BXD2 Mice Research Articles

Regulation of autoimmune germinal center reactions in lupus-prone BXD2 mice by follicular helper T cells.

BXD2 mice spontaneously develop autoantibodies and subsequent glomerulonephritis, offering a useful animal model to study autoimmune lupus. Although initial studies showed a critical contribution of IL-17 and Th17 cells in mediating autoimmune B cell responses in BXD2 mice, the role of follicular helper T (Tfh) cells remains incompletely understood. We found that both the frequency of Th17 cells and the levels of IL-17 in circulation in BXD2 mice were comparable to those of wild-type. By contrast, the frequency of PD-1+CXCR5+ Tfh cells was significantly increased in BXD2 mice compared with wild-type mice, while the frequency of PD-1+CXCR5+Foxp3+ follicular regulatory T (Tfr) cells was reduced in the former group. The frequency of Tfh cells rather than that of Th17 cells was positively correlated with the frequency of germinal center B cells as well as the levels of autoantibodies to dsDNA. More importantly, CXCR5+ CD4+ T cells isolated from BXD2 mice induced the production of IgG from naïve B cells in an IL-21-dependent manner, while CCR6+ CD4+ T cells failed to do so. These results together demonstrate that Tfh cells rather than Th17 cells contribute to the autoimmune germinal center reactions in BXD2 mice.

Identification of follicular T helper cells in tissue sections.

Follicular T helper (Tfh) cells are a critical population of CD4 T helper cells that are primarily localized in the germinal centers (GCs) to help B cell maturation and antibody production. Tfh cells can be identified in tissue sections based on the expression of a panel of classical Tfh surface makers, transcription marker(s), and effector-function cytokines, as well as by their unique anatomic proximity to other GC cells, including follicular dendritic cells (FDC) and GC B cells. Here, we describe an immunofluorescence staining method for visualization of GC Tfh cells in frozen spleen tissue sections of the autoimmune BXD2 mouse using a confocal imaging strategy. Tfh cells were characterized based on the expression of CD4, CXCR5, Bcl6, IL-21, and IL-17.

Interleukin-21 promotes germinal center reaction by skewing the follicular regulatory T cell to follicular helper T cell balance in autoimmune BXD2 mice.

Follicular regulatory T (Tfr) cells act as the regulatory counterpart of follicular helper T (Tfh) cells to suppress germinal center (GC) B cell differentiation. We recently showed that interleukin-21 (IL-21) promoted Tfh cell differentiation in autoimmune BXD2 mice that develop spontaneous GCs. This study was undertaken to determine the modulatory effects of IL-21 on Tfr cells and the Tfr cell to Tfh cell balance in BXD2 mice. The percentage and phenotype of Tfr cells were determined in BXD2 and BXD2-IL21(-/-) mice. The effects of IL-21 on Tfr cells and the Tfr cell:Tfh cell ratio were evaluated. Sorted Tfr cells from BXD2-IL21(-/-) mice were cocultured with Tfh cells and B cells, or transferred into BXD2 mice to determine their function. The percentages and numbers of GC B cells and Tfh cells were significantly reduced, but the percentage of Tfr cells was 2-fold higher in BXD2-IL21(-/-) mice than in wild-type BXD2 mice. Administration of AdIL-21 to BXD2-IL21(-/-) mice decreased the percentages and numbers of Tfr cells and the Tfr cell:Tfh cell ratio but increased the number of GC B cells in the spleen. Recombinant murine IL-21 suppressed FoxP3 and significantly reduced Tgfb1, Il2, and Gitr but enhanced Il21, Il6, Pd1, Cxcr5, and Icos expression in Tfr cells. IL-21 also counteracted Tfr cell-mediated inhibition of antibody secretion in the Tfh cell-B cell coculture system. Transfer of Tfr cells into young BXD2 mice reduced GC size and decreased the numbers of autoantibody-producing B cells. Our findings indicate that high levels of IL-21 selectively enhance Tfh cell differentiation but inhibit Tfr cell commitment and the suppressive function of Tfr cells on Tfh cells and B cells, suggesting that IL-21 skews the balance from Tfr cells to Tfh cells to promote autoreactive GC reactions in BXD2 mice.

