Figures were created with Biorender

Figures were created with Biorender. Funding Statement This study was supported by the following grants: Fas C- Terminal Tripeptide Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)grant no. Fas C- Terminal Tripeptide analysis, making biological interpretation challenging. Here, we perform a representative single-cell immune repertoire analysis in experimental murine AAA and show a reliable bioinformatic processing pipeline highlighting opportunities and limitations of this approach. Methods We performed scRNA TCR and BCR sequencing of isolated lymphocytes from the infrarenal aorta of male C57BL/6J mice 3, 7, 14, and 28 days after AAA induction via elastase perfusion of the aorta. Sham-operated mice at days 3 and 28 and non-operated mice served as controls. Results Comparison of complementarity-determining region (CDR3) length distribution of 179 B cells and 796 T cells revealed neither differences between AAA and control nor between the disease stages. We found no clonal expansion of B cells in AAA. For T cells, we identified several clones in 11 Fas C- Terminal Tripeptide of 16 AAA samples and one of eight control samples. Immune receptor repertoire comparison indicated that only a few clones were shared between the individual AAA samples. The most frequently used V-genes in the TCR beta chain in AAA were TRBV3, TRBV19, and the splicing variant TRBV12-2?+?TRBV13-2. Conclusion We found no clonal expansion of B cells but evidence for clonal expansion of T cells in elastase-induced AAA in mice. Our findings imply that a more precise characterization of TCR and BCR distribution requires a more extensive number of lymphocytes to prevent undersampling and potentially detect rare clones. Thus, further experiments are necessary to confirm our findings. In summary, this paper examines TCR and BCR sequencing results, identifies limitations and pitfalls, and offers guidance for future studies. Keywords: aortic aneurysm, single-cell sequencing (scRNA-seq), T cell receptor (TCR), B cell receptor (BCR), clonality analysis Introduction An abdominal aortic aneurysm (AAA) is a cardiovascular disease characterized by a permanent dilation of the abdominal aorta greater than 50% or 3?cm. Most AAAs develop in the infrarenal region between the renal veins and the aortic bifurcation (1). The prevalence of AAA is 4%C8% in men older than 60 years and 0.5%C1.5% in women, with the rupture of AAA conferring a high mortality rate (2). AAA is a multifactorial and progressive disease. Genetic factors and inflammation strongly contribute to AAA development (3), Fas C- Terminal Tripeptide and several studies have revealed recently that autoimmunity may contribute to the pathogenesis of AAA (4C8). Inflammation and immune cell recruitment are characteristics of AAA. Accordingly, T and B cells are among the predominant infiltrating immune cells in human AAA tissue (4, 8, 9). The presence of these lymphocytes in AAA tissue was confirmed in several experimental mouse models of AAA including the porcine pancreatic elastase (PPE) perfusion model, which was used for this study (10C14). The PPE model produces infrarenal aortic aneurysms and is considered the experimental mouse model most resembling human AAA, although the aneurysms do not form intraluminal thrombus or rupture (15). Several experimental interventions (e.g., HIF-1 inhibitors, PI3K inhibitors) preventing elastase-induced AAA are associated with decreased numbers of lymphocytes in the aneurysmal tissue (16C18) (Supplementary Table S1), suggesting an important role for lymphocytes in AAA development in this model. Previous reports have implicated both T helper-1 (Th1) and T helper-2 (Th2) cells in various stages of AAA development (19). Th2 cells release inflammatory mediators and cytokines such as interleukins 4, 5, 9, 10, and 13 and Fas ligand that may contribute to the regulation of AAA progression, whereas Th1-derived interferon-gamma and CD40 ligand are associated with macrophage activation, regulation of vascular smooth muscle cell apoptosis, and aortic wall remodeling (2, 19C22). Investigation of the T cell receptor (TCR)/antigen/human leukocyte antigen (HLA) complex revealed evidence that AAA encompasses a specific antigen-driven T cell response (4, 5). Studies discovered the clonal expansion of T cells in AAA Hoxa lesions, linked AAA to specific HLA Class I and Class II types, and identified self- or non-self-antigens that may be associated with AAA (4, 6, 23). The role of B cells in AAA is controversially discussed. B cell-derived immunoglobulins (Ig), such as IgM and IgG, localize in AAA tissue, where they promote inflammation.