BXD2 Mice Research Articles

Increased development of T-bet+CD11c+ B cells predisposes to lupus in females: Analysis in BXD2 mouse and genetic crosses

Cardinal features of lupus include elevated B cell activation and autoantibody production with a female sex preponderance. We quantified interactions of sex and genetic variation on the development of autoimmune B-cell phenotypes and autoantibodies in the BXD2 murine model of lupus using a cohort of backcrossed progeny (BXD2 x C57BL/6J) x BXD2. Sex was the key factor leading to increased total IgG, IgG2b, and autoantibodies. The percentage of T-bet+CD11c+ IgD+ activated naive B cells (aNAV) was higher in females and was associated with increased T-bet+CD11c+ IgD− age-related B cells, Fas+GL7+ germinal center B cells, Cxcr5−Icos+ peripheral T-helper cells, and Cxcr5+Icos+ follicular T-helper cells. IFN-β was elevated in females. Variation in aNAV cells was mapped to Chr 7 in a locus that shows significant interactions between the female sex and heterozygous B/D variant. Our results suggest that activation of naive B cells forms the basis for the female-predominant development of autoantibodies in lupus-susceptible BXD2 mice.

17β-estradiol and B-cell intrinsic type I interferon amplify toll-like receptor 7 signaling loop in B cells of female lupus prone BXD2 mice

Abstract While systemic lupus erythematosus (SLE) disproportionally affects women versus men, elevation in 17β -estradiol (E2) alone is not sufficient to promote the development of autoantibody producing B cells. The objective of the present study is to determine if B-cell intrinsic mechanisms contribute to the increased TLR7 response to E2 stimulation. We identified that there were elevated circulating levels of E2 in young African American (AA) SLE patients (24–41 yr-old), compared to older AA (42–56 yr-old) and European American SLE patients (24–66 yr-old). Circulating E2 levels positively correlated with the levels of anti-Smith (Sm) and the expression of interferon-beta (IFNβ) in naïve B cells of SLE patients (n=39). Mouse studies were used to determine if E2 stimulates the expression of IFNβ in B cells and if sex plays a role to influence B cell responses to E2. Serum levels of Sm/RNP and RNP autoantibodies positively correlated with the levels of E2 in female lupus prone BXD2 mice post-puberty (12-wk-old) but not pre-puberty (4–6-wk-old). At the post-puberty stage (>12 wk-old), there were significantly elevated levels of anti-DNA and anti-Sm in female BXD2 mice, compared to male mice. This was associated with an increased TLR7-induced expression of IFNβ and CD69 in transitional stage 1 (T1: CD23 −IgM +CD93 +) B cells in female BXD2 mice, compared to male mice. Interestingly, E2 stimulation promoted intracellular levels of IFNβ and TLR7 in T1 B cells from female but not male BXD2 mice. Together, our results suggest that elevation of E2 in combination with an increased B-cell susceptibility to E2 induction of IFNβ may play a role in the increased B cell responses to TLR7 stimulation in individuals predisposed to the development of SLE. This study was supported by grants from VA Merit Review grant (I01BX004049), NIH grants R01-AI-071110, R01 AI134023, and Lupus Research Alliance Distinguished Innovator Award to J.D.M, the LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311

IL-4 acts through aryl hydrocarbon receptor to antagonize TLR7 induced double negative 2 B cells in lupus

Abstract We recently showed that in systemic lupus erythematosus (SLE), IL-4R signaling is a powerful antagonist that can effectively suppress the development of activated naïve (aNAV) and CD11c +T-bet +IgD −CD27 −double negative 2 (DN2) B cells promoted by both type I and type II IFNs. In the present study, we used the BXD2 mouse model of lupus to determine the mechanism of IL-4 in suppressing the development of DN2 B cells in vivo. Administration of IL-4 significantly inhibited the development of anti-Smith, anti-DNA, and anti-histone autoantibodies induced by TLR7 agonist R848 in BXD2 mice. This was associated with a decreased percentages of CD11c +T-bet +IgD −B cells. Feature-barcoding single cell RNA-sequencing analysis showed that IL-4 modulated B cell development at the transitional stage 2 (T2) and skewed naïve B cells to develop into the CD23 +CD21 −follicular B cells. IL-4 induced the gene encoding interleukin-4-induced1 (IL4i1), an enzyme that metabolizes aromatic amino acids and this was associated with the upregulation of aryl hydrocarbon receptor (AhR) and downstream genes. Metabolomics analysis revealed IL-4 induced AhR agonistic metabolites in B cells including kynurenine (Kyn), indole-3-acetic acid, and indole-3-lactic acid. In the absence of IL-4, Kyn and a potent AhR agonist, formylindolo[3,2-b]carbazole (FICZ), significantly suppressed TLR7 plus IFNβ-induced DN2 B-cell development in vitro. Our results suggest that IL-4 acts through the IL4i1-AhR pathway to inhibit B-cell regulatory response to TLR7 and type I IFN. Identification of small molecular metabolites that act directly in B cells to induce homeostasis may lead to development of orally dosed metabolome modulating therapeutics efficacious in the treatment of SLE. This study was supported by grants from VA Merit Review grant (I01BX004049), NIH grants R01 AI134023, and Lupus Research Alliance Distinguished Innovator Award to J.D.M, the LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311.

