M Leuner(1,2,3), A Lübken(4), A Fähnrich(5), J von Esebeck(2), M Spielmann(1), I Eitel(3,6), OJ Müller(2,3), M Meusel(2,3), H Langer(7,8), H Nording(2,3)

1. Institute of Human Genetics, Universitätsklinikum Schleswig-Holstein, University of Lübeck and University of Kiel, 23562, Lübeck, Germany; 2. Department of Internal Medicine V (Cardiology and Angiology), University of Kiel, and German Centre for Cardiovascular Research, Partner Site Hamburg/Kiel/Lübeck, Kiel, 24105, Germany; 3. DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Lübeck/Kiel, Lübeck, 23562, Germany; 4. Cardioimmunology Group, Medical Clinic II, University Heart Center Lübeck, Lübeck, 23538, Germany; 5. Institute of Experimental Dermatology, University of Luebeck, Lübeck, 23538, Germany; 6. University Heart Center Lübeck, Medical Clinic II (Cardiology/Angiology/Intensive Care Medicine), University Hospital Schleswig-Holstein, Lübeck, 23538, Germany; 7. Department of Cardiology, Angiology, Haemostaseology, and Medical Intensive Care, University Medical Centre Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, 68167, Germany; 8. German Centre for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim, Germany.

The vascular system can respond to atherosclerotic occlusion by forming bypass circuits (collaterals). Clinical studies demonstrate that the degree of collateralization varies greatly between individuals. In asymptomatic patients with peripheral artery disease (PAD), vascular adaptations are so advanced that, despite significant arterial occlusion, no ischemic pain or tissue damage occur.

To elucidate immune mechanisms involved in collateral formation, a cohort of symptomatic and asymptomatic PAD patients was collected. In the LUERPAD (Luebeck Registry of Peripheral Artery Disease)-IMMUNO study, we analysed the cellular transcriptomes of 16 patients using single-cell RNA sequencing. A novel lymphocyte subtype patients was identified, specific to asymptomatic patients. It is characterized by striking differential expression of GABBR2 and by surface proteins involved in endothelial adhesion.

To further investigate this population and uncover differences on the protein level, we performed full-spectrum flow cytometry using a custom-designed 43-colour panel. AI-assisted clustering and annotation strengthened the hypothesis of a “GABBR2”-Lymphocyte, which allowed us to uncover the identity of GABBR2 lymphocytes differentially regulated between asymptomatic versus symptomatic patients to be NK cells. As validation, we developed a FACS sorting approach. T cells, B cells, NK cells, and monocytes were isolated, followed by RNA extraction and digital PCR. Interestingly, for asymptomatic patients NK cells display the highest GABBR2-expression.

Finally, in vitro experiments showed that PBMCs stimulated with GABBR2 agonist release proangiogenic agents.

Our findings indicate that asymptomatic PAD patients may possess a unique immune profile favouring collateral vessel formation. Proangiogenic GABBR2 NK cells may be key contributors to this unique immunologic signature.