Btk regulates localization, in vivo activation, and class switching of anti-DNA B cells

Abstract

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk-/- mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk-/- mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk-/- mice. A transgene expressing a low level of Btk in B cells (Btk(lo)) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btk(lo) B cells. anti-DNA IgG was not observed in 56R.Btk(lo) mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached.

Figure 1. B cells with potential for anti-DNA reactivity are present in 56R.Btk−/− but are not preferentially localized to the marginal zone

Splenocytes from 3–6 month old mice were stained with antibodies against A) B220 and IgMa and B,C) B220, CD21, CD23, and Igλ. All B220+ cells (B) or B220+ Igλ+ cells (C) were analyzed for expression of CD21 and CD23. The frequency of gated cells in the marginal zone (CD23-CD21+) population is indicated. D) The mean +/− SD percentage of marginal zone cells among B220+ cells in mice without the 56R transgene and among B220+ and B220+Igλ+ cells in mice with the 56R transgene (D) is shown. n = 3. * = p< 0.05, wt vs. Btk−/−.

Figure 2. Btk−/− B cells proliferate and differentiate in response to CpG DNA but demonstrate impaired upregulation of IL-10

A) Purified B cells from 3–6 month old mice were stimulated with 1 ug/ml ODN 1826. Proliferation was measured by ³H-thymidine incorporation during the final 6 hrs of a 48 hr culture. Data represent mean +/− SEM, n = 6–8, p = 0.5 (not significant). B) Purified B cells were stimulated with media alone (thin line) or 1 ug/ml ODN 1826 (bold line) for 72 hours and stained with antibodies against CD86. Data are representative of 3 independent experiments. C) Purified B cells were stimulated with 1 ug/ml ODN 1826 for 72 hours and stained with antibodies against B220 and CD138, a plasma cell marker. The frequency of CD138^hi cells is indicated. Data are representative of 4–7 individual mice per genotype. D) Purified B cells were stimulated with 1 ug/ml ODN 1826 for 72 or 96 hours. Total IgM levels in the culture supernatants were measured by ELISA. The 72 hr. and 96 hr. time points were obtained in separate experiments, and each time point represents the mean +/− SD of 2 mice per genotype, for a total of 4 mice analyzed per genotype. E) Purified splenic B cells were stimulated with 1 ug/ml ODN 1826 for 24 hours. Culture supernatants were analyzed for IL-10 by ELISA. Data represent mean +/−SD, n = 4–5. * = p< 0.05, ** = p < 0.01. No IL-10 was detected in supernatant from cells stimulated with media alone.

Figure 3. Btk is required for some, but not all, interactions between BCR and TLR9 signals

A) Purified B cells were stimulated with 1 ug/ml ODN 1826, 0.1 ug/ml ODN 1826, 2 ug/ml anti-IgM F(ab)’₂ fragments, or 0.1 ug/ml ODN 1826 plus 2 ug/ml anti-IgM F(ab)’₂ fragments for 48 hours. Proliferation was measured by ³H-thymidine incorporation. Data represent mean +/− SD of triplicate wells and are representative of 4 independent comparisons. * = p < 0.01 vs. both 0.1 ug/ml ODN 1826 alone and 2 ug/ml anti-IgM alone. B) Purified B cells were stimulated with 15 ug/ml anti-IgM F(ab)’ ₂ fragments, 1 ug/ml ODN 1826, or both for 6 hours. cDNA was prepared and subjected to quantitative real time PCR for IL-2, using GAPDH as a loading control. Data are presented as relative IL-2 expression compared to wild type cells stimulated with anti-IgM + ODN 1826, mean +/− SD, n=2–3. * = p < 0.05 vs. wt cells treated with anti-IgM + ODN 1826. C) Purified Btk−/− B cells were stimulated with 15 ug/ml anti-IgM F(ab)’₂ fragments, 1 ug/ml ODN 1826, or both for 48 hours. Cultures were photographed with a Zeiss Axiovert microscope at 10X magnification.

Figure 4. 56R B cells proliferate in response to CpG-containing DNA fragments independently of Btk

A) Total splenocytes were stained on ice with FITC-labeled donkey anti-mouse Ig F(ab)’₂ fragments. Cells were then incubated at 37°C for the indicated times to induce BCR signaling and internalization and analyzed by flow cytometry. The mean fluorescence intensity of FITC+ cells was determined and is presented as % of the starting value. Internalization of the BCR is accompanied by a reduction in fluorescence due to entry of the FITC label into acidic compartments (Aluvihare et al, 1997). Data represent the mean +/− SD for two experiments. Similar results were also obtained with xid mice, which have an inactivating point mutation in Btk (data not shown). B, C) Purified B cells were stimulated with media alone, 20 ug/ml anti-IgM F(ab)’₂ fragments, 0.1 ug/ml CG50 fragment +/− 4 ug/ml inhibitory ODN 2088, or 0.1 ug/ml HIV (CG+) fragment. Proliferation was measured by ³H-thymidine incorporation during the final 12 hrs of a 36 hr culture. Cpm are presented as mean +/− SEM, n = 3–6. * = p < 0.05, ** = p < 0.01 56R vs. no 56R. D) Stimulation index = avg cpm stimulated/avg cpm media from the data in B and C. Note that the response of wt cells to anti-IgM is off scale in B and D. The legend in panel C is also for panels B and D.

Figure 5. Differential effects of reduced Btk signaling on anti-DNA IgM and IgG production

6–12 month old wild type or 56R mice with the indicated Btk genotypes were bled and the serum levels of anti-ssDNA (A,B) and anti-dsDNA (C,D) IgM (A,C) and IgG (B,D) measured by ELISA The OD405 for a 1:100 dilution of serum is indicated. Each diamond represents an individual mouse. Horizontal bars represent the mean. * = p < .02 vs wild type and 56R.Btk−/−. Btk hi mice express both the endogenous Btk gene and the Btk lo transgene. E) Purified splenic B cells were stimulated with 1 ug/ml ODN 1826 for 24 hours. Cells were harvested for RNA and analyzed by real time PCR for expression of Tbx21 (T-bet) and Aicda (AID), using GAPDH as a loading control. Data represent the percentage of the average wild type level in ODN 1826 stimulated cells and are plotted as mean +/− SD, n = 2–4. * = p< 0.05, ** = p < 0.01.