Host Genetics But Not Commensal Microbiota Determines the Initial Development of Systemic Autoimmune Disease in BXD2 Mice.

To determine the extent to which the gut microbiome influences systemic autoimmunity in a mouse model of lupus. We generated germ-free (GF) lupus-prone BXD2 mice, which under normal conditions develop spontaneous germinal centers (GCs) and high titers of serum autoantibodies. GF status was confirmed by gut bacterial culture. The autoimmune phenotypes of 6- and 12-month-old gnotobiotic GF BXD2 mice and specific pathogen-free (SPF) BXD2 mice were compared. Serum levels of autoantibodies were measured by enzyme-linked immunosorbent assay. Histologic sections of the mouse kidney and joints were evaluated. Flow cytometry was used to analyze GCs and age-associated B cells. CD4+ T cells were analyzed for PD-1+ICOS+ activated T cells, T follicular regulatory (Tfr) cells (Foxp3+CD25+ PD-1+CXCR5+), and PD-1+ICOS+ T cells expressing interleukin-17A (IL-17A) or interferon-γ (IFNγ) after stimulation with phorbol myristate acetate (PMA)/ionomycin. In 6-month-old mice, GF status did not affect splenomegaly, GC B cells, age-associated B cells, or serum autoantibody levels, except for IgG antihistone. GF BXD2 mice exhibited a significantly higher percentage of Tfr cells compared to their SPF counterparts (P < 0.05). At 12 months of age, however, GF BXD2 mice had significantly diminished IgG autoantibody levels and a lower percentage of GC B cells and age-associated B cells (P < 0.05). Following stimulation with PMA/ionomycin, PD-1+ICOS+ CD4+ T cells expressed significantly lower IL-17A, but not IFNγ, levels in GF BXD2 mice compared to SPF BXD2 mice (P < 0.01). SPF BXD2 mice and GF BXD2 mice developed equivalent renal and joint disease with no significant differences in severity. Our results suggest a model in which genetics plays a dominant role in determining the initial development of autoimmunity. In contrast, gut microbiomes may regulate the persistence of certain aspects of systemic autoimmunity.

IL-17 receptor A signaling on B cells promotes B-cell responses to TLR7 and enhances autoreactive germinal center B-cell development

Abstract IL-17 receptor A (IL-17RA) expression has been identified in B cells and macrophages. There was low expression of IL-17RA on T cells. Surprisingly, we previously showed that IL-17RA signaling through NF-κB p65/p50 has a complex effect on both B and T-cell localization and germinal center (GC) architecture, making it difficult to dissect the specific effects of IL-17RA on B cells. To isolate the effects of the absence of IL-17RA specifically on B cells, we have generated a B-cell specific IL-17RA knockout (Il17rafloxed/floxed x Cd19.Cre) BXD2 mice. Flow cytometry confirmed the loss of IL-17RA expression in only CD19+ B cells but not CD3+ T cells or CD11b+ macrophages. There was a 2-fold decreased spontaneous GC B cell population in Il17rafloxed/floxed x Cd19.Cre BXD2, compared to Il17rawt/floxed x Cd19.Cre BXD2 mice. Interestingly, the abnormal activation and expansion of T-follicular helper cell (Tfh) phenotype in BXD2-Il17ra−/− mice was abrogated in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. In the BXD2-Il17ra−/− mice, B cells were anergic to TLR7 stimulation and exhibited a strong stimulus-specific transcription repressor p50/p50 homodimer and a lower NF-κB phospho-p65. To demonstrate that this anergy is due specifically to IL-17RA on B cells but not other cells, we have identified that there was a lower TLR7-induced CD69 in vitro in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. Taken together, our results suggest that IL-17RA signaling directly regulates B cell autoimmune phenotype in the BXD2 mice through an enhancement of B-cell responses stimulated by the NF-κB signaling pathway. A deficiency of such signal enforces B cell anergy and prevents autoreactive GC formation.

