Abstract 19642: Immuno-Peptidome Profiling Identifies Human Cathelicidin hCAP18 as a Potential CD8+ T Cell Auto-Immune Protein Involved in Atherosclerosis
Background: Auto-immunity is believed to contribute to inflammation in atherosclerosis. Mass spectrometry (MS) enables analysis of patient immuno-peptidomes (IPs). We profiled the IP of a patient with known coronary artery disease (CAD) and tested the relevance of disease-related IPs in a mouse model of atherosclerosis.
Methods and Results: HLA-I/peptide complexes were immuno-precipitated from the plasma of a male CAD patient and an age-matched healthy control. HLA-I/peptide complexes were confirmed by Western blotting. Peptides were extracted by denaturation and size-exclusion, and subjected to MS analyses using rapid Collisional-Induced Dissociation (rCID) and High-energy Collisional Dissociation (HCD). Results of the MS showed congruence of signal intensities of peptides common to both control and patient samples using the rCID output (R2=0.498; P<0.0001). Among peptides unique to the CAD patient, the highest intensity on both rCID and HCD methods was a fragment of hCAP18. The biological relevance of this finding was tested in vitro using a truncated (t) form of the mouse homolog of hCAP18, CRAMP, on splenocytes from athero-prone ApoE-/- mice. Stimulation with tCRAMP (20μg/ml) for 48h resulted in an increase in CD8+IFN-γ+ cells (P<0.05, N=4, Figure) and CD8+ Central Memory (CM) cells (P<0.05). CD8+ T cell proliferation was increased with tCRAMP stimulation for 5 days (P<0.05). High fat diet feeding (HC) for 6 weeks resulted in an expansion of CD8+ Effector Memory (EM) cells compared to normal chow feeding (NC) with a further increase after in vitro stimulation with tCRAMP (P<0.05, N=5,Figure). There were no observed increases in the CD4+ T cell subpopulation in the parameters tested.
Conclusion: IP profiling in a patient with CAD using HLA-I/peptide immune-precipitation and MS along with in vitro testing identifies hCAP18 as a potential auto-immune protein involved in the CD8+ T cell response in atherosclerosis.
Author Disclosures: P.M. Mihailovic: None. W. Lio: None. J. Yano: None. X. Zhao: None. J. Zhou: None. B. Zhou: None. W. Yang: None. M. Freeman: None. K. Chyu: None. P.K. Shah: None. B. Cercek: None. P. Dimayuga: None.
- © 2016 by American Heart Association, Inc.