Anti-scavenger receptor immune complexes disrupted marginal zone macrophage integrity via bruton's tyrosine kinase (BA4P.213)

Abstract Macrophage receptor with collagenous structure (MARCO) and scavenger receptor A (SR-A) are marginal zone macrophage (MZM) restricted scavenger receptors that play important roles in apoptotic cell (AC) clearance in the spleens. We identified that in SLE patients, higher serologic levels of anti-scavenger antibodies at early stage were associated with the late development of end-stage-renal disease. In lupus prone BXD2 mice, elevated anti-MARCO/anti-SR-A antibodies in the sera correlated with the loss of MZMs in the spleens. To determine the molecular connection between anti-MARCO/anti-SR-A and MZM loss, immune complexes (ICs) generated from a mixture of AC-Ags and autoantibodies (autoAbs) produced from BXD2 mouse were administrated to B6 mice. Interestingly, disruption of MZMs was observed in mice administered ICs generated from AC-Ags plus polyreactive autoAbs that exhibit high reactivity to both MARCO and SR-A. Confocal imaging analysis showed that, in these mice, there were high levels of bruton's tyrosine kinase (Btk) phosphorylation (pBtk) in the MZMs. Increased pBtk was also observed in MZMs of BXD2. Delivery of Ibrutinib, a Btk inhibitor. systemically or Ibrutinib-liposome as MZM targeted, prevented MZM loss and attenuated autoantibodies and IC-mediated glomerulonephritis in BXD2 mice. As we also identified the loss of MARCO+ MZMs in SLE patient spleens, the present study suggests the potential in development of anti-Btk in MZMs as a novel therapy for lupus.

Lymphotoxin-expressing marginal zone B cells support marginal zone integrity by maintaining the megakaryoblastic leukemia-1 expression (BA4P.221)

Abstract Marginal zone macrophages (MZMs) engulf apoptotic debris and play a key role in preventing autoimmunity. We previously reported that in autoimmune BXD2 mice, there is a progressive reduction in the function and numbers of MZMs associated with a gradual decline in MZ B cells. MZ B cells, compared with follicular B cells, expressed higher levels of membrane bound lymphotoxin (mLT). A mixed bone marrow reconstitution(Ly5.1+Ltβr+ : Ly5.2+Ltβr-=1:1) in Rag1-/- mice with an biased repopulation of MZMs (88:12), demonstrated that LT was essential for the maintenance of MZMs. To identify the mechanism of mLT on the maintenance of MZMs, MZM gene expression of BXD2 mice treated with PBS or soluble LTβ receptor (LTβR-Ig) was analyzed by the Illumina MouseWG-6 chips. Pathway analysis revealed that megakaryoblastic leukemia-1(MKL1) mediated genes were the most significantly down-regulated by LTβR-Ig. MKL1 is a Rho kinase mechanosensing signal that mediates cell elasticity. There was dramatic reduction of MKL1 in MZMs of BXD2 and CD19.Cre LTβf/f mice, compared to normal B6 mice. Similarly, there were reduced MZMs as well as spontaneous development of germinal centers and autoantibodies in B6-Mkl1+/- mice, compared to Mkl1+/+ mice. Administration of CCG1423 (0.15 mg/kg), a Rho/MKL1inhibitor, to BXD2 mice exacerbated the loss of MZMs. Together, our results suggest a novel immune tolerance mechanism that LT+ B cells in the MZ support MZM integrity by maintaining cell elastic signal, MKL1

Autoreactive B cell tetramer analysis reveals a type I interferon-mediated marginal zone precursor B cell defect in autoimmune BXD2 mice (BA12P.102)

