B cell activating factor (BAFF) and T cells cooperate to breach B cell tolerance in lupus-prone New Zealand Black (NZB) mice

Abstract

The presence of autoantibodies in New Zealand Black (NZB) mice suggests a B cell tolerance defect however the nature of this defect is unknown. To determine whether defects in B cell anergy contribute to the autoimmune phenotype in NZB mice, soluble hen egg lysozyme (sHEL) and anti-HEL Ig transgenes were bred onto the NZB background to generate double transgenic (dTg) mice. NZB dTg mice had elevated levels of anti-HEL antibodies, despite apparently normal B cell functional anergy in-vitro. NZB dTg B cells also demonstrated increased survival and abnormal entry into the follicular compartment following transfer into sHEL mice. Since this process is dependent on BAFF, BAFF serum and mRNA levels were assessed and were found to be significantly elevated in NZB dTg mice. Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process. Although NZB mice had modestly elevated BAFF, the enhanced NZB B cell survival response appeared to result from an altered response to BAFF. In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production. The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help.

Figure 1. Breach of B cell anergy in NZB dTg mice.

(A) Serum levels of IgM^a anti-HEL antibodies in 8–12 wk old B6 and NZB non-Tg (nTg), IgTg and dTg mice. (B) Presence of anti-HEL Ab-producing cells in NZB dTg mice. The number of anti-HEL Ab-forming cells was determined by ELISpot in 8-12 wk old B6 and NZB IgTg and dTg mice. Horizontal lines represent the mean for each group examined. (C) Presence of an increased proportion of early (CD21⁺CD138^int) and late (CD138^high) pre-plasma cells in NZB dTg mice. Bone marrow cells from 8 wk old B6 and NZB IgTg and dTg mice were stained with anti-B220, -CD138, and -CD21 Abs and analyzed by flow cytometry. Dot plots are gated on the B220⁺ population and numbers indicate the percentage of gated cells in each region. Asterisks indicate the significance level for comparison between B6 and NZB mice as determined by the Mann-Whitney test: * p<0.05, ** p<0.005, *** p<0.0005.

Figure 2. NZB dTg B cells appear functionally anergic in-vitro.

(A) Sorted B cells from IgTg and dTg B6 or NZB mice were stimulated in-vitro with increasing concentrations of HEL (0 to 1 µg/ml) together with a submitogenic concentration of LPS (50 ng/mL). B cell proliferation was measured by [³H]-thymidine incorporation at 36 h by pulsing the cells overnight with 1 µCi/well. Uptake of [³H]-thymidine was quantified using a scintillation counter and expressed as mean cpm ± SD of triplicate wells. Results are representative of three independent experiments. (B) The percentage of CD86⁺ cells was measured 16 h after stimulation with 1 µg/ml HEL, gating on the B220⁺IgM^a+ population.

Figure 3. Increased survival of NZB dTg B cells following transfer into sHEL recipients.

(A) Percent B220⁺ CFSE⁺ B cells surviving in sHEL recipient mice expressed as a percentage of survival in non-Tg mice, 3 d following transfer of T cell-depleted CFSE-labelled splenocytes from B6 or NZB, IgTg or dTg mice. Open circles indicate B6 mice and filled circles NZB mice, with horizontal lines representing the mean. Asterisks indicate the significance level as determined by the Mann-Whitney test: * p<0.05, ** p<0.005, *** p<0.0005. (B) Immunofluorescence microscopy of spleens from sHEL B6 or NZB mice, 3 or 7 d following transfer of T cell-depleted CFSE-labelled splenocytes from dTg mice. Sections were stained with anti-B220 or PNA, and anti-IgM^a. Arrows indicate IgM^a+ cells in the B cell follicle (d 3) or germinal center (d 7). (C) B cell survival following transfer into NZB sHEL recipient mice injected with PBS alone (No Ab) or anti-CD4 Ab. Open circles indicate NZB IgTg mice and filled circles NZB dTg mice. % differences in the survival of transferred cells due to CD4⁺ T cell depletion: NZB IgTg  = 20.17±1.18; NZB dTg  = 4.55±0.67. (D) Serum IgM^a anti-HEL Ab production following depletion of CD4⁺ T cells in NZB dTg mice. Mice were injected with anti-CD4 mAb or PBS beginning 8 wks of age and bled every 2 weeks until 14 wks of age. P values were calculated using a two way ANOVA followed by Bonferroni post-hoc analysis.

Figure 4. Elevated BAFF levels in NZB mice enhance survival of transferred NZB dTg B cells.

(A) Serum BAFF levels in 6–12 wk old non-Tg (nTg), IgTg and dTg, B6 and NZB mice. (B) Baff mRNA expression in the splenocytes of 8–14 wk old non-Tg (nTg), IgTg and dTg, B6 and NZB mice. Horizontal lines represent the mean for each group examined. Significant p values for the difference between B6 and NZB mice are shown and were determined by one way ANOVA test (Kruskal-Wallis test) followed by Dunns' post test. (C) NZB sHEL recipient mice were injected with TACI-Ig or PBS alone, 1 d before transfer of CFSE-labelled NZB dTg B cells. Splenocytes were analyzed 3 d later by flow cytometry. Numbers indicate the percentage of surviving cells, determined by the ratio of CFSE⁺ B220⁺ cells to CFSE⁺B220^- cells as compared to a NZB non-Tg control. Asterisks indicate the significance level for comparison between B6 and NZB mice as determined by the Mann-Whitney test: * p<0.05, ** p<0.005, *** p<0.0005.

Figure 5. Heightened survival response of NZB dTg B cells to BAFF in-vitro.

(A) Up-regulation of Bcl-2 in NZB IgTg and dTg B cells. B cells isolated from B6 (open bars) or NZB (filled bars) IgTg or dTg mice were incubated with media alone or media containing HEL (100 ng/ml), BAFF (40 ng/ml), or a combination of both, for 20 or 96 h at 37°C. The percent Bcl-2 positive cells was determined by flow cytometry gating on the B220⁺ population. Results shown are for a single mouse from each strain and are representative experiment of four independent experiments in which a total of 2 nTg, 5 IgTg, 7 dTg B6 mice, and 3 nTg, 6 IgTg, 4 dTg NZB mice were examined. (B) Enhanced expression of Bcl-2 in all B cell subsets of NZB IgTg or dTg B cells after incubation with HEL and BAFF. Cells were isolated and incubated for 96 h at 37°C, as outlined in (A). The percent Bcl-2 positive cells was determined by flow cytometry gating on the B220⁺ population and using CD21 and CD24 antibodies to subset B cells, as shown in Table 1. (C) Scatterplots showing the proportion of BAFF-R⁺ cells in various B cell subsets in B6 (open circles) and NZB (closed circles), IgTg and dTg mice, gated as in (B). Background staining with a relevant isotype control was extremely low, and similar in B6 and NZB mice. Asterisks indicate the significance level for comparison between B6 and NZB mice as determined by the Mann-Whitney test: * p<0.05, ** p<0.005, *** p<0.0005.