Somatic hypermutation as a generator of antinuclear antibodies in a murine model of systemic autoimmunity

Abstract

Systemic lupus erythematosus (SLE) is characterized by high-avidity IgG antinuclear antibodies (ANAs) that are almost certainly products of T cell-dependent immune responses. Whether critical amino acids in the third complementarity-determining region (CDR3) of the ANA originate from V(D)J recombination or somatic hypermutation (SHM) is not known. We studied a mouse model of SLE in which all somatic mutations within ANA V regions, including those in CDR3, could be unequivocally identified. Mutation reversion analyses revealed that ANA arose predominantly from nonautoreactive B cells that diversified immunoglobulin genes via SHM. The resolution afforded by this model allowed us to demonstrate that one ANA clone was generated by SHM after a V(H) gene replacement event. Mutations producing arginine substitutions were frequent and arose largely (66%) from base changes in just two codons: AGC and AGT. These codons are abundant in the repertoires of mouse and human V genes. Our findings reveal the predominant role of SHM in the development of ANA and underscore the importance of self-tolerance checkpoints at the postmutational stage of B cell differentiation.

Figure 1.

Chromatin-binding IgG antibodies in B6.Nba2 Tdt^−/− Igh^+/− Igk^+/− mice. (A) Serum titers of IgG anti-chromatin antibodies from B6.Nba2 Tdt^−/− Igh^+/− Igk^+/− mice (8–10 mo old). Red line, representative serum from a nonautoimmune C57BL/6 mouse. Standard errors are shown. (B) Chromatin binding assay with purified monoclonal antibodies generated from a spontaneously autoimmune B6.Nba2 Tdt^−/− Igh^+/− Igk^+/− mouse (female, 12 mo old). A prototypical ANA, 3H9/Vκ4, is shown in red. Bound antibodies were detected in a solid-phase europium (Eu³⁺)-based fluoroimmunometric assay (as counts per second). The figure shows one of two experiments with similar results.

Figure 2.

HCDR3 sequences for clones subjected to mutation-reversion analysis. Germline-encoded D_H sequences are shown above and germline-encoded V_H and J_H sequences are shown below hybridoma (shaded) sequences. Somatic mutations are underlined. The blue lowercase t in J6.1 is a templated nucleotide (P element) added during V_H(D)J_H recombination. In lineage #4, somatic mutations that eliminated Arg codons are explicitly indicated for both members.

Figure 3.

Dendrograms of multimember lineages. Numbers of somatic mutations are indicated along each branch, where r denotes an amino acid replacement and red indicates a mutation shared by all members of the lineage. The length of a branch is proportional to the number of somatic mutations. Clones from the first animal subjected to reversion are colored green, and those from a second autoimmune mouse are in yellow. Four of seven clones from the first animal that were represented by single hybridomas and subjected to reversion are shown.

Figure 4.

Chromatin binding assays for monoclonal antibodies and engineered revertants lacking somatic mutations. Standard errors are shown. R and red font color denote germline revertants. Two of the original monoclonal antibodies (J3.4 and J9.11) demonstrated binding activity against several chromatin components as shown in B–D. All antibodies were purified by a stringent affinity method designed to remove potentially contaminating nuclear antigens (Guth et al., 2003). A prototypical ANA, 3H9/Vκ4, is shown in blue in A. Bound antibodies were detected in a solid-phase europium (Eu³⁺)-based fluoroimmunometric assay (as counts per second). One of three experiments with similar results is shown.

Figure 5.

Immunofluorescence staining of HEp-2 cells and whole frozen sections of neonatal mouse with monoclonal antibodies and engineered revertants without somatic mutations. (A) Stains of fixed HEp-2 cells. R and red font color denote revertant. Note higher concentrations used for revertant antibodies. 3H9/Vκ4 and 36–71 served as positive and negative controls, respectively. The experiment was performed three times. Bar, 100 µm. (B) Stains of whole frozen sections of neonatal mice. Mutant antibody J6.1 served as positive control. Experimental section was stained with a mixture of revertant antibodies (RJ3.4, RJ9.11, and RJ6.1), each at a concentration equivalent to that of the positive control J6.1 mAb. Bound positive antibodies were detected with an FITC-coupled sheep anti–mouse IgG (γ-chain specific). Sections were counterstained with DAPI (blue) to highlight organs (In, intestine; Li, liver; K, kidney; Lu, lung; H, heart; Br, brain; E, eye). One of two experiments with similar results is shown. Bar, 3 mm.

Figure 6.

Functional Igh gene for the Q10.4 hybridoma created by V_H replacement. (A) The Q10.4 V_H/D/J_H boundary sequence illustrating an 8-base sequence remaining from the original V_H12.1.78 rearrangement in red. (B) The original V_H12.1.78 rearrangement showing the nonproductive translational reading frame ending in consecutive stop codons (red). The cryptic heptamer recombination signal sequence within V_H12.1.78 is shown in blue. The 8-base sequence of V_H12.1.78 that is destined to remain after replacement by V_HJ558.26.116 is also shown in red.