A soluble BAFF antagonist, BR3-Fc, decreases peripheral blood B cells and lymphoid tissue marginal zone and follicular B cells in cynomolgus monkeys

Abstract

BAFF (also known as BLyS), a member of the tumor necrosis factor superfamily, plays a critical role in the maturation and development of B cells. BAFF has three receptors on B cells, the most crucial of which is BR3. In this study, we demonstrate the biological outcome of BAFF blockade in cynomolgus monkeys using a soluble fusion protein consisting of human BR3 and human IgG1 Fc. In vitro, BR3-Fc blocked BAFF-mediated survival and proliferation of cynomolgus monkey B cells. Weekly treatment of cynomolgus monkeys with BR3-Fc for 13 to 18 weeks resulted in significant B-cell reduction in the peripheral blood and in lymphoid organs. CD21(high) B cells in lymphoid tissues, a subset analogous to human marginal zone B cells, expressed nearly twofold higher BR3 levels than did CD21(med) B cells. Lymphoid tissue flow cytometric analysis showed that BR3-Fc reduced this CD21(high) B-cell subset to a greater extent than it reduced CD21(med) B cells. Dual-label immunohistochemistry and morphometric image analysis supported these results by demonstrating that BR3-Fc reduced a significant proportion of the B cells within the splenic inner and outer marginal zones. These findings should prove very useful in guiding the desired therapeutic use of BR3-Fc for autoimmune diseases in the clinic.

Figure 1

Histomorphology of a normal cynomolgus monkey splenic lymphoid follicle. Sections of cynomolgus splenic lymphoid follicle stained for the B-cell marker CD20 (blue) and α-SMA (brown) (A), CD20 (blue) and the T-cell marker CD3 (brown) (B), and IgD (green) and F-actin (red) (C). A and B: Serial paraffin-embedded sections; C: frozen section. A: The band of SMA⁺ myofibroblasts (MFs) divides the IMZ from the OMZ. B: Periarteriolar lymphoid sheath (PALS) of CD3⁺ T cells that surrounds the central arteriole (CA) adjacent to the CD20⁺ lymphoid follicle. C: IgD expression differentiates between the GC⁻ IgD⁻ mantle zone (strongly IgD⁺) and the IMZ (weakly to moderately strongly IgD⁺). CA, central arteriole; MZ, mantle zone; PALS, periarteriolar lymphoid sheath; PZ, peripheral zone. Bars = 100 μm.

Figure 2

BR3-Fc blocks BAFF-mediated survival and proliferation of cynomolgus monkeys B cells in vitro. Cynomolgus monkey B cells were isolated from PBMCs by MACS sorting. A: B cells were cultured with human CD40L (1 μg/ml) and IL-2 (2 ng/ml) for 4 days. B cells were then washed and cultured for 0 to 8 days with human IL-2 in the presence or absence (as indicated) of soluble murine BAFF (5 μg/ml), BR3-Fc (50 μg/ml), or human IgG (50 μg/ml). Viability was assayed at the indicated time points by trypan blue exclusion. Data show average viability (n = 3/time point); error bars show ±SD in this value. B: B cells were cultured in the presence or absence of BAFF (5 μg/ml), anti-IgM (10 μg/ml), and BR3-Fc or human IgG, as indicated. Proliferation was assessed by incorporation of tritiated thymidine. Shown are mean cpm (n = 3) ± SD.

Figure 3

Flow cytometric analysis illustrates BR3-Fc-mediated reduction and subsequent recovery of peripheral blood B cells in cynomolgus monkeys. Cynomolgus monkeys were treated weekly with 0, 2, or 20 mg/kg BR3-Fc for 18 weeks (127 days) and allowed to recover until week 41 (day 287). The total absolute CD20⁺ B-cell count and B-cell subset counts (as indicated) were assessed by FACS at the specified number of days after the first dose, as described in the text. A: BR3-Fc induced a non-dose-dependent decrease in the peripheral blood absolute CD20⁺ B-cell count through dosing on day 127, with eventual gradual recovery by day 287. The reduction in total CD20⁺ B-cell counts relative to the control group was statistically significant from day 29 through the terminal necropsy at day 127 (week 18) for the 2-mg/kg dose group (all 2-mg/kg dose group animals were necropsied at week 18) and from day 64 through day 225 (14 weeks after the end of dosing) for the 20-mg/kg dose group. B: BR3-Fc had no effect on peripheral blood CD20⁺ CD21⁻ B cells. C: BR3-Fc had no effect on peripheral blood CD21⁺CD27⁺ (memory) B cells, compared with the control group. D: BR3-Fc induced a non-dose-dependent decrease in the peripheral blood CD21⁺CD27⁻ (naïve) B-cell count, with eventual gradual recovery by day 287, as was seen for the total CD20⁺ B cells in A. The reduction in peripheral blood CD21⁺CD27⁻ B cells was a statistically significant decrease relative to the control group from day 15 through the terminal necropsy at day 127 (week 18) for the 2-mg/kg dose group (all 2-mg/kg dose group animals were necropsied at week 18), and from days 15 to 211 for the 20-mg/kg dose group. Total absolute cell counts (×10³/ml) ± SD are shown at each time point.

