The pathogenesis of Systemic lupus erythematosus (SLE) is driven by autoreactive B and T cells and autoinflammation. Impaired apoptosis in T and B cells has been shown to result in autoimmune disease, and we recently demonstrated that reducing apoptosis in B cells alone, by B cell-specific deletion of Bim (B.Bim f/f) could lead to SLE with prominent features of Sjogren’s Syndrome in C57BL/6 mice. Consistently, B cell targeting therapies are successful in reducing SLE pathogenesis in mouse models, however, their clinical success requires a better understanding of the contributions of B cells and the mechanisms that drive inflammation to autoimmunity. To address the role of inflammation in the development and progression of autoimmunity we generated a novel mouse model that lacks ZFAND6 (Zfand6 −/−) and crossed with the B.Bim f/fmice. ZFAND6 is an A-20 like ZF domain-containing protein with a potential role in the negative regulation of NF-kB activation, however, its physiological function is unknown. The compound mutant mice displayed accelerated and exacerbated overlapping lupus and Sjogren’s autoimmune symptoms with a higher incidence of splenomegaly and kidney pathology than either single mutant alone including the early appearance of anti-SSA, anti-SSB, and anti-RNP autoantibodies. The compound mutant mice displayed increased spontaneous and TLR-induced TNFa and IL-6 and B cells from these mice produced much greater levels of these cytokines in response to TLR7 and TLR9. These results suggest that inappropriate B cell survival and persistent inflammation cooperate in the initiation and severity of lupus pathogenesis. Sylvester Comprehensive Cancer Center, University of Miami, Miami FL

While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl ) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren’s Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton’s tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.

Systemic lupus erythematosus (SLE) afflicts more than 1.5 million individuals in the United States with very limited and debilitating therapeutic options. The etiology of the disease remains unclear. It is hypothesized that in SLE, peripheral B cell tolerance is breached by inappropriate survival of autoreactive cells. Consistently, overexpression of B cell activating factor (BAFF), a key regulator of B cell survival can rescue autoreactive B cells and contribute to autoimmune disease. BAFF counters apoptosis in part through decreasing BH3-only pro-apoptotic protein Bim. Notably, both BAFF and Bim are physiological regulators of B cell tolerance. Therefore, to define the role of B cells and B cell tolerance in the initiation and progression of autoimmune disease we created a mouse model wherein gene encoding Bim is selectively deleted in B cells (B.Bimf/f). Initial analyses of these mice suggest that B cells can initiate an autoimmune pathogenesis phenotypically similar to SLE, with mice exhibiting splenomegaly and increased CD21loCD23lo B cells. Given that persistent activity of NF-kB in B cells can also result in autoimmune diseases, we are now investigating whether the persistent NF-kB and dysregulated apoptosis can synergize in SLE pathogenesis. To that end, we have crossed B.Bimf/f mice with a mouse line that lacks a recently discovered potential negative regulator of NF-kB, ZFAND6 (B.Bimf/f x ZfanD6−/−). Preliminary findings suggest that the compound mutant mice display exacerbated autoimmune symptoms with higher incidence of splenomegaly and kidney pathology. These preliminary results suggest that a breach in tolerance (B.Bimf/f ) and persistent NF-kB activity cooperate in the initiation and severity of SLE pathogenesis.

Splenic transitional B-cells (T1 and T2) are selected to avoid self-reactivity and to safeguard against autoimmunity, then differentiate into mature follicular (FO-I and FO-II) and marginal zone (MZ) subsets. Transcriptomic analysis by RNA-seq of the five B-cell subsets revealed T1 cell signature genes included RAG suggesting a potential for receptor revision. T1 to T2 B-cell differentiation was marked by a switch from Myb to Myc, increased expression of the PI3K adapter DAP10 and MHC class II. FO-II may be an intermediate in FO-I differentiation and may also become MZ B-cells as suggested by principle component analysis. MZ B-cells possessed the most distinct transcriptome including down-regulation of CD45 phosphatase-associated protein (CD45-AP/PTPRC-AP), as well as upregulation of IL-9R and innate molecules TLR3, TLR7, and bactericidal Perforin-2 (MPEG1). Among the endosomal TLRs, stimulation via TLR3 further enhanced Perforin-2 expression exclusively in MZ B-cells. Using gene-deleted and overexpressing transgenic mice we show that IL-9/IL-9R interaction resulted in rapid activation of STAT1, 3, and 5, primarily in MZ B-cells. Importantly, CD45-AP mutant mice had reduced transitional and increased mature MZ and FO B-cells, suggesting that it prevents premature entry of transitional B-cells to the mature B-cell pool or their survival and proliferation. Together, these findings suggest, developmental plasticity among splenic B-cell subsets, potential for receptor revision in peripheral tolerance whereas enhanced metabolism coincides with T2 to mature B-cell differentiation. Further, unique core transcriptional signatures in MZ B-cells may control their innate features.

