The NZB/NZW F1 spontaneous murine model of SLE is characterized by both polyclonal activation of naive B cells and excessive activation of T cells that occur early in life. Disease onset occurs at the age of 4–6 months when autoreactive B cells become further activated and their immunoglobulin genes undergo class switching and somatic mutation to produce high-affinity pathogenic autoantibodies (28
). This process is dependent on costimulation via B7/CD28 (8
We have engineered a nonreplicating adenovirus that expresses high levels of murine CTLA4Ig, allowing us to achieve long-term blockade of the B7/CD28 pathway in NZB/NZW F1 mice after a single intravenous injection of virus. Mice that received a low dose of this virus expressed CTLA4Ig in the serum for an average of 103 days and had a delay in appearance of high titers of anti-DNA antibodies, but this did not result in a better disease outcome compared with untreated controls. In mice treated with high-dose Ad-CTLA4Ig, there was a marked delay in disease onset as assessed by anti-DNA antibody titer, development of fixed proteinuria, histological evidence of renal damage, and survival. These results are similar to those reported by Finck et al. using repeated administration of CTLA4Ig protein (15
Both murine and human SLE are characterized by early T cell–independent B-cell hyperreactivity that leads to production of polyclonal IgM antibodies including dsDNA-binding antibodies (33
). The underlying B-cell hyperreactivity of NZB/NZW F1 mice appeared to be unaffected by CTLA4Ig as evidenced by the persistence of high serum IgM levels and of IgM anti-DNA antibody–secreting B cells over the course of treatment. In accordance with these observations, there was no alteration of expression of the autoreactive VH
BW-16 gene in the IgM compartment. However, CTLA4Ig did attenuate the age-related proliferation of cells producing IgM anti-DNA antibodies, suggesting that the activation and proliferation of these cells that are associated with disease onset and progression are costimulation-dependent.
The number of IgG-secreting B cells in the spleens of treated mice was markedly affected by CTLA4Ig treatment. This effect did not occur within the first 8 days; however, in spleens harvested 21 days after treatment, there was a 90% reduction in the frequency of IgG-secreting cells that was maintained for the duration of treatment. This effect was associated with a block in class switching to IgG. CD28-deficient and CTLA4Ig-transgenic mice have been reported to have a block in class switching associated with decreased serum IgG levels (35
). In contrast, serum IgG levels in our Ad-CTLA4Ig–treated NZB/NZW F1 mice were maintained up to 30 weeks after treatment. Because the frequency of IgG-secreting B cells in the bone marrow was not affected by CTLA4Ig, serum IgG levels in our treated mice may have been maintained by bone marrow plasma cells. These cells are thought to be responsible for maintaining long-term antibody synthesis (37
) and appear not to require either T cells or the continuing presence of antigen to survive long term and secrete antibody (38
). Alternatively, there may be a subpopulation of B cells that is able to mature and maintain serum IgG levels in the autoimmune background even in the absence of costimulation.
The results of the ELISpot assays further indicated that the decrease in the frequency of B cells producing IgG anti-DNA antibodies induced by CTLA4Ig was out of proportion to the decrease in overall numbers of IgG-producing B cells. This did not appear to be due to B-cell anergy because we were unable to rescue autoreactive IgG-producing hybridomas from treated mice using the NSO-bcl2 fusion partner, nor could we detect secretion of IgG anti-DNA antibodies in the supernatants of LPS-treated B cells from these mice. These findings suggest either that CTLA4Ig blocks the activation of anti-DNA antibody–producing B cells so that they do not undergo germinal center maturation or that deletion of high-affinity IgG anti-DNA antibody–secreting B cells has occurred.