Figure 4

FACS analysis illustrates BR3-Fc-mediated reduction and subsequent recovery of total B cells (CD20⁺) in lymphoid tissue of cynomolgus monkeys. Cynomolgus monkeys were treated weekly with 0, 2, or 20 mg/kg BR3-Fc for 18 weeks (127 days) and allowed to recover until week 41 (day 287). The B-cell fraction of tissue lymphocytes was assessed by FACS at the specified number of weeks after the first dose, as described in the text. CD20⁺ B cells in the bone marrow (BM); spleen; and inguinal (LN-Ing), mandibular (LN-Man), and mesenteric (LN-Mesen) lymph nodes were analyzed by FACS at week 13 (A), week 18 (B), and week 41 (C) after the first dose. A: BR3-Fc at 20 mg/kg induced a decrease in the total B-cell fraction in the spleen and the inguinal and mandibular lymph nodes; this decrease was statistically significant in the spleen (P = 0.0003). B: Both doses of BR3-Fc induced a decrease in the total B-cell fraction in all tissues examined without a clear dose-dependent effect at week 18. Statistically significant decreases are noted with P values. C:By the end of recovery period at week 41, all tissue B cells had essentially returned to baseline levels. The mean total B-cell fraction (CD20⁺) of lymphocytes ± SD is shown.

Figure 5

Dual-label IHC for CD20 (blue) and smooth muscle actin (SMA) (brown) on week-18 spleens from BR3-Fc-treated cynomolgus monkeys illustrates a decrease in follicular CD20⁺ immunostaining as well as in the OMZ of BR3-Fc-treated spleens. Representative paraffin-embedded sections of spleen dual-labeled for CD20 (blue) and SMA (brown) from two untreated control animals (left), two animals treated for 18 weeks with 2 mg/kg BR3-Fc (middle), and two animals treated with 20 mg/kg BR3-Fc (right). These panels illustrate that both doses of BR3-Fc induced a decrease in both total follicle size, particularly in the CD20⁺ follicular lymphocytes between the GC and the SMA⁺ band, indicated by double-headed arrows, and in the outer marginal zone area (CD20⁺ lymphocytes outside the SMA⁺ band identified by asterisks). Bars = 200 μm.

Figure 6

Dual-label fluorescent IHC for IgD (green) and F-actin (red) on week-18 spleens from BR3-Fc-treated cynomolgus monkeys illustrates a decrease in IgD⁺ lymphocytes in BR3-Fc-treated spleens. Representative frozen sections of spleen dual-labeled for IgD (green) and F-actin (red) from two untreated control animals (left), two animals treated for 18 weeks with 2 mg/kg BR3-Fc (middle), and two animals treated with 20 mg/kg BR3-Fc (right). These panels illustrate that both doses of BR3-Fc induced a decrease in the follicular area expressing IgD, particularly in the lymphocytes that were strongly IgD⁺ (the mantle zone). In addition, BR3-Fc also induced a decrease in the IgD⁺ lymphocytes in the weakly to moderately strongly IgD⁺ lymphocytes in the IMZ (illustrated by double-headed arrows as the area between the strongly IgD⁺ mantle zone and the red band of myofibroblasts). MF, myofibroblasts. Bars = 100 μm.

Figure 7

Higher BR3 expression in CD21^high (marginal zone) B cells and BR3-Fc-mediated targeting of these CD21^high B cells in the spleen and lymph nodes of cynomolgus monkeys. Cynomolgus monkeys were treated weekly with 0, 2, or 20 mg/kg BR3-Fc for 18 weeks. A and B: Spleen samples from animals in the 0-mg/kg control group were stained with antibodies to CD20, CD21, and BR3 and assessed for expression of BR3. A: Representative FACS expression of BR3 for B cells expressing CD21^med (dotted line) or CD21^high (bold solid line). B: A summary of BR3 MFI for the two different B-cell subsets. Lines correspond to the average BR3 MFI for each subset (CD21^med and CD21^high) and demonstrate that the CD21^high B cells have a higher level of BR3 expression than do CD21^med B cells. C: The CD21^high (marginal zone) B-cell fraction of lymphocytes was assessed by flow cytometry at 18 weeks after the first dose, as described in the text. The mean CD21^high B-cell fraction ± SD is shown. LN-Ing, LN-Man, and LN-Mesen are inguinal, mandibular, and mesenteric lymph nodes, respectively. P values denote decreases that are statistically different from control.