Summary In aged mice, new B‐cell development is diminished and the antibody repertoire becomes more autoreactive. Our studies suggest that (i) apoptosis contributes to reduced B lymphopoiesis in old age and preferentially eliminates those B‐cell precursors with higher levels of the surrogate light chain (SLC) proteins (λ5/VpreB) and (ii) λ5 low B‐cell precursors generate new B cells which show increased reactivity to the self‐antigen/bacterial antigen phosphorylcholine (PC). Pro‐B cells in old bone marrow as well as pro‐B cells from young adult λ5‐deficient mice are resistant to cytokine‐induced apoptosis (TNFα; TGFβ), indicating that low λ5 expression in pro‐B cells is sufficient to cause increased survival. Transfer of TNFα‐producing ‘age‐associated B cells’ (ABC; CD21/35 − CD23 − ) or follicular (FO) B cells from aged mice into RAG‐2 KO recipients led to preferential loss of λ5 high pro‐B cells, but retention of λ5 low , apoptosis‐resistant pro‐B cells. In old mice, there is increased reactivity to PC in both immature bone marrow B cells and mature splenic FO B cells. In young mice, absence of λ5 expression led to a similar increase in PC reactivity among bone marrow and splenic B cells. We propose that in old age, increased apoptosis, mediated in part by TNFα‐producing B cells, results in preferential loss of SLC high pro‐B cells within the bone marrow. Further B‐cell development then occurs via an ‘SLC low ’ pathway that not only impairs B‐cell generation, but promotes autoreactivity within the naïve antibody repertoires in the bone marrow and periphery.

Abstract Age-associated B cells (ABC, CD21/35- CD23-) accumulate in spleen and bone marrow of ~2 yrs. old mice and induce TNF-α-dependent apoptosis of pro-B cells. The remaining B cell precursors in aged mice exhibit reductions in surrogate light chain (SLC) and compromise of the preBCR checkpoint. Notably, the residual pro-B cells in aged bone marrow are resistant to ABC/TNF-α-mediated apoptosis. SLC is associated with cadherin 17 on the surface of pro-B cells. Like SLC-low aged pro-B cells, pro-B cells from λ5 SLC-deficient young adult mice were also resistant to TNF-α-induced apoptosis. This implies that expression of the SLC/cadherin 17 complex is required for normal susceptibility to apoptosis in pro-B cells. Resistance to apoptosis in aged pro-B cells and λ5-deficient young pro-B cells coincided with increased phosphorylation of the pro-apoptotic protein Bim, a modification that tags Bim for degradation. We hypothesize that, in old mice, expanded pro-inflammatory ABC within the bone marrow cause apoptosis of pro-B cells. However, this cell death occurs preferentially in pro-B cells which express relatively high levels of SLC. The remaining pro-B cells in aged mice are consequently skewed to lower SLC expression. We suggest that ABC, by inducing the loss of “high SLC” pro-B cells, deviate B lymphopoiesis into an ”SLC low pathway” with inefficient B cell production and altered B cell antibody repertoire.

B-lymphocytes are integral to host defense against microbial pathogens and are associated with many autoimmune diseases. The B-cell receptor implements B-cell self-tolerance based on the antigen specificity, and B-cell-activating factor receptor (BAFF-R) imposes homeostatic control. While shaping the repertoire, the immune tolerance process also culls mature B cells into distinct populations. The activation response of B cells is tailored to the type of pathogen attack and is facilitated by T-cell help via CD40/CD40L interaction and/or innate cell help via toll-like receptors in conjunction with BAFF receptors and ligands. Activated effector B cells not only produce antibodies, but also produce a variety of cytokines to enhance and suppress the immune response. Not surprisingly, B cells play multiple roles in both humoral and cellular immune responses during infection and autoimmune pathogenesis. Here, we discuss how gene expression and signaling networks regulate peripheral B-cell tolerance, B-cell effector functions and emerging therapies targeting B-cell signaling in autoimmune diseases.

Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on BAFF for survival. BAFF activates the canonical and noncanonical NF-κB pathways, both of which are required for B cell survival. In this study we report that deficiency of the adaptor protein B cell lymphoma 10 (Bcl10) impaired the ability of BAFF to support B cell survival in vitro, and it specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. Bcl10-dependent survival of self-reactive anergic B cells was confirmed in the Ig hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model of B cell anergy. Furthermore, we found that BAFF stimulation induced Bcl10 association with IκB kinase β, a key component of the canonical NF-κB pathway. Consistently, Bcl10-deficient B cells were impaired in BAFF-induced IκBα phosphorylation and formation of nuclear p50/c-Rel complexes. Bcl10-deficient B cells also displayed reduced expression of NF-κB2/p100, severely reducing BAFF-induced nuclear accumulation of noncanonical p52/RelB complexes. Consequently, Bcl10-deficient B cells failed to express Bcl-xL, a BAFF-induced NF-κB target gene. Taken together, these data demonstrate that Bcl10 controls BAFF-induced canonical NF-κB activation directly and noncanonical NF-κB activation indirectly. The BAFF-R/Bcl10/NF-κB signaling axis plays a critical role in peripheral B cell tolerance by regulating the survival of self-reactive anergic B cells.

Autoreactive B cells are known to contribute to the onset and progression of a variety of autoimmune diseases including systemic lupus erythematosus (SLE). To prevent this, the B cell receptor (BCR) exerts specificity-based quality control and B cell activating factor receptor (BAFF-R) maintains homeostasis primarily through controlling apoptosis. An appropriate balance in the levels of the pro- and anti-apoptotic members of the Bcl-2 family is central to this process. Using mice with B lineage-specific deletion of the pro-apoptotic gene Bim (B-bim-/-), we showed that B cell autonomous dysregulation of apoptosis is sufficient to cause systemic autoimmune disease with lymphoproliferation. B-bim-/- mice display progressive splenomegally and lymphoadenopathy with a significant increase in B cells as well as an overall increase in innate and T cells which display markers of activation. B-bim-/- mice had higher serum levels of autoantibodies and proinflammatory cytokines. These mice also displayed increased lymphocyte proliferation and infiltration of the salivary gland, lung and liver. Furthermore, accumulation of lymphocytes in the B-bim-/- mice results in the formation of tertiary lymphoid structures. These results suggest that apoptosis resistant B-bim-/- B cells can acquire an activated phenotype and become pathogenic, culminating in the activation of T cells and the generation of a pro-inflammatory microenvironment leading to systemic autoimmunity. In addition, massive lymphoproliferation and inflammation may predispose B-bim-/- mice to B cell and other hematopoietic malignancies.

PROBLEM:Hepatocyte Growth Factor (HGF) secretion facilitates epithelial cell growth and development in the female reproductive tract (FRT) and may contribute to pathological conditions such as cancer and endometriosis. We hypothesized that estradiol and poly (I:C), a synthetic RNA mimic, may have a regulatory effect on HGF secretion by stromal fibroblasts from FRT tissues. METHOD OF STUDY:Following hysterectomies, normal tissue from the uterus, endocervix, and ectocervix were dispersed into stromal cell fractions by enzymatic digestion and differential filtering. Stromal fibroblasts were cultured and treated with estradiol and/or poly (I:C), and conditioned media were analyzed for HGF via enzyme-linked immunosorbent assay. RESULTS:Treating uterine fibroblasts with estradiol or poly (I:C) significantly increased HGF secretion. When uterine fibroblasts were co-treated with estradiol and poly (I:C), the effect on HGF secretion was additive. In contrast, stromal fibroblasts from endo- and ecto-cervix were unresponsive to estradiol, but were stimulated to secrete HGF by poly (I:C). CONCLUSION:HGF secretion is uniquely regulated in the uterus, but not in ecto- and endo-cervix, by estradiol. Moreover, potential viral pathogens further induce HGF. These findings have potential applications in understanding both hormonal regulation of normal tissue as well as the role of HGF in tumorogenesis, endometriosis, and human immunodeficiency virus infection.

