Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02087-y

Investigating the Role of Adaptive Immunity in Calcific Aortic Valve Disease via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat

M. R. Christopher1, B. Alushi2, L. Curini3, A. Lauten4

1CC 11: Med. Klinik für Kardiologie, Charité - Universitätsmedizin Berlin, Berlin; 2Interventionelle Kardiologie, Klinik Vincentinum, Augsburg; 3Department of Experimental and Clinical Medicine, University of Florence, Firenze, IT; 4Allgemeine und Interventionelle Kardiologie und Rhythmologie, Helios-Klinikum Erfurt, Erfurt;

Aortic stenosis is the most frequent valve disease requiring surgery, with rising prevalence due to the aging population. Calcific aortic valve disease (CAVD) impacts 2.8% of adults over 75 years and poses high burden on healthcare, due to the high associated mortality. Effective treatment of severe aortic stenosis remains limited to surgical or transcatheter aortic valve replacement. The pathogenesis of CAVD is multifaceted, with key factors involving chronic inflammation, deposition of lipids and mineralization of the aortic valve interstitial cells. Calcification has been previously viewed as a passive, age-related phenomenon, however, recent evidence suggests that it is an active process involving both the innate and adaptive immunity in the pathways that begin with inflammation and lead to subsequent stenosis. However, the underlying mechanisms remain unclear. With longitudinal study of disease progression studies being limited due to difficulty obtaining tissue samples for histopathological analysis, it is essential to explore alternative ways to study CAVD.  The assaying of T cells from fresh valve tissue may be an efficient way to clarify the role of T cells in the development of calcification, as it provides a close representation of the aortic valve cells in vivo. Here we present a protocol for the isolation of T cells from fresh samples of calcified aortic valves and the use of fluorescence activated cell sorting (FACS) to analyze T cell clones, as a means to explore the role of adaptive immunity in CAVD pathophysiology. We show how the assaying of T cells from fresh valve tissue may be an effective way to reveal the role of T cells in the development of calcification. In this two-centre study, stenotic aortic valve samples from CAVD patients undergoing valve replacement surgery were collected and underwent our extraction protocol. The high level of leukocytes in the valve tissue, in addition to the strong presence of CD4+ cells implicated in propagating chronic inflammation, as well the presence of CD8+ cells, support our hypothesis of the adaptive immunity implication in CAVD. Based on our findings we speculate that after activation, regulatory T cells become dysfunctional, with a Th1- initiated response driving inflammation and secretion of pro-calcific factors. The valve cells then acquire a phenotype similar to osteoblasts and calcific nodules form in the aortic valve. While further investigation may be required, our methodology can be used to capture changes in T cell concentrations and assess activation states of specific T-cell subtypes. Unveiling the mechanisms and role of immune cells in valve disease may aid in the development of non-invasive preventative treatments and novel pharmaceutical targets.

https://dgk.org/kongress_programme/ht2022/aP245.html