Abstract. Goodpasture's disease is a severe nephritis characterized by autoantibodies to the ␣3 chain of type IV collagen, ␣3(IV)NC1, in the glomerular basement membrane. The disease is very strongly associated with HLA-DR15, the affinities of ␣3(IV)NC1 peptides for DR15 are known, and elution experiments have identified major naturally processed sequences. Here, the fine specificity and cytokine profile of ␣3(IV)NC1-reactive T cells from patients with Goodpasture's disease is defined. Peripheral blood mononuclear cells from patients at diagnosis proliferated in response to significantly more peptides ( 2 ϭ 8.6, P ϭ 0.004) from a panel spanning the sequence of ␣3(IV)NC1 than did those from control DR15-positive donors and were highly focused (P ϭ 0.0002, binomial distribution) on two peptides, ␣3 71-90 and ␣3 131-150 .

Biochemical analysis of HLA class II-associated peptides from antigen-pulsed cells is a potentially useful approach to the analysis of antigen processing and presentation because it examines directly which antigen-derived peptides are presented. This is especially advantageous in the analysis of self-antigen presentation where conventional approaches utilizing antigen-specific T cells may be biased by the presence of self-tolerance. However, successful biochemical analysis has been reported for only one exogenous antigen and no autoantigens. We have used a novel analytical approach coupling biochemical data with the reported properties of class II-associated peptides to characterize the peptides derived from a clinically relevant autoantigen presented on the disease-associated class II type. Incubating the target of autoimmune attack in patients with Goodpasture's disease, the 230-amino acid NC1 domain of the alpha3 chain of type IV collagen (Goodpasture antigen, alpha3(IV)NC1), with human B cells homozygous for HLA-DR15, the allele carried by 80% of patients, we find that alpha3(IV)NC1 is presented as at least two sets of three to five peptides centered on common core sequences (nested sets). Synthetic peptides containing these core sequences bind to HLA-DR15 with intermediate affinity (IC50, 1.1-6 microM).