Search

A human La/Sjögren’s syndrome-B (hLa)–specific TCR/hLa neo–self-Ag double-transgenic (Tg) mouse model was developed and used to investigate cellular tolerance and autoimmunity to the ubiquitous RNA-binding La Ag often targeted in systemic lupus erythematosus and Sjögren’s syndrome. Extensive thymic clonal deletion of CD4+ T cells occurred in H-2k/k double-Tg mice presenting high levels of the I-Ek–restricted hLa T cell epitope. In contrast, deletion was less extensive in H-2k/b double-Tg mice presenting lower levels of the epitope, and some surviving thymocytes were positively selected as thymic regulatory T cells (tTreg). These mice remained serologically tolerant to hLa and healthy. H-2k/b double-Tg mice deficient of all endogenous Tcra genes, a deficiency known to impair Treg development and function, produced IgG anti-hLa autoantibodies and displayed defective tTreg development. These autoimmune mice had interstitial lung disease characterized by lymphocytic aggregates containing Tg T cells with an activated, effector memory phenotype. Salivary gland infiltrates were notably absent. Thus, expression of nuclear hLa Ag induces thymic clonal deletion and tTreg selection, and lymphocytic infiltration of the lung is a consequence of La-specific CD4+ T cell autoimmunity.

Thrombotic thrombocytopenic purpura (TTP) is a rare life-threaten vascular autoimmune disease. There is no effective method to treat it in clinical trials since pathogenesis of TTP has not been fully elucidated. Here we investigate the role of NK cells in relapse development of TTP. Our results showed that the frequencies of CD3 − CD56 dim CD16 − and CD3 − CD56 bri CD16 − NK cells are increased in TTP patients with a history of relapse. Expression of CD107a, granzyme A and IFN-γ by CD3 − CD56 dim NK cells following in vitro stimulation with PMA/ionomycin / monensin is improved in the relapse group, compared with those on NK cells derived from TTP patients without relapse development. NK cells isolated from TTP patients with a history of relapse indicated stronger cytotoxicity to target K562 cells than those of NK cells derived from TTP patients without relapse development, suggesting prior activation of NK cells in vivo . Treatment with anti-human CD16 antibody up-regulates cytotoxicity of NK cells derived from TTP patients without relapse development. However, Anti-human CD16 antibody treatment does not affect cytotoxicity of NK cells isolated from TTP patients with a history of relapse, suggesting inability of CD16-mediated signaling in NK cells derived from TTP patients with relapse development. These data provide evidence of altered NK cell activation and/or licensing in TTP patients with a history of relapse modulated by CD16-mediated signaling and a new avenue of investigation into mechanisms of TTP immunopathogenesis.

We investigated the consequences of altering the form and valence of defined autodeterminants on the initiation and spreading of experimentally induced La/Ro autoimmunity. Anti-La and Ro (SS-A) Ab responses were monitored following immunization of healthy mice with defined immunodominant and subdominant T cell determinants of the La (SS-B) autoantigen synthesized as either monomeric or multiple antigenic (MAP) peptides. Abs to mouse La (mLa) developed faster and were of higher titer in mice immunized with the subdominant mLa25–44 MAP compared with mice immunized with the 25–44 monomer. Rapid intermolecular spreading of the autoimmune response to 60-kDa Ro was observed in AKR/J mice immunized with mLa25–44 MAP, but not in mice immunized repeatedly with monomeric peptide. A/J mice immunized and boosted with the known tolerogenic mLa287–301 determinant delivered as monomeric peptide failed to develop Abs to either intact mLa or mLa287–301 peptide. However, immunization with the multivalent mLa287–301 peptide led to the rapid production of high titer mLa autoantibodies associated with a proliferative T cell response to the mLa287–301 peptide. The data suggested that the enhanced immunogenicity of MAPs was not due to augmented Ag presentation or T cell stimulation. However, MAP-, but not monomer peptide-, containing immune complexes were potent substrates for Ab-dependent fixation of complement. These results demonstrate that the form of Ag responsible for inducing autoimmunity can profoundly influence the nature and magnitude of the immune response. Thus, molecular mimicry of tolerogenic and nontolerogenic self determinants might trigger autoimmunity under conditions of altered valence.