Abstract Splenic immature B cells traverse through the early transitional (T1) and late transitional (T2) stages to produce the mature B cell repertoire. BCR specificity-based selection ensures elimination of pathogenic B cells by apoptosis, receptor editing or anergy. These processes are tightly regulated by anti- and pro-apoptotic members of the Bcl-2 family. Conventional gene deletion of the pro-apoptotic member Bim lead to prolonged life-span of autoreactive B cells, defective anergy and production of autoantibodies. Whether these pathophysiological conditions arise as a result of B cell autonomous function of Bim remains unclear. We have found that T1 cells express higher levels of Bim and display increased susceptibility to BCR-induced apoptosis than more mature B cells. These properties are shared by BCR signaling defective Bruton’s tyrosine kinase (Btk) deficient B cells. We investigated the B cell-intrinsic role of Bim in the regulation of B cell maturation and survival in mice carrying floxed Bim and CD19-Cre in Btk-sufficient or deficient background. B lineage-specific deletion of Bim increased the B cell compartment through selective expansion of mature B cells and significantly restored mature B cells in Btk-mutant mice. These studies demonstrate a B cell autonomous role for Bim in BCR-dependent regulation of B cell homeostasis. Analysis of these mice in the context of BCR transgene will reveal the B cell autonomous function of Bim in tolerance and autoimmunity.

BACKGROUND:DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. METHODOLOGY AND PRINCIPAL FINDINGS:Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8+ and/or CD4+ T cells. Finally adjuvants SP-D-OX40L and SP-D-CD70 increased T(H)1 (IgG2a) but not T(H)2 (IgG1) antibody responses in the vaccinated animals. Surprisingly, the B cell-activating protein BAFF did not enhance anti-Gag antibody responses when given as an SP-D fusion adjuvant, but nonetheless enhanced CD4+ and CD8+ T cell responses. CONCLUSIONS:We present evidence that various SP-D-TNFSFL fusion constructs can enhance immune responses following DNA vaccination with HIV-1 Gag expression plasmid. These data support the continued evaluation of SP-D-TNFSFL fusion proteins as molecular adjuvants for DNA and/or viral vector vaccines. Constructs of particular interest included SP-D-OX40L, SP-D-4-1BBL, SP-D-LIGHT, and SP-D-CD70. SP-D-BAFF was surprisingly effective at enhancing T cell responses, despite its inability to enhance anti-Gag antibody secretion.

B cell receptor (BCR) and B cell activating factor receptor (BAFF-R) signaling are critical for the generation of mature splenic B cells. BCR expressing Transitional type 1 (T1) cells migrate from the bone marrow to the spleen where they must successfully pass BCR mediated checkpoints to become Transitional type 2 (T2) cells and subsequently mature follicular B (FoB) cells. T1 and T2 cells are fundamentally different in their response to BCR or BAFF-R stimulation; T1 cells do not survive whereas T2 cells do when triggered through BCR or BAFF-R. Our results demonstrate that Bruton’s tyrosine kinase (Btk) plays a critical role in both BCR and BAFF-R mediated T2 and mature B cell survival. These results suggest that a potential mechanism that provides T2 cells with a survival advantage is their ability to sustain c-Rel expression in response to BCR engagement via mechanisms involving Btk. The increased c-Rel expression coincides precisely with the up-regulation of anti-apoptotic members of the Bcl-2 family as well as BAFF-R and its substrate p100 (NF-κB2). Thus, BCR indirectly enhances the survival function of BAFF-R in part through c-Rel. Consistent with a supporting role for BCR/c-Rel signaling in the activation of alternative NF-κB pathway, c-Rel-deficient B cells are impaired for BCR-induced expression of p100 as well as RelB.

Signaling from the BCR and B cell activating factor receptor (BAFF-R or BR3) differentially regulates apoptosis within early transitional (T1) and late transitional (T2; CD21int-T2) B cells during selection processes to generate mature B lymphocytes. However, molecular mechanisms underlying the differential sensitivity of transitional B cells to apoptosis remain unclear. In this study, we demonstrate that BCR signaling induced more long-term c-Rel activation in T2 and mature than in T1 B cells leading to increased expression of anti-apoptotic genes as well as prosurvival BAFF-R and its downstream substrate p100 (NF-κB2). Sustained c-Rel activation required de novo c-Rel gene transcription and translation via Btk-dependent mechanisms. Like T1 cells, mature B cells from Btk- and c-Rel-deficient mice also failed to activate these genes. These findings suggest that the gain of survival potential within transitional B cells is dependent on the ability to produce a long-term c-Rel response, which plays a critical role in T2 B cell survival and differentiation in vivo by inducing anti-apoptotic genes, BAFF-R and NF-κB2, an essential component for BAFF-R survival signaling. Thus, acquisition of resistance to apoptosis during transitional B cell maturation is achieved by integration of BCR and BAFF-R signals.

