Abstract
Apolipoprotein B (apoB)-100-derived peptides previously used by others in vaccine preparations to treat atherosclerosis were shown to reduce atherosclerotic plaque development by 50% in experimental animals. This was associated with the induction of T helper-2 (Th2) immune responses. This study aimed to characterise some of the innate and T cell-mediated mechanisms underpinning these responses, including the role of regulatory T cells (Tregs). The presence of peptide-specific antibodies in the plasma of well-defined patient groups and its correlation with disease status, gender and race was also studied. Apolipoprotein E deficient (apoE-/-) mice were immunised with apoB-100-derived peptides P2, P45 and P210, either conjugated to BSA or loaded onto bone marrow-derived dendritic cells (DCs). The numbers of spleen-derived CD4+ and CD8+ Tregs in these animals were measured by flow cytometry and enzyme-linked immunosorbent assays (ELISA) was used to assess the synthesis of anti-peptide IgGl and IgG2a, as well as the levels of interleukin (IL)-10 and interferon gamma (IFN-y) in the plasma of immunised animals. Immunisation with peptides and peptide-loaded DCs resulted in significantly increased levels of peptide-specific immunoglobulins and IL-10. Our experiments also showed that Tregs induced by treatment with apoB-100-derived peptides were able to reduce mixed leukocyte proliferation induced by DCs treated with low density lipoprotein (ox-LDL) and Staphylococcal enterotoxin B (SEB), suggesting a tolerogenic mechanism. We also showed that these peptides can also modulate the homing properties of DCs, can decrease the expression of Toll like receptors (TLRs) and can increase the numbers of Tregs in vivo. Depletion of CD4+CD25+ Tregs with PC61, an anti-CD25 monoclonal antibody, showed an increase in the synthesis of interferon gamma (IFN-γ), a decrease in IL-10 secretion and an increase in the levels of IL-4 consistent with a pro-inflammatory effect. The treatment was also accompanied by an increase in cholesterol and triglyceride plasma levels and a decrease in HDL. PC61-treated mice also showed larger atherosclerotic plaques demonstrating the athero-protective effect of Tregs. The study using patient samples revealed strong correlation between plasma levels of anti-P45 and anti-P210 and CVD. East Indian-descendents were found to have higher levels of anti-P45, while levels of anti-P210 were higher in African descendents. The presence of anti-P210 antibodies was significantly higher in all age groups. No significant differences in anti-peptide antibody levels were found between gender groups. The findings support the hypothesis that epitope-specific immune responses are characteristic of specific disease status and may be linked to genetic differences in the population. Overall, the study provides evidence for the induction of antigen-specific T cell-mediated immuno-suppressive effect by the apoB-100-derived peptides and also highlights the role of Tregs in athero-protection. In addition, the study highlights that potential differences of the immune response elicited by these peptides in vivo may be explained by genetic differences in the population. These features could be used to propose the use of these peptides as biomarkers to study and understand differences in susceptibility to CVD in different population groups.