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called “telomeres” to the growing ends of chromosomal DNA. Characterization of mammalian telomerase has been elusive because of its low level of expression. We describe a bioinformatics approach to enrich and characterize the human telomerase complex. Using local sequence homology search methods, we detected similarity of the Tetrahymena p80 subunit of telomerase with the autoantigen Ro60. Antibodies to Ro60 immunoprecipitated the telomerase activity. Ro60 and p80 proteins were cross-recognizable by antibodies to either protein. Telomerase activity and the RNA component of telomerase complex were localized to a doublet in a native gel from the Ro60 antibody-precipitated material. The enriched material showed specific binding to a TTA GGG probe in vitro in an RNA template-dependent manner. Polyclonal antibodies to the doublet also immunoprecipitated the telomerase activity. These results suggest an evolutionary conservation of the telomerase proteins.

Objective To compare the performance of the American–European Consensus Group (AECG) and the newly proposed American College of Rheumatology (ACR) classification criteria for Sjogren9s Syndrome (SS) in a well-characterised sicca cohort, given ongoing efforts to resolve discrepancies and weaknesses in the systems. Methods In a multidisciplinary clinic for the evaluation of sicca, we assessed features of salivary and lacrimal gland dysfunction and autoimmunity as defined by tests of both AECG and ACR criteria in 646 participants. Global gene expression profiles were compared in a subset of 180 participants. Results Application of the AECG and ACR criteria resulted in classification of 279 and 268 participants with SS, respectively. Both criteria were met by 244 participants (81%). In 26 of the 35 AECG+/ACR participants, the minor salivary gland biopsy focal score was ≥1 (74%), while nine had positive anti-Ro/La (26%). There were 24 AECG−/ACR+ who met ACR criteria mainly due to differences in the scoring of corneal staining. All patients with SS, regardless of classification, had similar gene expression profiles, which were distinct from the healthy controls. Conclusions The two sets of classification criteria yield concordant results in the majority of cases and gene expression profiling suggests that patients meeting either set of criteria are more similar to other SS participants than to healthy controls. Thus, there is no clear evidence for increased value of the new ACR criteria over the old AECG criteria from the clinical or biological perspective. It is our contention, supported by this report, that improvements in diagnostic acumen will require a more fundamental understanding of the pathogenic mechanisms than is at present available.

The serologic hallmark of primary Sjögren's syndrome (SS) is the presence of IgG antibodies specific for Ro (SSA) and La (SSB). The molecular characteristics of gland-derived B cells at the site of primary SS inflammation have been described previously; however, parallels between glandular antibody-secreting cells (ASCs) and serologic antibody specificities have not been evaluated. We used recombinant monoclonal antibody (mAb) technology to study the specificities of salivary gland (SG)-derived ASCs, evaluate their molecular characteristics, and identify IgG antibody specificity.Human antibodies were generated from glandular IgG ASCs. Heavy chain and light chain use and immunoglobulin subclass were analyzed by sequencing. Enzyme-linked immunosorbent assay, indirect immunofluorescence, enzyme immunoassay, and (35) S-labeled protein immunoprecipitation analysis were used to determine antibody specificity.Evaluation of single ASCs in SG biopsy specimens from a patient with primary SS and a patient with SS and overlapping systemic lupus erythematosus revealed significant concordance between serum autoantibody and glandular ASC specificities. Gland-derived ASC heavy chains and light chains were extensively somatically hypermutated, which is indicative of antigen-driven responses. Specifically, we produced the first fully human mAb derived from SGs.In patients with SS, the SGs are a site for the production of antibodies that extend beyond the canonical Ro and/or La SS specificities. Glandular antibody production strongly reflected the serologic humoral response in the 2 patients whom we studied.

