TARGETING EPITOPE-SPECIFIC T CELLS AND MODELLING PROTEIN HYPERCITRULLINATION IN RHEUMATOID ARTHRITIS

Abstract

Introduction: Rheumatoid arthritis (RA) is a debilitating, autoimmune disease of the synovial joints, involving a complex interplay of genetic and environmental risk factors. In RA, an overactive peptidyl-arginine deiminase (PAD) enzyme, citrullinates ‘self-proteins’, converting positively charged arginine residues to neutral citrulline, generating new immunogenic peptides. These peptides interact favourably with a positively charged pocket within the binding groove of HLA-DR molecules. Peptides are subsequently presented to and activate autoreactive T cells, driving an inflammatory response. The Aims of this research were (1) to demonstrate proof-of-principal for a therapeutic mRNA vaccine substituting glutamine residues for citrulline (non-coded amino acid). T cell responses to native/citrullinated/glutamine-substituted peptides were compared, and (2) to overexpress the PAD4 enzyme and investigate the consequences of protein hypercitrullination in RA pathogenesis. Methods: In Aim 1, 17 RA patients and healthy controls were recruited from a local Rheumatology clinic. Participants’ peripheral blood mononuclear cells were isolated from whole blood to assess T cell responses to native, citrullinated, and glutamine-substituted peptides. In Aim 2, an adeno-associated virus (AAV), encoding a murine PAD4, was transduced into human and mouse cells. Protein hypercitrullination was investigated by western blotting, immunocytochemistry, flow cytometry, and ELISA. Results: In Aim 1, 4/17 participants demonstrated an equivalent frequency of T cell responses to citrulline and glutamine epitopes. Antigen-specific T cell responses to unmodified peptides were also detected. In Aim 2, the AAV-encoding PAD4 was expressed in human and mouse cells. Cytoplasmic, rather than the expected nuclear and endoplasmic reticulum distribution was observed. Protein hypercitrullination was not detected suggesting the engineered protein was non-functional. Discussion/Conclusion: For Aim 1, it was apparent that glutamine was not an appropriate surrogate for citrulline (in vitro) in the design of a therapeutic mRNA vaccine. While, for Aim 2, an AAV-expression system to assess PAD4-mediated hypercitrullination led to production of a non-functional enzyme.