Autoreactive IgG memory antibodies in patients with systemic lupus erythematosus arise from nonreactive and polyreactive precursors

Abstract

Persistent autoantibody production in patients with systemic lupus erythematosus (SLE) suggests the existence of autoreactive humoral memory, but the frequency of self-reactive memory B cells in SLE has not been determined. Here, we report on the reactivity of 200 monoclonal antibodies from single IgG+ memory B cells of four SLE patients. The overall frequency of polyreactive and HEp-2 self-reactive antibodies in this compartment was similar to controls. We found 15% of IgG memory B cell antibodies highly reactive and specific for SLE-associated extractable nuclear antigens (ENA) Ro52 and La in one patient with serum autoantibody titers of the same specificity but not in the other three patients or healthy individuals. The germ-line forms of the ENA antibodies were non-self-reactive or polyreactive with low binding to Ro52, supporting the idea that somatic mutations contributed to autoantibody specificity and reactivity. Heterogeneity in the frequency of memory B cells expressing SLE-associated autoantibodies suggests that this variable may be important in the outcome of therapies that ablate this compartment.

Fig. 1.

IgH and IgL chain gene features from IgG memory B cell antibodies of SLE patients and HC. Ig gene repertoire and Ig gene features of IgG memory B cell antibodies cloned from three published (24) and one unpublished HC (JH) and four patients with SLE (169, 174, 175, and 176). P values compare data from the patients to the HC. (A) Pie charts depict VH and JH gene usage. The absolute number of sequences analyzed is indicated in the center of each pie chart. (B) Pie charts depict Vκ/Jκ (Left) and Vλ/Jλ gene usage (Right). The absolute number of sequences analyzed is indicated in the center of each pie chart. The frequency of Igκ- and Igλ-positive antibodies is indicated. (C) Bar graphs show IgG1 (white), IgG2 (black), IgG3 (dark gray), and IgG4 (light gray) subclass usage. (D) The absolute number of mutations (nucleotide exchanges as compared with germ line) in individual VH, Vκ, and Vλ genes (FWR1 - FWR3) of antibodies from IgG+ memory B cells of four healthy HC (filled circles) and four SLE patients (open circles) is shown. Horizontal lines represent the average number of mutations and gray bars represent the standard deviation.

Fig. 2.

Polyreactivity and self-reactivity of IgG memory B cell antibodies from SLE patients. (A) IgG memory B cell antibodies from HC JH and SLE patients were tested for polyreactivity with ssDNA, dsDNA, insulin, and LPS by ELISA. Representative ELISA graphs for reactivity with ssDNA and insulin are shown. Dotted lines represent the high positive control antibody ED38 (61). Horizontal lines show cut-off OD₄₀₅ for positive reactivity. Green and red lines show the negative control antibody mGO53 and low positive control antibody eiJB40, respectively (26). (B) Pie charts summarize the frequency of polyreactive (black) and nonpolyreactive (white) IgG+ memory B cell clones from four HC and SLE patients (24). The number of tested antibodies is indicated in the pie chart center. P values are compared with four HC (24). (C) IgG memory B cell antibodies from HC JH and patients with SLE were tested for self-reactivity by HEp-2 cell ELISA. The horizontal line shows ELISA cut-off OD₄₀₅ for positive reactivity, and the red line shows low positive control serum. (D) Pie charts summarize the frequency of self-reactive IgG memory B cell antibodies from HC and SLE patients with nuclear (black), nuclear plus cytoplasmic (dark gray), and cytoplasmic (light gray) HEp-2 cell IFA staining patterns and the frequency of nonreactive antibodies (white). The number of tested antibodies is indicated in each pie chart center. P values are compared with IgG memory B cell antibodies from four HC (24).

Fig. 3.

Ro52/SSA-reactive and La/SSB-reactive SLE IgG memory B cell antibodies. (A) LIA with 13 SLE-associated autoantigens (SmB, SmD, RNP-70k, RNP-A, RNP-C, Ro52/SSA, Ro60/SSA, La/SSB, Cenp-B, Topo-1/Scl-70, Jo-1/HRS, Ribosomal P, Histones) identified four La/SSB-reactive (29, 162, 264, and 276) and two Ro52/SSA-reactive (107 and 128) antibodies among 200 tested IgG memory B cell antibodies from SLE patients and 84 tested IgG memory B cell antibodies from HC. Anti-Ro52/SSB and anti-La/SSB antibodies were all from SLE175. LIA result obtained with serum from the same patient is shown for comparison. HEp-2 cell IFA staining patterns of the same antibodies are shown. (B) Germ-line versions of the two anti-Ro52/SSA and four anti-La/SSB reactive IgG memory B cell antibodies from SLE175 were tested by LIA and IFA as in A. (C) HEp-2 ELISA results of recombinant mutated (filled symbols) anti-La/SSB (29, 162, 264, 276; Left) and anti-Ro52/SSA (107, 128; Right) antibodies and their germ-line counterparts (open symbols). Red lines in the ELISA graphs represent low positive control serum, dotted lines represent ED38 (61), and horizontal lines show cut-off OD₄₀₅ for positive reactivity.