A substantial fraction of individuals vaccinated against anthrax have low to immeasurable levels of serum Lethal Toxin (LeTx)-neutralizing activity. The only known correlate of protection against Bacillus anthracis in the currently licensed vaccine is magnitude of the IgG response to Protective Antigen (PA); however, some individuals producing high serum levels of anti-PA IgG fail to neutralize LeTx in vitro. This suggests that non-protective humoral responses to PA may be immunodominant in some individuals. Therefore, to better understand why anthrax vaccination elicits heterogeneous levels of protection, this study was designed to elucidate the relationship between anti-PA fine specificity and LeTx neutralization in response to PA vaccination. Inbred mice immunized with recombinant PA produced high levels of anti-PA IgG and neutralized LeTx in vitro and in vivo. Decapeptide binding studies using pooled sera reproducibly identified the same 9 epitopes. Unexpectedly, sera from individual mice revealed substantial heterogeneity in the anti-PA IgG and LeTx neutralization responses, despite relative genetic homogeneity, shared environment and exposure to the same immunogen. This heterogeneity permitted the identification of specificities that correlate with LeTx-neutralizing activity. IgG binding to six decapeptides comprising two PA epitopes, located in domains I and IV, significantly correlate with seroconversion to LeTx neutralization. These results indicate that stochastic variation in humoral immunity is likely to be a major contributor to the general problem of heterogeneity in vaccine responsiveness and suggest that vaccine effectiveness could be improved by approaches that focus the humoral response toward protective epitopes in a greater fraction of vaccinees.

Anthrax Lethal Toxin consists of Protective Antigen (PA) and Lethal Factor (LF), and current vaccination strategies focus on eliciting antibodies to PA. In human vaccination, the response to PA can vary greatly, and the response is often directed toward non-neutralizing epitopes. Variable vaccine responses have been shown to be due in part to genetic differences in individuals, with both MHC class II and other genes playing roles. Here, we investigated the relative contribution of MHC class II versus non-MHC class II genes in the humoral response to PA and LF immunization using three immunized strains of inbred mice: A/J (H-2k at the MHC class II locus), B6 (H-2b), and B6.H2k (H-2k). IgG antibody titers to LF were controlled primarily by the MHC class II locus, whereas IgG titers to PA were strongly influenced by the non-MHC class II genetic background. Conversely, the humoral fine specificity of reactivity to LF appeared to be controlled primarily through non-MHC class II genes, while the specificity of reactivity to PA was more dependent on MHC class II. Common epitopes, reactive in all strains, occurred in both LF and PA responses. These results demonstrate that MHC class II differentially influences humoral immune responses to LF and PA.

Sjögren's syndrome is a common autoimmune disease (affecting ?0.7% of European Americans) that typically presents as keratoconjunctivitis sicca and xerostomia. Here we report results of a large-scale association study of Sjögren's syndrome. In addition to strong association within the human leukocyte antigen (HLA) region at 6p21 (P meta = 7.65 × 10 -114), we establish associations with IRF5-TNPO3 (P meta = 2.73 × 10 -19), STAT4 (P meta = 6.80 × 10 -15), IL12A (P meta = 1.17 × 10 -10), FAM167A-BLK (P meta = 4.97 × 10 -10), DDX6-CXCR5 (P meta = 1.10 × 10 -8) and TNIP1 (P meta = 3.30 × 10 -8). We also observed suggestive associations (P meta less than 5 × 10 -5) with variants in 29 other regions, including TNFAIP3, PTTG1, PRDM1, DGKQ, FCGR2A, IRAK1BP1, ITSN2 and PHIP, among others. These results highlight the importance of genes that are involved in both innate and adaptive immunity in Sjögren's syndrome. © 2013 Nature America, Inc. All rights reserved.

