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。尽管已在动脉粥样硬化病变中检测到T细胞（T-cell），但免疫耐受功能异常作为疾病驱动因素的相关机制仍未得到深入探索。本研究针对罹患晚期动脉粥样硬化的小鼠，通过单细胞RNA测序（single-cell RNA sequencing）结合单细胞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（artery tertiary lymphoid organs, ATLOs）及引流淋巴结（regional lymph nodes, RLNs）中的免疫耐受检查点展开了系统性分析。本研究使用C57BL/6背景的载脂蛋白E敲除（ApoE-/-）小鼠与野生型（wild-type, WT）C57BL/6小鼠，所有动物均饲养于慕尼黑大学的无特定病原体（specific pathogen-free, SPF）动物实验设施中，饲养环境为12小时光暗循环，环境温度控制在23℃，相对湿度为60%。所有小鼠年龄为78至85周，全程饲喂标准啮齿类颗粒饲料。本研究的动物实验方案已通过上巴伐利亚政府依据当地动物使用与护理委员会指南及国家动物福利法完成审批。小鼠采用盐酸氯胺酮（ketamine hydrochloride）与盐酸甲苯噻嗪（xylazine hydrochloride）实施安乐死。通过心脏穿刺（cardiac puncture）采集血液样本。随后从左心室依次灌注10 mL 5 mM EDTA缓冲液（EDTA buffer）、20 mL磷酸盐缓冲液（phosphate-buffered saline, PBS）及20 mL流式细胞术缓冲液（FACS buffer）。在体视显微镜（dissecting microscope）下采集引流淋巴结（RLNs）。为收集动脉粥样硬化斑块与ATLOs，需小心剥离主动脉周围的脂肪组织与主动脉旁淋巴结。完整剥离主动脉并将其置于预冷的FACS缓冲液的细胞培养皿中。将主动脉沿纵轴剪开，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，剩余的主动脉组织则作为ATLOs进行收集。最终收集的样本包括RLNs、ATLOs与动脉粥样硬化斑块。本研究共使用3组独立的小鼠队列：(i) 第一队列中，将3只ApoE-/-小鼠与3只WT小鼠混合以收集斑块、ATLOs及RLNs，另取5只ApoE-/-小鼠与5只WT小鼠混合以采集血液样本；(ii) 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WT小鼠，以收集ATLOs与RLNs；(iii) 第三队列将5只ApoE-/-小鼠混合以收集斑块组织。制备单细胞悬液后，使用荧光抗体染色以分选存活的白细胞。随后将合格的单细胞悬液加载至10x Genomics芯片中，以制备凝胶珠乳液微滴（gel bead in emulsions）。