Glomerulopathy Induced by Immunization with a Peptide Derived from the Goodpasture Antigen α3IV-NC1(1)
The major autoantigen is the noncollagenous domain 1 of the a3-chain of type IV collagen (a3IV-NC1), which is targeted by both Abs and T cells. Interestingly, the T cell response to heterologous human a3IV-NC1 in DBA/1 mice and found a biphasic disease course. Autoantibodies are deposited early along the GBM with a membranous phenotype whereas the necrotizing/crescentic GN develops rapidly during the late phase.
Mice 6–8 wk of age were immunized s.c. at two sites in the back with 25 mg ∼1 nmol) recombinant m-a3IVNC1 in 50 ul PBS emulsified in an equal volume of CFA containing 50 ug Mycobacterium tuberculosis H37RA. Booster immunizations using the same dose of Ag emulsified in IFA were given s.c. at days 21, 35, and 49. The nephritogenicity of immunodominant T cell epitopes, the peptides P05 (RHSQTTAIPSCPEGT), P32 (MDMAPISGRALEPYI), and P71 (STVKAGDLEKIISRC) as well as a control peptide P15 (KRAHGQDLGTLGSCL) were dissolved in distilled water at a concentration of 1 mM. Mice were immunized s.c. with a total of 50 nmol peptide in 50 ul PBS emulsified in an equal volume of CFA. Subcutaneous booster immu- nizations were performed with the same dose emulsified in IFA on days 21, 35, and 49.
The immunization of m-a3IVNC1 developed mild proteinuria at 8 wk and moderate to severe proteinuria at 10 wk in DBA/1 mice.
T cell epitopes within the m-a3IV-NC1 domain were evaluated using splenoytes prepared 10 d after immunization with m-a3IV-NC1stimulated in vitro with a set of overlapping 15mer peptides spanning the whole m-a3IV-NC1 domain. Interestingly, P05 includes the dominant epitope found in the WKY rat EAG model.
None of the mice developed clinical signs of EAG within the 6-mo observation period. However, three of six mice immunized with P71 developed mild proteinuria starting at week 18. No proteinuria was detected in the other groups of peptide-immunized mice
No peptide- or m-a3IV-NC1–specific Abs could be detected in the other peptide-immunized groups. The IgG isotype pattern was similar to the pattern in m-a3IV-NC1–immunized mice. As with total IgG, the proteinuric P71 mice had higher titers than did the nonproteinuric mice. Ab binding to m-a3IV-NC1 did not change after preincubation of the sera with excess P71 peptide. In contrast, binding to P71 was almost completely blocked.
Immunization of DBA/1 mice with m-α3IV-NC1 caused fatal autoimmunity in the kidney. Depending on the number of immunizations, mice developed membranous glomerulopathy (two immunizations) or a focal necrotizing GN superimposed on a membranous glomerulopathy (four immunizations). 2) Immunization of mice resulted in the generation of α3IV-NC1–specific autoantibodies as well as low frequencies of α3IV-NC1–specific CD4+ T cells, which accumulated in the kidney in the necrotizing/crescentic stage of disease. 3) A peptide corresponding to one of three identified T cell epitopes from m-α3IV-NC1 was capable of inducing a membranous glomerulopathy after generating an Ab response directed against m-α3IV-NC1. The nephrotoxic nephritis models suggest a role of both Th1 and Th17 cells in the development of crescentic GN.
In mouse EAG, Fcgr2b has a protective effect, as C57BL/6 mice deficient in Fcgr2b develop crescentic GN after immunization with α3IV-NC1 and peptide immunization in the HLA-DRB1*15:01 transgenic mice, which also carried the Fcgr2b knockout, resulted in focal segmental necrotizing GN.
Detection of anti-P71 Abs together with anti–α3IV-NC1 Abs rather suggests that the Ab response was secondary to damage inflicted by P71-specific T cells. α3IV-NC1 released from basement membranes could subsequently provoke autoantibodies against the protein, including anti-P71 Abs. It was supported that P71-specific CD4+ T cells produce TNF-α, IFN-γ, or IL-17A in spleens of P71-immunized mice, although at relatively low frequencies. T-cells can be activated by a short peptide but protein requires uptake and processing by APCs. These make a difference in T-cell frequencies.
This is the key point in this paper: Peptide immunization resulted in glomerular damage with the emergence of a secondary α3IV-NC1–specific Ab response in the draining lymph nodes