Abstract In human systemic lupus erythematosus and rheumatoid arthritis, development of self-reactive B cells has been associated with defective B-cell checkpoints prior to somatic hypermutation and class-switch recombination in germinal centers (GCs). To identify mechanisms leading to checkpoint defects in the periphery, sera from BXD2 autoimmune mice were screened on a PEPperPRINT Microarray. Highly specific autoepitopes were narrowed down via Discotope epitope analysis and high-throughput ELISA and ELISPOT analyses. Three peptides La, snRNP, and fibromodulin-citrulline (fib-cit) were used to make tetramers (tet) for characterization of B cell subsets in the spleen. Analysis of fib-cit tet+ B cells revealed an age-dependent 5 fold increase in tet+ B cells, especially within the marginal zone-precursor (MZ-P) (10.6% vs 4.85%) and GC (7.3% vs 0%) subsets, in BXD2 vs B6 mice. While the total frequency of La and snRNP tet+ B cells was not different in BXD2 vs B6, there was also an increase in the MZ-P (7.66% vs 1.51%) phenotype. Sorted tet+ B cells exhibited a >100-fold enhanced ability to secrete specific autoAb compared to tet- B cells. Additionally, there was significantly higher CD69 expression (75.5% vs 5.71%), a type I interferon (IFN)-induced protein in MZ-P B cells in BXD2 vs B6 mice. Together, these results establish a general method for characterization of autoAg specific B cells and suggest a checkpoint defect at the MZ-P stage as a result of type I IFN stimulation.

Extension of the germinal center stage of B cell development promotes autoantibodies in BXD2 mice.

Regulator of G protein signaling (RGS) proteins inhibit chemokine signaling by desensitizing G protein-coupled receptor signals. This study was undertaken to determine the mechanisms by which RGS13 promotes the generation of pathogenic autoantibodies in germinal centers (GCs), using BXD2-Rgs13-/- mice. Confocal and light microscopy imaging techniques were used to determine the location of cells that express RGS13 and activation-induced cytidine deaminase (AID) in the mouse spleen, and the number of plasmablasts. The levels of GC and plasma cell program transcripts in GC B cells were determined by real-time quantitative polymerase chain reaction (qPCR). Differential interleukin-17 (IL-17)-mediated expression of RGS13 in GC versus non-GC B cells was analyzed using A20 and 70Z/3 B cells. In the spleens of BXD2 mice, RGS13 was mainly expressed by GC B cells and was stimulated by IL-17 but not IL-21. IL-17 up-regulated RGS13 in A20 GC cells but not 70Z/3 non-GC B cells. BXD2- Rgs13-/- mice exhibited smaller GCs and lower AID levels, suggesting lower somatic hypermutation and affinity maturation. However, GC B cells from BXD2- Rgs13-/- mice showed increased levels of IgMbright plasmablasts, up-regulation of the genes encoding plasma program, including interferon regulatory factor 4, B lymphocyte-induced maturation protein 1, and X-box binding protein 1 and the p-CREB target genes Fosb and Obf1, and down-regulation of the GC program genes Aid, Pax5, and Bach2 compared to BXD2 mice. BXD2-Rgs13-/- mice had lower titers of IgG autoantibodies and IgG deposits in the glomeruli, suggesting reduced autoantibody pathogenicity. RGS13 deficiency is associated with a reduction in GC program genes and the exit of fewer pathogenic IgM plasmablasts in BXD2 mice. Our findings indicate that prolonged GC program, mediated by up-regulation of RGS13, enhances AID expression and enables the generation of pathogenic autoantibodies in autoreactive GCs.

IL-17RA Is Essential for Optimal Localization of Follicular Th Cells in the Germinal Center Light Zone To Promote Autoantibody-Producing B Cells

Germinal centers (GCs) provide a microenvironment that promotes and regulates the interactions of B cells with follicular Th (TFH) cells. In this study, we show that there are significantly higher frequencies of CXCR5(+)ICOS(+) TFH cells in autoimmune BXD2 mice, and these cells express both IL-21R and IL-17RA. Although IL-17 and IL-21 are both important for the formation of spontaneous GCs and development of pathogenic autoantibodies, IL-21, but not IL-17, is required for the proper development of TFH cells in BXD2 mice. The total numbers of TFH cells and their ability to induce B cell responses in vitro were not affected by a deficiency of IL-17RA in BXD2-Il17ra(-/-) mice, the majority of CXCR5(+) TFH cells from BXD2-Il17ra(-/-) mice were, however, not localized in the GC light zone (LZ). Interruption of IL-17 signaling, either acutely by AdIL-17R:Fc or chronically by Il17ra(-/-), disrupted TFH-B interactions and abrogated the generation of autoantibody-forming B cells in BXD2 mice. IL-17 upregulated the expression of regulator of G-protein signaling 16 (RGS16) to promote the ability of TFH to form conjugates with B cells, which was abolished in TFH cells from BXD2-Rgs16(-/-) mice. The results suggests that IL-17 is an extrinsic stop signal that it acts on postdifferentiated IL-17RA(+) TFH to enable its interaction with responder B cells in the LZ niche. These data suggest a novel concept that TFH differentiation and its stabilization in the LZ are two separate checkpoints and that IL-21 and IL-17 act at each checkpoint to enable pathogenic GC development.

