Abstract 150: A Population of ApoB-100 Peptide-specific CD8+ T cells Tracks Atherosclerosis in ApoE-/- Mice

Abstract

Background: Growing evidence supports a potential role for CD8+ T cells in atherogenesis, yet CD8+T cell antigens relevant to the disease remain to be defined. The current study was undertaken to identify and characterize endogenous, apoB-100 antigen-specific CD8+ T cells in atherosclerosis using fluorescently tagged, synthetic soluble MHC-I/peptide complexes called Pentamers (Pent). Methods and Results: Self-reactive CD8+ T cells were tested for recall response to a 20-mer peptide derived from apoB-100 under investigation as a potential vaccine candidate. CD8+IFN-γ+ T cells in splenocytes of apoE-/- mice were significantly increased after 48 hours of peptide stimulation, which was enhanced by a 6-week high fat diet. We then tested the apoB-100 peptide for potential binding to mouse MHC-I alleles H2Db and H2Kb. Binding assays identified 13 sequential fragments of 8-mer lengths with potential H2Kb binding. Pents corresponding to the 13 peptide fragments were then synthesized and were screened for reactivity to apoB-100-specific CD8+ T cells from 13 week-old wild type and apoE-/- mice fed normal chow (NC). Flow cytometric analysis detected higher binding to CD8+ T cells from apoE-/- mice for 3 Pents. The first of the 3, called Pent 2, was then selected as prototype for further analysis. The OVA Pentamer SIINFEKL confirmed Pent 2 specificity. Pent 2+CD8+ T cells were significantly increased in 13 week-old apoE-/- mice fed a high fat diet (HC) for 6 weeks compared to NC fed mice (0.99±0.15% vs. 0.51±0.02%, respectively, N=5 each; P<0.05). Pent 2+CD8+ T cells correlated with plaque size of apoE-/- mice fed NC or HC for 6 weeks (R 2 =0.645, P=0.005, N=10) or for 15-22 weeks (R 2 =0.623, P=0.001, N=13). Pent 2 blocked CD8+ cytolytic activity against oxLDL-primed macrophages by 50% (N=6; P<0.05). There was a shift in the Pent 2+CD8+ Vβ repertoire after HC feeding for 6 weeks compared to NC, with significantly increased Pent 2+CD8+ Effector Memory cells (65.0±5.7% vs. 48.8±6.0%, respectively; P<0.01). Flow cytometry of digested aortic sinus plaques confirmed presence of Pent 2+CD8+ T cells in atherosclerotic plaques. Conclusion: Our study provides evidence of a self-reactive, apoB-100-specific CD8+ T cell population that tracks atherosclerotic disease in apoE-/- mice.