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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Serum B lymphocyte stimulator (BLyS) is increased in autoimmune diseases, both in animal models and in humans. This study examined the effect of BLyS blockade in 3 animal models of lupus.

Methods

Antibodies and lupus-like disease manifestations were examined in mice after administration of a single injection of an adenoviral construct for the transmembrane activator and CAML interactor receptor (AdTACI) that produces high serum levels of TACI-Fc fusion protein.

Results

In C57BL/6 (B6) lpr/lpr mice (B6.lpr/lpr), which were used to model autoimmunity in the absence of severe disease, treatment of younger mice with AdTACI prevented the development of hypergammaglobulinemia. In contrast, use of AdTACI for BLyS blockade had only transient effects on the levels of IgG in normal B6 mice. AdTACI blocked the development of autoantibodies in younger B6.lpr/lpr mice and reversed the production of autoantibodies in older B6.lpr/lpr mice, and also reduced the numbers of splenic plasma cells. In MRL.lpr/lpr mice, which were used to examine disease manifestations, AdTACI reduced the extent of glomerulonephritis and proteinuria and improved survival, but had little effect on T cell infiltration and interstitial nephritis. However, in (NZB × NZW)F1 mice, AdTACI induced neutralizing anti-TACI antibodies and failed to reduce the numbers of B cells.

Conclusion

BLyS blockade has little effect on IgG levels in normal mice, but reverses the production of spontaneously produced IgM and IgG autoantibodies in the setting of established autoimmunity. Blockade of BLyS ameliorates B cell–dependent disease manifestations even in the MRL.lpr/lpr model, but its effectiveness on autonomous T cell aspects of the disease is limited. Moreover, its effectiveness is neutralized by anti-TACI antibodies when present. These results provide a basis for understanding the potential effects of BLyS blockade in human disease.

B lymphocyte stimulator (BLyS; also known as TALL-1, THANK, zTNF4, and BAFF) regulates the survival of B cells in the periphery (1–5). Three receptors for BLyS have been identified; these are transmembrane activator and CAML interactor (TACI), B cell maturation antigen (BCMA), and BAFF receptor (BAFF-R), each of which is expressed in B cells (6–12). TACI and BCMA also bind APRIL. BAFF-R is specific for BLyS and mediates its effect on B cell survival (12–14).

Forced overexpression of BLyS in otherwise-normal mice induces a lupus-like disease, with autoantibodies and immune complex deposits in the kidney (15, 16). Serum levels of BLyS are significantly higher in mouse models of lupus than in normal mice (17). Patients with systemic lupus erythematosus (SLE) have increased serum levels of BLyS compared with matched, healthy controls (18, 19). These observations suggest a role for BLyS in the production of autoantibodies and autoimmune disease in murine and human SLE.

Initial trials of BLyS blockade are underway in subjects with SLE. Animal models of lupus-like disease give support for such a treatment strategy, but the evidence regarding the mechanism of its effect has been contradictory. Injection of a soluble TACI-Fc fusion protein in (NZB × NZW)F1 mice resulted in a modest delay in the appearance of proteinuria and in mortality, but serum autoantibody levels were not altered (17). However, BAFF-R-Fc inhibited anti-DNA antibody production in the same mouse model (14). The difference in the effect of the 2 inhibitors raises the question as to whether different approaches to blockade have different effects on autoantibody production. In addition, given that TACI-Fc reduced the numbers of B cells but did not reduce the autoantibody levels, an additional question arising from these previous studies is whether BLyS blockade alters the survival of plasma cells as well as that of mature B cells, a potentially important point in considering different B cell–directed therapies for autoimmunity. A related question relevant to human therapy is whether BLyS blockade, rather than acting simply as a preventative measure, could be effective in reducing the levels of circulating autoantibodies in older mice that have established autoantibody production.

We used an adenoviral approach to determine the effect of stable, high levels of soluble TACI in 3 different models of lupus-like B cell autoimmunity. Experiments that focused on B cell autoimmunity more than disease pathogenesis used C57BL/6 (B6) lpr/lpr (B6.lpr/lpr) mice, which develop autoantibodies, including anti–double-stranded DNA (dsDNA) and rheumatoid factor (RF), and glomerulonephritis but not the rapidly progressive disease observed in MRL.lpr/lpr mice (20). Both the (NZB × NZW)F1 and MRL.lpr/lpr models were examined in our experiments to determine the effects of BLyS blockade on disease manifestations.

