Background: Carotid atherosclerosis is a chronic inflammatory disorder and is responsible for the vast majority of ischemic strokes. Inappropriate innate and adaptive immune responses synergize with malfunctional vascular wall cells to cause atherosclerotic lesions. Yet, functional characteristics of specific immune and endothelial cell subsets associated with atherosclerosis and cerebrovascular events are poorly understood. Methods: Here, using single-cell RNA sequencing, the unprecedentedly largest data set from 20 patients' carotid artery plaques and paired peripheral blood mononuclear cells was generated, with which an ultra-high-precision cellular landscape of the atherosclerotic microenvironment involving 372 070 cells was depicted. Results: Compared with peripheral blood mononuclear cells, 3 plaque-specific T-cell subsets exhibiting proatherogenic features of both activation and exhaustion were identified. Strikingly, usually antiatherogenic, CD4+FOXP3+ regulatory T cells from plaques of patients with symptomatic disease acquired proinflammatory properties by probably converting to T helper 17 and T helper 9 cells, while CD4+NR4A1+/C0 and CD8+SLC4A10+ T cells related to cerebrovascular events possessed atherogenic attributes including proinflammation, polarization, and exhaustion. In addition, monocyte-macrophage dynamics dominated innate immune response. Two plaque-specific monocyte subsets performed diametrically opposed functions, EREG+ monocytes promoted cerebrovascular events while C3+ monocytes are anti-inflammatory. Similarly, IGF1+ and HS3ST2+ macrophages with classical proinflammatory M1 macrophage features were annotated and contributed to cerebrovascular events. Moreover, SULF1+ (sulfatase-1) endothelial cells were also found to participate in cerebrovascular events through affecting plaque vulnerability. Conclusions: This compendium of single-cell transcriptome data provides valuable insights into the cellular heterogeneity of the atherosclerotic microenvironment and the development of more precise cardiovascular immunotherapies. Keywords: carotid artery diseases; endothelial cells; immunotherapy; macrophages; monocytes.

本周为大家带来的文献为发表Cancer Discov. (IF: 29.7)的” An Inflammatory Checkpoint Generated by IL1RN Splicing Offers Therapeutic Opportunity for KRAS-Mutant Intrahepatic Cholangiocarcinoma”。本文使用了LabEx提供的Luminex检测服务。

 

近年来肝内胆管细胞癌（iCCA）作为全球主要癌症问题的现状及相关研究进展。尽管免疫检查点抑制剂（ICIs）和肿瘤微环境（TME）相关的治疗策略在多种实体癌症中取得了突破性进展，但在iCCA中的初步临床结果令人失望，即使ICIs联合化疗在不可切除的iCCA患者中带来了适度的生存益处。为了开发新的治疗策略，亟需深入了解iCCA的发病机制。

 

高通量测序分析扩展了对iCCA分子特征的认识，研究表明KRAS突变是iCCA的主要驱动因素，并且这种突变在炎症亚型中显著富集。炎症能加速肿瘤进展并严重削弱免疫疗法的效果。相反，以前的研究发现白细胞介素-1受体拮抗剂（IL1RN）是一种内源性拮抗剂，通过阻断白细胞介素-1（IL-1）途径来抑制炎症级联反应。然而，KRAS突变如何正面或负面影响iCCA中的炎症和抗肿瘤免疫的分子机制仍不清楚。

 

值得注意的是，越来越多的研究支持替代mRNA剪接（AS）在炎症过程中的关键作用。AS是一个重要的转录后调控过程，通过选择性剪接和重新组装RNA前体来生成多样化的RNA转录本。然而，目前尚未有大规模的iCCA转录组学研究或对KRAS突变相关炎症的深入探索。

 

LabEx提供的Luminex检测服务

除单片段分析外，本实验在 PDTF 细胞培养的上清液中收集了 48 小时后的样品，用于研究细胞因子、趋化因子和细胞毒性介质。这些上清液被立即冷冻并储存在 -80°C。解冻并混合后的上清液使用 LabEX 试剂盒测定其中适当的细胞因子和趋化因子。此外，使用 ELISA 试剂盒（MultiSciences Biotech Co. Ltd.）测定组织上清液中的 CXCL3 和 GZMB 水平，操作按照制造商的说明进行。

  

与接受抗PD-1单药治疗的PDTF相比，接受anakinra加抗PD-1 Ab联合治疗的6名KRAS突变患者的PDTF显示出明显较高的CXCL9、CXCL10和GZMB水平，同时CXCL3等免疫抑制因子也有所下降(图F)。

 

 

重要发现

研究者利用从大量患者中收集的多组学数据，发现 KRAS 突变与肝内胆管癌(iCCA)中与髓系炎症相关的特定 mRNA 替代剪接景观有关。然后，我们发现了一种负反馈机制，在这种机制中，替代剪接导致的白细胞介素1受体拮抗剂(IL1RN)-201/203的上调在KRAS突变的iCCA中具有重要的抗炎作用。在KRAS突变型iCCA小鼠中，IL1RN-201/203的上调和anakinra治疗都能通过改变中性粒细胞的募集和表型来激发显著的抗肿瘤免疫反应。此外，anakinra治疗还能协同增强抗PD-1疗法，激活KRAS突变iCCA小鼠瘤内的GZMB+ CD8+ T细胞。在临床上，我们发现KRAS突变型iCCA患者体内高水平的IL1RN-201/203与抗PD-1免疫疗法的良好反应显著相关。