Type I interferon enhanced double negative 2 B cell differentiation and lupus autoantibody production

Abstract Systemic Lupus Erythematosus (SLE) is characterized by high level of type I interferon (IFN) and abnormal expansion of effector B cells including IgD−CD27−double negative (DN), CD21−T-bet+DN2 B cells, and antibody secreting cells (ASCs). It remains unclear as to the role of type I and type II interferon (IFN) to promote DN2 B cell differentiation. The in vivo effects of type I IFN signaling in promoting DN B cells were accessed using the BXD2 mouse model of lupus which exhibit a low Th1 response, compared to normal B6 mice. Compared to normal B6 mouse, there was a significantly elevated percent of DN B cells in the BXD2 mice, but this was reduced in BXD2 mice that are deficient in IFNAR1 (BXD2-Ifnar1−/−). Compared to wild-type BXD2 mice, there was a lower level of IgG autoantibodies specific to DNA, histone and Smith (Sm) in BXD2-Ifnar1−/− mice. BXD2-Ifnar1−/− B cells presented impaired differentiation into the DN and the DN2 populations under an in vitro stimulation condition in the presence of IFNb and a TLR7 agonist. To determine if type I IFN stimulation can direct differentiation of healthy B cells to these effector B cells without the influence of type II IFN, B cells purified from healthy individuals were stimulated under a DN stimulation condition (anti-Ig+IL-21+BAFF+TLR7) in the presence and absence of IFNb. We found that IFNb significantly increased the percentage and of DN and DN2 B cells, but decreased the percentage of CD27+CD38+ plasmablasts/plasma. Under the same condition, IFNb increased the secretion of autoantibodies specific to DNA, histone and Sm. Our results suggest that type I IFN signaling stimulates DN2 B cell differentiation and can promote the abnormal expansion of these B cells in vivo independent of type II IFN.

IL-23 Promotes a Coordinated B Cell Germinal Center Program for Class-Switch Recombination to IgG2b in BXD2 Mice.

IL-23 promotes autoimmune disease, including Th17 CD4 T cell development and autoantibody production. In this study, we show that a deficiency of the p19 component of IL-23 in the autoimmune BXD2 (BXD2-p19-/- ) mouse leads to a shift of the follicular T helper cell program from follicular T helper (Tfh)-IL-17 to Tfh-IFN-γ. Although the germinal center (GC) size and the number of GC B cells remained the same, BXD2-p19-/- mice exhibited a lower class-switch recombination (CSR) in the GC B cells, leading to lower serum levels of IgG2b. Single-cell transcriptomics analysis of GC B cells revealed that whereas Ifngr1, Il21r, and Il4r genes exhibited a synchronized expression pattern with Cxcr5 and plasma cell program genes, Il17ra exhibited a synchronized expression pattern with Cxcr4 and GC program genes. Downregulation of Ighg2b in BXD2-p19-/- GC B cells was associated with decreased expression of CSR-related novel base excision repair genes that were otherwise predominantly expressed by Il17ra + GC B cells in BXD2 mice. Together, these results suggest that although IL-23 is dispensable for GC formation, it is essential to promote a population of Tfh-IL-17 cells. IL-23 acts indirectly on Il17ra + GC B cells to facilitate CSR-related base excision repair genes during the dark zone phase of GC B cell development.