Our results demonstrated that blockade with soluble TACI, similar to soluble BAFF-R, is able to block autoantibody production, and this observation could be extended to the B6.lpr/lpr and MRL.lpr/lpr models. Three clinically important results arise from this study. In comparisons of normal B6 mice and B6.lpr/lpr mice, high-dose TACI-Fc reduced the levels of serum autoantibodies and the number of plasma cells in the autoimmune setting, but did not eliminate circulating IgG in the normal mice, suggesting that BLyS blockade can reduce the number of autoantibody-producing cells without eliminating long-lived, nonautoreactive plasma cells. In the MRL.lpr/lpr mice, BLyS blockade did not reduce T cell infiltrates in the kidney, but nevertheless reduced proteinuria and improved survival, indicating that this approach has selective effects on different aspects of the disease. In female (NZB × NZW)F1 mice, spontaneously produced antibodies to TACI were detected, which correlated with reduced serum levels of TACI, raising the possibility that some lupus patients may also be resistant to such therapy through a similar mechanism. Thus, our analysis of BLyS blockade in 3 experimental models shows the promise and the pitfalls of this therapeutic approach.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Recombinant adenoviral vectors encoding TACI-Fc (AdTACI) and green fluorescent protein (AdGFP).

A fusion gene construct was obtained from Human Genome Sciences (Rockville, MD), in which the extracellular domain of human TACI was fused with the Fc portion of human IgG1. The extracellular domain of human TACI was amplified as a Bam HI–Xba I fragment and cloned into a human IgG Fc cassette. The complementary DNA (cDNA) encoding the TACI-Fc fusion protein was cloned into pcDNA3 at Bam HI–Xho restriction sites. The fusion protein was then expressed in transfected Cos-7 cells. The TACI-Fc cDNA was cloned into an adenoviral shuttle vector purchased from Quantum Biotechnologies (Carlsbad, CA). The recombinant adenoviral vector AdTACI was then made by cotransfection of 293 cells with the linearized shuttle vector containing the TACI-Fc with a large fragment of the adenovirus DNA. Recombinant adenoviral vectors that transduce expression of soluble TACI were screened by in situ immunofluorescence staining of the plaques with an anti-human IgG1 antibody. A control adenoviral vector, AdGFP, was similarly constructed with the cDNA for GFP.

Analysis of TACI-Fc by immunoprecipitation and immunoblot.

The culture supernatants of Cos-7 cells were transduced with pcDNA3. TACI-Fc or AdTACI were immunoprecipitated with protein A–conjugated Sepharose 4B. The immunoprecipitated proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto the nitrocellulose membrane. The blots were probed with peroxidase-conjugated anti-human IgG1.

Treatment of mice with AdTACI.

B6 and MRL strains of +/+ and lpr/lpr mice and NZB × NZW mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained at the Animal Resource Program of the University of Alabama at Birmingham in accordance with institutional policies for animal care and use. Mice were intravenously injected with a single dose of 109 plaque-forming units (PFU) of AdTACI or AdGFP.

Measurement of TACI and specific and total antibodies in serum.

Enzyme-linked immunosorbent assay (ELISA) was used for measurement of serum levels of TACI. ELISA plates (Costar, Cambridge, MA) were coated with 2 μg/ml polyclonal anti-human IgG (mouse IgG absorbed; Southern Biotechnology, Birmingham, AL) in phosphate buffered saline and blocked with 3% bovine serum albumin. Sera diluted 1:100 were added and the plates were incubated for 30 minutes. After washing, 0.2 μg/ml biotin-conjugated recombinant BLyS (BLyS protein, a trademark of Human Genome Sciences) was added and incubated for another 30 minutes. After incubation with 0.1 μg/ml peroxidase–streptavidin (Southern Biotechnology), the reaction was developed with tetramethylbenzidine substrate buffer (Sigma, St. Louis, MO) and read in an ELISA plate reader (Bio-Rad, Richmond, CA) at 450/655-nm wavelength. A standard curve was made using serially diluted affinity-purified TACI-Fc, and serum levels of TACI-Fc were calculated. Serum antibodies to TACI were measured in plates precoated with purified His-tagged human TACI and detected with anti-mouse IgG. A protein encoded by the same vector (pET-32a; Novagen, Madison, WI), containing all elements except the TACI sequence, was used as a control.

For measurement of responses to specific antigens, mice were injected intraperitoneally with 100 μg trinitrophenyl–keyhole limpet hemocyanin (TNP-KLH)–conjugated protein, and serum levels of IgM and IgG anti-TNP antibody were measured as previously described (21). Serum levels of total Ig, anti-dsDNA antibodies, and RF in the IgM, IgG1, and IgG2a subclasses were measured by ELISA as previously described (20). Briefly, 96-well ELISA plates were coated with rabbit or human IgG for RF assays or thymus dsDNA for anti-dsDNA assays, and then incubated with dilutions of serum. Bound antibodies were detected with antibodies to the appropriate isotype of mouse Ig (Southern Biotechnology).