YRNAs are small cytoplasmic RNAs that are components of the Ro ribonucleoprotein complex. This complex, which also includes the 60-kDa Ro protein, is a human autoantigen which is conserved among vertebrates, and is of unknown function. Multiple sequences with YRNA homology, known as YRNA-like sequences, have been detected in rabbit, mouse, duck, iguana and frog genomes with human Y cDNA probes. As judged by Northern blots of total RNA, however, not all of these genomic YRNA-like sequences are expressed. Complementary DNA and putative gene sequences for iguana Y3 (iY3) and iguana Y4 (iY4) Ro RNAs have been determined and used, along with previously sequenced human and frog Ro YRNA sequences, to construct the most likely Y3 and Y4 RNA secondary structures. The data presented indicate that Y3 is the most conserved Ro RNA, not only by its more consistent presence in other species, but also at the levels of sequence divergence and secondary structure similarity. The differences observed between the secondary structure solutions for the Y3 and Y4 Ro RNAs are consistent with the possibility that these RNAs perform different cellular functions.

Toll-like receptors have attracted increased attention in recent years, not only for their role in sensing conserved microbial components, but also in the realm of autoimmunity. Though TLRs are most widely known for their capacity to detect conserved motifs of infectious agents, mounting evidence indicates that these innate receptors also promote autoimmune conditions by causing uncontrolled auto-inflammation as a result of chronic recognition of self. In response to the need for modern approaches for treatment of autoimmune diseases, several groups have begun investigating ways to target TLRs as new therapeutic options for autoimmune conditions. Here we discuss recent data describing advancements in Toll-like receptors as therapeutic targets for treatment of autoimmune diseases, with a focus on systemic lupus erythematosus.

Publisher Summary This chapter focuses on examining roles for infection in the initiation and pathogenesis of systemic lupus erythematosus (SLE). It presents accumulating data for a role of Epstein-Barr virus (EBV) in lupus initiation, followed by work in other pathogens linked with SLE autoimmunity. Infections may act as major environmental triggers inducing, promoting, and complicating SLE. This chapter provides a discussion on how environmental exposures, particularly EBV, may influence SLE development in select individuals as well as other processes that exacerbate SLE. Molecular mimicry, epitope spreading, exaggerated local activation of antigen presenting cells, and priming of large numbers of T cells with broad specificities may all have important roles in lupus autoimmunity. The interplay of infections and select lupus genetic polymorphisms are also beginning to be evaluated. Polyclonal activation of infected B cells resulting in B-cell proliferation, enhanced antibody production, and generation of circulating immune complexes, as well as bystander T-cell activation, where enhanced cytokine production induces the expansion of autoreactive T cells, may promote the development of SLE, an illness termed by clinicians and researchers alike as one of the most complex among all chronic multisystem diseases.

The efficacy biomarker of the currently licensed anthrax vaccine (AVA) is based on quantity and neutralizing capacity of anti-protective antigen (anti-PA) antibodies. However, animal studies have demonstrated that antibodies to lethal factor (LF) can provide protection against in vivo bacterial spore challenges. Improved understanding of the fine specificities of humoral immune responses that provide optimum neutralization capacity may enhance the efficacy of future passive immune globulin preparations to treat and prevent inhalation anthrax morbidity and mortality. This study ( n = 1000) was designed to identify AVA vaccinated individuals who generate neutralizing antibodies and to determine what specificities correlate with protection. The number of vaccine doses, years post vaccination, and PA titer were associated with in vitro neutralization, reinforcing previous reports. In addition, African American individuals had lower serologic neutralizing activity than European Americans, suggesting a genetic role in the generation of these neutralizing antibodies. Of the vaccinated individuals, only 69 (6.9%) had moderate levels of anti-LF IgG compared to 244 (24.4%) with low and 687 (68.7%) with extremely low levels of IgG antibodies to LF. Using overlapping decapeptide analysis, we identified six common LF antigenic regions targeted by those individuals with moderate levels of antibodies to LF and high in vitro toxin neutralizing activity. Affinity purified antibodies directed against antigenic epitopes within the PA binding and ADP-ribotransferase-like domains of LF were able to protect mice against lethal toxin challenge. Findings from these studies have important implications for vaccine design and immunotherapeutic development.