Genetics but not intestinal microbiota determines the onset of systemic autoimmune disease in BXD2 mice

Abstract The onset and severity of autoimmunity can be attributed to both genetic predisposition and environmental triggers including intestinal microbiota dysbiosis. To determine if development of spontaneous autoimmunity is dependent on gut microbiota, we generated germ-free (GF) BXD2 lupus mice which otherwise develop spontaneous germinal centers (GCs) and high titers of serum autoantibodies. The microbiome and immune phenotypes in specific pathogen free (SPF) and GF BXD2 and normal B6 strains of mice were analyzed. The GF status was confirmed by gut bacterial culture. Surprisingly, the GF status did not affect disease onset and severity in 6-mo-old mice. There were comparable serum levels of IgG or IgM autoantibodies against lupus autoantigens (DNA, histone, and RNP) in SPF versus GF BXD2 mice. Flow cytometry analysis showed that the frequency and absolute numbers of GC B cells, CD4+ T cells, follicular T-helper (Tfh) cells in the spleen were also indistinguishable between SPF and GF BXD2 mice although GF BXD2 mice exhibited a higher number of CD19+ B cells and Tregs, compared to the SPF counterpart. Histologic examination of kidney and lung also showed similar disease scores between SPF and GF BXD2 mice. At older age (12-mo-old), however, there were diminished GC B cells and serum levels of autoantibodies in GF BXD2 mice compared to SPF mice. The results suggest a model in which genetic factors play a dominant role in determining the onset and susceptibility of autoimmunity and that endogenous self-antigens are the primary driver of autoreactive T and B cells leading to spontaneous GCs in BXD2 mice. In contrast, environmental factors such as gut microbiomes may play a modifying role in regulating the persistence of systemic autoimmunity.

IL-23 is essential for the switching between Tfh-IFN-γ and Tfh-IL-17 in lupus

Abstract The role of IL-23 in promoting pathogenic autoantibody development in autoimmune disease is unclear. The purposes of this study are to use the IL-23p19 knockout in the BXD2, a lupus mouse strain, to determine: (i) if IL-23 is required for germinal center (GC) and pathogenic autoantibody development; and (ii) if IL-23 acts through follicular T-helper cells (Tfh) lineage switching to induce autoantibody formation. Sera autoantibodies were measured by ELISA. GC development and the expression of AID were determined by confocal imaging. IL-17 and IFN-γ producing Tfh cells were detected by intracellular staining. The expression of GC program genes and T cell program genes was measured by qRT-PCR. Exogenous IL-23 was administered to B6 mice using an IL-23 expressing adenovirus (AdIL-23). There was significantly increased IgM but decreased IgG autoantibodies in BXD2-p19−/− mice compared to BXD2 mice. The expression of AID was decreased in BXD2-p19−/− mice. Surprisingly, the size and number of GC were increased in BXD2-p19−/− mice. There was also similar percentage of PD1+CXCR5+Tfh cells in BXD2 and BXD2-p19−/− mice. However, the percentage of IL-17A expressing Tfh cells was suppressed but the IFN-γ producing Tfh was increased in BXD2-p19−/− mice. Consistent with this, AdIL-23 administration suppressed the expression of Tbet, Ifng, Bcl6, Il21, Gata3, and Il4 but enhanced the expression Il17a, Il17f in Tfh cells. Our data suggest Tfh-IFN-γ and Tfh-IL-17 each plays an important role to determine the development of autoantibody producing B cells. While IL-23-mediated Tfh-IL-17 does not promote the size of autoreactive GCs, it regulates the induction of AID and thereby plays an important role to determine autoantibody affinity maturation.

A novel mechanism for IL-23 to up-regulate AICDA and promote pathogenic autoantibodies in lupus

Abstract Targeting IL-23 to treat autoimmune disease and chronic inflammation is under development based on its proinflammatory function and pro-Th17 cell effects. IL-23 was previously shown to be important for promoting pathogenic autoantibody production in B6-Faslpr/lpr mice. In contrast, the lack of IL-23 did not influence germinal center (GC) formation and IgG anti-CII autoantibodies in type II collagen immunized DBA/1 mice. We determined if IL-23 regulated development of spontaneous GCs and autoantibody production in lupus-prone BXD2 mice by generating IL-23 deficient BXD2-p19−/− mice. There was significantly decreased total IgG, but increased total IgM and IgM anti-DNA and RF autoantibodies in BXD2-p19−/− mice compared to BXD2 mice. Flow cytometry and confocal imaging analyses showed that both the size and number of GL-7+Fas+ GC B cells were increased, yet there was a decreased development of GC dark zone (DZ) (CD86−CXCR4+) B cells in BXD2-p19−/− mice. Interestingly, the expression of activation-induced cytidine deaminase (AICDA or Aicda) was significantly decreased in GC B cells in BXD2-p19−/− mice. However, the expression of GC program genes (Bach2, Pax5) was increased except Bcl6. In contrast, the expression of key plasma cell program genes Prdm1 in GC B cells was diminished in the absence of IL-23. Our data suggest a novel pathway for IL-23 to promote IgG autoantibody production by upregulation of AICDA in the GC DZ. Importantly, such pathway is independent of the canonical GC program genes that have been implicated for the induction of AICDA. These results suggest a potential novel action of anti-IL-23 therapies to inhibit adaptive immune responses in addition to its known inhibitory role of innate response in autoimmune diseases.