Flow cytometry and immunohistochemistry.

Immunohistology and flow cytometry were conducted as previously described (22, 23). These analyses were performed with antibodies to mouse CD3, CD4, CD8, CD23, CD138, B220 (PharMingen, San Diego, CA), IgA, IgG, IgM (Southern Biotechnology), CD21 (a gift from Dr. Michael Holers), CD19 (a gift from Dr. Douglas Fearon), and metallophilic macrophages 1 (MOMA-1; a gift from Dr. Georg Kraal).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Characterization of the recombinant adenoviral vector encoding TACI-Fc.

To examine the effect of sustained, high-level blockade of BLyS and APRIL on autoimmune disease, we constructed a recombinant adenoviral vector, AdTACI, that encodes a human TACI-Fc fusion protein. Expression of a protein of appropriate molecular weight that bound the recombinant BLyS protein was confirmed by transduction of Cos-7 cells (results not shown).

In the B6.+/+ (normal) and B6.lpr/lpr mice, intravenous inoculation with AdTACI, but not control AdGFP, resulted in serum levels of TACI-Fc that were higher than 100 μg/ml for at least 10 weeks (Figure 1). To confirm the biologic activity of the adenovirus-encoded TACI-Fc, B6.+/+ and B6.lpr/lpr mice were inoculated intravenously with AdTACI or AdGFP and then challenged with a T cell–dependent antigen, TNP-KLH. Inoculation of the mice with AdTACI, but not AdGFP, blocked the production of IgM and IgG anti-TNP antibodies (results not shown), as has been reported previously in mice receiving repeated intraperitoneal injections (5). These results indicate that inoculation with AdTACI produces high levels of soluble, functional TACI-Fc in vivo.

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Figure 1. Serum levels of the transmembrane activator and CAML interactor (TACI)-Fc fusion protein after adenoviral TACI (AdTACI) inoculation. Six-week-old B6.+/+ and B6.lpr/lpr mice were intravenously inoculated with 109 plaque-forming units of control adenoviral green fluorescent protein (AdGFP) or AdTACI. Serum levels of TACI-Fc were measured weekly by enzyme-linked immunosorbent assay. The results are presented as the mean ± SD μg/ml in 8–10 mice per group.

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Blockade of hypergammaglobulinemia by TACI-Fc in lpr/lpr mice.

Fas-deficient lpr/lpr mice develop an age-dependent increase in serum Ig. To determine whether TACI-Fc expression could inhibit the development of hypergammaglobulinemia or reverse it, 6-week-old B6.+/+ mice, 6-week-old B6.lpr/lpr mice, which had not developed high levels of serum Ig, and 16-week-old B6.lpr/lpr mice, which had established hypergammaglobulinemia, were inoculated intravenously with AdTACI. The serum levels of total IgM, IgG1, and IgG2a were followed up for up to 10 weeks after treatment. In B6.+/+ mice, serum levels of IgM were rapidly reduced 1 week after the inoculation with AdTACI, and were maintained at low levels over the 7-week course of the experiment (Figure 2A). In contrast, levels of IgG1 and IgG2a never dropped lower than 50% of the initial levels and increased back up to 75% and 95% of baseline levels, respectively, by 7 weeks, at which time serum levels of TACI-Fc remained higher than 100 μg/ml. Thus, in normal B6 mice, BLyS blockade with high-level TACI-Fc had only a mild and transient effect on the serum IgG levels.

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Figure 2. Effect of TACI-Fc on the serum levels of immunoglobulins. Eight-week-old B6.+/+ (A), 8-week-old B6.lpr/lpr (B), and 16-week-old B6.lpr/lpr (C) mice were inoculated intravenously with AdGFP (open circles) or AdTACI (solid circles). Serum levels of total immunoglobulin isotypes were determined by enzyme-linked immunosorbent assay. The results are presented as the mean ± SEM μg/ml in 8–10 mice per group. See Figure 1 for definitions.

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Six-week-old B6.lpr/lpr mice treated with control AdGFP developed an age-dependent hypergammaglobulinemia, as expected (Figure 2B). There were ∼5-fold, ∼10-fold, and ∼4-fold increases in the serum levels of IgM, IgG1, and IgG2a, respectively, up to 7 weeks after injection of the virus. In contrast, the increase in serum IgM, IgG1, and IgG2a was blocked when 6-week-old B6.lpr/lpr mice were treated with AdTACI. These results demonstrate that inoculation of young mice with AdTACI blocks the development of increased serum levels of Ig.

