Similarities and differences between selective and nonselective BAFF blockade in murine SLE

Abstract

B cells have multiple roles in immune activation and inflammation separate from their capacity to produce antibodies. B cell depletion is currently under intense investigation as a therapeutic strategy for autoimmune diseases. The TNF family members B cell-activating factor of the TNF family (BAFF) and its homolog A proliferation-inducing ligand (APRIL) are B cell survival and differentiation factors and are therefore rational therapeutic targets. We compared the effects of BAFF receptor-Ig, which blocks only BAFF, with those of transmembrane activator and calcium modulator ligand interactor-Ig, which blocks both BAFF and APRIL, in a murine SLE model. Both reagents prolonged the life of NZB/W F1 mice when given either before or after disease onset. Many immunologic effects of the 2 reagents were similar, including B cell and B cell subset depletion and prevention of the progressive T cell activation and dendritic cell accumulation that occurs with age in NZB/W mice without substantial effects on the emergence of the IgG anti-double-stranded DNA response. Furthermore, both reagents inhibited the T cell-independent marginal zone B cell response to particulate antigen delivered i.v., but not the B1 B cell response to the same antigen delivered i.p. In contrast, blockade of both BAFF and APRIL, but not blockade of BAFF alone, reduced the serum levels of IgM antibodies, decreased the frequency of plasma cells in the spleen, and inhibited the IgM response to a T cell-dependent antigen. The differences between selective and nonselective BAFF blockade are relevant to the choice of a BAFF blocking agent for the treatment of autoimmune and malignant diseases.

Figure 1

BAFF-R–Ig serum levels in SCID mice. (A) SDS-PAGE of immunoprecipitated control mouse IgG2a (lane 1) and sera from SCID mice injected with either Ad–TACI-Ig (lane 2) or Ad-BAFF-R–Ig (lane 3). TACI-Ig is a 94-kDa dimer; BAFF-R–Ig is a 37-kDa monomer. (B) Mean ± SD serum BAFF-R–Ig levels over time in NZB/W F1 mice (n = 10 per group) injected at day 0 with 10⁹ pfu Ad–BAFF-R–Ig alone or together with 6 doses of 100 μg CTLA4Ig.

Figure 2

Ad–BAFF-R–Ig prolongs survival of NZB/W F1 mice. Kaplan-Meier plots of proteinuria-free survival (A) and overall survival (B) in treated NZB/W F1 mice aged 18–20 weeks. Mice treated with Ad–BAFF-R–Ig alone or together with CTLA4Ig had prolonged survival (P < 0.0001, Ad–BAFF-R–Ig versus control; P < 0.003, Ad–BAFF-R–Ig/CTLA4Ig versus Ad-LacZ/CTLA4Ig) and prolonged proteinuria-free survival compared with untreated or irrelevant adenovirus controls (P < 0.001, Ad–BAFF-R–Ig versus untreated; P < 0.01, Ad–BAFF-R–Ig/CTLA4Ig versus Ad-LacZ/CTLA4Ig). (C) Time of appearance of high-titer IgG anti-dsDNA antibodies. In both Ad–BAFF-R–Ig– and Ad–BAFF-R–Ig/CTLA4Ig–treated groups (P < 0.0013 and P < 0.0139, respectively), there was a delay of approximately 6 weeks in the appearance of IgG anti-dsDNA antibodies compared with the relevant control.

Figure 3

Ad–BAFF-R–Ig does not alter serum Ig levels. Mean ± SD serum IgM (A) and IgG (B) in Ad–BAFF-R–Ig–treated NZB/W F1 mice were not significantly different from control Ad-LacZ–treated mice. Low IgG levels at 150 days in control mice reflect losses due to proteinuria in these mice. (C) The frequency of IgG-secreting cells per 10³ or 10⁵ total spleen cells (4–5 per group) was determined by ELISpot assay and was not significantly different in treated mice compared with controls. (D) Mean + SD number of IgM or IgG antibody–secreting B cells per spleen from control and treated NZB/W F1 mice. There was a significant reduction in the number of IgG-secreting cells in both Ad–BAFF-R–Ig–treated groups (P < 0.03) and of IgG anti-dsDNA–secreting cells in the Ad–BAFF-R–Ig/CTLA4Ig group (P < 0.04) compared with the relevant controls. Asterisks indicate significant differences.