Atherogenic dyslipidemia promotes autoimmune follicular helper T cell responses via IL-27.

The incidence of atherosclerosis is higher among patients with systemic lupus erythematosus (SLE); however, the mechanism by which an atherogenic environment affects autoimmunity remains unclear. We found that reconstitution of atherosclerosis-prone Apoe-/- and Ldlr-/- mice with bone marrow from lupus-prone BXD2 mice resulted in increased autoantibody production and glomerulonephritis. This enhanced disease was associated with an increase in CXCR3+ follicular helper T cells (TFH cells). TFH cells isolated from Apoe-/- mice had higher expression of genes associated with inflammatory responses and SLE and were more potent in inducing production of the immunoglobulin IgG2c. Mechanistically, the atherogenic environment induced the cytokine IL-27 from dendritic cells in a Toll-like receptor 4 (TLR4)-dependent manner, which in turn triggered the differentiation of CXCR3+ TFH cells while inhibiting the differentiation of follicular regulatory T cells. Blockade of IL-27 signals diminished the increased TFH cell responses in atherogenic mice. Thus, atherogenic dyslipidemia augments autoimmune TFH cell responses and subsequent IgG2c production in a TLR4- and IL-27-dependent manner.

Single cell analysis of transitional B cells reveals autocrine IFNβ sustains a dynamic type I IFN network in lupus

Abstract Increased selection of transitional B cells reactive with nucleic acid antigens is a prominent feature of systemic lupus erythematosus (SLE). We found that transitional stage 1 (T1) B cells from both human SLE patients and BXD2 lupus mice intrinsically express significantly higher levels of IFNα and IFNβ compared to normal individuals. IFNβ expression in T1 B cells was significantly correlated with the development of mature autoreactive 9G4+ B cells in SLE patients and La+ B cells in BXD2 mice. IFNβ was also specifically required for optimal B cell survival and TLR7 responses. In single T1 B cells isolated from Ifnb−/− vs. WT B6 bone marrow chimeras, upregulation of Ifnα7 was completely abrogated in single Ifnb−/− T1 B cells, while other genes (Ifna1, Ifna4, Cd86, Tlr7) were significantly but incompletely diminished implicating both autocrine and paracrine IFNβ activity. To resolve these signals in an autoimmune environment, single cell gene expression analysis was carried out on sorted BXD2 T1 B cells. Hierarchical clustering revealed three distinct gene expression patterns. Cluster 1 cells were “IFN producers” characterized by high expression of type I IFN genes, but low co-expression of IFN receptor genes, Ifnar1 and Ifnar2. Cluster 2 “IFN responders” exhibited the highest co-expression of both Ifnar1 and Ifnar2 as well as immunomodulatory IFN response genes (Irg1, IL6, Cd69, Cd86). Cluster 3 had low expression of type I IFN and IFNAR genes. Clusters 1 and 2 exhibited the highest Tlr7 expression, while Cluster 3 had the highest expression of Baffr and Tlr9. The heterogeneity of IFN signatures at the T1 stage suggests that varying interferonogenic phenomena can differentially prime T1 B cells for subsequent responses and cell fates.

Type I interferon signaling acts directly on transitional T2 B cells to promote RNP-Ag tolerance loss in BXD2 mice