To test the effect of AdTACI on established hypergammaglobulinemia, 16-week-old B6.lpr/lpr mice were given a single inoculation of AdGFP or AdTACI. Mice injected with control AdGFP exhibited a continued increase in IgM, IgG1, or IgG2a (Figure 2C). In contrast, in mice inoculated with AdTACI, serum levels of IgM and IgG1 were reduced to <30% and <10% of initial values, respectively, during the 7-week course of the study. The reduction in serum IgG2a in these B6.lpr/lpr mice treated with AdTACI was more modest, compared with initial levels, but levels were reduced to 30% of those in age-matched B6.lpr/lpr mice treated with AdGFP. In the older B6.lpr/lpr mice treated with AdTACI, serum IgM and IgG were reduced to levels equivalent to those in B6.+/+ or 6-week-old B6.lpr/lpr mice. These results demonstrate that inoculation of B6.lpr/lpr mice with AdTACI both blocks the development of increased serum levels of Ig in younger mice and reverses it in older mice, but does not significantly reduce serum IgG levels in normal mice.

Prevention of autoantibody production by TACI-Fc.

B6. lpr/lpr mice develop autoantibodies after 8–10 weeks of age. In B6.lpr/lpr mice treated with the control AdGFP, there was an age-dependent increase in production of anti-dsDNA and RF antibodies (Figures 3A and B, respectively), as expected. In contrast, in B6.lpr/lpr mice treated with AdTACI, only 20% of mice developed IgM anti-dsDNA or RF, and IgG1 or IgG2a autoantibodies were present in ≤10% of the mice. These results indicate that TACI-Fc effectively prevents the production of autoantibodies.

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Figure 3. Prevention of the development of autoantibodies by TACI-Fc in 6–8-week-old B6.lpr/lpr mice. The mice were inoculated with AdGFP (open circles) or AdTACI (solid circles). Serum levels of anti–double-stranded DNA antibody (anti-dsDNA) (A) and rheumatoid factor (RF) (B) were determined by enzyme-linked immunosorbent assay. The positive cutoff value was defined as the mean +3 SD of more than 10 age-matched B6.+/+ mice. The percentages were determined in groups of 10 mice each. See Figure 1 for other definitions.

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To determine whether inoculation with AdTACI reverses established autoantibody production, 16-week-old B6.lpr/lpr were treated with either AdGFP or AdTACI. Levels of IgM, IgG1, and IgG2a anti-dsDNA (Figure 4A) and RF (Figure 4B) antibodies remained constant or increased in the control AdGFP-treated mice. However, after inoculation with AdTACI, serum IgM anti-dsDNA and RF antibodies were rapidly reduced to levels that were only slightly higher than those in B6.+/+ mice. These results indicate that TACI-Fc reduces the production of autoantibodies in lpr/lpr mice with established autoimmunity.

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Figure 4. Reduction in the production of autoantibodies by TACI-Fc in mice with established autoimmunity. Sixteen-week-old B6.lpr/lpr mice were inoculated with AdGFP (open circles) or AdTACI (solid circles). Serum levels of anti-dsDNA antibody (A) and RF (B) were determined by enzyme-linked immunosorbent assay. The results are presented as the mean ± SEM of serum concentrations (in μg/ml) from 5–10 mice per group. See Figures 1 and 3 for definitions.

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The reduction of serum autoantibodies in B6.lpr/lpr mice treated with AdTACI differs from the lack of a reduction seen in (NZB × NZW)F1 mice injected with TACI-Fc in a previous study (17). To determine whether the AdTACI system would be effective in (NZB × NZW)F1 mice, 12-week-old female mice were injected with either AdTACI or AdGFP. AdTACI inoculation resulted in only a transient (1 week) reduction in the number of circulating B cells and failed to block the development of anti-dsDNA antibodies or proteinuria in these mice (results not shown). In ELISAs, immunoreactive TACI-Fc was detectable in the serum of these mice 7 days after inoculation, but never reached more than 1 μg/ml, which was 2 orders of magnitude less than in the B6 mice, and declined to near baseline by day 14.

Extensive reports in the literature document the presence of autoantibodies against lymphocyte surface molecules in SLE. Therefore, to determine whether antibodies to TACI might be present, we performed ELISAs using His-tagged human TACI as the capture reagent, with anti-mouse IgG1 as the detection antibody. We found that mouse IgG antibody to human TACI was induced in (NZB × NZW)F1 mice and peaked at 4 weeks after AdTACI inoculation (in 10 mice tested, the mean ± SD concentration of IgG was 108 ± 65 μg/ml). Specificity was demonstrated by the lack of reactivity to a parental construct containing all peptide sequences except the TACI insert. Although there was wide variability between mice, the antibodies to human TACI were sufficient to neutralize, in a dose-dependent manner, the ability of the TACI-Fc to block binding of BLyS. We conclude that antibodies reactive to human TACI neutralized TACI-Fc and prevented the blocking of autoantibody production in the (NZB × NZW)F1 model.

