contig_annotations.zip

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.