Repertoire analysis of anergic B cells from patients with systemic lupus erythematosus

Patients with active systemic lupus erythematosus (SLE) have atypical CD19loIgD⁺ peripheral blood B cells that require 100-fold more anti-immunoglobulin or recombinant CD154 to induce in vitro proliferation. Additional phenotypic and in vitro functional analyses strongly suggested that the CD19loIgD⁺ cells are anergic. Since anergy is one of the mechanisms by which the function of autoreactive B cells can be downregulated, a comparison of the productive B-cell repertoires of CD19loIgD⁺ and CD19hiIgD⁺ subsets from the same lupus patient was undertaken to determine whether the anergic B cells were oligoclonal or otherwise enriched in cells expressing genes known to encode autoantibodies. To accomplish this, the usage of V_HDJ_H, V_kJ_k and V_λ J_λ gene elements by individual B cells was determined for each subset. The two subsets exhibited low mutational frequencies (CD19hiIgD⁺, V_H = 0.6%, V_k = 0.05%, V_λ = 0.1%; CD19loIgD⁺, V_H = 0.2%, V_k = 0.05%, V_λ = 0.2%) and involved substitutions that rarely increased the basic amino acid content. The immunoglobulin heavy and light chain repertoires of each subset were polyclonal and very similar. However, some differences from the normal repertoire were noted. For example, the V_k1 family was under-represented and V_k4 B3 and V_k5 B2 were over-represented in both subsets. Multiple small clones (average size 3.5) were present in the V_H, V_k and V_λ repertoires of both subsets. A large (n = 32) clone using V_H 4–34, D3-22 and J_H 6 gene elements, containing FR mutations, was found. Notably, it was split two-thirds within the CD19hiIgD⁺ subset and one-third within the CD19loIgD⁺ subset. In spite of the overwhelming similarities between subsets, there were three distinct differences. The V_H4 family was significantly (37% versus 17%) more abundant in the CD19loIgD⁺subset. Second, CD19loIgD⁺ V_λ J_λ clones were two-fold more numerous and more diverse in gene utilization than the CD19hiIgD⁺ subset. Third, B cells utilizing V_k5 B2 genes were significantly (16% versus 7%) more abundant in the CD19hiIgD⁺ subset. Because of the polyclonal distribution of immunoglobulin heavy and light chain genes, and the overall similarity between the two subsets, it is likely that anergy in the CD19loIgD⁺ subset is not antigen specific.