Abstract In lupus, autoreactive B cells fail to be removed at the transitional B cell stage. Lupus prone BXD2 mice exhibit transitional B cell tolerance loss and production of autoAb against RNA binding proteins (RNP). The purpose of this study was to determine direct effects of IFNα on developing transitional B cells. Analysis of IFNαR expression in B6 and BXD2 mice revealed the highest expression levels on transitional B cells with lower levels on mature FO, MZ, and GC B cells. Interestingly, both in vitro and in vivo, peripheral B cell responsiveness to IFNα started at the IgMhiCD23lo T1 stage and was significantly upregulated at the T2 stage, as measured by CD69 induction (76±5% in B6 vs 89±3% in BXD2 T2). In vivo treatment of B6 and BXD2 mice with IFNα promoted B cell upregulation of CD69 and CD86 on T2 B cells. Using an antigen (Ag) tetramer approach for isolation and characterization of La and RNP (La13-27 and snRNP357-373) autoreactive B cells, we found that IFNα stimulation increased maturation of transitional RNP autoreactive B cells and expansion of the autoantigen engaged IgMlo T3 compartment in BXD2 mice. Consistent with abnormal signaling and activation in vivo, transitional B cells from spleens of BXD2 mice exhibited elevated basal phospho-tyrosine and BCR induced activation, beginning at the T2 stage. Single cell analysis of transitional T2 B cells from BXD2 mice revealed a heterogeneity of gene expression within this population and a negative correlation between the expression of IFNαR gene and Id3, an important transcription factor for B-cell anergy. Our results suggest that B-cell tolerance loss in BXD2 mice occurs at the transitional stage, and IFNα exhibits the potential to break tolerance to RNP in transitional B cells.

Endogenous dsRNA and dsDNA sensing is increased in transitional B cells in BXD2 autoimmune-prone mice

Abstract Enhanced nucleic acid-sensing (NS) pathways has been associated with development of autoimmune disease. Increased type I IFN signaling is identified in lupus prone BXD2 mouse B cells and there is a predominant production of autoantibodies against RNA binding autoantigens in BXD2 mice. Determination of the expression of Usp18 and Oasl2 in early CD93+ transitional B cells (T1 and T2) revealed a surprising finding that early transitional B cell exhibited elevated type I IFN signature gene expression, compared to CD93− mature B cells. Consistent with this, there is also elevated expression of NS pathway genes in early transitional B cells. The most highly expressed were the dsRNA sensing Rig1 and transcription factors Irf3 and Irf7, which were expressed at higher levels compared to pDCs. Interestingly, although Usp18 and Oasl2 levels were undetectable in BXD2-Ifnar−/− transitional B cells, dsRNA sensing gene expression were highly expressed suggesting an IFNAR-independent up-regulation of dsRNA NS pathway in transitional B cells. There was lower expression of Mda5 and Pkr5, and genes involved in DNA sensing including Zbp1 and Irf9. Stimulation of B cells with a RIG1 agonist, poly(I:C)-LMW/LyoVec leads to significantly elevated induction of NS pathway genes in both WT and IFNAR deficient transitional B cells from BXD2 mice. These results suggest that transitional B cells have an intrinsic enhanced nucleic acid-sensing pathway, allowing them to rapidly react to nucleic acid stimulation. Dysregulation of this pathway as a result of apoptotic cell clearance defects can act together with type I IFN stimulation to lead to survival and maturation of B cells that by-pass tolerance checkpoint 2 and produce anti-nucleic acid autoantibodies in lupus.

IL-17 receptor A signaling impedes NF-κB p50/p50 repressor and subverts B-cell anergy in BXD2 mice

Abstract B-cell fate decision checkpoint 2 at the transitional T2 stage represents a critical checkpoint defect in the development of autoimmune disease. We have found that there is a failure of anergy induction in the CD93+IgM+CD23+ transitional T2 B cells in the spleen of lupus prone BXD2 mice, which is associated with B-cell tolerance loss to anti-IgM or TLR7 in vitro. Interestingly, there was an enhanced BXD2-Il17ra−/− T2 B cell anergy phenotype in vivo, which is associated with a strong B cell anergic response to BCR or TLR7 stimulation in vitro. Abnormal survival of T2 B cells is thought to be regulated through a strong BAFF-R signaling. Surprisingly, despite normal expression of BAFF-R, BlyS cannot reverse the anergic response of BXD2-Il17ra−/− B cells to BCR stimulation. Analysis of the expression of IL-17RA in the B-cell subsets in BXD2 mice shows that only T2 and germinal center (PNA+Fas+) B cells, but not T1, T3 or mature (CD93−) B cells express surface IL-17RA. Single cell analysis of T2 B cells reveals the co-expression of Il17ra with B-cell activation gene, Bclxl. The strong anergic phenotype of BXD2-Il17ra−/− mouse B cells is associated with a dramatically enhanced nuclear expression of NF-κB1 (p50) and down-modulation of NF-κB phospho-p65. Our results suggest that, in BXD2-Il17ra−/− B cells, the anergy phenotype is established at the T2 stage. In these B cells, the stimulus-specific transcription repressor p50/p50 homodimer may act as a master transcriptional regulator to counteract pro-activation/survival NF-κB signaling provided by other major B-cell stimulators to enforce B cell anergy. Reagents that can promote the NF-κB p50/p50 repressome complex may be a novel strategy to enhance B-cell tolerance for autoimmunity.

