Spatiotemporal dynamics of the lymph adaptive immune repertoire in response to viral infection. Spatiotemporal dynamics of the lymph adaptive immune repertoire in response to viral infection

The spatial organization of adaptive immune cells within lymph nodes is critical for understanding immune responses during infection and disease. Here, we investigated the spatial and temporal changes in the adaptive immune repertoire (AIR) in the draining lymph node after footpad infection with Vaccinia virus (VACV) in mice. To achieve this, we integrated high-resolution spatial transcriptomics with high-fidelity long-read adaptive immune receptor sequencing of T and B cell receptors. This approach enables simultaneous analysis of whole transcriptomes and AIR in their spatial context. We mapped the spatial distribution and temporal dynamics of immune phenotypes and AIRs at 3, 7, 10, 14, and 21 days post-infection. Our analysis revealed heterogeneous activation niches characterized by Interferon-gamma (IFN-γ) during the early stages of infection. We observed sub-anatomical structures within the germinal center (GC) and provided direct evidence that antibody-producing plasma cells differentiate and exit the GC from the dark zone. Furthermore, we traced the spatial lineage trajectory of B cell clones and found evidence of B cell maturation across multiple GCs. Overall, this method offers valuable insights into the spatial dynamics of immune responses within lymphoid organs, with relevance for spatiotemporal studies of the immune system response to an infectious agent. Overall design: Six popliteal lymph nodes were collected from 6 individual mice with different conditions including one mock sample as control. We performed spatial transcriptomics on these lymph nodes by using the Curio Seeker platform and followed their protocol to get the complimentary cDNA library from it. Each cDNA library was split into short read (SR) for unbiased spatial trancriptomics or long read (LR) for TCR/BCR transcripts enrichment. So in total we have 12 samples library.

淋巴结内适应性免疫细胞的空间组织形式，对于解析感染与疾病进程中的免疫应答具有关键意义。本研究针对小鼠足垫感染痘苗病毒（Vaccinia virus, VACV）后，其引流淋巴结内适应性免疫组库（adaptive immune repertoire, AIR）的时空变化展开探究。
为此，我们将高分辨率空间转录组学（spatial transcriptomics）技术，与针对T细胞受体和B细胞受体的高保真长读长适应性免疫受体测序技术相结合。该方法可在空间背景下同步分析完整转录组与适应性免疫组库。我们绘制了感染后3、7、10、14及21天，免疫表型与适应性免疫组库的空间分布及时间动态图谱。
我们的分析揭示，感染早期存在以γ干扰素（Interferon-gamma, IFN-γ）为特征的异质性激活生态位。我们在生发中心（germinal center, GC）内观测到亚解剖结构，并提供直接证据表明，抗体产生型浆细胞可从暗区分化并迁出该生发中心。此外，我们追踪了B细胞克隆的空间谱系轨迹，并发现多个生发中心内存在B细胞成熟的相关证据。
综上，本方法为解析淋巴器官内免疫应答的空间动态特征提供了重要见解，可为免疫系统针对病原体的应答时空研究提供参考。
整体实验设计：从6只接受不同处理的小鼠体内采集6个腘淋巴结，其中包含1个模拟感染样本作为空白对照。我们使用Curio Seeker平台对这些淋巴结开展空间转录组学实验，并遵循其官方实验流程制备互补DNA（complementary DNA, cDNA）文库。将每份cDNA文库分为两部分：一部分用于开展无偏倚空间转录组学的短读长（short read, SR）测序，另一部分用于富集T细胞受体/B细胞受体（T cell receptor/B cell receptor, TCR/BCR）转录本的长读长（long read, LR）测序。最终共计获得12个样本文库。