Reduction in the number of B cells by TACI-Fc.

The numbers of circulating B cells were measured by flow cytometric analysis of CD19+ cells in the peripheral blood. Inoculation with AdTACI resulted in an ∼50% reduction in the number of circulating B cells in B6.+/+ mice, compared with the same mice treated with AdGFP (Figure 5A). The number of B cells in B6.lpr/lpr mice was 25% higher than in B6.+/+ mice. This increase persisted after treatment with AdGFP. In contrast, the numbers of B cells were reduced by 80% in B6.lpr/lpr treated with AdTACI. Thus, AdTACI inoculation resulted in a greater proportional decrease in B cells in the lpr/lpr mice (80%) than in the normal mice (50%). This suggests that the circulating B cells in the autoimmune mice are more dependent on BLyS than are those in normal mice.

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Figure 5. Effect of TACI-Fc on B cells. A, To determine the number of circulating B cells, 6-week-old B6.+/+ mice (squares) and lpr/lpr mice (circles) were inoculated with AdGFP (open squares and open circles) or AdTACI (solid squares and solid circles). Total peripheral blood leukocytes were counted and then stained with phycoerythrin-conjugated anti-CD19 antibody, and the number of circulating B cells calculated. The results are presented as the mean ± SEM in 8–10 mice per group. B, The percentage of circulating B cells (left panel) was determined in 8-week-old B6.lpr/lpr mice that were inoculated with AdGFP or AdTACI and killed 2 weeks later. The spleen cells were then stained with antibodies to CD19, CD21, and either IgM or CD23 and analyzed by flow cytometry (right panel). CD19+ lymphocytes were gated and divided into subsets as shown. FO = follicular mantle; MZ = marginal zone; T = transitional cells. The results are representative of 3–4 mice per group. See Figure 1 for other definitions.

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A similar effect was observed in the spleen. In B6.lpr/lpr mice, treatment with AdTACI reduced the percentage of splenic CD19+ B cells to <12% of the number of B cells in mice treated with control vector (Figure 5B). The total number of splenic CD19+ B cells in AdTACI-inoculated mice was reduced to 1.6 × 106, which represented 5% of the mean 30 × 106 CD19+ B cells present in the mice treated with AdGFP (Table 1).

Table 1. Effect of AdTACI on spleen cells in B6.lpr/lpr mice*
 Spleen cells/106CD19+ B cells/106 (% lymphocytes)CD5high T cells/106 (% lymphocytes)Syndecan-1+ plasma cells/104 (%)
  • *

    Values are the mean ± SEM total number of cells (% of total spleen cells). The percentage of cells within the lymphocyte scatter gate was measured by flow cytometry. The total number was calculated from the percentage of spleen cells in the lymphocyte gate and the total number of spleen cells. AdGFP = adenoviral vector for green fluorescent protein; AdTACI = adenoviral vector for transmembrane activator and CAML interactor.

B6.lpr AdGFP (n = 3)73 ± 330 ± 2 (41 ± 1.9)26.2 ± 2 (36 ± 3)46 ± 3 (0.63 ± 0.05)
B6.lpr AdTACI (n = 4)33 ± 81.6 ± 0.6 (5 ± 1.7)22 ± 1 (67 ± 3)8 ± 0.9 (0.26 ± 0.04)

The absolute numbers of B cells of all subsets were reduced in the mice treated with AdTACI. The percentage of cells with the phenotype of transitional cells (Figure 5B) increased in mice inoculated with AdTACI, compared with that in mice treated with the control AdGFP. In contrast, the percentage of circulating T cells, as determined by CD3 staining, or the percentage of circulating abnormal T cells in lpr/lpr mice, as determined by CD3 and B220 double staining, were not affected by TACI-Fc (results not shown), and the absolute number of T cells was reduced by only 16% (Table 1). Thus, in mice with loss of B cell tolerance but little overt disease, inoculation with AdTACI resulted in depletion of circulating and splenic B cells but had little effect on the numbers of T cells.

Sections of spleen were examined by immunohistochemistry to determine the effect of the AdTACI on B cell subsets in situ. In mice treated with AdGFP control virus, a normal follicular structure was apparent (Figure 6, top left), with the central T cell periarteriolar sheath (in red) surrounded by the B cell follicular mantle (in green), itself lined by the MOMA-1+ macrophages (in blue) in the marginal sinuses. In the animals treated with AdTACI, the follicular mantle had collapsed and only scattered B cells were observed (Figure 6, top right).

