Regulation of the B cell receptor repertoire and self-reactivity by BAFF

Abstract

The TNF-family cytokine BAFF (BLyS) promotes B lymphocyte survival and is overexpressed in individuals with systemic lupus erythematosus and Sjögren's Syndrome. BAFF can rescue anergic autoreactive B cells from death, but only when competition from nonautoreactive B cells is lacking. Yet, high BAFF levels promote autoantibody formation in individuals possessing diverse B cells. To better understand how excess BAFF promotes autoimmunity in a polyclonal immune system, Ig L chain usage was analyzed in 3H9 site-directed IgH chain transgenic mice, whose B cells recognize DNA and chromatin when they express certain endogenous L chains. BAFF levels were manipulated in 3H9 mice by introducing transgenes expressing either BAFF or its natural inhibitor ΔBAFF. B cells in BAFF/3H9 mice were elevated in number, used a broad L chain repertoire, including L chains generating high-affinity autoreactivity, and produced abundant autoantibodies. Comparison of spleen and lymph node B cells suggested that highly autoreactive B cells were expanded. By contrast, ΔBAFF/3H9 mice had reduced B cell numbers with a repertoire similar to that of 3H9 mice, but lacking usage of a subset of Vκ genes. The results show that limiting BAFF signaling only slightly selects against higher affinity autoreactive B cells, whereas its overexpression leads to broad tolerance escape and positive selection of autoreactive cells. The results have positive implications for the clinical use of BAFF-depleting therapy.

Figure 1. Influence of BAFF bioactivity levels on the B cell compartment of 3H9 Tg mice

A) 12 to 16-week-old mice were assessed for total numbers of B220⁺ B cells, MZ, κ⁺ and λ⁺ B cell subsets in spleen (SP) and lymph nodes (LN). Colored bars indicate genotypes as follows: WT, white; 3H9, green; BAFF/3H9, red; DBF/3H9, blue. Data for 4 mice per group (mean ± standard error (SE)). Asterisks show p values calculated by two-tailed Student’s t test. *; p<0.05, **; p <0.01, ***; p <0.001, ns; not significant. B) FACS plots of SP and LN κ⁺ and λ⁺ B cells gated on CD4⁻CD8⁻B220⁺ population. One representative experiment out of four is shown.

Figure 2. Effects of BAFF bioactivity on serum anti-dsDNA antibodies and CD93+ splenic B cell subsets

Mice of the indicated genotypes were analyzed by ELISA for serum anti-DNA activity and by flow cytometry for markers of B cell maturity. A) IgM, IgG, IgA and λ anti-dsDNA antibody production in sera from 12 to 16-week-old mice of the indicated genotypes. Diamonds represent values obtained in individual mice, with the mean for each group indicated by a horizontal bar. B) FACS plots of splenic CD93hi immature B cells (upper panel box) and transitional B cells (lower panel). T1; upper left, T2; upper right, T3; lower right, T3’; lower left. One representative experiment out of four is shown. C) Frequency of each B cell subset from 3H9 (green, n=5), BAFF/3H9 (red, n=5) and DBF/3H9 (blue, n=7) analyzed as in B. Shown is mean±SE. Asterisks show p values calculated by two-tailed Students t test. *; p<0.05, **; p <0.01, ***; p <0.001, ns; not significant.

Figure 3. Jκ usage

Frequencies of Jκ usage among κ L-chain cDNAs from B cells of the indicated mouse genotypes are shown. A) Jκ usage in spleen and B) in lymph nodes. Jκ1; white, Jκ2; green, Jκ4; red, Jκ5; blue. Asterisks show p values calculated by Chi-square test. ***; p <0.001. Data for 4 mice per group. 12 to 16-week-old mice were used.

Figure 4. Pyrosequence analysis of Vκ usage in mice of differing BAFF activity

κ light chain genes observed in B cells isolated from spleen A) and lymph nodes B) in each strain. Only genes used at >0.5% frequency are shown. Colored bars indicate genotypes as follows, 3H9; green, BAFF/3H9; red, DBF/3H9; blue. Data for 4 mice per group. 12 to 16-week-old mice were used.

Figure 5. Vκ usage depicted as estimated absolute cell number

Data shown estimate total cells in the indicated tissues that express the indicated Vκ genes. A) spleen. B) lymph nodes. Values shown are fractional usage taken from Figure 4 multiplied by total B220⁺ B cell number from Figure 1A.

Figure 6. Vκs that are significantly skewed by altered BAFF levels and analysis of corresponding hybridoma proteins for DNA reactivity

Increased κ light chain genes in lymph nodes from each strain are shown. Genes increased in A) BAFF/3H9, B) only in 3H9, and C) in DBF/3H9. Colored bars indicate genotypes as follows: 3H9, green; BAFF/3H9, red; DBF/3H9, blue. Data for 4 mice per group. 12 to 16-week-old mice were used. Asterisks show p values calculated by Chi-square test. ***; p <0.001.

Figure 7. Effect of BAFF activity level on Jκ usage and CDR3 amino acids frequencies of particular Vκ genes

A) B cells with Vκ19-93 (red) or Vκ12-46 (blue) selected in each strain depends on Jκ usages in lymph nodes. Each strain has different pattern of Jκ usage. B) B cells with Vκ12-44 (black), Vκ8-28 (yellow), Vκ6-23 (green), Vκ8-19 (red) and Vκ6-15 (blue) showed similar pattern of Jκ usage in LNs. C) Analysis of CDR amino acid sequences revealed different selection of B cells with unbiased Jκ usage shown in B. Colored bars indicate genotypes as follows: 3H9, green; BAFF/3H9, red; DBF/3H9, blue.

Figure 8. Hybridoma analysis

Shown are κ L-chain family usages of hybridomas produced from spleen and lymph nodes previously stimulated for 3 days with LPS. Upper left, SP from BAFF/3H9; upper right, SP from DBF/3H9; lower left, lymph nodes from BAFF/3H9; and lower right, lymph nodes from DBF/3H9. Colored bars indicate J usage as follows, Jκ1; yellow, Jκ2; green, Jκ4; red and Jκ5; blue. A) κ L-chain family usage observed in dsDNA reactive clones. Asterisks show p values calculated by Chi-square tests between Vk8 family usage in lymph nodes from BAFF/3H9 and DBF/3H9. *; p<0.05. B) κ light chain family usages observed in clones without binding affinity against dsDNA.