IL-21 promotes autoreactive germinal center development in BXD2 mice partially by regulating TFR. (P4116)

Abstract Follicular regulatory T cells (TFR) are a regulatory CD4 (Treg) subset that suppress germinal center (GC) B cell differentiation. Interleukin 21 (IL-21) promotes T follicular helper (TFH) differentiation. In the present study we investigated the modulatory effects of IL-21 on TFR in autoimmune BXD2 mice that develop spontaneous GCs with accumulation of IL-21+TFH cells in the spleen. In BXD2-Il21-/- mice, GL-7+Fas+GC B cells and CXCR5+ICOS+TFHs were significantly reduced, but the frequency of TFR cells was 2-fold higher than that in wild-type BXD2 mice. Most CXCR5+ICOS+CD4 T cells in BXD2-Il21-/- mice highly expressed Treg markers including FoxP3, CTLA4, TGF-β1 and GITR. FoxP3+TFR cells were localized in spleen follicular areas of BXD2-Il21-/- mice. Surprisingly, transfer of TFRs from BXD2-Il21-/- mice into BXD2 mice reduced IgM but not IgG autoantibody producing B cells in the spleen. This may be due to the suppressive effects of IL-21 on TFRs as administration of AdIL-21 increased GC B cells and decreased the ratio of TFR/TFH in the spleen of BXD2-Il21-/- mice in vivo. In vitro, IL-21 increased TFH but decreased TFR cells and significantly reduced Tgfb expression by TFRs. IL-21 also counteracted TFR-induced B cells death and inhibition of TFHs. The present study suggests that IL-21 positively promotes autoreactive GC reactions in BXD2 mice at least partially by decreasing TFRs and compromising the suppressive function of TFRs on B cells and TFHs.

IL-23 controls autoimmunity by maintaining the integrity of marginal zone barrier that clears apoptotic bodies (P4178)

Abstract Th17 cells, which are maintained by IL-23, are important for the spontaneous germinal center (GC) response in autoimmune BXD2 mice. Here, we unexpectedly found that BXD2-Il23-/- mice exhibit accelerated development of spontaneous GCs and production of pathogenic autoantibodies compared with wild-type (WT) BXD2 mice. Administration of adenovirus expressing IL-23 to BXD2-Il23-/- mice at early age delayed the disease onset. Despite a decline of Th17 cells in the spleen of BXD2-Il23-/- mice, there was a severe impairment in clearance of apoptotic bodies and an accelerated age-related loss of marginal zone macrophages (MZMs). In the spleens of both B6 and BXD2 mice, IL-23R was expressed primarily by MZMs which is the major cell that clears apoptotic debris in the spleens. Absence of IL-23 and loss of MZMs in BXD2-Il23-/- mice are associated with elevated production of type I interferons (IFNs) from plasmacytoid dendritic cells (pDCs). Selective depletion of MZMs by repeated injection of clondronate liposome to BXD2-WT, but not BXD2-Ifnar-/- mice, mimicked the phenotypes of BXD2-Il23-/-. Our results suggest a novel mechanism that IL-23 can act as a double edge sword to control autoimmunity in that over-expression of IL-23 can provoke autoimmunity through the maintenance of Th17 yet a complete deficiency of IL-23 induced autoimmunity through the gradual loss of marginal zone barrier, defective clearance of apopototic debris and elevated induction of type I IFNs.