OBJECTIVE:To examine the expression of toll-like receptors (TLR) by primary human Fallopian tube epithelial cells (FTEC) and to determine whether exposure to the TLR3 agonist poly(I:C) induces an antiviral response. DESIGN:Tissue culture study. SETTING:University medical center. PATIENT(S):Premenopausal women undergoing hysterectomy. INTERVENTION(S):Primary human FTEC were grown to confluence and high transepithelial resistance and treated with TLR agonists. Conditioned media was collected and RNA was extracted and analyzed for the expression of cytokines, chemokines, and antimicrobial genes. MAIN OUTCOME MEASURE(S):The RNA was analyzed by real-time polymerase chain reaction and protein levels were assessed by enzyme-linked immunosorbent assay. RESULT(S):The FTEC were demonstrated to express TLR1-9 but not 10. Treatment of FTEC with TLR3 agonist poly(I:C) resulted in increased expression of interleukin-8, tumor-necrosis factor alpha, human beta-defensin 2, interferon beta, and interferon stimulated genes myxovirus resistance gene 1, 2',5'-oligoadenylate synthetase, and protein kinase R. Additionally, FTEC exposed to poly(I:C) also resulted in the induction of TLR2, TLR3, and TLR7. CONCLUSION(S):Our results suggest that FTEC are sensitive to viral infection and/or exposure to viral double-stranded RNA and can respond by secreting proinflammatory cytokines that mediate the initiation of an inflammatory response as well as expressing genes that can directly inhibit viral replication.

The uterine endometrium coordinates a wide spectrum of physiologic and immunologic functions, including endometrial receptivity and implantation as well as defense against sexually transmitted pathogens. Macrophages and epithelial cells cooperatively mediate innate host defense against bacterial invasion through the generation of immunologic effectors, including cytokines and antimicrobial peptides. In this study, we demonstrate that stimulation of peripheral blood monocytes and uterine macrophages with bacterial LPS induces the production of biologically active proinflammatory IL-1beta. High doses of estradiol enhance LPS-induced IL-1beta expression in an estrogen receptor-dependent manner. Furthermore, both peripheral blood monocyte- and uterine macrophage-derived IL-1beta induce secretion of antimicrobial human beta-defensin 2 by uterine epithelial cells. These data indicate dynamic immunologic interaction between uterine macrophages and epithelial cells and implicate a role for estradiol in the modulation of the immune response.

BACKGROUNDDuring pregnancy, the placenta may become exposed to micro-organisms, such as viruses, which may pose a substantial threat to the embryo/fetus well-being. Recent insight into the immunological capabilities of the trophoblast suggests that the placenta may function as an active barrier by recognizing and responding to pathogens through Toll-like receptors (TLRs).METHODSThe objective of this study was to determine whether the engagement of TLR-3 with viral dsRNA by first-trimester trophoblast could induce the production of factors necessary to generate an antiviral response. Therefore, trophoblast cells were exposed to the TLR-3 agonist, Poly(I : C).RESULTSWe report that following stimulation with Poly(I : C), first-trimester trophoblast cells produce interferon beta (IFNbeta) and secretory leukocyte protease inhibitor (SLPI), as well as the intracellular factors 2',5'-oligoadenylate synthetase (OAS), Myxovirus-resistance A (MxA) and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G). This response is TLR-3 specific because the TLR-4 ligand, lipopolysaccharide (LPS), had no effect on the production of these antimicrobial factors. Furthermore, we describe a positive feedback mechanism in which IFNbeta enhances the antiviral response by promoting the production of OAS, MxA and APOBEC3G.CONCLUSIONSThese findings suggest that trophoblast cells are able to recognize and specifically respond to viral products in a highly regulated fashion and that the placenta may be pivotal in the control of viral infections at the maternal-fetal interface.