Enhanced IFN-γ signals and STAT5-phosphatase dependent attenuated IL-2 signals in the pathogenesis of rheumatoid arthritis.

Abstract The importance of type I interferons as a driver of pathogenesis in autoimmunity, including rheumatoid arthritis (RA) is well recognized. However, in this context the contribution of type II interferons (IFN-γ) has been equivocal. This study developed from our recent discovery that the expression of IFN-γ receptor gene Ifngr1 strongly associates with RA disease and expression of Ifngr2 associates with radiographic severity. In this study we show that IFN-γ induced activation is significantly greater in naïve, central memory, Tfh and Treg subsets of CD4+ T cell populations from RA patients compared to healthy controls (HC). Remarkably, such enhanced IFN-γ induced STAT1 activation was not observed in CD8 T cell populations from RA patients. The activation of STAT1 and consequently, IFN-γ signals are regulated by tyrosine phosphatases. Treatment with phenyl-arsine oxide (PAO), a broadly active phosphatase inhibitor, had no effect on IFN-γ induced STAT1 activation in any T cell population from RA or HC. In contrast, such treatment with PAO greatly enhanced IL-2 induced activation of STAT5 in RA CD4 T cell populations (naïve, central memory effector memory, Treg, Tfh) but not from HC or in CD8 T cell populations. This result suggests that the regulatory activity of IL-2 in RA is dampened by a STAT5-specific phosphatase. We further determined that such a STAT5-phosphatase was also expressed and active in regulatory and effector CD4 T cell populations from the spontaneously autoimmune BXD2 mice. Overall our results indicate that CD4 T cell subpopulation dependent enhanced IFN-γ signals and dampened IL2-STAT5 signals contributes to pathogenesis in RA.

General Approach for Tetramer-Based Identification of Autoantigen-Reactive B Cells: Characterization of La- and snRNP-Reactive B Cells in Autoimmune BXD2 Mice.

Autoreactive B cells are associated with the development of several autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. The low frequency of these cells represents a major barrier to their analysis. Ag tetramers prepared from linear epitopes represent a promising strategy for the identification of small subsets of Ag-reactive immune cells. This is challenging given the requirement for identification and validation of linear epitopes and the complexity of autoantibody responses, including the broad spectrum of autoantibody specificities and the contribution of isotype to pathogenicity. Therefore, we tested a two-tiered peptide microarray approach, coupled with epitope mapping of known autoantigens, to identify and characterize autoepitopes using the BXD2 autoimmune mouse model. Microarray results were verified through comparison with established age-associated profiles of autoantigen specificities and autoantibody class switching in BXD2 and control (C57BL/6) mice and high-throughput ELISA and ELISPOT analyses of synthetic peptides. Tetramers were prepared from two linear peptides derived from two RNA-binding proteins (RBPs): lupus La and 70-kDa U1 small nuclear ribonucleoprotein. Flow cytometric analysis of tetramer-reactive B cell subsets revealed a significantly higher frequency and greater numbers of RBP-reactive marginal zone precursor, transitional T3, and PDL-2(+)CD80(+) memory B cells, with significantly elevated CD69 and CD86 observed in RBP(+) marginal zone precursor B cells in the spleens of BXD2 mice compared with C57BL/6 mice, suggesting a regulatory defect. This study establishes a feasible strategy for the characterization of autoantigen-specific B cell subsets in different models of autoimmunity and, potentially, in humans.

