T-cell toelrance in atherosclerosis

Atherosclerotic plaques form in the inner layer of arteries triggering heart attacks and strokes. Although T-cells have been detected in atherosclerosis, tolerance dysfunction as a disease driver remains unexplored. Here, we examined tolerance checkpoints in atherosclerotic plaques, artery tertiary lymphoid organs, and lymph nodes in mice burdened by advanced atherosclerosis, via single-cell RNA sequencing paired with single T-cell receptor sequencing. In this study, ApoE-/- mice on a C57BL/6 background and C57BL/6 WT mice were housed in the specific pathogen-free animal facilities of Munich University with a 12 h light/dark cycle, air-conditioned (23° C and 60% relative humidity). All mice were 78-85 weeks old and had been maintained on a standard rodent chow. Animal procedures were approved by the Regierung of Oberbayern according to the guidelines of the local Animal Use and Care Committee and the National Animal Welfare Laws. Mice were euthanized by ketamine hydrochloride and xylazine hydrochloride. Blood was collected by cardiac puncture. Perfusion was performed from the left ventricle with 10 ml 5 mM EDTA buffer, 20 ml PBS and 20 ml FACS buffer, respectively. RLNs were collected under a dissecting microscope. To collect plaques and ATLOs, adipose tissue and paraaortic LNs were carefully removed. The whole aorta was dissected and collected in cell culture dishes with pre-cooled FACS buffer. The aorta was opened in the longitudinal direction, the plaque tissues were carefully removed using curved forceps (Dumont #5/45, Fine Science Tools) under the dissecting microscope. The remaining aorta was collected as ATLOs. RLNs, ATLOs and plaques. Three separated cohorts of mice were used: (i) pools of 3 ApoE-/- and 3 WT mice were used to collect plaques, ATLOs, and RLNs, and pools of 5 ApoE-/- and 5 WT mice were used to collect blood in the first cohort; (ii) Cell hashtags (hashtags, Biolegend, TotalSeq™ C) were used to label 4 ApoE-/- and 4 WT mice individually to collect ATLOs and RLNs; (iii) Pools of 5 ApoE-/- mice to collect plaque tissues. Single cell suspensions are prepared and stained with fluorescent antibodies for single live leukocyte sorting Next, appropriate single cell suspension are loaded onto 10x Genomics chip to generate gel bead in emulsions.

动脉粥样硬化斑块（atherosclerotic plaques）形成于动脉内膜，可诱发心肌梗死与脑卒中。尽管已有研究在动脉粥样硬化病变中检测到T细胞（T-cell），但作为疾病驱动因素的免疫耐受功能异常仍未得到充分探索。本研究针对罹患晚期动脉粥样硬化的小鼠，结合单细胞RNA测序（single-cell RNA sequencing）与单细胞T细胞受体测序（single T-cell receptor sequencing），对动脉粥样硬化斑块、动脉三级淋巴器官及淋巴结中的免疫耐受检查点进行了系统分析。本研究使用C57BL/6背景的ApoE-/-小鼠与野生型（WT）C57BL/6小鼠，饲养于慕尼黑大学无特定病原体（specific pathogen-free, SPF）动物实验设施中，饲养环境为12小时光暗循环、温度23℃、相对湿度60%。所有小鼠均为78~85周龄，全程饲喂标准啮齿类饲料。动物实验方案经上巴伐利亚行政区政府批准，符合当地动物使用与护理委员会指南及国家动物福利法相关规定。小鼠通过盐酸氯胺酮与盐酸赛拉嗪实施安乐死，经心脏穿刺采集血液；随后经左心室依次灌注10ml 5mM EDTA缓冲液、20ml磷酸盐缓冲液（PBS）与20ml流式细胞术缓冲液（FACS buffer）。在体视显微镜下采集区域淋巴结（RLNs）；剥离脂肪组织与主动脉旁淋巴结以分离目标组织：完整剥离主动脉并收集于预冷的流式细胞术缓冲液培养皿中，沿纵轴切开主动脉，在体视显微镜下使用弯形镊子（Dumont #5/45，Fine Science Tools）小心分离斑块组织，剩余主动脉组织则作为动脉三级淋巴器官（ATLOs）进行收集。本研究分为三个独立的小鼠队列：① 第一队列：将3只ApoE-/-小鼠与3只野生型（WT）小鼠混合，用于采集斑块、ATLOs与RLNs，另取5只ApoE-/-小鼠与5只野生型（WT）小鼠混合以采集血液；② 第二队列：使用细胞标签（Cell hashtags，Biolegend，TotalSeq™ C）分别标记4只ApoE-/-小鼠与4只野生型（WT）小鼠，用于采集ATLOs与RLNs；③ 第三队列：取5只ApoE-/-小鼠混合以采集斑块组织。随后制备单细胞悬液，使用荧光标记抗体染色以分选存活的白细胞群体；将合格的单细胞悬液加载至10x Genomics芯片中，以制备凝胶微珠乳剂（gel bead in emulsions）。