Clin Res Cardiol (2022).

Development of an assay to detect ApoB-specific CD4+ T cells in human blood samples
S. Hansen1, H. Horstmann1, A.-S. Burkard1, M. C. Gissler1, T. Marchini1, C. Bode1, D. Wolf1
1Klinik für Kardiologie und Angiologie I, Universitäts-Herzzentrum Freiburg - Bad Krozingen GmbH, Freiburg im Breisgau;

Atherosclerosis is a chronic inflammatory disease of arteries that is accompanied by a strong adaptive immune response. Previous findings of our group show that human atherosclerotic lesions are dominated by CD4+  T helper cells and suggest that antigen-specific CD4+ T-helper cells recognizing Apolipoprotein B (ApoB), the core protein of low-density lipoprotein (LDL) play a key role in its development.  Here, we developed a novel in vitro cell assay that allows to detect circulating ApoB-specific CD4+ T cells in human blood samples.


Methods and results: 
To detect ApoB-specific CD4+ T-cells, we designed a restimulation assay with peripheral mononuclear blood cells (PBMCs) isolated from human blood samples and a pool of 30 ApoB-derived peptides selected in an in-silico assay. We defined reactive T-cells after a co-incubation for 6 hours by the expression of CD40L (CD154), a co-stimulatory molecule and established activation marker of T-cells. To circumvent transient expression of CD40L, we co-incubated PBMCs with fluorochrome labeled anti-CD40L during restimulation. In a clinical cohort with 100 patients that underwent coronary angiography, we observed that background-subtracted percentages suggest that 1,28 +/- 1,68% of CD4+ T cells were ApoB-specific. We observed no regulation of the fraction of ApoB-specific T cells in patients without or with coronary disease but observed an upregulation in females and a positive correlation with LDL-plasma levels and BMI (r=0,3, p=0,004 and r=0,16, p=0,13). 


A novel restimulation assay allows for the first time to detect the population of ApoB-specific CD4+ T-cells in human PBMCs. This tool will be instrumental to define the dynamics of the autoimmune response against ApoB/LDL in clinical atherosclerosis.