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Figure 6. Histologic analysis of the effects of TACI-Fc. Sections of spleen from the B6.lpr/lpr mice described in Figure 5B, which had been inoculated with either AdGFP or AdTACI, were stained with antibodies to metallophilic macrophages 1 (in blue), IgM (B cells, in green), and CD4 and CD8 (T cells, in red) (top panels) or IgM (in blue), IgG1 (in green), and IgA (in red) (bottom panels). Image brightness was adjusted to reveal all stained cells (top) or only intensely staining plasma cells (bottom). (Original magnification × 160.) See Figure 1 for definitions.

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This technique was also used to examine plasma cells, which are located in the red pulp and stain more intensely due to the large amount of cytoplasmic antibodies. In the spleens of B6.lpr/lpr mice treated with AdGFP (Figure 6, bottom left), brightly staining IgM (in blue), IgG1 (in green), and IgA (in red) plasma cells can be seen outside of the follicles. In contrast, the density of these cells was reduced in the spleens of mice inoculated with AdTACI (Figure 6, bottom right).

The effect of BLyS blockade on plasmablasts and plasma cells was measured more quantitatively by flow cytometry (Figure 7). As illustrated in Figure 7A, we used syndecan-1 to gate on these cells (left plot), and then subsetted the cells by the class of Ig expressed intracellularly (middle and right plots). The relative reduction in absolute numbers of cells expressing syndecan-1 after treatment with AdTACI was greater in the B6.lpr/lpr mice than in the B6.+/+ mice (P < 0.05) (Figure 7B). Interestingly, the number of plasmablasts/plasma cells expressing IgG was not reduced by BLyS blockade in the B6.+/+ mice. IgM, IgG, and IgA syndecan-1–postive cells were all reduced in the autoimmune mice (Table 1 and Figure 7C).

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Figure 7. Effect of TACI-Fc on plasmablasts/plasma cells. Spleen mononuclear cells from B6.+/+ and B6.lpr/lpr mice inoculated with either AdGFP or AdTACI as described in Figure 6 were stained for expression of syndecan-1 (syn-1) and B220 and intracellular antibodies (IgM and either IgG or IgA). Plasmablasts/plasma cells were gated (left plot) (A) and analyzed for intracellular Ig (middle and right plots) (A). Absolute numbers of total plasmablasts/plasma (syn-1+) cells (B) or of syn-1+ cells with intracellular IgM, IgG, or IgA (C) were calculated. Bars show the mean and SEM. See Figure 1 for other definitions.

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These data, taken together, demonstrate that treatment of autoimmune mice with adenovirus-delivered TACI-Fc blocks the development of mature B2-lineage subsets as well as the generation and/or survival of antibody-forming cells. The dramatic reduction in plasmablasts/plasma cells paralleled the decrease in serum Ig levels in the B6.lpr/lpr mice with established autoimmunity. However, IgG-expressing plasmablasts/plasma cells were preserved in nonautoimmune mice after BLyS blockade.

Inhibition of kidney damage by TACI-Fc in lpr/lpr mice.

Inoculation of B6.lpr/lpr mice with AdTACI prevented the deposition of IgG in the glomeruli of all 6 treated mice when kidney sections were examined 3 months later, while all 6 mice that were injected with AdGFP were strongly positive for IgG (results not shown). To determine whether treatment with AdTACI could be used to treat kidney disease in a model with more extensive disease, female MRL.lpr/lpr mice were intravenously inoculated at 12 weeks of age with 109 PFU of either AdTACI or AdGFP. The mice were followed up for changes in circulating B and T cells and increases in autoantibodies and proteinuria, until they were killed for renal histopathology.

The single inoculation resulted in a rapid drop in circulating B cells, which persisted for 3 months (Figure 8A). However, BLyS blockade had no apparent effect on the expansion of the B220+ T cells characteristic of this disease. At this age, nearly all mice had established autoantibodies, which were similar in the 2 groups at the time of virus inoculation. MRL.lpr/lpr mice that received AdGFP showed a progressive increase in the serum levels of anti-dsDNA (Figure 8B). In contrast, serum levels of anti-dsDNA antibody were reduced in MRL.lpr/lpr mice at 2 and 4 weeks after treatment with AdTACI. Thus, AdTACI was effective in reducing autoantibodies in the disease-model mice as well in the B6.lpr/lpr mice.

