Nan-Hua Chang

Aberrant IgM signaling promotes survival of transitional T1 B cells and prevents tolerance induction in lupus-prone New Zealand black mice.

New Zealand Black (NZB) mice develop a lupus-like syndrome. Although the precise immune defects leading to autoantibody production in these mice have not been characterized, they possess a number of immunologic abnormalities suggesting that B cell tolerance may be defective. In the bone marrow, immature self-reactive B cells that have failed to edit their receptors undergo apoptosis as a consequence of Ig receptor engagement. Splenic transitional T1 B cells are recent bone marrow emigrants that retain these signaling properties, ensuring that B cells recognizing self-Ags expressed only in the periphery are deleted from the naive B cell repertoire. In this study we report that this mechanism of tolerance is defective in NZB mice. We show that NZB T1 B cells are resistant to apoptosis after IgM cross-linking in vitro. Although extensive IgM cross-linking usually leads to deletion of T1 B cells, in NZB T1 B cells we found that it prevents mitochondrial membrane damage, inhibits activation of caspase-3, and promotes cell survival. Increased survival of NZB T1 B cells was associated with aberrant up-regulation of Bcl-2 after Ig receptor engagement. We also show that there is a markedly increased proportion of NZB T1 B cells that express elevated levels of Bcl-2 in vivo and provide evidence that up-regulation of Bcl-2 follows encounter with self-Ag in vivo. Thus, we propose that aberrant cell signaling in NZB T1 B cells leads to the survival of autoreactive B cells, which predisposes NZB mice to the development of autoimmunity.

Autoreactive B Cells in Lupus-Prone New Zealand Black Mice Exhibit Aberrant Survival and Proliferation in the Presence of Self-Antigen In Vivo

To identify defects in B cell tolerance that may contribute to the production of autoantibodies in New Zealand Black (NZB) mice, we crossed soluble hen egg white lysozyme (sHEL) and anti-HEL Ig transgenes (Ig Tg) onto the NZB background. In this study, we have examined one of the first checkpoints involved in maintenance of peripheral B cell tolerance, follicular exclusion and elimination of self-reactive B cells in the absence of T cell help. Freshly isolated anti-HEL Ig Tg B cells were labeled with CFSE, adoptively transferred into sHEL recipients, and the fate of self-reactive anti-HEL Ig Tg B cells was followed using flow cytometry and immunofluorescence microscopy. Although anti-HEL Ig Tg B cells from NZB mice are appropriately excluded from B cell follicles in NZB sHEL recipient mice, they demonstrate aberrant survival, proliferation, and generation of anti-HEL Ab-producing cells. This abnormal response results from an intrinsic defect in NZB B cells, requires the presence of CD4+ T cells, and is facilitated by the splenic environment in NZB mice. Thus, NZB mice have immune defects that interact synergistically to allow autoreactive B cells to become activated despite the presence of tolerizing autoantigens.

B Cell Activating Factor (BAFF) and T Cells Cooperate to Breach B Cell Tolerance in Lupus-Prone New Zealand Black (NZB) Mice

The presence of autoantibodies in New Zealand Black (NZB) mice suggests a B cell tolerance defect however the nature of this defect is unknown. To determine whether defects in B cell anergy contribute to the autoimmune phenotype in NZB mice, soluble hen egg lysozyme (sHEL) and anti-HEL Ig transgenes were bred onto the NZB background to generate double transgenic (dTg) mice. NZB dTg mice had elevated levels of anti-HEL antibodies, despite apparently normal B cell functional anergy in-vitro. NZB dTg B cells also demonstrated increased survival and abnormal entry into the follicular compartment following transfer into sHEL mice. Since this process is dependent on BAFF, BAFF serum and mRNA levels were assessed and were found to be significantly elevated in NZB dTg mice. Treatment of NZB sHEL recipient mice with TACI-Ig reduced NZB dTg B cell survival following adoptive transfer, confirming the role of BAFF in this process. Although NZB mice had modestly elevated BAFF, the enhanced NZB B cell survival response appeared to result from an altered response to BAFF. In contrast, T cell blockade had a minimal effect on B cell survival, but inhibited anti-HEL antibody production. The findings suggest that the modest BAFF elevations in NZB mice are sufficient to perturb B cell tolerance, particularly when acting in concert with B cell functional abnormalities and T cell help.

