Rationale:
Atherosclerosis is a chronic inflammatory disease that involves auto-antibodies and autoreactive CD4+ T helper cells that recognize Apolipoprotein B (ApoB), the core protein of low-density lipoprotein (LDL) cholesterol. ApoB is a known auto-antigen in atherosclerosis, but whether ApoB-specific autoreactive T cells exist in humans is not well established. Here, we established a novel in vitro restimulation assay to detect and characterize circulating ApoB specific CD4+ in human peripheral blood. 

Methods and results:
Peripheral blood mononuclear cells (PBMCs) were isolated from arterial blood samples from patients at high risk for atherosclerosis during coronary angiography. ApoB-reactive CD4+ T cells specifically recognize ApoB-peptides bound on MHC-ll of antigen presenting cells (APCs) by the T cell receptor (TCR). We selected a pool of 30 ApoB-peptides with a high MHC-II binding affinity towards a number of highly frequent MHC-ll alleles in a Caucasian population. PBMCs containing CD4+ T cells and APCs were co-incubated with the pool of ApoB-peptides for 6 hours. We defined ApoB-reactive T cells as cells expressing CD40L (CD154), a co-stimulatory molecule and established activation marker of T-cells. To circumvent transient expression of CD40L, we co-incubated the cell suspension with anti-CD40L to avoid internalization of CD40L. We used different peptide-pools as controls, including MHC-II binding peptides from bacteria and viruses, Actin-peptides and CD8+ MHC-I peptides as negative controls. Background-adjusted CD40L-expression indicated that approximately 0.8% of circulating CD4+ T cells in human peripheral blood were reactive to ApoB in a cohort of 250 patients. Central-memory T cells and effector-memory T cells were more frequent among CD40L+ T cells than among non-reactive T cells. Notably, we did not detect an association of ApoB-specific T cell frequencies with clinical disease status, age, gender, or lipoproteins. The frequency among healthy patients and those affected by coronary artery disease (CAD) was comparable. 

Conclusions:
A novel in vitro restimulation assay enabled the detection of ApoB specific T cells in a clinical cohort with 250 subjects. The number of ApoB specific T cells did not correlate with clinically apparent atherosclerosis suggesting a relative importance not of frequency but of the immunophenotype. Here, we link cardiovascular risk factors and clinical patient profiles to ApoB specific T-cells. This translational approach will allow to characterize cellular autoimmunity in a clinical setting.