Autoantibody production in lpr/lpr gld/gld mice reflects accumulation of CD4+ effector cells that are resistant to regulatory T cell activity.

In Fas/FasL-deficient mice anti-chromatin Ab production is T cell dependent and is not apparent until after 10 weeks of age. Early control of anti-chromatin antibodies may be due to the counterbalancing influence of Treg cells. Here we show that Treg cells block lpr/lpr gld/gld Th cells from providing help to anti-chromatin B cells in an in vivo transfer system. Interestingly, the percentage and absolute numbers of Foxp3+ Treg cells is elevated in BALB/c-lpr/lpr gld/gld mice and increases with age compared to BALB/c mice. The majority of Foxp3 expression is found in the B220- CD4+ T cell population, and Foxp3-expressing cells are localized in the splenic PALS (periarteriolar lymphocyte sheath). Strikingly, although the lack of functional Fas/FasL does not affect the ability of Treg cells to block Th cell proliferation, Treg cells can block the IFN-gamma differentiation of Th cells from BALB/c or young BALB-lpr/lpr gld/gld mice but not of pre-existing Th1 cells from older BALB/c-lpr/lpr gld/gld mice. Thus, we suggest autoantibody production is not caused by the lack of Treg cells but by a defect in activation-induced cell death that leads to the accumulation of T effector cells that are resistant to regulatory T cell activity.

Exogenous and endogenous TLR ligands activate anti-chromatin and polyreactive B cells.

Autoreactive B cells may become activated in a T-independent manner via synergistic engagement of the BCR and TLRs. Using the VH3H9 Ig H chain transgene to track anti-chromatin B cells, we demonstrate that VH3H9/Vlambda1 anti-chromatin B cells proliferate in response to stimulatory oligodeoxynucleotides containing CpG motifs, suggesting that these autoreactive B cells are responsive to TLR9 signaling. Strikingly, some VH3H9 B cells, but not the well-characterized VH3H9/Vlambda1 B cells, proliferate spontaneously in culture medium. This proliferation is blocked by inhibitory CpG oligodeoxynucleotides, implicating the TLR9 (or possibly TLR7) pathway. Most hybridomas generated from the proliferating cells are polyreactive, and one exhibits binding to nuclear Ags but not to the other Ags tested. Thus, B cells carrying autoreactive and/or polyreactive specificities may be susceptible to T cell-independent activation via dual engagement of the BCR and TLRs.

CD4+ CD25+ regulatory T cells inhibit the maturation but not the initiation of an autoantibody response.

To investigate the mechanism by which T regulatory (Treg) cells may control the early onset of autoimmunity, we have used an adoptive transfer model to track Treg, Th, and anti-chromatin B cell interactions in vivo. We show that anti-chromatin B cells secrete Abs by day 8 in vivo upon provision of undeviated, Th1- or Th2-type CD4+ T cell help, but this secretion is blocked by the coinjection of CD4+ CD25+ Treg cells. Although Treg cells do not interfere with the initial follicular entry or activation of Th or B cells at day 3, ICOS levels on Th cells are decreased. Furthermore, Treg cells must be administered during the initial phases of the Ab response to exert full suppression of autoantibody production. These studies indicate that CD25+ Treg cells act to inhibit the maturation, rather than the initiation, of autoantibody responses.

The influence of effector T cells and Fas ligand on lupus-associated B cells.

Circulating autoantibodies against dsDNA and chromatin are a characteristic of systemic lupus erythematosus in humans and many mouse models of this disease. B cells expressing these autoantibodies are normally regulated in nonautoimmune-prone mice but are induced to secrete Abs following T cell help. Likewise, anti-chromatin autoantibody production is T cell-dependent in Fas/Fas ligand (FasL)-deficient (lpr/lpr or gld/gld) mice. In this study, we demonstrate that Th2 cells promote anti-chromatin B cell survival and autoantibody production in vivo. FasL influences the ability of Th2 cells to help B cells, as Th2-gld/gld cells support higher titers of anti-chromatin Abs than their FasL-sufficient counterparts and promote anti-chromatin B cell participation in germinal centers. Th1 cells induce anti-chromatin B cell germinal centers regardless of FasL status; however, their ability to stimulate anti-chromatin Ab production positively correlates with their level of IFN-gamma production. This distinction is lost if FasL-deficient T cells are used: Th1-gld/gld cells promote significant titers of anti-chromatin Abs regardless of IFN-gamma production levels. Thus, FasL from effector T cells plays an important role in determining the fate of anti-chromatin B cells.

The regulation and activation of lupus-associated B cells.