Figure 4

Ad–BAFF-R–Ig prevents renal inflammation. Histologic analysis of kidneys from 36-week-old control (A, C, and E) and treated (B, D, and F) NZB/W mice. Control kidneys had perivascular interstitial infiltrates (arrows), glomerulonephritis, and tubular casts (A) as well as Ig deposits (E). Ad–BAFF-R–Ig treatment at 18–20 weeks prevented glomerulonephritis and interstitial inflammation (B and D) but did not prevent the deposition of IgG in the glomeruli (F). Magnification, ×40 (A–D); ×10 (E and F).

Figure 5

Ad–BAFF-R–Ig/CTLA4Ig induces disease remission. Kaplan-Meier plots of proteinuria-free (A) and overall survival (B) in NZB/W F1 mice treated at age 28–30 weeks (arrows) with Ad–BAFF-R–Ig, Ad–TACI-Ig, or Ad-LacZ, each given with 6 doses of CTLA4Ig, and untreated controls. Ad–BAFF-R–Ig or Ad–TACI-Ig treatment delayed proteinuria (A; P < 0.0032, Ad–BAFF-R–Ig/CTLA4Ig versus Ad-LacZ/CTLA4Ig; P < 0.0006, Ad–TACI-Ig/CTLA4Ig versus Ad-LacZ/CTLA4Ig) and prolonged survival (B; P < 0.0035, Ad–BAFF-R–Ig/CTLA4Ig and Ad–TACI-Ig/CTLA4Ig versus Ad-LacZ/CTLA4Ig). (C) Histologic analysis of kidneys showed significantly less glomerular damage (Glom; P < 0.01) and interstitial inflammation (Int; P < 0.05) in the kidneys of both sets of treated mice compared with Ad-LacZ controls. A score of 4.5 indicates death of an animal due to uremia. Horizontal lines denote the mean score for each group.

Figure 6

IgM, IgG1, and IgG2a anti-oxazolone titers. Mean ± SD anti-oxazolone (Anti-ox) titers in Ad-LacZ–, Ad–BAFF-R–Ig–, and Ad–TACI-Ig–treated NZB/W F1 mice (n = 8 per group). Titers were measured 1 and 2 weeks after immunization (IgM: P < 0.003, Ad–TACI-Ig versus Ad-LacZ and Ad–BAFF-R–Ig; IgG1 and IgG2a: P < 0.006, Ad–BAFF-R–Ig and Ad–TACI-Ig versus Ad-LacZ; P < 0.004, Ad–BAFF-R–Ig and Ad–TACI-Ig versus naive). Asterisks indicate significant differences.

Figure 7

Ad–BAFF-R–Ig and Ad–TACI-Ig inhibit the splenic response to bacteria but have no effect on peritoneal B1 cells. Anti-PC response in NZB/W F1 mice following i.p. or i.v. immunization with heat-killed streptococci. Following i.p. injection, serum IgM anti-PC antibody levels (A) and IgM ELISpot analysis of the peritoneal cells (B) showed no difference between treatment groups and controls. Following i.v. injection, serum IgM anti-PC antibody levels failed to increase following immunization in treated mice (C; P < 0.01, Ad–BAFF-R–Ig and Ad–TACI-Ig versus Ad-LacZ), and ELISpot analysis of the spleen cells (D) showed that both Ad–BAFF-R–Ig and Ad–TACI-Ig suppressed the IgM anti-PC response (P < 0.01, Ad–TACI-Ig versus Ad-LacZ for both IgM and anti-PC; P < 0.04, Ad–BAFF-R–Ig versus Ad-LacZ for both IgM and anti-PC). Asterisks indicate significant differences.

