The meninges host a unique compartment of regulatory T cells that bulwarks adult hippocampal neurogenesis [scRNA-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. We investigated the transcriptional heterogeneity of meningeal CD4+ T cells via single-cell RNA and Tcr sequencing (scRNA-seq/ scTcr-seq), and investigate the changes in the hippocampal niche after punctual Treg cell depletion via diphtheria toxin (DT) injections into Foxp3-DTR+ and littermate Foxp3-DTR- controls (scRNA-seq) .For standard genome-wide scRNA-seq analyses, non neuronal non microglia (Live CD45- Thy1-) were sorted by flow cytometry and encapsulated using the Chromium Single Cell 3' v2 platform. For coupled scRNA-seq and scTcr-seq, CD4+ T cells from meninges of Foxp3Gfp mice and b6 wildtype mice were sorted and encapsulated using the Chromium Single Cell 5' v3 and V(D)J platform (10x Genomics).我们关于脑膜免疫系统的认知近年来蓬勃发展，尤其是先天免疫与适应性免疫效应细胞如何响应脑部刺激而被动员。然而，关于脑膜免疫细胞在健康个体中如何维持脑部稳态的相关信息仍较为匮乏。 本研究聚焦于小鼠脑膜中具有异质性且功能多样的调节性T（regulatory T, Treg）细胞区室。两类特征鲜明的Treg亚群分别专司调控Th1细胞应答与B细胞滤泡内免疫应答，构成了该细胞区室的核心组成部分，这也预示了后续的关键发现：精准清除Treg细胞会快速触发脑膜淋巴细胞分泌干扰素-γ，使其得以侵入脑实质，并改变脑膜B细胞的表型谱。 在远端脑部区域，海马体呈现反应性状态，多种胶质细胞类型被激活；在齿状回中，神经干细胞的死亡进程加剧且停止进一步分化，这与空间参考记忆的受损密切相关。 综上，脑膜Treg细胞是稳态条件下维持脑部稳态的多维度防御屏障。 本研究通过单细胞RNA测序与T细胞受体测序（single-cell RNA and T cell receptor sequencing, scRNA-seq/scTcr-seq）分析了脑膜CD4+ T细胞的转录异质性，并通过向Foxp3-DTR+小鼠及其同窝Foxp3-DTR-对照小鼠注射白喉毒素（diphtheria toxin, DT）实现精准Treg细胞清除，随后借助单细胞RNA测序分析了海马微环境的变化。 针对标准全基因组单细胞RNA测序分析，研究人员通过流式细胞术（flow cytometry）分选得到非神经元、非小胶质细胞的活细胞（Live CD45⁻ Thy1⁻），并使用10x Genomics的Chromium单细胞3' v2平台（Chromium Single Cell 3' v2 platform）进行单细胞封装。 对于联合单细胞RNA测序与T细胞受体测序实验，我们分选了Foxp3GFP小鼠及C57BL/6野生型小鼠脑膜来源的CD4+ T细胞，采用10x Genomics的Chromium单细胞5' v3及V(D)J测序平台（Chromium Single Cell 5' v3 and V(D)J platform）进行封装。