Functional Interplay between Intrinsic B and T Cell Defects Leads to Amplification of Autoimmune Disease in New Zealand Black Chromosome 1 Congenic Mice

Genetic loci on New Zealand Black (NZB) chromosome 1 play an important role in the development of lupus-like autoimmune disease. We have shown previously that C57BL/6 mice with an introgressed NZB chromosome 1 interval extending from ∼35 to 106 cM have significantly more severe autoimmunity than mice with a shorter interval extending from ∼82 to 106 cM. Comparison of the cellular phenotype in these mice revealed that both mouse strains had evidence of increased T cell activation; however, activation was more pronounced in mice with the longer interval. Mice with the longer interval also had increased B cell activation, leading us to hypothesize that there were at least two independent lupus susceptibility loci on chromosome 1. In this study, we have used mixed hemopoietic radiation chimeras to demonstrate that autoimmunity in these mice arises from intrinsic B and T cell functional defects. We further show that a T cell defect, localized to the shorter interval, leads to spontaneous activation of T cells specific for nucleosome histone components. Despite activation of self-reactive T cells in mixed chimeric mice, only chromosome 1 congenic B cells produce anti-nuclear Abs and undergo class switching, indicating impaired B cell tolerance mechanisms. In mice with the longer chromosome 1 interval, an additional susceptibility locus exacerbates autoimmune disease by producing a positive feedback loop between T and B cell activation. Thus, T and B cell defects act in concert to produce and amplify the autoimmune phenotype.

Abrogation of pathogenic IgG autoantibody production in CD40L gene-deleted lupus-prone New Zealand Black mice.

New Zealand Black (NZB) mice spontaneously develop a lupus-like autoimmune disease. Since CD40-CD40L interactions are important for B cell class-switch recombination and germinal center formation, we sought to understand the impact of these interactions on the immune abnormalities in NZB CD40L gene-deleted (CD40L(-/-)) mice in vivo. NZB.CD40L(-/-) mice demonstrated abrogation of all IgG autoantibodies tested and attenuated kidney disease. However, polyclonal B cell activation in vivo and B cell proliferation and class-switching in response to TLR ligands in vitro were preserved in the absence of CD40L in NZB mice. Although, plasmacytoid dendritic cell expansion and elevated BAFF production were unaffected by the absence of CD40L, there was some evidence that IFN-α-induced gene expression was reduced in the bone marrow of NZB.CD40L(-/-) mice. Our results suggest that CD40-CD40L interactions play an important role in promoting pathogenic IgG autoantibody production and kidney disease in NZB mice.

An intrinsic B-cell defect supports autoimmunity in New Zealand black chromosome 13 congenic mice.

Introgression of a New Zealand Black (NZB) chromosome 13 interval onto a C57BL/6 (B6) background (B6.NZBc13) is sufficient to produce many hallmarks of lupus, including high‐titre anti‐chromatin antibody production, abnormal B‐ and T‐cell activation, and renal disease. In this study we sought to characterize the immune defects leading to these abnormalities. By generating hematopoietic chimeras and BCR transgenic mice, we show that the congenic autoimmune phenotype can be transferred by BM cells and requires the presence of autoreactive B cells. Using the hen egg white lysozyme immunoglobulin transgenic mouse model, we demonstrate that B‐cell anergy, deletion, and receptor editing are intact. Nevertheless, congenic B cells exhibit altered peripheral B‐cell selection, as demonstrated by enhanced survival and activation of endogenous B cells with autoreactivity to chromatin and Sm/ribonucleoprotein. Given the autoantibody specificities to nuclear antigens, TLR signalling was assessed. B6.NZBc13 B cells were hyper‐responsive to poly(I:C), a TLR3 ligand, demonstrating enhanced proliferation and survival as compared to B6 B cells. Our findings indicate the presence of an intrinsic B‐cell defect on NZB chromosome 13 that results in hyper‐responsiveness to a dsRNA analogue and implicates its potential supporting role in the generation of autoimmunity in B6.NZBc13 mice.

Epistatic Suppression of Fatal Autoimmunity in New Zealand Black Bicongenic Mice

Numerous mapping studies have implicated genetic intervals from lupus-prone New Zealand Black (NZB) chromosomes 1 and 4 as contributing to lupus pathogenesis. By introgressing NZB chromosomal intervals onto a non–lupus-prone B6 background, we determined that: NZB chromosome 1 congenic mice (denoted B6.NZBc1) developed fatal autoimmune-mediated kidney disease, and NZB chromosome 4 congenic mice (denoted B6.NZBc4) exhibited a marked expansion of B1a and NKT cells in the surprising absence of autoimmunity. In this study, we sought to examine whether epistatic interactions between these two loci would affect lupus autoimmunity by generating bicongenic mice that carry both NZB chromosomal intervals. Compared with B6.NZBc1 mice, bicongenic mice demonstrated significantly decreased mortality, kidney disease, Th1-biased IgG autoantibody isotypes, and differentiation of IFN-γ–producing T cells. Furthermore, a subset of bicongenic mice exhibited a paucity of CD21+CD1d+ B cells and an altered NKT cell activation profile that correlated with greater disease inhibition. Thus, NZBc4 contains suppressive epistatic modifiers that appear to inhibit the development of fatal NZBc1 autoimmunity by promoting a shift away from a proinflammatory cytokine profile, which in some mice may involve NKT cells.

