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doi: 10.1371/journal.pone.0005456. Epub 2009 May 6.

Crucial role for BAFF-BAFF-R signaling in the survival and maintenance of mature B cells

Affiliations

Crucial role for BAFF-BAFF-R signaling in the survival and maintenance of mature B cells

Melanie Rauch et al. PLoS One.2009.

Abstract

Defects in the expression of either BAFF (B cell activating factor) or BAFF-R impairs B cell development beyond the immature, transitional type-1 stage and thus, prevents the formation of follicular and marginal zone B cells, whereas B-1 B cells remain unaffected. The expression of BAFF-R on all mature B cells might suggest a role for BAFF-R signaling also for their in vivo maintenance. Here, we show that, 14 days following a single injection of an anti-BAFF-R mAb that prevents BAFF binding, both follicular and marginal zone B cell numbers are drastically reduced, whereas B-1 cells are not affected. Injection of control, isotype-matched but non-blocking anti-BAFF-R mAbs does not result in B cell depletion. We also show that this depletion is neither due to antibody-dependent cellular cytotoxicity nor to complement-mediated lysis. Moreover, prevention of BAFF binding leads to a decrease in the size of the B cell follicles, an impairment of a T cell dependent humoral immune response and a reduction in the formation of memory B cells. Collectively, these results establish a central role for BAFF-BAFF-R signaling in the in vivo survival and maintenance of both follicular and marginal zone B cell pools.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Binding of anti-BAFF-R antibodies to BAFF-R expressing Y3 rat myeloma cells.
Panel A. FACS analysis of a 1∶1 mixture of BAFF-R-IRES-GFP transfected and un-transfected Y3 myeloma cells. Upper plot: irrelevant isotype control antibody. Lower plots: the anti-BAFF-R antibodies 9B9 and 5A12 stained the BAFF-R expressing BAFF-R-IRES-GFP transfected Y3 myeloma cells but not the un-transfected GFP negative cells. Panel B. The same mixture of BAFF-R-IRES-GFP transfected and un-transfected Y3 myeloma cells was pre-incubated with or without the four different anti BAFF-R antibodies as indicated: 5A12, 9B6, 9B9 and 5H10 followed by HA-tagged BAFF, which was revealed by a PE labeled anti-HA antibody (upper plot right). 9B9 and 5H10 (lower plots), but not 5A12 and 9B6 (middle plots) were preventing the binding of HA-tagged BAFF to BAFF-R, revealing blocking capacity. Panel C. FACS analysis on the different B and T cell subsets for BAFF-R surface expression, as indicated for each plot. Dotted histograms represent isotype control stainings.
Figure 2
Figure 2. Circulating mature B cells and kinetic analysis of peripheral blood B and T cell depletion.
Upper graph: C57BL/6 mice were injected i.v. at day 0 with 0.5 mg of a given anti-BAFF-R antibody, as indicated. At day 14 the percentage of CD93−CD19+ mature B cells was determined by FACS analysis on the peripheral blood mononucleated cells. Each symbol represents an individual mouse. Statistical analysis revealed a significant difference for control versus 9B6, 9B9 and 5H10 (p<0.05). Lower graph: C57BL/6 mice were injected with either 0.5 mg of the anti-BAFF-R mAb 9B9 (white bars), or 0.5 mg of the anti-CD4 mAb GK1.5 (grey bars). Black bars represent PBS injected controls. The mAbs were injected at day 0 and the percentages of immature B cells (CD93+CD19+), mature B cells (CD93−CD19+), CD4 and CD8 T cells were determined by FACS at days 4, 7 and 10. Each column represents a time point: day 4 left column, day 7 middle and day 10 right column. Statistical analysis revealed a significant difference between PBS treated mice as compared to anti-BAFF-R mAb at each time point analyzed for mature B cells but not for immature B cells.
Figure 3
Figure 3. Circulating mature B cells in FcRγ deficient and Bcl2-transgenic mice following anti BAFF-R 9B9 injection.
Panel A. FcRγ−/− mice were injected at day 0 and the percentage of CD93−CD19+ mature B cells in the blood was determined at day 14 by FACS analysis. Non-injected FcRγ−/− mice were used as controls. The difference between FcRγ−/− treated versus untreated mice was statistically significant (p<0.05). Panel B. The percentage of mature B cells in the blood of C57BL/6 and Bcl2 transgenic mice at day 14 following the injection of 9B9 mAb. The difference between C57BL/6 and Bcl2 transgenic mice following 9B9 treatment was statistically significant (p<0.01).
