Antigen selection of anti-DSG1 autoantibodies during and before the onset of endemic pemphigus foliaceus

Abstract

Fogo selvagem (FS), the endemic form of pemphigus foliaceus (PF), is characterized by pathogenic anti-desmoglein 1 (DSG1) autoantibodies. To study the etiology of FS, hybridomas that secrete either IgM or IgG (predominantly IgG1 subclass) autoantibodies were generated from the B cells of eight FS patients and one individual 4 years before FS onset, and the H and L chain V genes of anti-DSG1 autoantibodies were analyzed. Multiple lines of evidence suggest that these anti-DSG1 autoantibodies are antigen selected. First, clonally related sets of anti-DSG1 hybridomas characterize the response in individual FS patients. Second, H and L chain V gene use seems to be biased, particularly among IgG hybridomas, and third, most hybridomas are mutants and exhibit a bias in favor of CDR (complementary determining region) amino acid replacement (R) mutations. Strikingly, pre-FS hybridomas also exhibit evidence of antigen selection, including an overlap in V(H) gene use and shared multiple R mutations with anti-DSG1 FS hybridomas, suggesting selection by the same or a similar antigen. We conclude that the anti-DSG1 response in FS is antigen driven and that selection for mutant anti-DSG1 B cells begins well before the onset of disease.

Figure 1

Anti-Dsg1 hybridoma antibodies are specific for Dsg1. a. Representative anti-Dsg1 mAbs (all are IgG1, except FGS-4E4 and GCDS-2D11 (IgG4)) from different FS patients were used to immunoprecipitate Dsg1. Hybridoma antibodies bound to protein G agarose beads were used to immunoprecipitate His-tagged Dsg1 ectodomain. The immmunoprecipitated samples were used in Western blots using an anti-His HRP conjugate to detect Dsg1 protein. b. Similar to serum from FS patient, anti-Dsg1 mAbs (FGS-4E4) show typical indirect immunofluorescence staining patterns using monkey esophagus as the substrate.

Figure 2

V gene use by FS and pre-FS clonally independent anti-Dsg1 hybridomas. a. V_H gene use. b. V_L gene use. IgM and IgG are summarized separately for FS (upper panels) and pre-FS (lower panels). The number of hybridomas identified by sequence similarity to use each gene is indicated. Genes used by both FS and pre-FS hybridomas are marked by an asterisk.

Figure 3

The expressed V_H genes from FS and pre-FS hybridomas share replacement mutations. Sequence comparisons were conducted using Vector NTI (Invitrogen Life Technologies). All the sequences shown are from independent clones. FWRs (1, 2, and 3) and CDRs (1 and 2) are boxed and replacement mutations are highlighted. Positions in which no mutations occurred are in yellow, while conservative replacement mutations are highlighted in blue and non-conservative replacement mutations highlighted in white. Positions where shared mutations occurred are indicated with arrows. Germline genes are shown at the bottoms of each V_H gene set. The amino acid differences at position 33 (G or A) and 98 (K or R) of IGHV3-30 may be polymorphisms and are not considered in this analysis.