T-Cells Specific for a Self-Peptide of ApoB-100 Exacerbate Aortic Atheroma in Murine Atherosclerosis

Abstract

On the basis of mouse I-A^b-binding motifs, two sequences of the murine apolipoprotein B-100 (mApoB-100), mApoB-100_3501-3515 (designated P3) and mApoB-100_978-992 (designated P6), were found to be immunogenic. In this report, we show that P6 is also atherogenic. Immunization of Apoe^-/- mice fed a high-fat diet (HFD) with P6 resulted in enhanced development of aortic atheroma as compared to control mice immunized with an irrelevant peptide MOG_35-55 or with complete Freund's adjuvant alone. Adoptive transfer of lymph node cells from P6-immunized donor mice to recipients fed an HFD caused exacerbated aortic atheromas, correlating P6-primed cells with disease development. Finally, P6-specific T cell clones were generated and adoptive transfer of T cell clones into recipients fed an HFD led to significant increase in aortic plaque coverage when compared to control animals receiving a MOG_35-55-specific T cell line. Recipient mice not fed an HFD, however, did not exhibit such enhancement, indicating that an inflammatory environment facilitated the atherogenic activity of P6-specific T cells. That P6 is identical to or cross-reacts with a naturally processed peptide of ApoB-100 is evidenced by the ability of P6 to stimulate the proliferation of T cells in the lymph node of mice primed by full-length human ApoB-100. By identifying an atherogenic T cell epitope of ApoB-100 and establishing specific T cell clones, our studies open up new and hitherto unavailable avenues to study the nature of atherogenic T cells and their functions in the atherosclerotic disease process.

Keywords: T-cell; adoptive transfer; atherosclerosis; clones; epitopes; peptides.

Figure 1

Titration curves of T cell proliferative responses to peptide3 (ApoB-100p3501–3516) and peptide6 (ApoB-100p978–993). C57BL/6 or Apoe^−/− mice were immunized with P3 or P6. Ten days later, draining lymph nodes were isolated and teased into single cell suspension. A total of 2 × 10⁵ lymph node cells were cultured with no or titrating concentrations of their respective priming peptides for 5 days. Sixteen hours before the termination of the cultures, 1 μCi of tritiated thymidine was added to each well. Cultures were harvested in an automatic cell harvester, and the incorporation of ³H was counted in a liquid scintillation counter.

Figure 2

Experimental scheme for immunization of ApoE^−/− mice with apolipoprotein B-100 peptides.

Figure 3

Immunization with P6 enhanced development of atherosclerosis in Western diet-fed Apoe^−/− mice. Apoe^−/− mice were fed a high-fat diet for 5 weeks. Groups of mice were then immunized with P3, P6, MOG_35–55, or saline emulsified with complete Freund’s adjuvant by procedures described in Table 1. Two weeks later, mice were given a boost with 100 μg of the same priming peptide subcutaneously at the back. Three weeks later, aortas were isolated according to the procedures developed in the Eitzman Lab (29). After staining with Oil Red O, the aorta was pinned on wax. Pinned aortic tissue was imaged, and the surface area occupied by atherosclerotic plaques was quantified at the aortic arch and major branches with Image-Pro Plus software. The lesion area was expressed as a percentage of total surface area examined. (A) Representative aortas from each group. (B) Dot plot of data.

Figure 4

High-fat diet feeding was necessary for enhanced atherosclerotic lesions. Apoe^−/− mice were fed a high-fat diet or regular mouse chow. Five weeks later, the two groups of mice received 5 × 10⁷ lymph node cells from donor Apoe^−/− mice immunized with either P6 or MOG_35–55 peptides. The primed donor lymph node cells were cultured with their respective antigens for 5 days before adoptive transfer. The HFD-P6 and HFD-MOG groups continued to be fed a HFD for another 5 weeks. The chow-P6 and chow-MOG groups were fed regular mouse chow. After 5 weeks, mouse aortas were isolated and stained with Oil Red O, and the areas of atherosclerotic plaques were quantified. The lesion area was expressed as a percentage of total surface area examined.

Figure 5

P6-specific T cell clone adoptively transferred exacerbation of atherosclerotic development T cell clones were derived by the limiting dilution technique (31). Apoe^−/− mice were fed a high-fat diet for 5 weeks. The mice were divided into two groups. A total of 2 × 10⁷ T cell clone specific for P6 or T cell line specific for MOG_35–55 were adoptively transferred into each group. Five weeks later, the mice were analyzed for the extent of plaque lesions via en face staining of the aortic samples with Oil Red O. *p = 0.001.