Figure 8

Both Ad–BAFF-R–Ig and Ad–TACI-Ig deplete marginal zone B cells in M167 mice. Mice were immunized with heat-killed streptococci after pretreatment with Ad–BAFF-R–Ig or Ad–TACI-Ig. Following immunization, an increase in serum IgM levels (A) and IgM anti-PC antibodies (B) was seen only in the control group (P < 0.01, Ad-LacZ versus Ad–BAFF-R–Ig and Ad–TACI-Ig for both IgM and anti-PC). (C) FACS analysis showed that both Ad–BAFF-R–Ig and Ad–TACI-Ig depleted marginal zone B cells (CD19gated/IgM⁺/CD1d^hi: P < 0.03 for either Ad-TACI-Ig or Ad-BAFF-R-Ig treatment versus untreated controls). Similar depletion was observed in treated mice that were not immunized (not shown). (D) Both treatments prevented the expansion of M167⁺B220^lo plasmablasts (left half of red box, P < 0.01 for either Ad-TACI-Ig or Ad-BAFF-R-Ig treatment versus untreated controls; numbers represent total number of M167⁺ cells). Representative FACS plots are shown.

Figure 9

TACI-Ig protein inhibits the response of marginal zone B cells. Anti-PC response following i.v. immunization of M167 mice with heat-killed streptococci after pretreatment with BAFF-R–Ig or TACI-Ig protein as described in Methods. Only the TACI-Ig–treated group manifested a significant reduction in serum IgM levels (A; P < 0.01 versus IgG2a and BAFF-R–Ig), serum IgM anti-PC antibodies (B; P < 0.02 versus IgG2a), the number of IgM- and anti-PC antibody–secreting cells in spleen (C; P < 0.02 versus IgG2a and BAFF-R–Ig), and the number of M167⁺B220^lo B cells (D; P < 0.03 versus BAFF-R–Ig; P < 0.02 versus IgG2a) and plasmablasts quantitated as in Figure 8 (P < 0.02 versus BAFF-R–Ig and IgG2a). Asterisks indicate significant differences.

Figure 10

TACI-Ig depletes plasma cells in NZM2410 mice. NZM2410 mice (14 weeks old) were injected with a single dose of Ad–TACI-Ig, Ad–BAFF-R–Ig, or Ad-LacZ. Serum IgM (A) and IgG (B) were decreased only in the Ad–TACI-Ig–treated group (P < 0.004 for both IgM and IgG, ANOVA). (C) ELISpot analysis of spleen and bone marrow cells 4 weeks after treatment showed a significant reduction in the frequency of IgM and IgG producing cells in the spleen and bone marrow only in the Ad–TACI-Ig–treated group (P < 0.003, Ad–TACI-Ig versus Ad-LacZ for both IgM and IgG).

Figure 11

Depletion of splenic plasma cells by Ad–TACI-Ig. Immunohistochemistry of spleens stained with anti-IgM (green) and -IgD (red) from NZB/W F1 mice treated with Ad-LacZ (top panels), Ad–BAFF-R–Ig (middle panels), and Ad–TACI-Ig (bottom panels). IgM-containing plasma cells were abundant in the red pulp of control and Ad–BAFF-R–Ig–treated mice but were smaller and less frequent in Ad–TACI-Ig–treated mice. Anti-IgD and anti-IgM or anti-IgG staining of spleens from NZM2410 mice that were treated 1 week previously with Ad-LacZ, Ad–BAFF-R–Ig, Ad–TACI-Ig (bottom center panel), or left untreated (bottom right panel) similarly shows that both IgM and IgG plasma cells were depleted only in the spleens of Ad–TACI-Ig–treated mice. Magnification, ×10.

Figure 12

TACI-Ig and BAFF-R–Ig have similar effects on the germinal center response. Following NP-KLH immunization in NZM2410 mice, the serum IgM anti-NP response was significantly reduced only in the Ad–TACI-Ig–treated mice [A; NP(30)-BSA, P < 0.005, Ad–TACI-Ig versus Ad-LacZ and Ad–BAFF-R–Ig], while the IgG anti-NP response [B; NP(3)-BSA]was variable and not significantly different between treated mice and controls. Results are shown at week 2 for IgM and week 3 for IgG.