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. 2010 Aug;62(8):2443-57.
doi: 10.1002/art.27515.

Prolonged effects of short-term anti-CD20 B cell depletion therapy in murine systemic lupus erythematosus

Kai W Bekar  1 , Teresa Owen, Robert Dunn, Travis Ichikawa, Wensheng Wang, Roger Wang, Jennifer Barnard, Sean Brady, Sarah Nevarez, Bruce I Goldman, Marilyn Kehry, Jennifer H Anolik
Affiliations

Prolonged effects of short-term anti-CD20 B cell depletion therapy in murine systemic lupus erythematosus

Kai W Bekar et al. Arthritis Rheum. 2010 Aug.

Abstract

Objective: Although B cells are implicated in the pathogenesis of systemic lupus erythematosus, the role of B cell depletion (BCD) as a treatment is controversial, given the variable benefit in human disease. This study was undertaken to test the effects of BCD therapy in a murine lupus model to better understand the mechanisms, heterogeneity, and effects on disease outcomes.

Methods: (NZB x NZW)F(1) female mice with varying degrees of disease severity were treated with an anti-mouse CD20 (anti-mCD20) antibody (IgG2a), BR3-Fc fusion protein (for BAFF blockade), or control anti-human CD20 monoclonal antibody (approximately 10 mg/kg each). Tissue samples were harvested and analyzed by flow cytometry. The development and extent of nephritis were assessed by monitoring proteinuria (using a urine dipstick) and by immunohistochemical analysis of the kidneys. Serum immunoglobulin levels were measured by enzyme-linked immunosorbent assay.

Results: After a single injection of anti-mCD20, BCD was more efficient in the peripheral blood, lymph nodes, and spleen compared with the bone marrow and peritoneum of normal mice as well as younger mice with lupus. Since depletion of the marginal zone and peritoneal B cells was incomplete and variable, particularly in older mice with established nephritis, a strategy of sequential weekly dosing was subsequently used, which improved the extent of depletion. BAFF blockade further enhanced depletion in the spleen and lymph nodes. Early BCD therapy delayed disease onset, whereas BCD therapy in mice with advanced disease reduced the progression of nephritis. These effects were long-lasting, even after B cell reconstitution occurred, and were associated with a reduction in T cell activation but no significant change in autoantibody production.

Conclusion: The lasting benefit of a short course of BCD therapy in lupus-prone mice with an intact immune system and established disease highlights the validity of this treatment approach.