Increased type I interferon disrupts marginal zone barrier integrity via dissociation of lymphotoxin-expressing B cells and marginal zone macrophages in systemic autoimmunity (P4160)

Abstract In the spleen, membrane expressed Lymphotoxin (LT) on B cells maintains the integrity of marginal zone macrophages (MZM) which acts as the final barrier to prevent follicular entry of autoantigens derived from apoptotic debris. In this study, we found that there was an age-dependent decrease in the number of MZMs and clearance of apoptotic debris in the spleens of autoimmune BXD2 mice. Selective depletion of MZMs by repeated injection of recombinant LTR fusion protein to BXD2 mice accelerated autoimmune phenotypes. Interestingly, in BXD2 spleen, the major LT producing MZ and MZ precursor B cells are primarily localized in the follicles due to type I IFN-induced upregulation of CD69. Adoptive transfer of B cells from BXD2-Ifnar-/- but not BXD2 to BXD2-Rag2-/- mice resulted in retention of B cells in the MZ, leading to repopulation of MZMs. Consistent with this, there was no age-related loss of MZMs in the spleens of BXD2-Ifnar-/- mice. Reconstitution of BXD2-Rag2-/- with a mixed bone marrow of BXD2-GFP+Ifnar+ and BXD2-GFP-Ifnar- (1:1) resulted in a 50:50 repopulation of donor MZMs in recipient mice, thereby excluding the direct effect of type I IFNs on MZM maintenance. Our present study suggests that one novel mechanism associated with type I IFN-induced autoimmunity is related to the effects on promoting follicular migration of LT expressing B cells. The dissociation of LT+ B cells and MZMs disrupts MZ barrier and enables follicular entry of apoptotic antigens.

Cutting Edge: Defective Follicular Exclusion of Apoptotic Antigens Due to Marginal Zone Macrophage Defects in Autoimmune BXD2 Mice

Marginal zone macrophages (MZMs) act as a barrier to entry of circulating apoptotic debris into the follicles of secondary lymphoid organs. In autoimmune BXD2 mice, there is a progressive reduction in the function and numbers of MZMs. Absence of MZMs results in retention of apoptotic cell (AC) debris within the marginal zone (MZ) and increased loading of AC Ags on MZ B cells and MZ-precursor (MZ-P) B cells. The MZ-P B cells are capable of translocating the AC Ags to the follicular zone and stimulating T cells. Both MZMs and MZ-P B cells from BXD2 mice express low levels of tolerogenic signals and high levels of inflammatory signals. Thus, the current study suggests a multifaceted mechanism in which MZMs maintain tolerance to apoptotic autoantigens and suppress their translocation to follicles. Lack of clearance of apoptotic debris by MZMs drives follicular Ag-transportation by MZ-P B cells to stimulate an autoimmune response.

IL-17-induced Regulator of G-protein Signaling orchestrates germinal center kinetics in the onset of autoimmunity in BXD2 mice (159.24)

Abstract Development of germinal centers (GCs) is a hallmark of BXD2 autoimmunity. We have shown that IL-17+ CD4 cell frequencies are significantly elevated in the spleen of BXD2 mice, leading to the induction of Regulator of G-protein Signaling (RGS) 13 and 16 and decreased sensitivity to CXCL12 and CXCL13. The resulting chemotactic arrest within the GC promotes extensive somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID). Here we explore mechanisms underlying this phenotype using BXD2-Rgs13-/- and BXD2-Rgs16-/- mice. BXD2-Rgs13-/- mice exhibited higher autoantibody titers in early age compared to wild-type (WT) mice though smaller GCs and lower AID levels suggested a lesser extent of SHM and affinity maturation. There was upregulation of IRF4 in B cells of BXD2-Rgs13-/- mice, suggesting these B cells develop into plasma cells more rapidly than WT B cells. At older ages, BXD2-Rgs13-/- mice showed less IgG deposits in the glomeruli, suggesting reduced autoantibody pathogenicity. RGS16 deficiency led to aberrantly localized T cell zones and reduced aggregation of a population of CD86hi marginal zone precursor (MZP) B cells, accompanied by significantly fewer GCs, suggesting a role in localization of MZP in GC light-zone and B-T co-stimulation events. Taken together, these results suggest a unique role for IL-17 in inducing pathogenic GCs and development of autoimmune disease in BXD2 mice through RGS13 and RGS16-dependent pathways.

