于体外以Env126-138肽段进行再刺激。对分泌干扰素-γ（interferon-γ, IFN-γ）的CD4⁺ T细胞进行单细胞分选（single-cell sorting）及TCR测序（TCR sequencing），鉴定出编码TCRα链与β链的两种不同可变区基因。将上述基因克隆至可高效表达TCR的载体中，随后将该载体共注射至受精的C57BL/6小鼠卵中，获得两条分别表达Env126-138 TCRα链与β链的转基因小鼠品系。将这两个品系杂交，最终得到HBV特异性T细胞受体（TCR）转基因小鼠，命名为Env126小鼠。