Dr Lani Shochet1,2, CHUNNI LU2, MAI TRAN3, CLAERWEN JONES3, PEEMAPAT PRAKONGTHAM2, TIING JEN LOH3, JIA JIA LIM3, JESSICA RYAN1,4, LARS FUGGER5, DIETER JENNE6, NICOLE LA GRUTA3, HUGH REID3, JAMIE ROSSJOHN3, A RICHARD KITCHING1,2,7
1Department of Nephrology, Monash Health, Clayton, Australia, 2Monash University Centre for Inflammatory Diseases, Department of Medicine, Clayton, Australia, 3Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia, 4Monash University Department of Medicine, Clayton, Australia, 5Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom, 6Max-Planck-Institut for Biological Intelligence, Planegg-Martinsried, Germany, 7Department of Paediatric Nephrology, Monash Health, Clayton, Australia
Biography:
Bio to come
Aim:
To define the immunodominant and nephritogenic CD4+ T cell proteinase 3 (PR3) epitope in the context of HLA-DR15.
Background:
PR3-AAV is associated with HLA-DR15 and HLA-DP4. Little is known about anti-PR3 CD4+ T cell responses, in part due to poor sequence homology between rodent and human PR3.
Methods:
Human PR3 knock-in mice expressing HLA-DR15 (hPR3.DR15 mice) were immunised with human PR3 (hPR3) peptides (45 16mers, overlapping by 11 amino acids). Recall responses were studied 10 days post-immunisation (IL-17A/IFN-γ ELISPOTS). HLA-DR15/PR3 peptide structures were solved by crystallography. T cell receptors (TCRs) were sequenced from single PR3-specific T cells from mice and HLA-DR15+ PR3-AAV patients. Anti-PR3 glomerulonephritis was modelled using hPR3.DR15 mice.
Results:
Immunization with hPR3 peptide pools/subpools demonstrated CD4+ T cell autoreactivity (IL-17A/IFN-γ ELISPOT) only within the hPR3₁₉₆-₂₅₆ region. Single peptide and hPR3 protein immunization studies defined hPR3₂₁₆-₂₃₁ as the only peptide consistently inducing autoimmunity. However, experiments shortening PR3₂₁₆-₂₃₁ to 12-15 mers showed reduced (13-15 mers), or no reactivity and poor HLA-DR15 binding (12 mers). The HLA-DR15/hPR3₂₁₆-₂₃₁ structure revealed a unique binding conformation, with ten (rather than nine) amino acids within the HLA-DR15 antigen binding cleft. hPR3₂₁₆-₂₃₁-specific CD4+ TCRs of HLA-DR15-PR3₂₁₆-₂₃₁ tetramer sorted T cells from hPR3₂₁₆-₂₃₁-immunized hPR3.DR15 mice and five HLA-DR15+ PR3-AAV patients demonstrated clonal expansion, biased TCR usage and similarities between humans and mice. Compared with OVA₃₂₃-₃₃₉-immunized mice, PR3₂₁₆-₂₃₁-immunised hPR3.DR15 mice challenged with anti-basement membrane globulin developed more glomerular lesions (5.9±2.1 vs 37.1±14.3% glomeruli affected, p<0.05) with increased glomerular and interstitial CD4+ T cells and macrophages.
Conclusion:
PR3₂₁₆-₂₃₁ is the nephritogenic CD4+ epitope in HLA-DR15+ PR3-AAV. PR3₂₁₆-₂₃₁’s unique binding to HLA-DR15 has fundamental implications for predicting epitopes and for CD4+T cell responses.