Th2 single-cell heterogeneity and clonal interorgan distribution in helminth-infected mice

Th2 cells provide effector functions in type 2 immune responses to helminths and allergens. Despite substantial knowledge about molecular mechanisms of Th2 cell differentiation, there is little information on Th2 cell heterogeneity and clonal distribution between organs mainly due to technical limitations. To address this issue, we performed combined single-cell transcriptome and TCR clonotype analysis on murine Th2 cells in mesenteric lymph nodes (MLN) and lung after infection with Nippostrongylus brasiliensis (Nb) as a model of human hookworm infection. We identified strong organ-specific expression profiles, but also found populations with conserved effector or migration signatures. A substantial MLN subpopulation with an interferon response signature suggests a role for interferon-signaling in Th2 cell differentiation or diversification. RNA-inferred developmental directions further implied proliferation as a hub for differentiation decisions. Although the TCR repertoire appeared to be highly heterogeneous, we identified expanded Th2 clones and CDR3 motifs. Clonal relatedness between distant organs confirmed the effective exchange of Th2 effector cells. However, locally expanded clones dominated the response, as the most expanded clones in MLN and lung did not overlap. This new insight in Th2 cell subsets and clonal relatedness in distant organs demonstrates their heterogeneity and suggests that they serve distinct effector functions. Overall design: We performed combined transcriptome and TCR clonotype analysis using the chromium 10xGenomics and Illumina single cell sequencing platform with Th2 cells isolated from lung and MLN of two IL-4eGFP reporter (4get) mice (Mohrs et al. Immunity 2001) that had been infected 10 days before with Nb. Th2 cells (CD4+IL-4eGFP+) were sorted from single cell suspensions of both organs and were directly subjected to scRNA library preparation. Additionally, we compared the transcriptional profile of the lung draining mediastinal LN and MLN in a second single cell sequencing run.

Th2细胞（Th2 cells）可在针对蠕虫与变应原的2型免疫应答中发挥效应功能。尽管目前对于Th2细胞分化的分子机制已有较为充分的认知，但受限于技术瓶颈，学界关于Th2细胞异质性及其在不同器官间的克隆分布特征的相关信息仍十分匮乏。为解决这一问题，本研究以巴西日圆线虫（Nippostrongylus brasiliensis, Nb）——一种模拟人体钩虫感染的模型——感染小鼠后，对其肠系膜淋巴结（mesenteric lymph nodes, MLN）与肺部的Th2细胞开展了单细胞转录组与T细胞受体（TCR）克隆型联合分析。本研究不仅鉴定出显著的器官特异性表达谱，同时也发现了携带保守效应或迁移特征的细胞亚群。肠系膜淋巴结中存在大量携带干扰素应答特征的细胞亚群，这提示干扰素信号通路在Th2细胞分化与功能多样化中发挥了一定作用。基于转录组数据推断的发育轨迹进一步表明，细胞增殖是Th2细胞分化决策的核心调控枢纽。尽管T细胞受体库（TCR repertoire）呈现出高度异质性，本研究仍鉴定出了扩增的Th2细胞克隆与互补决定区3（CDR3）基序。不同远端器官间的克隆相关性分析证实了Th2效应细胞可在体内进行有效迁移交换。但局部扩增的克隆主导了免疫应答，肠系膜淋巴结与肺部的优势扩增克隆并无重叠。本研究关于Th2细胞亚群及远端器官克隆相关性的新发现，揭示了Th2细胞的异质性特征，并提示其可发挥不同的效应功能。

整体实验设计：本研究使用10x Genomics Chromium与Illumina单细胞测序平台，对两只感染巴西日圆线虫10天的IL-4eGFP报告基因小鼠（4get小鼠，Mohrs等人于2001年发表于《Immunity》期刊）的肺部与肠系膜淋巴结中分离的Th2细胞开展转录组与T细胞受体克隆型联合分析。Th2细胞（CD4+IL-4eGFP+）从两种器官的单细胞悬液中分选得到，并直接用于单细胞RNA测序文库构建。此外，本研究在第二轮单细胞测序中还比较了肺引流纵隔淋巴结与肠系膜淋巴结的转录谱。