AUTHOR=Elster Christin , Ommer-Bläsius Miriam , Lang Alexander , Vajen Tanja , Pfeiler Susanne , Feige Milena , Yau Pang Tin , Böttenberg Marius , Verheyen Sarah , Lê Quý Khang , Chernigovskaya Maria , Kelm Malte , Winkels Holger , Schmidt Susanne V. , Greiff Victor , Gerdes Norbert 

TITLE=Application and challenges of TCR and BCR sequencing to investigate T- and B-cell clonality in elastase-induced experimental murine abdominal aortic aneurysm

JOURNAL=Frontiers in Cardiovascular Medicine

VOLUME=Volume 10 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/cardiovascular-medicine/articles/10.3389/fcvm.2023.1221620

DOI=10.3389/fcvm.2023.1221620

ISSN=2297-055X

ABSTRACT=Background: Abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. Although its pathogenesis is still poorly understood, recent evidence suggests that AAA displays characteristics of an autoimmune disease. Particularly T cells responding to AAA-related antigens in the aortic wall may contribute to the initial immune response. Single-cell RNA (scRNA) T-and B-cell receptor (TCR and BCR) sequencing is a powerful tool to investigate clonality. However, difficulties such as limited numbers of isolated cells must be considered during implementation and data 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 day 3 and 28 as well as 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 of 16 AAA samples and in 1 of 8 control samples. Immune receptor repertoire comparison indicated that only few clones were shared between the individual AAA samples. The most frequently used Vgenes in the TCR beta chain in AAA were TRBV3, TRBV19, and the splicing variant TRBV12-2+TRBV13-2.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 to 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.