The pathogenicity of T cell epitopes on human Goodpasture antigen and its critical amino acid motif

Abstract

Goodpasture antigen, the non‐collagenous domain of α3 chain of type IV collagen [α3(IV)NC1], is the target antigen of anti‐glomerular basement membrane (GBM) antibodies. The pathogenicity of T cell epitopes is not elucidated clearly. In this study, we aim to define the nephritogenic T cell epitopes and its critical amino acid residues. Twenty‐four overlapping linear peptides were synthesized covering the whole sequence of human α3(IV)NC1. Wistar–Kyoto rats were immunized with linear peptides, and experimental autoimmune glomerulonephritis was evaluated. Critical amino acid was identified by the loss of nephritogenic function after each amino acid substitution by alanine. Of the 24 peptides, P14 (α3127‐148) could induce 90.5% (19/21) of WKY rats developing anti‐GBM glomerulonephritis with proteinuria, elevated serum urea and creatinine, IgG linear deposit on GBM and substantial (in average 82.4 ± 5.6%) crescent formation in glomeruli. Lymphocytes of immunized rats proliferated in response to α3127‐148 and α3(IV)NC1 in vitro. Sera of these rats recognized α3127‐148 and later on together with intact human α3(IV)NC1. Antibodies towards α3127‐148 and intact α3(IV)NC1 could also be detected from the kidney elutes. These antibodies showed no cross‐reaction with each other, which implies intramolecular epitope spreading during disease progress. After sequential amino acid substitution, the α3127‐148 with substitution of tryptophan136, isoleucine137, leucine139 or tryptophan140 lost its nephritogenicity. Human α3127‐148 is a nephritogenic T cell epitope in WKY rats, with the critical amino acids as W136I137xL139W140. These findings might facilitate future investigation on microbial aetiology and potential specific immunotherapy of anti‐GBM disease.

The Diagnostic and Clinical Utility of Autoantibodies in Systemic Vasculitis

“…Furthermore, there is evidence for T-cell component in anti-GBM disease. Recently, a nephritogenic T cell epitope (P14 a3127-148) on human Goodpasture antigen was identified and this epitope not only induced severe anti-GBM nephritis, but also initiated epitope spreading in WKY rats [60].…”

A Modified Peptide Derived from Goodpasture Autoantigen Arrested and Attenuated Kidney Injuries in a Rat Model of Anti-GBM Glomerulonephritis

“…Twenty-four hour urine samples and blood samples were collected before and after immunization at each week. All rats were euthanized at the end of week 6 after immunization according to our previous experience, 15 after which, renal tissues, spleens, and blood samples were collected.…”

“…IgG deposited in kidneys were eluted with glycine as described previously. 15 Serum antibodies and kidney elutes against All of the WKY rats were immunized with 200 mg/kg a3-P14, administered subcutaneously at hind footpads at day 0. For early-treatment groups, m-P14 or s-P14 were injected intraperitoneally at 30 mg/kg (early-treatment group A) or 10 mg/kg (early-treatment group B) daily from day 0 to 14, and every other day from day 15 to 28.…”

“…peptides and a3(IV)NC1 were detected by ELISA. 15 Peptides (5 mg/ml) and a3(IV)NC1 (2 mg/ml) were coated overnight to the 96-well plates.…”

“…13,14 By sequential amino acid substitution, we found that tryptophan (W 136 ), isoleucine (I 137 ), leucine (L 139 ), and tryptophan (W 140 ) were crucial for inducing anti-GBM GN in Wistar Kyoto (WKY) rats. 15 The immunodominant epitope a3 135-145 is presented by HLA-DRB1*1501, the predisposing allele for Goodpasture disease, 16,17 with the anchor residues I 137 , W 140 , phenylalanine (F 143 ), and F 145 in the binding pocket 1, 4, 7, 9 of DR15 molecule. 18 T cells specific for this epitope from DR15 + transgenic mice were largely CD4 + Foxp3 2 T conv cells and produced the pathogenic T helper cell (Th) Th1 and Th17 cytokines.…”