IL-23 inhibits autoimmunity by facilitating clearance of apoptotic bodies in the marginal zone (171.26)

Abstract IL-23 helps expansion of Th17 cells which are important in the pathogenesis of spontaneous autoimmune disease in BXD2 mice. In this study, we surprisingly found that IL-23p19-deficient BXD2 mice (BXD2-Il23-/-) exhibit accelerated development of spontaneous germinal centers (GCs) and production of pathogenic autoantibodies, compared with wild-type BXD2 mice. Expression of IL-23 by adenovirus in BXD2-Il23-/- mice reversed these phenotypes. IL-23R was mainly expressed by marginal zone macrophages (MZMs) in the spleen of mice, as indicated by the GFP-IL-23R reporter. In BXD2-Il23-/-, however, there was an age-related loss of both MZMs and CD21hiIgMhi marginal zone (MZ) B cells. MZMs are a small subset of specialized splenic macrophages essential for clearance of apoptotic cells entering to the spleen from circulation to prevent generation of immunogenic autoantigens. MZM specific depletion by clondronate liposome severely accelerated BXD2 autoimmune disease progression. Absence of IL-23 correlates with downregulated expression of BAFF by MZMs which is essential for the differentiation of MZ B cells. Our results suggest a unique mechanism for IL-23 to limit autoimmunity in that IL-23 supports the cross-talk between MZM and MZ B cells. In this model, IL-23 acts on MZM to upregulate BAFF to promote the survival of MZ B cells, and MZ B cells help to maintain the survival of MZM. Loss of MZM is associated with defective clearance of apoptotic cells and acceleration of autoimmunity.

IL-17-induced Rgs16 expression is essential for follicular T helper cell localization in the germinal centers of autoimmune BXD2 mice (123.44)

Abstract Autoimmune BXD2 mice developed spontaneous germinal centers (GCs) and displayed increased number of CXCR5+ICOS+follicular T helper (Tfh) cells in the spleen. Although IL-21 but not IL-17 has been reported to be essential for the development of Tfh, there were significantly reduced PNA+Fas+GC B cells in the spleen of BXD2-Il21-/- and BXD2-Il17ra-/- mice. Interestingly, the frequency of Tfhs was 2.5-fold lower in BXD2-Il21-/- but 1.5-fold higher in BXD2-Il17ra-/- mice compared to wild type. Although increased, CXCR5+CD4 T cells were not specifically localized to the GCs in BXD2-Il17ra-/- spleens. Administration of AdIL-21 increased IL-17 levels in Tfhs and serum but had minimal effect on GC B cell frequencies in BXD2-Il17ra-/- spleens. In contrast, administration of AdIL-17 to BXD2-Il21-/- mice partially expanded GC B cells, suggesting that IL-17 acts downstream of IL-21 to facilitate the GC response. IL-21 stimulation of spleen CD4 T cells in vitro increased the frequency of Tfhs and upregulated expression of IL17R on Tfhs. IL-17 stimulated expression of Rgs16 and diminished CXCL13-mediated chemotaxis of CD4 T cells in vitro. Consistent with this, there were fewer GC CD4 T cells in BXD2-Rgs16-/- spleens in vivo. Our study suggests that IL-21 and IL-17 are required to promote the maximal autoreactive GC responses. IL-21 is important for Tfh development whereas IL-17 induced Rgs16 expression is required for Tfh localization in GCs to promote GC B-cell development.

