The meninges host a unique compartment of regulatory T cells that bulwarks adult hippocampal neurogenesis [RNA-Seq]

Our knowledge about the meningeal immune system has recently burgeoned, particularly how innate and adaptive effector cells are mobilized in response to brain challenges. However, information on how meningeal immunocytes guard brain homeostasis in healthy individuals remains sparse. This study highlights the heterogeneous and polyfunctional regulatory T (Treg) cell compartment in mouse meninges. A Treg subtype specialized in controlling Th1-cell responses and another subtype devoted to controlling responses in B-cell follicles were substantial components of this compartment, foretelling the finding that punctual Treg-cell ablation rapidly unleashed interferon-gamma production by meningeal lymphocytes, unlocked their access to the brain parenchyma, and altered meningeal B-cell profiles. Distally, the hippocampus took on a reactive state, with activation of multiple glial-cell types; within the dentate gyrus, neural stem cells showed exacerbated death and desisted from further differentiation, associated with inhibition of spatial-reference memory. Thus, meningeal Treg cells are a multifaceted bulwark to brain homeostasis at steady-state. For characterizing meningeal Treg cell identity and changes upon physiological variations (sex, age), and anatomical location we double sorted Treg cells from the meninges, spleen and skull bone marrow at different ages. To study how Tregs regulate the meningeal niche, Tregs were punctually depleted in two different ways: via diphtheria toxin (DT) injections into Foxp3-DTR+ and littermate Foxp3-DTR- controls, or via intra-cisterna magna injection of Ultra-LEAF purified mouse anti-CD25 mAb (BioLegend, clone PC61) or the recommended rat IgG1lambda isotype control mAb (BioLegend); then the meninges were digested in QIAzol for RNA extraction, and followed whole-tissue RNA-seq. performed. To study how microglia responded upon punctual Treg depletion (DTR- and DTR+ littermates), we double sorted microglia from the hippocampus. To understand how different brain regions responded upon punctual Treg depletion, we isolated the hippocampus, cortex, thalamus and hypothalamus, and cerebellum, digested using QIAzol for RNA extraction, and followed whole-tissue RNA-seq.近年来，我们对脑膜免疫系统的认知快速拓展，尤其聚焦于固有免疫与适应性免疫效应细胞如何响应脑部应激而被动员激活。然而，针对健康个体中脑膜免疫细胞如何维持脑部稳态的相关研究仍较为匮乏。本研究聚焦小鼠脑膜中异质性且兼具多效性功能的调节性T细胞（regulatory T cell, Treg）亚群库，其中两类亚型构成了该亚群库的核心组分：一类专门调控Th1细胞免疫应答，另一类负责调控B细胞滤泡内的免疫应答。该发现预示：精准清除Treg细胞可快速触发脑膜淋巴细胞分泌干扰素-γ，解除其对脑实质的浸润限制，并改变脑膜B细胞的表型特征。从远端效应来看，海马体呈现反应性激活状态，多种胶质细胞类型被活化；在齿状回区域，神经干细胞死亡加剧且停止进一步分化，这与空间参考记忆功能受损密切相关。综上，稳态条件下脑膜Treg细胞是维持脑部稳态的多维度屏障。为解析脑膜Treg细胞的身份特征及其在生理变量（性别、年龄）与解剖位置影响下的表达变化，本研究对不同年龄小鼠的脑膜、脾脏及颅骨骨髓中的Treg细胞进行了双重分选。为探究Treg细胞如何调控脑膜微环境，本研究采用两种不同方式精准清除Treg细胞：一是向Foxp3-DTR阳性小鼠及其同窝阴性对照小鼠注射白喉毒素（DT）；二是通过小脑延髓池注射Ultra-LEAF纯化小鼠抗CD25单克隆抗体（BioLegend，克隆号PC61）或推荐的大鼠IgG1λ同型对照单克隆抗体（BioLegend）。随后使用QIAzol裂解脑膜组织以提取RNA，并完成全组织RNA测序。为探究精准清除Treg细胞后小胶质细胞的应答反应（以DTR阳性与同窝阴性小鼠为模型），本研究从海马体中双重分选出小胶质细胞。为明确不同脑区在精准清除Treg细胞后的应答情况，本研究分离了海马体、大脑皮层、丘脑、下丘脑及小脑，使用QIAzol裂解组织以提取RNA，并完成全组织RNA测序。