The studies in this proposal test the hypothesis that HLA alleles, HLA self-peptides and microbial mimicry, in concert, contribute to the pathogenesis of systemic sclerosis (SSc). Observations leading to the hypothesis point to a central role for the HLA-DRP I molecule and are at least fourfold. The first is the finding that a woman's risk of SSc is significantly increased by prior birth of a child who was HLA-compatible for DRbeta1. Second, particular HLA alleles increase risk of SSc, and include an amino acid sequence of the DR11 DRbeta1 chain (aa67-71, """"""""FLEDR"""""""") that is shared with DRbeta5. Third, human cytomegalovirus (HCMV) is known to negatively impact conditions of chimerism that arise from transplantation and spontaneously occurring microchimerism has recently been implicated in SSc, as has HCMV. Finally, HCMV has sequence identity with a conserved sequence of HLA-DRbeta1 (aa 53-57), that extends further (aa 53-58, """"""""LGRPDE"""""""") for DRB1 alleles that encode for DR11, which is associated with SSc. The proposed experiments will provide the initial test of the concept that HLA peptides derived from DRbeta1 function in cellular communications among host cells and between host and non-host cells. The first Specific Aim will design candidate peptides that encompass the identified sequences of the DRbeta1 molecule and will test them in binding and functional T cell assays. The second and third Specific Aims will identify, enumerate and characterize peptide specific T cells using artificial antigen presenting cells (aAPC) complexed with the HLA self-peptides and peptides derived from the homologous HCMV sequence. The aAPC is a very recently developed technique that offers an advantage over tetramers in capturing low affinity interactions due to permissive movement within a liposorne membrane. The aAPC will be used in a T cell capture assay to isolate peptide specific T cells which will then be phenotypically and functionally characterized in SSc patients and in controls. Analysis will be conducted for cytokine production and differential gene expression from sorted cells of SSc patients and controls. Finally, in the fourth Specific Aim, Real-Time PCR will be used to quantitatively assess microchimerism in peripheral blood mononuclear cell subsets from the same patients and controls. The results of the proposed experiments will permit the initial test of a model of pathogenesis that incorporates a concert of contributory factors including specific HLA alleles, HLA-relationships among host and non-host cells, and microbial mimicry in disease pathogenesis. Studies are designed so as to maximize the potential to advance understanding of disease pathogenesis in a way that could potentially lead to the development of new therapeutic modalities for this difficult disease.
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