Figure 4
Figure 4. Depletion of the B and T cell subsets following anti BAFF-R 9B9 antibody injection.
Panel A. Representative FACS plot of the immature (CD93+CD19+) and mature (CD93−CD19+) B cell compartments in the spleen of C57BL/6 mice; upper plots untreated control, lower plots day 14 after 0.5mg of anti-BAFF-R 9B9 injection. On the left side total splenocytes are depicted. On the right side, gated on mature B cells (CD93−CD19+) follicular (CD21+CD23+) and MZB (CD21highCD23low) B cells are shown. Panel B. Absolute numbers of splenic T1 and T2/3 immature B cells, B-2 and MZ B cells, CD4 and CD8 T cells in controls (black bars) and 9B9 injected C57BL/6 mice at day 14 after injection (white bars). 5 mice were analyzed for each group. A significant difference could be observed for T2/T3, B-2 and MZB cell numbers in control as compared to 9B9 injected mice. Panel C. Representative FACS plot analysis indicating the percentages of CD19+CD23+ B-2, CD19+CD11b+ B-1b and CD19+CD5+ B-1a B cells in the peritoneal cavity of control (upper dot plots) and C57BL/6 mice injected with 9B9 mAb at day 14 after injection (lower dot plots).
Figure 5
Figure 5. Turnover of splenic B cells following injection with anti-BAFF-R 9B9.
Panel A. Turnover of splenic B cell populations in control (black bars) and 9B9 injected C57BL/6 mice (white bars). C57BL/6 mice were injected with 1 mg of BrdU and BrdU was added to the drinking water. 10 days after splenic T1, T2/3, B-2 and MZ B cells were stained, sorted and the percentage of BrdU positive cells was determined by FACS analysis. Mean values with standard deviation are shown. 4 mice were analyzed for each group. Differences were statistically not significant. Panel B. Representative FACS plot analysis of the immature (CD93+CD19+) and mature (CD93−CD19+) B cell compartments in the spleen of C57BL/6 mice treated over a 5 months period with anti-BAFF-R 9B9 mAb. Depicted on the right side, CD21 and CD23 staining gated on immature B cells (upper plot) and on mature B cells (lower) plot. Indicated are the percentages of the cells represented in each quadrant.
Figure 6
Figure 6. Immunohistochemistry of spleen sections.
Spleen histology of C57BL/6 mice 14 days after injection of either the non-blocking 5A12 or blocking 9B9 anti-BAFF-R mAbs, as indicated. Cryosections were stained with anti-IgM (red) and CD90 (T cells) (green), left panels, and with anti-IgM (green) and anti-IgD (red), right panels, as indicated above. Magnification 240×.
Figure 7
Figure 7. Humoral immune response.
Panel A. 12 days after T dependent immunization serum levels of anti-NIP IgG were measured by ELISA in groups of 5 controls and 5 C57BL/6 mice injected either with anti-BAFF-R mAb 9B9 or anti-CD4 mAb GK1.5, as indicated. Immunization with NIP-ovalbumin was performed 10 days after injection of the 9B9 or the GK1.5 mAbs. The titer is defined as the serum dilution that gives OD values twice the background and is depicted on a logarithmic scale. Each symbol represents a mouse. A significant difference could be observed in the response of 9B9 as well as GK1.5 mAbs treated mice as compared to untreated. Panel B. Sera of mice subjected to different treatments was collected at different time points as indicated for each group. Mice received repeated injections of the mAb every third week, over the indicated period of time. Levels of anti-NIP IgG were measured. Each symbol represents a mouse. The titer is defined as the serum dilution that gives OD values twice the background and is depicted on a logarithmic scale. Groups 1 and 4: serum collected at day 1. Mice were treated with 5A12 mAb (non-blocking) and 9B9 mAb (blocking), respectively from day 1 to day 14. Mice were not immunized. Groups 2 and 5: serum was collected at day 74. Mice were treated with 5A12 mAb (non-blocking) 9B9 mAb (blocking), respectively from day 1 to day 74. Mice were only boosted at day 60. Groups 3 and 6 Serum was collected at day 74. Mice were treated with 5A12 mAb (non-blocking) and 9B9 mAb (blocking), respectively from day 1 to day 74. Mice were primed with NIP-ovalbumin at day 14 and boosted at day 60. Group 7. Serum was collected at day 94. Mice were treated with 9B9 mAb (blocking) from day 60 to day 74. Mice were primed with NIP-ovalbumin at day 14 and boosted at day 80. Statistical analysis revealed a significant difference for group 3 versus group 6, but not for group 3 versus 7.
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