Atheroprotective vaccination with MHC-II-restricted ApoB peptides induces peritoneal IL-10-producing CD4 T cells

Abstract

Although immunization with major histocompatibility complex (MHC) class II-restricted apolipoprotein B (ApoB) peptides has been shown to be atheroprotective, the mechanism is unclear. Here, we investigated CD4 T cell populations in immunized atherosclerotic mice. Peptides (16-mers) from mouse ApoB, the core protein of low-density lipoprotein (LDL), were screened for binding to I-A by computer prediction and confirmed by radiolabeled peptide competition. Three new peptides, P101 (FGKQGFFPDSVNKALY, 5.5 nM IC), P102 (TLYALSHAVNSYFDVD, 6.8 nM), and P103 (LYYKEDKTSLSASAAS, 95 nM), were tested in an atherosclerosis model ( mice on Western diet). Immunization with each of the three peptides (1 time in complete Freund's adjuvant subcuntaneously and 4 time in incomplete Freund's adjuvant intraperitoneally) but not with adjuvant alone showed significantly reduced atherosclerotic plaques in the aortic root by serial sections and in the whole aorta by en face staining. There were no differences in body weight, LDL cholesterol, or triglycerides. Peritoneal leukocytes from ApoB peptide-immunized mice, but not control mice, secreted significant amounts of IL-10 (150 pg/ml). Flow cytometry showed that peptide immunization induced IL-10 in 10% of peritoneal CD4 T cells, some of which also expressed chemokine (C-C motif) receptor 5 (CCR5). Vaccination with ApoB peptides expanded peritoneal FoxP3 regulatory CD4 T cells and more than tripled the number of CCR5FoxP3 cells. Similar trends were also seen in the draining mediastinal lymph nodes but not in the nondraining inguinal lymph nodes. We conclude that vaccination with MHC class II-restricted autologous ApoB peptides induces regulatory T cells (Tregs) and IL-10, suggesting a plausible mechanism for atheroprotection. Vaccination against apolipoprotein B (ApoB), the protein of LDL, attracts attention as a novel approach to prevent atherosclerosis. We discovered major histocompatibility complex class II-restricted ApoB peptides, which reduce atherosclerosis and induce IL-10-producing CD4 T cells and chemokine (C-C motif) receptor 5 expression on regulatory T cells, suggesting that immunization with ApoB peptides inhibits atherosclerosis by inducing anti-inflammatory cytokines.

A clinically applicable adjuvant for an atherosclerosis vaccine in mice

“…IL-10 is known to be antiatherogenic [10,11]. Recently, we showed that a similar vaccination scheme using three different MHC-II-restricted ApoB peptides induced robust production of IL-10 in peritoneal CD4 T cells [12]. Similar to other studies [13,14], we also see induction of regulatory T cell (Treg) responses.…”

Effector and Regulatory T Cells Roll at High Shear Stress by Inducible Tether and Sling Formation

“…Cytokine production and transcription factor staining was done as described previously (Kimura et al, 2017). For cytoplasmic cytokine staining, cells were stimulated with cell stimulation cocktail (eBioscience, San Diego, CA) with ionomycin and monensin for 5 hours at 37°C, fixed a nd permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience), and stained with PE/Cy7-IL-17A (eBio17B7), Brilliant Violet™ 605 IFN-γ (XMG1.2) and PE-IL-4 (11B11) for 30 min at RT.…”

Monocyte-Macrophages and T Cells in Atherosclerosis

“…Many preclinical studies have validated the concept of treating atherosclerosis with therapeutic vaccinations that tolerize against antigens that drive proatherogenic T cell responses. Recent progress in this area has included identification of apoB100 peptide epitopes that bind mouse or human class II MHC glycoproteins, and the demonstration that vaccination of mice expressing MHC molecules that can bind the peptide reduces lesion formation (Gistera et al, 2017; Kimura et al, 2017). Evidence is provided suggesting the mechanism of protection is induction of Treg cells(Kimura et al, 2017) or antibodies that block LDL activation of macrophages (Gistera et al., 2017).…”

“…Recent progress in this area has included identification of apoB100 peptide epitopes that bind mouse or human class II MHC glycoproteins, and the demonstration that vaccination of mice expressing MHC molecules that can bind the peptide reduces lesion formation (Gistera et al, 2017; Kimura et al, 2017). Evidence is provided suggesting the mechanism of protection is induction of Treg cells(Kimura et al, 2017) or antibodies that block LDL activation of macrophages (Gistera et al., 2017). Careful assessment of efficacy and adverse effects of these new therapies, as well as future ones that will undoubtedly be conceived as we learn more about the immune mechanisms that drive atherosclerosis, represent important ongoing efforts to complement apoB LP-lowering therapy for the prevention and treatment of atherothrombotic vascular disease.…”