Inhibition of the catalytic function of activation-induced cytidine deaminase promotes apoptosis of germinal center B cells in BXD2 mice

To determine whether functional suppression of the catalytic domain of activation-induced cytidine deaminase (AID) can suppress the hyperreactive germinal center (GC) responses in BXD2 mice. We generated transgenic BXD2 mice expressing a dominant-negative (DN) form of Aicda at the somatic hypermutation site (BXD2-Aicda-DN-transgenic mice). Real-time quantitative reverse transcriptase-polymerase chain reaction was used to determine the expression of Aicda and DNA damage/repair genes. Enzyme-linked immunosorbent assay was used to measure serum levels of autoantibodies and immune complexes (ICs). Development of GCs and antibody-containing ICs as well as numbers of proliferative and apoptotic cells were determined using flow cytometry and/or immunohistochemical analyses. Development of arthritis and kidney disease was evaluated histologically in 6-8-month-old mice. Suppression of the somatic hypermutation function of AID resulted in a significant decrease in autoantibody production without affecting the expression of DNA damage-related genes in GC B cells of BXD2-Aicda-DN-transgenic mice. There was decreased proliferation, increased apoptosis, increased expression of caspase 9 messenger RNA in GC B cells, and lower numbers of GCs in the spleens of BXD2-Aicda-DN-transgenic mice. Decreased GC response was associated with lower levels of IgG-containing ICs. Anti-IgM- and anti-CD40 plus anti-Ig-induced B cell proliferative responses were decreased in BXD2-Aicda-DN-transgenic mice. Inhibition of the AID somatic hypermutation function in BXD2 mice suppressed development of spontaneous GCs, generation of autoantibody-producing B cells, and autoimmunity in BXD2 mice. Suppression of AID catalytic function to limit selection-based survival of GC B cells could become a novel therapy for the treatment of autoimmune disease.

P53 Negatively Regulates RGS13 Protein Expression in Immune Cells

RGS13, a member of the regulator of G protein signaling (RGS) family, inhibits G protein-coupled receptor signaling in B cells and mast cells (MCs) and suppresses IgE-antigen-induced MC degranulation and anaphylaxis. Although RGS13 expression is induced by immune receptor and chemokine receptor stimulation, the molecular regulation of RGS13 transcription is unknown. Here, we investigated the role of two p53 response elements (REs) in the regulation of RGS13 promoter activity and expression. We found that a 1000-bp DNA fragment upstream of the ATG translation start site (TSS) had promoter activity in reporter gene assays, and deletion or mutation of a p53-binding motif nearest the TSS abolished promoter activity. Notably, p53 bound to both REs in the RGS13 promoter in vivo as assessed by chromatin immunoprecipitation, suggesting that the p53 RE most distal to the TSS is physiologically inactive. We detected reduced RGS13 expression in MCs exogenously expressing p53 or treated with doxorubicin, which induces genotoxic stress and leads to p53 accumulation. RNA silencing of p53 up-regulated RGS13 expression in B lymphocytes, and bone marrow-derived MCs from p53(-/-) mice had increased RGS13 expression. Finally, p53-depleted B cells with increased RGS13 expression had reduced Ca(2+) mobilization in response to sphingosine 1-phosphate. These studies indicate that p53 may modulate immune responses through suppression of RGS13 transcription in MCs and B cells.

Sequential effects of IL-21 and IL-17 to regulate spleen CXCR5+ follicular T helper cells in autoimmune BXD2 mice (101.44)

Abstract CXCR5+ follicular T helper (Tfh) cells locate in the light zone (LZ) of germinal centers (GCs) to help differentiation of antibody producing B cells. Both IL-17 and IL-21 have been associated with the development of GCs but their effects on Tfh have not been clear. Autoimmune BXD2 mice developed spontaneous GCs and displayed increased number of Tfh in the spleen. In BXD2-Il21-/- or BXD2-Il17ra-/- mice, there were significantly reduced PNA+Fas+ GC B cells in the spleen and autoantibody titers in sera. This is not exclusively related to Tfh development since the percentage of CXCR5+ICOS+ Tfhs is 2-fold lower in BXD2-Il21-/- but is 1.5-fold higher in BXD2-Il17ra-/- mice compared to wild type. Confocal images, however, showed dramatically diminished CD4 T cells in GC LZ of BXD2-Il17ra-/- spleen. In vitro treatment of CD4 T cells with IL-21 increased the percent of CXCR5+ICOS+ Tfhs and the expression of IL17R on them. qRT-PCR confirmed that IL-21 treatment increased the expression of Cxcr5, Icos and Il21 on CD4 T cells. In contrast, IL-17 treatment increased Rgs16 but decreased Cxcr5 and diminished CXCL13-mediated chemotaxis on CD4 T cells. Our study suggests that IL-21 and IL-17 act sequentially on Tfhs in BXD2 mice. IL-21 first promotes differentiation of Tfhs and prepared these cells for IL17 stimulation by upregulating IL-17R. Subsequently, IL-17 is required to upregulated the expression of Rgs16 and stabilizes Tfh in GC LZ to promote spontaneous development of GCs.