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Figure 8. Effect of TACI-Fc in the MRL.lpr/lpr model. Mice were inoculated intravenously with AdGFP (open symbols) or AdTACI (solid symbols) at 12 weeks of age and followed up for changes in the percentages of blood B cells (circles) and B220+ T cells (squares) or B220− T cells (triangles)(A), serum anti-dsDNA antibody titer (B), proteinuria (C) as measured by dipstick on a 0–4 scale, and mortality (D), with 10–12 mice per group (mean and SEM). The groups were statistically significantly different at 14 weeks of age (P < 0.05 by t-test) and thereafter (P < 0.01 by t-test) (B). Statistical differences in proteinuria are indicated by rank sums (C). The Kaplan-Meier survival curves are statistically significantly (P < 0.05) different by rank sums (D). See Figures 1 and 3 for definitions.

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Although the levels of anti-dsDNA antibody began to increase 8 weeks after AdTACI treatment, the levels remained at ∼35% of those in the AdGFP-treated mice. At 16 weeks of age (1 month after treatment), when proteinuria was present in 50% of control mice, it was present in only 10% of AdTACI-treated mice (Figure 8C). A long-term followup demonstrated that both the incidence and the severity of the kidney disease, as measured by proteinuria, were significantly reduced in AdTACI-treated MRL.lpr/lpr mice. By 6 months of age (3 months after treatment), only 20% of the AdTACI-treated mice developed severe nephritis (proteinuria ≥2+) compared with 90% of the control group. AdTACI treatment also improved survival in the MRL.lpr/lpr mice, since 90% and 70% of AdTACI-treated mice survived at 7 and 8 months of age, respectively, compared with 50% and 30% of the control group, respectively (Figure 8D).

On examination of the kidneys from the mice that had received AdGFP 3 months earlier, there was abundant glomerular deposition of IgG as well as increased glomerular cellularity and scarring, as expected in this model (Figure 9B). Extensive interstitial lymphocyte infiltration and tubular damage were also apparent. In the mice that received AdTACI, glomerular IgG deposition was either absent or greatly diminished (Figure 9A) in comparison with that in the mice treated with the control virus. Glomerular cellularity and scarring were also decreased. In contrast, the distribution of the T cell interstitial infiltrate was no different from that in the control mice (bottom rows in Figures 9A and B). The persistent T cell kidney interstitial infiltrate correlated with a failure of AdTACI to block the expansion of circulating abnormal (B220+,CD3+) T cells in the blood of MRL.lpr/lpr mice (Figure 8A). Thus, the single inoculation of AdTACI delayed the increase in autoantibodies in the highly penetrant MRL.lpr/lpr disease model, and this was associated with blocking of glomerular IgG deposition and preservation of glomerular architecture. Nevertheless, this did not prevent the increase in abnormal T cells or the interstitial T cell component of the disease.

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Figure 9. Effect of TACI-Fc on kidney disease. MRL.lpr/lpr mice were injected with a single inoculation of AdTACI (A) or AdGFP (B) at 16 weeks of age. Kidneys obtained from these mice 3 months later (28 weeks of age) were sectioned and stained by hematoxylin and eosin (top rows), for IgG by immunofluorescence (middle rows), or for T cells by immunohistochemistry (bottom rows). See Figure 1 for definitions.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Using a gene delivery system, a single viral injection produced serum levels of TACI-Fc that reached 150 μg/ml during the first 2 weeks after inoculation and were sustained at a level exceeding 100 μg/ml for 10 weeks. The TACI-Fc transduced by this system is functional as a blocker for BLyS, as demonstrated by its ability to specifically bind BLyS in vitro and to inhibit the antigen-stimulated B cell response in vivo. This system created an inducible but sustained blockade of BLyS, allowing us to study its effect on the development of B cell autoimmunity. Unlike studies of transgenic animals or knockout studies, viral delivery allowed timed intervention at different points in the course of the expression of autoimmunity. In contrast to repeated injections, virally transduced endogenous production results in stable and prolonged serum concentrations of TACI-Fc.

We used this system to demonstrate that BLyS blockade with TACI-Fc blocks production of spontaneously arising autoantibodies. Previous studies with TACI-Fc had demonstrated an effect on induced autoantibodies in collagen-induced arthritis, but this is not surprising given the known ability of BLyS blockade to suppress induced antibody responses (5, 24, 25). We have now shown that TACI-Fc blocks the development of spontaneous autoantibodies in younger lpr/lpr mice. More importantly in terms of designing strategies for treatment of human disease, we also demonstrated that BLyS blockade reversed the production of autoantibodies in older mice with established disease. The effect was observed in studies of both general hypergammaglobulinemia and specific anti-dsDNA and RF antibodies (Figures 2, 3, 4, and 8). Thus, our results suggest that BLyS blockade may be effective as an interventional strategy, rather than being simply a preventative measure, in humans presenting with established disease.

