Atheroprotective Vaccination with MHC-II Restricted Peptides from ApoB-100

Abstract

Background: Subsets of CD4(+) T-cells have been proposed to serve differential roles in the development of atherosclerosis. Some T-cell types are atherogenic (T-helper type 1), while others are thought to be protective (regulatory T-cells). Lineage commitment toward one type of helper T-cell versus another is strongly influenced by the inflammatory context in which antigens are recognized. Immunization of atherosclerosis-prone mice with low-density lipoprotein (LDL) or its oxidized derivative (ox-LDL) is known to be atheroprotective. However, the antigen specificity of the T-cells induced by vaccination and the mechanism of protection are not known.

Methods: Identification of two peptide fragments (ApoB3501-3516 and ApoB978-993) from murine ApoB-100 was facilitated using I-Ab prediction models, and their binding to I-Ab determined. Utilizing a vaccination scheme based on complete and incomplete Freund's adjuvant (CFA and IFA) [1 × CFA + 4 × IFA], we immunized Apoe(-/-)mice with ApoB3501-3516 or ApoB978-993 emulsified in CFA once and subsequently boosted in IFA four times over 15 weeks. Spleens, lymph nodes, and aortas were harvested and evaluated by flow cytometry and real time RT-PCR. Total atherosclerotic plaque burden was determined by aortic pinning and by aortic root histology.

Results: Mice immunized with ApoB3501-3516 or ApoB978-993 demonstrated 40% reduction in overall plaque burden when compared to adjuvant-only control mice. Aortic root frozen sections from ApoB3501-3516 immunized mice showed a >60% reduction in aortic sinus plaque development. Aortas from both ApoB3501-3516 and ApoB978-993 immunized mice contained significantly more mRNA for IL-10. Both antigen-specific IgG1 and IgG2c titers were elevated in ApoB3501-3516 or ApoB978-993 immunized mice, suggesting helper T-cell immune activity after immunization.

Conclusion: Our data show that MHC Class II restricted ApoB-100 peptides can be atheroprotective, potentially through a mechanism involving elevated IL-10.

Keywords: T-cell; atherosclerosis; inflammation; vaccination.

Figure 1

Atherosclerosis is decreased in ApoB_3501–3516 and ApoB_978–993-treated mice compared to controls. (A) Vaccination schedule: 8-week-old female Apoe^−/− mice were immunized once with either PBS or peptide in CFA, then boosted four more times with PBS or peptide in IFA. WD was maintained for 13 weeks. Mice were sacrificed and organs harvested at 23 weeks of age. (B,C) Results of aortic pinning analysis after Sudan IV staining are shown with representative photographs. N = 12–15 in each group, *p < 0.05 when compared to 1× CFA + 4× IFA group. (D) Representative aortic root staining sections after ORO staining, counter-stained with hematoxylin. (E) Plaque area from aortic roots stained from each group. Lesion sizes from 30 to 40 μm distal to start of the aortic valve were averaged per group. N = 5 in each group, *p < 0.05 when compared to 1× CFA + 1× IFA control group.

Figure 2

Specific antibody titers after immunization. Sera from 9 to 10 animals per group were pooled and formal antibody dilution curves were measured by chemiluminescent ELISA against each of the antigens. Shown here are comparative binding data at serum dilutions of 1:250 and in each case, values shown are the fold increase compared to values found with pooled sera from 1× CFA + 4× IFA group. (A) IgG1 titers against ApoB_3501–3516 and ApoB_978–993. (B) IgG2c titers against ApoB_3501–3516 and ApoB_978–993. (C) IgG1 and IgG2c titers against MOG35–55 in MOG_35–55 immunized mice. IgG1 and IgG2c titers against (D) native (unmodified) LDL and (E) MDA (oxidized)-LDL.

Figure 3

FoxP3⁺ T-cells are not found in increased numbers in ApoB_3501–3516 or ApoB_978–993 immunized mice. (A) The lymphocyte population was captured via forward scatter (FSC) and side scatter (SSC) gating as shown in Region 1 (R1). Live CD45⁺ cells were isolated (R2), of which TCRβ⁺/CD4⁺ cells were selected (R3) and examined for their expression of FoxP3⁺. Analysis of a representative aorta is shown. (B) Percentage of aortic FoxP3⁺ cells within the CD4⁺/TCRβ⁺ population. (C) Percentage of para-aortic lymph node FoxP3⁺ cells within the CD4⁺/TCRβ⁺ population. (D) Percentage of splenic FoxP3⁺ cells within the CD4⁺/TCRβ⁺ population. (E) Percentage of non-draining lymph nodes (pooled from inguinal, axillary, mesenteric) FoxP3⁺ cells within the CD4⁺/TCRβ⁺ population (N = 4–5 in each group).

Figure 4

Real time RT-PCR analysis of IL-10 mRNA expression. Organs were harvested and immediately placed into RNA stabilization reagent, and frozen at −80^°C. IL-10 mRNA expression from (A) aortas, (B) para-aortic lymph nodes, (C) spleens, and (D) non-draining lymph nodes are shown. *p < 0.05 compared to 1× CFA + 4× IFA. N = 3–5 in each group.