Germinal Center Dark Zone harbors ATR-dependent determinants of T-cell exclusion that are also identified in aggressive lymphoma – Visium_FFPE_Spatial_Trasciptomics_project

The germinal center (GC) dark zone (DZ) and light zone (LZ) regions spatially separate expansion and diversification from selection of antigen-specific B-cells to ensure antibody affinity maturation and B cell memory. How GC polarity is established is poorly understood. This study provides novel insights into signals controlling asymmetric T-cell distribution between DZ and LZ regions. We identify spatially-resolved DNA damage response and chromatin compaction molecular features precluding DZ T-cell infiltration. The DZ spatial transcriptional signature linked to T-cell immune evasion clustered aggressive Diffuse Large B-cell Lymphomas (DLBCL) for differential T cell infiltration. We reveal the dependence of the DZ transcriptional core signature on the ATR kinase and dissect its role in restraining inflammatory responses contributing to estabslish an immune-repulsive imprint in DLBCL. These insights may guide ATR-focused treatment strategies bolstering immunotherapy in tumors marked by DZ transcriptional and chromatin-associated features. Overall design: Whole-genome transcriptomic profiling was performed on formalin-fixed tissue sections of mouse FFPE mesenteric lymph nodes from two samples: one with a knocked Aicda gene (B6.129P2-Aicdatm1(cre)Mnz/J, Jackson, RRID no 007770) and the other under wild-type conditions for Aicda gene (B6N;129-Ilktm1Star/J, Jackson, RRID no 023310). grant ID : *22145* grant title: *Linking genetic evolution, stromal and immune contextures to unravel tumor heterogeneity in aggressive B-cell lymphomas* name of the funding source:* A.I.R.C. (Associazione Italiana Ricerca Cancro) *

生发中心（germinal center, GC）暗区（dark zone, DZ）与亮区（light zone, LZ）通过空间分隔，将抗原特异性B细胞的扩增、多样化过程与筛选过程相分离，以保障抗体亲和力成熟与B细胞记忆形成。目前学界对生发中心极性的建立机制尚缺乏深入认知。本研究为调控DZ与LZ区域间T细胞不对称分布的信号通路提供了全新见解。我们鉴定出可阻断DZ T细胞浸润的空间分辨DNA损伤应答与染色质压缩分子特征。与T细胞免疫逃逸相关的DZ空间转录特征可对侵袭性弥漫大B细胞淋巴瘤（diffuse large B-cell lymphoma, DLBCL）进行聚类分型，以区分其T细胞浸润程度的差异。本研究揭示了DZ转录核心特征对ATR激酶（ATR kinase）的依赖性，并解析了其在抑制炎症应答、进而在DLBCL中形成免疫排斥印记过程中的作用机制。上述研究成果可为以ATR为靶点的治疗策略提供指导，助力在携带DZ转录及染色质相关特征的肿瘤中增强免疫治疗效果。

整体实验设计：针对两种样本的小鼠福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）肠系膜淋巴结组织切片开展全基因组转录组分析：一组为Aicda基因敲除样本（B6.129P2-Aicdatm1(cre)Mnz/J，杰克逊实验室，RRID编号007770），另一组为Aicda基因野生型对照样本（B6N;129-Ilktm1Star/J，杰克逊实验室，RRID编号023310）。

资助信息：
资助编号：*22145*
资助标题：*将遗传进化、基质与免疫微环境关联以解析侵袭性B细胞淋巴瘤的肿瘤异质性*
资助方名称：*意大利癌症研究协会（Associazione Italiana Ricerca Cancro, A.I.R.C.）*