Prolonged T–DC macro-clustering in lymph nodes initiates site-specific Th2 differentiation

T helper 2 (Th2) responses protect against pathogens while also driving allergic inflammation, yet how large-scale Th2 responses are generated in tissue context remains unclear. Here, we used quantitative imaging to investigate early Th2 differentiation within lymph nodes (LNs) following cutaneous allergen administration. Contrary to current models, we observed extensive activation and 'macro-clustering' of early Th2 cells with migratory type-2 dendritic cells (cDC2s) generating specialized Th2-promoting microenvironments. Macro-clustering was integrin mediated and promoted localized cytokine exchange among T cells to reinforce differentiation, which contrasted behavior during Th1 responses. Unexpectedly, formation of Th2 macro-clusters was dependent on the site of skin sensitization. Differences between sites were driven by divergent activation states of migratory cDC2 from different dermal tissues, with enhanced costimulatory molecule expression by cDC2 in Th2-generating LNs promoting prolonged T cell activation, macro-clustering, and cytokine sensing. Thus, generation of dedicated Th2 priming microenvironments through enhanced costimulatory molecule signaling initiates Th2 responses in vivo and occurs in a skin site-specific manner. Overall design: To investigate transcriptional differences in dendritic cells between auricular (Au) and brachial (Br) draining lymph nodes (LNs), we immunized mice in the ear or forepaw with papain and EaGFP. LN tissues were harvested 48 hours later and mechanically disrupted and subject to digestion in PBS with 10% fetal bovine serum (FBS) with DNase I (100µg/mL; Sigma), Dispase II (800µg/mL; Sigma), and Collagenase P (200µg/mL; Sigma) at 37°C shaking at 150rpm for 30 minutes with periodic manual disruption. We then stained and FACS sorted cells from three populations of EaGFP+ MHC-II-hi CD11c-int antigen bearing cDC2 (CD301b+CD11b+, CD301b-CD11b+, and CD301b-CD11b-) from Au or Br draining LNs 48 hours post-immunization. We also sorted non-antigen bearing DCs of the same populations from naive mice. Cells were sorted from pooled dLNs from 3 individual mice for each group. 500 of each cDC2 cell type was sorted on an Aria III (BD Biosciences) directly into reaction buffer from the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara), and reverse transcription was performed followed by PCR amplification to generate full length amplified cDNA.

辅助性T细胞2型（Th2）应答可抵御病原体感染，同时也会介导过敏性炎症，但在组织微环境中大规模Th2应答的产生机制仍不明确。本研究借助定量成像技术，探究了皮肤过敏原致敏后，引流淋巴结（LNs）内早期Th2细胞的分化过程。与现有模型相悖的是，我们观察到早期Th2细胞与迁移性2型树突状细胞（cDC2s）发生广泛活化并形成“宏观聚集簇”，进而构建出特异性促进Th2分化的微环境。该聚集过程由整合素（integrin）介导，可促进T细胞间局部细胞因子（cytokine）交换以强化分化，这与Th1型应答中的细胞行为截然不同。出乎意料的是，Th2宏观聚集簇的形成依赖于皮肤致敏部位。不同部位的差异源于不同真皮组织来源的迁移性cDC2活化状态存在分歧：在产生Th2应答的引流淋巴结中，cDC2的共刺激分子（costimulatory molecules）表达水平更高，可延长T细胞活化时间、促进宏观聚集并增强细胞因子感知能力。综上，通过增强共刺激分子信号以构建特异性Th2致敏微环境，可在体内启动Th2应答，且该过程具有皮肤部位特异性。

实验设计：为探究耳廓（Au）与臂部（Br）引流淋巴结（LNs）内树突状细胞的转录差异，我们分别在小鼠耳部或前爪注射木瓜蛋白酶（papain）与EaGFP进行免疫。免疫48小时后采集淋巴结组织，经机械解离后，于含10%胎牛血清（FBS）、脱氧核糖核酸酶I（DNase I，100μg/mL；Sigma）、中性蛋白酶II（Dispase II，800μg/mL；Sigma）及胶原酶P（Collagenase P，200μg/mL；Sigma）的磷酸盐缓冲液（PBS）中，于37℃、150rpm振荡孵育30分钟，并辅以手动间断解离。随后，对免疫后48小时的耳廓或臂部引流淋巴结中，三类EaGFP+、主要组织相容性复合体II类高表达（MHC-IIhi）、CD11c中等表达的抗原呈递性cDC2（CD301b+CD11b+、CD301b-CD11b+及CD301b-CD11b-）进行荧光激活细胞分选（FACS）。同时，我们还分选了未免疫小鼠体内相同群体的非抗原呈递性树突状细胞。每组实验均取自3只小鼠的混合引流淋巴结细胞。使用Aria III细胞分选仪（BD Biosciences）将每种cDC2群体的500个细胞直接分选至SMART-Seq v4超低起始量RNA测序试剂盒（SMART-Seq v4 Ultra Low Input RNA Kit，Takara）的反应缓冲液中，随后进行逆转录与聚合酶链式反应（PCR）扩增，以获得全长扩增互补脱氧核糖核酸（cDNA）。