To distinguish between these possibilities, we performed molecular analysis of the VH
BW-16 immunoglobulin heavy chain gene that is strongly associated with pathogenic anti-dsDNA antibody activity in SLE-prone mice but is regulated in normal mouse strains (21
). The development of high-affinity anti-dsDNA antibody activity encoded by VH
BW-16 in NZB/NZW F1 mice is associated with class switching from IgM to IgG and with the presence of particular amino acids, particularly arginine, in the CDRs of antibodies using this gene (22
). We found that although the frequency of IgM VH
BW-16 clones was not altered by treatment, there was a decrease in the frequency of VH
BW-16 clones recovered from IgG cDNA libraries of treated mice compared with controls. Analysis of the IgG VH
BW-16 clones obtained from the treated mice revealed that related clones were present, as in control mice, and that positive selection of VH
BW-16 IgG with basic CDR3 regions did not appear to be altered by treatment. There was, however, a 53% reduction in the frequency of somatic mutation in VH
BW-16 IgG sequences obtained from the treated mice compared with those obtained from the age-matched untreated controls. Subtle differences were also observed in the VH
BW-16 mutation patterns between treated and control mice; however, it is not possible to know from these experiments whether these differences caused a decrease in the affinity of the expressed antibodies for DNA. In sum, our findings point to a decrease in activation and maturation of IgG VH
BW-16–encoded antibodies. The decrease in frequency of VH
BW-16–positive clones in the IgG cDNA libraries and the alterations in mutation patterns might also imply differences in selection of B cells expressing this heavy chain in the treated mice.
FACS® analysis of spleen cells from CTLA4Ig-treated and control NZB/NZW F1 mice revealed that excessive T-cell activation was already present in 22-week NZB/NZW F1 mice, and this increased with age. In both groups of treated mice, overexpression of the CD69 activation marker was lost and there was also attenuation of the decrease in CD8-positive T cells. The results in these studies suggest that long-term blockade of the CD28/B7 pathway in the NZB/NZW F1 mouse has a marked negative effect on the numbers of activated T cells. This does not appear to be due to failure of activation of naive T cells, as transition of CD4 T cells from the naive to the memory compartment was not attenuated by Ad-CTLA4Ig treatment. Whether deletion of activated autoreactive T cells occurred remains to be determined. The decrease in the numbers of activated T cells may in turn be responsible for the observed effects of CTLA4Ig predominantly on the T cell–dependent steps of B-cell activation.
The failure of CTLA4Ig-treated mice to mount a T-dependent humoral immune response to either the nonreplicating adenoviral vector or, in experiments not shown, to the hapten oxazolone, is consistent with the results just described and shows that continuous treatment with CTLA4Ig is immunosuppressive. We did not challenge the mice with a replicating virus; however, it has previously been reported that CTLA4Ig transgenic mice are able to mount an effective immune response to replicating but not to nonreplicating viruses (39
). It remains to be determined whether a there is an optimal dose of CTLA4Ig that will have beneficial effects on autoreactive T and B cells without compromising immunity to foreign antigens.
Suppression of anti–DNA antibody production and glomerular disease in the Ad-CTLA4Ig–treated mice appeared to be related to persistence of expression of CTLA4Ig in the serum, as similar effects on both B and T cells were observed both in the low-dose and high-dose groups at age 30–36 weeks, but disease became manifest within 2–8 weeks after CTLA4Ig was no longer detected in the serum. Despite the observation that CTLA4Ig can induce tolerance in some transplant models (40
), it is striking that CTLA4Ig, even in high dose, did not induce a permanent state of tolerance in NZB/NZW F1 mice.
Our findings are of direct relevance in assessing the potential role of CTLA4Ig in the treatment of B cell–mediated autoimmune diseases in humans. It is likely that in clinical practice, CTLA4Ig will be most effective when it is used in short-term combination with other immunosuppressive reagents (41
). It will be of great interest to analyze the safety profile and mechanism of action of CTLA4Ig when used synergistically with anti-CD40L or cyclophosphamide (42
) and to determine whether CTLA4Ig will have a steroid-sparing effect. Animal studies in appropriate disease models that lead to better understanding of the specific effects of costimulatory blockade alone or in combination with other immunomodulatory drugs should point the way to more effective and safe treatment for human autoimmune disease.