Relapse of lupus after B cell depletion therapy is associated with loss of apoptotic debris clearing marginal zone macrophages in BXD2 mice (THER5P.911)

Abstract B-cell depletion therapy (BCDT) is used to treat autoimmune diseases but development of autoreactive B cells can occur during repopulation. Marginal zone macrophages (MZMs) are essential for tolerogenic clearance of apoptotic debris. Maintenance of MZMs requires MZ B cells. To determine if BCDT using anti-CD20 depletes MZ B cells and promote the loss of MZMs, lupus prone BXD2 mice were administered α-CD20 or isotype control (250 µg/single dose i.v.). This resulted in depletion of &amp;gt;80% B cells in the spleen at week 2 in BCDT group. This was associated with increased uncleared apoptotic debris in the spleen MZ and a dramatic decline in MZMs. By 5 weeks of BCDT, the B-cell repopulation rate was &amp;gt;50%. However, there was a significantly elevated expression of type I IFN signature genes Usp18 and Oasl in repopulated MZ precursor B cells in anti-CD20 group. There was a 2.8-fold increase in PNA+Fas+ CD19+ germinal center B cells in the spleens of anti-CD20-treated BXD2 mice, compared to isotype-treated mice. At week 9, the serum titers of IgG autoantibodies against DNA, histone, and malondialdehyde, a lipid peroxidation product associated with apoptotic blebs, were markedly increased in the α-CD20-treated BXD2 mice than control group. These results indicate that BCDT in BXD2 mice promotes an environment with excessive uncleared apoptotic debris which enhanced the effects of type I IFNs on repopulating B cells, leading to the return of autoreactive B cells.

Increased PKC signaling in lupus La reactive B cells promotes development of the transitional T3 population and memory B cells in autoimmune BXD2 mice (BA3P.108)

Abstract We recently identified that lupus prone BXD2 mice exhibited defective clearance of apoptotic debris. In vivo administration of excessive autologous apoptotic cells lead to elevation of IgG anti-La13-27 responses in BXD2 mice but not normal B6 mice, suggesting chronic self-antigen stimulation plus B-cell tolerance defects may be related to the IgG anti-La13-27 responses. To interrogate if there was abnormal maturation of anti-La13-27 B cells in naïve BXD2 mice, a recently developed La13-27 tetramer was used with CD80 and PD-L2 co-staining to reveal that there was a significantly higher frequency of memory CD80+PD-L2+ La13-27+ B cells in BXD2 (17%), compared to that in B6 (3%) mouse spleens. Tetramer analysis of CD93+ transitional B cells showed that there was an abnormal skewing of the La13-27+ T3 population, but not T1 or T2 B cells, in BXD2 (40%) compared to that in B6 (18%) mouse spleens. Survival of anti-La13-27 producing B cells in BXD2 but not B6 mice was associated with increased protein kinase C (PKC) activation, as stimulation of B cells with PMA+ionomycin which promotes survival of BCR stimulated transitional B cells resulted in an increase in IgG. As T3 B cells are considered anergic B cells maintaining self-tolerance through rapid turnover in vivo, the abnormal expansion of these B cells coupled with increased BCR signaling response in BXD2 mice may present a risk for B-cell tolerance loss to La13-27, leading to maturation into IgG secreting cells.

Type I interferons promote lupus through disruption of the lymphotoxin receptor-mediated mechanosensing MKL1 pathway in marginal zone macrophages (BA4P.124)

Abstract Defective clearance of apoptotic cells (ACs) is a central pathogenic event in systemic lupus erythematosus (SLE). Here, we describe an underlying mechanism in which follicular translocation of marginal zone (MZ) B cells in the spleen of lupus BXD2 mice disrupts marginal zone macrophages (MZMs), which normally clear apoptotic debris and prevent entry of the AC antigens (AC-Ags) into the follicles. The phagocytosis of ACs by the spleen MZMs is enabled by a megakaryoblastic leukemia 1 (MKL1) transcription factor-mediated mechanosensing pathway. The MZ B cells maintain this pathway through their expression of membrane lymphotoxin-α1β2 (mLT). Follicular shuttling of mLT+ MZ B cells disengages their interaction with the LTβR+ MZMs, thereby downregulating MKL1 in the MZMs. This leads to defective F-actin polymerization, loss of their ability to clear ACs and eventually MZM dissipation. Type I interferon (IFN) signaling, a well-established clinical characteristic of SLE and is commonly considered as a downstream event of AC-Ag stimulation, is identified to play a key role in MZM erosion via promoting follicular shuttling of mLT+ B cells. Follicular translocation of MZ B cells, erosion of MZMs and loss of MKL1 were similarly identified in lupus B6.Sle1.Sle2.Sle3 mouse and human SLE spleens. Collectively, the results suggest that lupus might be interrupted by strategies that maintain or enhance mechanosensing signals in the MZM barrier to prevent follicular entry of AC-Ags.