Loss of CIKS/Act1, the signaling adaptor for IL-17 family cytokines, significantly ameliorates development of fatal disease in the Fcgamma RIIB-deficient mouse model for lupus (101.18)

Abstract CIKS (Act1 or TRAF3IP2) is an adaptor protein required for signaling by receptors of the IL-17 family of cytokines. CIKS has been shown to be vital for disease in autoimmune mouse models of CIA and EAE. IL-17A may promote spontaneous germinal centers formation in the lupus prone BXD2 mice. However, the significance of IL-17 cytokines in the clinical outcome of lupus disease has never been addressed. Here we show that Th17 cell numbers are increased in Fcgr2b-deficient mice and IL-17 cytokine signaling is critically involved in disease development in this lupus model. Loss of CIKS significantly reduced mortality rates in Fcgr2b-deficient mice. Unexpectedly, the reduced mortality was not correlated with lower levels of antibodies to ANA or anti-dsDNA. Nevertheless, loss of CIKS on an FcgR2b-deficient background did reduce the formation of spontaneous germinal centers, which correlated with a diminished rate of isotype switching and plasma cell differentiation; furthermore, the generation of T-cell effectors was slowed. Most importantly, loss of CIKS profoundly reduced severe glomerulonephritis and inflammatory cell infiltrations in kidneys, key events in the fatal outcome of the lupus-like disease in Fcgr2b-deficient mice. These novel findings suggest that IL-17 family cytokines signaling via CIKS play critical roles in the pathogenesis of lupus disease in Fcgr2b-deficient mice; these findings also position CIKS as a potential new target for therapeutic intervention in SLE.

Type I interferon–dependent CD86high marginal zone precursor B cells are potent T cell costimulators in mice

To investigate the role of CD86(high) marginal zone (MZ) precursor B cells in type I interferon (IFN)-induced T cell-dependent responses in autoimmune BXD2 mice. Confocal microscopic imaging was used to determine the location of plasmacytoid dendritic cells (DCs), MZ precursor B cells, and CD4 T cells in the spleens of BXD2 and BXD2-Ifnar(-/-) mice. Immunohistochemical staining was used to determine IgG(bright) cells in the spleens of BXD2 and BXD2-Ifnar(-/-) mice. Enzyme-linked immunosorbent assay was used to determine serum levels of IFNα and autoantibodies, and 4-hydroxy-3-nitrophenylacetyl hapten (NP)-chicken γ-globulin (CGG) (NP-CGG)- or NP-Ficoll-induced anti-NP2 antibody titers. Real-time quantitative polymerase chain reaction was used to determine the levels of type I IFN transcripts. T cell proliferation was measured using (3) H-thymidine. The expression of CD86 and CD80 was determined by fluorescence-activated cell sorting analysis. The deletion of type I IFN receptor abrogated the development of IgG(bright) cells and suppressed a T cell-dependent antibody response. Type I IFN signaling was associated with the expression of CD86, but not CD80, on follicular, MZ, and MZ precursor B cells. However, MZ precursor B cells demonstrated the highest expression of CD86 and the highest capacity for T cell costimulation with intact type I IFN receptor. This effect was blocked by an antibody that neutralizes CD86. In IFN receptor-intact BXD2 mouse spleens, MZ precursor B cells clustered at the T cell-B cell border. CD86 deletion suppressed germinal center formation, autoantibody production, and development of autoimmune diseases in BXD2 mice. Type I IFN can promote autoimmune responses in BXD2 mice through up-regulation of CD86(high) expression on MZ precursor B cells and trafficking of MZ precursor B cells to the T cell-B cell border to provide costimulation of CD4 T cells.