Anti-double-stranded DNA (anti-dsDNA) B cells are regulated in non-autoimmune mice. While some are deleted or undergo receptor editing, a population of anti-dsDNA (VH3H9/V lambda 1) B cells that emigrate into the periphery has also been identified. These cells have an altered phenotype relative to normal B cells in that they have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B-cell interface in the spleen. This phenotype may be the consequence of immature B cells encountering antigen in the absence of T-cell help. When provided with T-cell help, the anti-dsDNA B cells differentiate into antibody-forming cells. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, the VH3H9/V lambda 1 anti-dsDNA B cells populate the B-cell follicle and by 12 weeks of age produce serum autoantibodies. The early event of anti-dsDNA B-cell follicular entry, in the absence of autoantibody production, is dependent upon CD4(+) T cells. We hypothesize that control of autoantibody production in young autoimmune-prone mice may be regulated by the counterbalancing effect of T-regulatory (T(reg)) cells. Consistent with this model, we have demonstrated that T(reg) cells are able to prevent autoantibody production induced by T-cell help. Additional studies are aimed at investigating the mechanisms of this suppression as well as probing the impact of distinct forms of T-cell-dependent and -independent activation on anti-dsDNA B cells.

The regulation of lupus-associated autoantibodies: immunoglobulin transgenic models.

Several advances have been made regarding the regulation of lupus-associated autoantibodies, including the identification of factors that lead to their production in autoimmunity. Recent studies have further defined the receptor-editing mechanisms used to eliminate autoreactive B cells, and have shown that these may be altered in autoimmune mice. In addition, evidence that autoreactive B cells persisting in the periphery can be activated by both T-dependent and T-independent stimuli suggests potential pathways by which autoantibodies may arise in lupus.

The regulation and activation potential of autoreactive B cells.

Anti-double-stranded DNA (dsDNA) B cells persist even in nonautoimmune- prone animals. In this review, we summarize data regarding the activation potential of these cells. Provision of cognate CD4 T cell help to anti-dsDNA B cells in nonautoimmune mice not only drives their maturation and entry into the B cell follicle, but also leads to secretion of anti-dsDNA autoantibodies. Intriguingly, if T regulatory cells are provided along with T helper cells, the antibody response of anti-dsDNA B cells is diminished. We have also found that T-independent stimulation with CpG oligodeoxynucleotides leads to the proliferation and enhanced recovery of antidsDNA B cells in vitro. These data suggest that control of anti-dsDNA antibody production may rely on elements from both the innate and adaptive arms of the immune system.

The impact of T helper and T regulatory cells on the regulation of anti-double-stranded DNA B cells.

Autoreactive B cells that appear to be inactivated can be found in healthy individuals. In this study, we examined the potential of these anergic cells to become activated. We show that anergy of anti-double-stranded DNA (dsDNA) B cells in BALB/c mice is readily reversed, requiring only the provision of T cell help. We further show that spontaneous loss of anergy among anti-dsDNA B cells in autoimmune lpr/lpr mice occurs in two phases: an abortive initial response to T help followed by full loss of tolerance. Strikingly, the abortive response can be reproduced in nonautoimmune mice when CD4+CD25+ T regulatory cells are administered in conjunction with CD4+ T helper cells, suggesting that loss of B cell tolerance may require both the production of T cell help and the overcoming of T suppression.

Chronic graft-versus-host in Ig knockin transgenic mice abrogates B cell tolerance in anti-double-stranded DNA B cells.

Anti-dsDNA Abs are specific diagnostic markers of systemic lupus erythematosus, and are also implicated in kidney pathology. Anti-dsDNA B cells have been shown to be tolerized in nonautoimmune mice. The immunodysregulation that causes these cells to break tolerance is presumably part of the fundamental defects in systemic lupus erythematosus. To explore these mechanisms, we used the chronic graft-versus-host model mediated by MHC class II differences. Induction of chronic graft-vs-host in anti-DNA H chain knockin (3H9.KI) transgenic mice on a nonautoimmune background resulted in specific activation of anti-dsDNA B cells, as evidenced by high titers of soluble Ab in sera and a high frequency (70%) of anti-dsDNA B cell clones recovered as hybridomas. In addition, the lambda(+)-anti-dsDNA B cells developed increased expression of cell surface activation markers, and concentrated in the T cell area of the follicle with an Ab-forming cell-compatible phenotype. Genetic analysis of the hybridoma clones showed strong evidence of secondary rearrangements of the L chain associated with anti-dsDNA reactivity. Thus, our study indicates that alloreactive T cell help can break tolerance in a complex manner, involving several events.

CD23 defines two distinct subsets of immature B cells which differ in their responses to T cell help signals.

Transitional immature B cells undergo apoptosis and fail to proliferate in response to BCR cross-linking, thus representing a target for negative selection of potentially autoreactive B cells in vivo. In agreement with recent reports, transitional B cells were divided into developmentally contiguous subsets based on their surface expression of CD23. When transferred, CD23(+) transitional B cells readily localized to the splenic follicles and the outer PALS. Compared with CD23(-) transitional B cells, CD23(+) transitional B cells proliferated more vigorously and were rescued from BCR-induced apoptosis to a greater degree, by T cell help signals. However, both CD23(-) and CD23(+) transitional B cells failed to up-regulate CD86 (B7-2) in response to BCR ligation. These findings demonstrate that phenotypically defined subsets within the transitional B cell population are functionally distinct. Specifically, responsiveness to T cell help is a late acquisition corresponding to the stage when the B cells gain access to peripheral compartments enriched in antigen and activated T cells. The failure of transitional B cells to up-regulate CD86 to BCR-mediated stimulation suggests a unique interaction between transitional B cells and T cells with implications for tolerance in the T cell compartment.