Two Distinct Pathways in Mice Generate Antinuclear Antigen-Reactive B Cell Repertoires

Abstract

The escape of anti-self B cells from tolerance mechanisms like clonal deletion, receptor editing, and anergy results in the production of autoantibodies, which is a hallmark of many autoimmune disorders. In this study, we demonstrate that both germline sequences and somatic mutations contribute to autospecificity of B cell clones. For this issue, we investigated the development of antinuclear autoantibodies (ANAs) and their repertoire in two different mouse models. First, in aging mice that were shown to gain several autoimmune features over time including ANAs. Second, in mice undergoing a chronic graft-versus-host disease (GVHD), thereby developing systemic lupus erythematosus-like symptoms. Detailed repertoire analysis revealed that somatic hypermutations (SHM) were present in all Vh and practically all Vl regions of ANAs generated in these two models. The ANA B cell repertoire in aging mice was restricted, dominated by clonally related Vh1-26/Vk4-74 antibodies. In the collection of GVHD-derived ANAs, the repertoire was less restricted, but the usage of the Vh1-26/Vk4-74 combination was still apparent. Germline conversion showed that the SHM in the 4-74 light chain are deterministic for autoreactivity. Detailed analysis revealed that antinuclear reactivity of these antibodies could be induced by a single amino acid substitution in the CDR1 of the Vk4-74. In both aging B6 and young GVHD mice, conversion of the somatic mutations in the Vh and Vl regions of non Vh1-26/Vk4-74 using antibodies showed that B cells with a germline-encoded V gene could also contribute to the ANA-reactive B cell repertoire. These findings indicate that two distinct pathways generate ANA-producing B cells in both model systems. In one pathway, they are generated by Vh1-26/Vk4-74 expressing B cells in the course of immune responses to an antigen that is neither a nuclear antigen nor any other self-antigen. In the other pathway, ANA-producing B cells are derived from progenitors in the bone marrow that express B cell receptors (BCRs), which bind to nuclear antigens and that escape tolerance induction, possibly as a result of crosslinking of their BCRs by multivalent determinants of nuclear antigens.

Keywords: antinuclear antibodies; autoantibodies; monoclonal antibodies; mouse model; somatic hypermutation; systemic lupus erythematosus-like disease.

Figure 1

ANA reactivity is directed against histones. Histone, dsDNA, ssDNA, Sm, or SS-B/La binding of ANA-reactive mAbs derived from (A) aging B6 (Table 1) or (B) young B6 × bm12 mice undergoing a cGVH reaction (Table 2). Significant binding was defined as an O.D. of five times over background in ELISA. (−), no significant binding at 5 μg/ml; (+), binding at 5 μg/ml; (++), binding at 1.7 µg/ml; (+++), binding at 0.55 µg/ml; and (++++), binding at 0.19 µg/ml or lower.

Figure 2

ANA reactivity of Vh1-26/Vk4-74 using mAbs is caused by mutations in the light chain. ANA titers of different Vh1-26/Vk4-74 mAbs derived from (A) aging B6 (Table 1) or (B) young B6 × bm12 mice undergoing a cGvH reaction (Table 2). The histograms depict titers of individual mAbs, either with the original SHM, or Vh and/or Vk in germline configuration (GL), or (C) having a single mutation in their Vk CDR1 resulting in a serine to arginine conversion. A titer of one was defined as binding at a concentration of 5 µg/ml mAb, followed by 1:2 serial dilution steps and recording the last dilution where binding still was possible. SHM, somatic hypermutation; GL, germline; n.b., no binding detectable at 5 µg/ml of mAb.

Figure 3

ANA reactivity of non-Vh1-26/Vk4-74 using mAbs is mainly germline encoded. ANA titers of different non-Vh1-26/Vk4-74 mAbs derived from (A) aging B6 or (B) young B6 × bm12 mice undergoing a chronic graft-versus-host disease. The histograms depict titers of individual mAbs listed in Table 3 containing V-regions, either with the original somatic hypermutations (SHM), or Vh and/or Vk in germline configuration (GL), or (C) containing a Vh CDR2 with conversions of aspartic and glutamic acids to glycine. Titers and legends as defined in Figure 2.

Figure 4

Two distinct pathways generate ANA producing B cells. [(A) red background] B cells with a B-cell receptor (BCR) using a germline-encoded Vh1-26 in combination with a germline-encoded Vk4-74 gets stimulated by a non-self (NS) antigen/hapten. Due to somatic hypermutations in the Vk4-74 gene, ANA-reactive memory B cells are generated. In aging B6 mice, these memory cells are then activated by nuclear antigens and Th1 cells to produce ANA. In the GVHD mice, nuclear antigens and alloreactive T cells activate these memory cells, resulting in ANA formation. [(B) blue background] Immature B cells in the bone marrow with germline-encoded BCRs specific for nuclear antigens escape tolerance induction mechanisms through BCR cross-linking by a T cell independent type 2-antigen (TI-2) antigens or a signal from alloreactive T cells. In the periphery of aging B6 mice, these cells get stimulated by nuclear antigens and Th1 cells to produce ANAs. In the periphery of GVHD mice, nuclear antigens and alloreactive T cells activate these cells to ANA formation.