3A; 16.0 ± 2.1% versus 10.4 ± 0.1%, P < 0.05). In order to study the specificity of CD8+ cytotoxic T cells, spleen cells from vaccinated and control mice were co-cultured with murine fibroblasts that were co-transfected with pcDNA3.1-IL-15 and pcDNA3.1-GFP. The number of surviving IL-15 expressing target cells was determined by counting GFP positive cells. The number of IL-15 expressing target cells was reduced by 50% after incubation with spleen cells from IL-15 vaccinated mice, whereas spleen cells from control vaccinated mice, did not significantly lyse IL-15 expressing cells ( Fig. 3B; 49 ± 1% in vaccinated group versus GSK1349572 81 ± 4% in control
group, P < 0.01). Atherosclerosis was determined in control and IL-15 vaccinated mice 6 weeks after collar placement. IL-15 vaccination did not affect plasma cholesterol levels during the experiment (Fig. 3C). Quantification of Hematoxylin–Eosin (HE) stained atherosclerotic plaques showed that vaccination AUY-922 mouse against IL-15 resulted in a 75% decrease in lesion size as compared to the control group (Fig. 4A–C; 13722 ± 3116 μm2 versus 53977 ± 15332 μm2, P < 0.05). Immunohistochemical
staining for macrophages showed a significant change in plaque composition ( Fig. 4F). The relative number of macrophages per plaque area was 2-fold higher in IL-15 vaccinated mice ( Fig. 4E) than that in control vaccinated mice ( Fig. 4D), indicative for a less advanced state of the lesions in the vaccinated mice. As hypercholesterolemia
induced surface expression of IL-15 on PBMCs and spleen cells (Fig. 1B) we evaluated the effect of IL-15 vaccination on the percentage of IL-15 positive cells within the spleen and PBMCs. Spleen cells and PBMCs were stained for IL-15 and for the macrophage marker F4/80 and analyzed by FACS. Upon IL-15 vaccination, the surface expression 3-mercaptopyruvate sulfurtransferase of IL-15 on spleen cells was almost completely reduced to a level comparable to that determined in mice before the start of the Western-type diet (Fig. 5A, P < 0.05). Within the PBMC population IL-15 surface expression was also decreased ( Fig. 5A, P < 0.05). Within the macrophage population we observed an almost 70% reduction in the percentage of IL-15 positive macrophages ( Fig. 5B, P < 0.01), while the CD4/CD8 ratio in blood, indicative of the inflammatoruy status of the mice, was 3-fold lower in the IL-15 vaccinated mice ( Fig. 5, P < 0.01). Atherosclerosis is considered a dyslipidemia-induced chronic inflammatory disease of the arterial wall. During atherosclerotic lesion formation, monocytes and subsequently T cells infiltrate the arterial wall . DNA vaccination against IL-15 leads in LDLr−/− mice to a blocked atherosclerotic lesion development, indicating that IL-15 accelerates lesion formation. Upon the start of a hypercholesterolemic diet in LDLr−/− mice the mRNA expression of IL-15 is increased within the spleen.