Use of the B6.lpr/lpr model allowed us to compare the effects of BLyS blockade in autoimmune mice with the effects in normal, congenic B6.+/+ mice. BLyS blockade had less effect on serum IgG levels in the normal mice than in the autoimmune mice. Thus, BLyS blockade may potentially reduce autoantibodies but will not eliminate the normal, protective serum IgG. As previously shown by others and confirmed in the present study, BLyS blockade will prevent immediately induced antibody responses, and thus presumably would block antibody responses to acute infections when used therapeutically in humans. However, the preservation of the serum IgG levels in normal mice suggests that IgG produced in response to previous infections or vaccines will be preserved.

BLyS/APRIL blockade reduces the number of plasmablasts/plasma cells in the red pulp (Table 1) in the autoimmune mice, in addition to the loss of mature B cells in the blood and splenic white pulp (Figures 5, 6, and 8). The reduction in plasmablasts/plasma cells is coincident with the reduction in serum autoantibody levels. Together with the observations that serum IgG is only mildly reduced and splenic IgG-expressing plasmablasts/plasma cells are preserved after BLyS blockade in normal mice, this suggests that plasmablasts/plasma cells produced in the autoimmune process are more sensitive to BLyS blockade. The acute activation of plasmablasts is dependent on BLyS/APRIL (26, 27). Further studies will be required, but the hypothesis derived from the present study is that the bulk of serum autoantibodies are dependent on ongoing activation of plasmablasts. Sensitivity of autoreactive plasmablasts/plasma cells to BLyS blockade would distinguish this approach from other B cell–directed therapies, e.g., anti-CD20 (rituximab), because many such B cell surface molecules are down-regulated during final maturation to the plasma cell stage. The preservation of normal IgG in serum and of IgG-expressing plasmablasts/plasma cells suggests that it may be possible to design strategies for BLyS blockade that ameliorate autoimmunity but preserve normal B cell memory.

BLyS blockade reversed B cell–mediated lupus-like pathology even in the setting of severe disease in the MRL.lpr/lpr model. Treatment with AdTACI resulted in a delay in the disease onset and a partial suppression of serum autoantibody levels and proteinuria. Glomerulonephritis was reduced in conjunction with the improvement in proteinuria (Figures 8 and 9). This was associated with improved survival in these mice, which is remarkable given the severe phenotype of the disease in the MRL.lpr/lpr model. The delayed mortality with BLyS blockade could result from reduced autoantibodies and/or from reduced numbers of B cells driving the infiltrative process (28). However, despite the requirement for B cells for the development of disease in this model (29), BLyS blockade failed to reduce the expansion of T cells, and interstitial kidney disease persisted. In these mice, the infiltrating T cells included both CD4+ cells and CD4−,CD8−,B220+ cells, and both appear required for pathogenesis (30–34). Thus, despite the requirement for B cells for nephritis in this model, BLyS blockade is an insufficient method of blocking the activation and expansion of T cells. These observations define potential limitations of therapy with BLyS inhibitors in human lupus. The likely determinants of the effectiveness of this therapy include the degree to which T cell abnormalities are intrinsic and the type of T cell activation (e.g., Th1 versus Th2), which might be differentially sensitive to B cell antigen presentation.

BLyS blockade with AdTACI had little effect on autoantibodies in the (NZB × NZW)F1 mice. The detection of antibodies that react with human TACI, produced in female (NZB × NZW)F1 mice after inoculation of AdTACI, provides a mechanism for our results. The absence of antibodies that react with BAFF-R-Fc would explain the different results with this form of BLyS blockade (14). Nonspecific immune stimuli induce increased autoantibody production in young (NZB × NZW)F1 mice (35). The development of antibodies to the therapeutic TACI could also occur in human lupus. Further studies are needed to determine whether (NZB × NZW)F1 mice have true autoantibodies to murine TACI, and indeed whether some human subjects with SLE might have similar antibodies.

In summary, BLyS blockade was effective in both reducing the development of autoantibodies and reversing established autoantibody production. This correlated with reduced glomerulonephritis and improved survival even in the MRL.lpr/lpr model. However, BLyS blockade did not reverse the T cell expansion or interstitial infiltrate in the MRL.lpr/lpr mice. BLyS blockade induced a greater reduction in serum antibodies in autoimmune mice than in the normal, congenic animals, and this was associated with a reduction in plasma cells in the spleen. However, these studies revealed a potential limitation of therapies targeting BLyS, in that the (NZB × NZW)F1 mice developed antibodies to the fusion construct. Each of these observations has implications for therapeutic targeting of BLyS in human patients with lupus.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank Human Genome Sciences for providing the BLyS protein and the cDNA for the TACI-Fc.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES