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=10 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

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease. Although its pathogenesis is still poorly understood, recent evidence suggests that AAA displays autoimmune disease characteristics. Particularly, T cells responding to AAA-related antigens in the aortic wall may contribute to an initial immune response. Single-cell RNA (scRNA) T cell receptor (TCR) and B cell receptor (BCR) sequencing is a powerful tool for investigating 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 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 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.