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Figures

Figure 1
Figure 1. B cell depletion in lupus prone mice improves with prolonged treatment
10 wk old NZB/NZWF1 mice were treated with (A) a single dose of anti-mCD20 (300 μg) or control anti-human CD20 mAb intravenously and tissues were harvested at 1,2,3,4 weeks following treatment or (B) 1,2,3 or 4 weekly doses of anti-mCD20 and tissues were harvested one week after the last antibody injection. B cells were enumerated by flow cytometry, with subset definitions as defined in the Materials and Methods. B cell subsets are depicted as a normalized ratio (mean +/− SE) compared to control treated group with n=4 animals per group and time point. PBL, peripheral blood. SPLN, spleen. LN, lymph node. BM, bone marrow. PERIT, peritoneum. MZ, marginal zone. FO, follicular.
Figure 2
Figure 2. B cell depletion with anti-CD20 in older lupus mice is more variable
Six month old NZB/NZWF1 mice were treated with anti-mCD20 (n=8) or control antibody (IgG2a) (n=8) (300 μg) once per week for 4 weeks. Mice were selected based on two consecutive measures of proteinuria ≥ 100 mg/dl (2+) and < 300 mg/dl (3+). Younger NZB/NZWF1 mice (10 wk) (n=4) and C57BL6 mice (n=6) were treated with anti-mCD20 for comparison. (A) Splenic B cells (B220+) were quantitated by flow cytometry 1 week after the fourth and final antibody injection and displayed as a % of the total lymphocyte gate. Astericks indicate significant differences: **, p<0.001 by student’s t-test. *, p<0.05 by student’s t-test. (B) Serum anti-mCD20 concentration was measured at day 14 (closed diamonds) and day 28 (open circles) during the course of weekly injections (x 4) of anti-mCD20 (300 μg). Mice that were able to maintain good serum drug levels had more effective B cell depletion (splenic depletion at day 28 shown). Mice with 3+ or greater proteinuria at the time of treatment are depicted with squares; the remaining mice began treatment at 2+ proteinuria.
Figure 3
Figure 3. Residual B cells after anti-CD20 mediated BCD
Six month old NZB/NZW F1 mice (≥ 100 mg/dl proteinuria) were treated as in Figure 2 with anti-mCD20 or control antibody (IgG2a) (300 μg) once per week for 4 weeks. (A) Immunohistochemistry of spleens stained with GL-7 (GC stain) and B220 antibody (B cell marker) or antibodies against MOMA (green), IgM (red), and IgD (blue) from NZB/NZWF1 mice treated with anti-CD20 (top panels) or control antibody (middle panels). Magnification, x10. (B) Enumeration of GCs, based on size as calculated from IHC staining for GL7+, and flow cytometry detection. (C) Depletion of B cells is depicted in spleen, with additional characterization of residual B cells via flow cytometry expression of the indicated markers. GC B cells are defined as GL7+ cells in the B220+ gate. Fractions I, II, and III are defined based on CD21 and IgM expression, gated through B220+. MZ B cells are defined here as CD21+CD1d+, gated through B220+. Representative dot plots are shown.
Figure 4
Figure 4. B cell depletion is improved by combination BAFF blockade
Six month old NZB/NZWF1 mice (≥ 100 mg/dl proteinuria) were treated with anti-mCD20 (n=9), anti-mCD20 with BR3-Fc (n=4) or control antibody (IgG2a) (n=8) (300 μg) once per week for 4 weeks. B220+ B cells were enumerated 1 week after the final antibody injection in multiple tissues by flow cytometry. (A) Depletion of B cells (B220+) in spleen (SPLN), bone marrow (BM), peritoneum (PERIT), peripheral blood (PBL), and lymph node (LN), reported as the B220+ B cell % of the lymphocyte gate (mean +/− SE). (B) Depletion in B cell subsets in spleen, peritoneum and bone marrow. B cell subsets were defined as in the Methods.
Figure 5
Figure 5. B cell depletion prevents the progression of nephritis
(A) Six month old NZB/NZW F1 mice were treated with control antibody (IgG2a), anti-mCD20 alone (300 mg) or anti-mCD20 combined with BR3-Fc (8 mg/kg) once per week for 4 weeks. Mice were selected based on two consecutive measures of proteinuria ≥ 100 mg/dl and < 300 mg/dl. Proteinuria monitored with urine dipstick during the treatment. Mean +/−SE.*p<0.05. (B) Hematoxylin and eosin stained kidney sections anti-mCD20 treated and untreated NZB/NZWF1 mice (20x). Deposition of IgG in the glomeruli is unchanged with anti-CD20 (10x). (C) Kidney pathology was scored according to anatomic site as described in the Materials and Methods. GN, glomerulonephritis. IN, interstitial nephritis. VI, perivascular infiltration. (D) Prolonged follow-up. NZB/NZW F1 mice with 2+ protein (100 mg/dl) were treated at 24–28 weeks with control antibody (IgG2a,n=15), anti-mCD20 (300 mg, n=8) once per week for 4 weeks (Group A), anti-mCD20 combined with BR3-Fc (n=7) for 4 weeks (Group B), or anti-mCD20 (300 mg, n=5) 2x/week for 4 weeks (Group C). The difference in proteinuria free survival was significant between the groups (p=0.01). The timing of reconstitution is indicated by the bar. (E) Early B cell depletion delays lupus disease onset. 18 weeks old pre-diseased NZB/NZW F1 mice were treated with control antibody (IgG2a,n=5) or anti-mCD20 (300 mg, n=6) once per week for 4 weeks (start of treatment and beginning of reconstitution indicated by arrows). Proteinuria was monitored and is depicted here as the percentage of proteinuria <300 mg/dl in both treatment groups. The difference in proteinuria free survival was significant between the two groups (p=0.0007). The dot plots on the right indicate that immature transitional B cells predominate during early reconstitution after BCD. Analysis of splenocytes with total lymphocytes on the left and B220+ gated B cells on the right. In the CD23 v IgM dot plots red cells are AA4.1+ and indicative of ‘immature’ status, also seen on the IgM v B220 plots as a high IgM population.
Figure 6
Figure 6. B cell depletion has minimal impact on autoantibody production
(A) Effects on serum autoantibodies: NZB/NZWF1 mice with 2+ proteinuria were treated with anti-mCD20 (n=8), anti-mCD20 combined with BR3-Fc (CoTx) (n=7) or control antibody (IgG2a,n=6) (300 mg) once per week for 4 weeks. Immunoglobulin levels (mean +−SE) were assessed by ELISA before (baseline) and after treatment. The plot on the left shows anti-dsDNA levels before and 1 week after completion of treatment (4 wks after treatment start). There was no significant difference between baseline and any one of the treatment groups by student’s t-test. The middle graph depicts serum IgG, IgM, and anti-dsDNA in mice treated for 4 weeks with anti-CD20, or on the right anti-CD20 and BR3-Fc, and then followed as in Fig. 5D. Even with prolonged follow-up serum immunoglobulin levels do not decrease. The point of maximal B cell reconstitution rate is indicated by the arrow. (B) Effects on antibody secreting cells: ELISPOT results for IgG to dsDNA (anti-dsDNA)-secreting cells and total IgG-secreting cells in the spleens and bone marrows 1 week after 4 weekly treatments of anti-mCD20 (time point shown by asterick in A) (n=5) vs. control antibody (IgG2a,n=6) (time point shown by asterick in A). Each point represents an individual mouse with mean+/−SE also depicted.
Figure 7
Figure 7. Autoantibody independent effects of B cell depletion
Six month old NZB/NZWF1 mice were treated with anti-mCD20 (n=9), anti-mCD20 combined with BR3-Fc (n=4) or control antibody (IgG2a,n=8) (300 μg) once per week for 4 weeks as in Figure 5. (A) T cells were analyzed by FACS 1 week after the completion of 4 weeks of treatment. Activated (CD69+ of the CD4+ T cells) and memory (CD44hiCD62Llow) T cells in the spleen are reduced by B cell depletion therapy. Representative dot plots are shown. Data not shown for combination treatment. (B) The decrease in activated and memory T cells for each cohort in the spleen or LN. *, p=0.002, **, p=0.0002, ***, p=0.001, ****, p=0.03, *****, p=0.01 by student’s t-test
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