Suppression of autoimmunity by CD5 + IL-10-producing B cells in lupus-prone mice

Systemic lupus erythematosus is a complex autoimmune disorder characterized by the production of pathogenic anti-nuclear antibodies. Previous work from our laboratory has shown that the introgression of a New Zealand Black-derived chromosome 4 interval onto a lupus-prone background suppresses the disease. Interestingly, the same genetic interval promoted the expansion of both Natural Killer T- and CD5+ B cells in suppressed mice. In this study, we show that ablation of NKT cells with a CD1d knockout had no impact on either the suppression of lupus or the expansion of CD5+ B cells. On the other hand, suppressed mice had an expanded population of IL-10-producing B cells that predominantly localized to the CD5+CD1dlow compartment. The expansion of CD5+ B cells negatively correlated with the frequency of pro-inflammatory IL-17 A-producing T-cells and kidney damage. Adoptive transfer with a single injection of total B cells with an enriched CD5+ compartment reduced the frequency of memory/activated, IFNγ-producing, and IL-17 A-producing CD4 T-cells but did not significantly reduce autoantibody levels. Taken together, these data suggest that the expansion of CD5+ IL-10-producing B cells and not NKT cells protects against lupus in these mice, by limiting the expansion of pro-inflammatory IL-17 A- and IFNγ-producing CD4 T-cells.

IL-10 Production Is Critical for Sustaining the Expansion of CD5+ B and NKT Cells and Restraining Autoantibody Production in Congenic Lupus-Prone Mice

The development and progression of systemic lupus erythematosus is mediated by the complex interaction of genetic and environmental factors. To decipher the genetics that contribute to pathogenesis and the production of pathogenic autoantibodies, our lab has focused on the generation of congenic lupus-prone mice derived from the New Zealand Black (NZB) strain. Previous work has shown that an NZB-derived chromosome 4 interval spanning 32 to 151 Mb led to expansion of CD5+ B and Natural Killer T (NKT) cells, and could suppress autoimmunity when crossed with a lupus-prone mouse strain. Subsequently, it was shown that CD5+ B cells but not NKT cells derived from these mice could suppress the development of pro-inflammatory T cells. In this paper, we aimed to further resolve the genetics that leads to expansion of these two innate-like populations through the creation of additional sub-congenic mice and to characterize the role of IL-10 in the suppression of autoimmunity through the generation of IL-10 knockout mice. We show that expansion of CD5+ B cells and NKT cells localizes to a chromosome 4 interval spanning 91 to 123 Mb, which is distinct from the region that mediates the majority of the suppressive phenotype. We also demonstrate that IL-10 is critical to restraining autoantibody production and surprisingly plays a vital role in supporting the expansion of innate-like populations.

Bone marrow-derived human hematopoietic stem cells engraft NOD/SCID mice and traffic appropriately to an inflammatory stimulus in the joint.

Objective. Studies of human inflammatory arthritis would be significantly aided by the development of better animal models. Our hypothesis is that it is possible to develop humanized arthritis models through novel techniques of hematopoietic stem and progenitor cell (HSPC) delivery. Methods. Bone marrow was obtained from patients with osteoarthritis who were undergoing total hip replacement. HSPC were enriched by negative selection and injected into the femur of irradiated anti-CD122 treated nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Human cell engraftment was analyzed by flow cytometry. Arthritis was induced by an intraarticular injection of Chlamydia trachomatis and injected knee joints were examined 5 days later by histology and immunohistochemistry. Results. Human bone marrow HSPC successfully engrafted NOD/SCID mice, with some mice showing up to 90% engrafted human cells. Human B lymphoid and myeloid cells were detected in the bone marrow and spleen 6 weeks following transfer of HSPC, and engrafted recipient mice remained healthy up to 12 weeks postinjection. Chlamydia-injected mice that had been repopulated with HSPC had synovial inflammation, consisting of human neutrophils and macrophages. Conclusion. Bone marrow-derived human HSPC engraft NOD/SCID mice and traffic appropriately to an inflammatory stimulus in the joint, thus offering the potential for direct studies on the immunopathogenesis and treatment of human arthritis.