Bacillus anthracis remains a serious bioterrorism concern, and the currently licensed vaccine remains an incomplete solution for population protection from inhalation anthrax and has been associated with concerns regarding efficacy and safety. Thus, understanding how to generate long lasting protective immunity with reduced immunizations or providing protection through post exposure immunotherapeutics are long sought goals. Through evaluation of a large military cohort, we characterized the levels of antibodies against protective antigen and found that over half of anthrax vaccinees had low levels of in vitro toxin neutralization capacity in their sera. Using solid phase epitope mapping and confirmatory assays, we identified several neutralization-associated humoral epitopes and demonstrated that select anti-peptide responses mediated protection in vitro. Finally, passively transferred antibodies specific for select epitopes provided protection in an in vivo lethal toxin mouse model. Identification of these antigenic regions has important implications for vaccine design and the development of directed immunotherapeutics.

In the inflamed retina, CD4+T cells can cause retinal damage when they are not properly regulated. Since tissue expression of major histocompatibility complex (MHC) class II and costimulatory molecules is a key mechanism for regulating effector T cells, we tested the hypothesis that upregulation of these proteins in the retina contributes to the regulation of CD4 T cells. Here we report that in retinas infected with the protozoan parasiteToxoplasma gondii, MHC class II is upregulated on infiltrating leukocytes as well as on resident retinal cells, including photoreceptors. Flow cytometric analysis indicated that B7 costimulatory family members (CD80, CD86, ICOS-L, and programmed death ligand 2 [PD-L2]) were not expressed on class II+cells. In contrast, PD-L1 (also named B7-H1 or CD274) was expressed on the majority of both hematopoietic and resident retinal MHC class II-expressing cells. Retinal cells fromToxoplasma-infected animals were able to suppress T-cell activation in a PD-L1-dependent manner. Finally, we demonstrate that the expression of MHC class II and PD-L1 was critically dependent on gamma interferon (IFN-γ) expression. These data suggest that retinal MHC class II and PD-L1 expression is a novel mechanism by which the retina protects itself from CD4 T-cell-mediated immune damage in ocular toxoplasmosis and other types of retinal immune responses.

Toll-like receptors (TLRs) are important innate immune receptors for the identification and clearance of invading pathogens. Twelve TLRs that recognize various conserved components of microorganisms are currently known. Among these, the endosomal TLRs 3, 7/8, and 9 recognize dsRNA, ssRNA, and CpG DNA, respectively. Nucleic acid-sensing TLRs, TLR 7 in particular, have been implicated in systemic lupus erythematosus (SLE) and are thought to exacerbate disease pathology. Activation of these TLRs results in the production of inflammatory cytokines and type I interferon. Genome-wide association studies, single nucleotide polymorphism analyses as well as experimental mouse models have provided evidence of TLR signaling involvement in SLE and other autoimmune diseases. Since activation of these receptor pathways promotes autoimmune phenotypes, inhibitory drugs that target these pathways constitute important new therapeutic strategies for the treatment of systemic autoimmunity.

Anthrax lethal and edema toxins (LeTx and EdTx, respectively) form by binding of lethal factor (LF) or edema factor (EF) to the pore-forming moiety protective antigen (PA). Immunity to LF and EF protects animals from anthrax spore challenge and neutralizes anthrax toxins. The goal of the present study is to identify linear B-cell epitopes of EF and to determine the relative contributions of cross-reactive antibodies of EF and LF to LeTx and EdTx neutralization. A/J mice were immunized with recombinant LF (rLF) or rEF. Pools of LF or EF immune sera were tested for reactivity to rLF or rEF by enzyme-linked immunosorbent assays, in vitro neutralization of LeTx and EdTx, and binding to solid-phase LF and EF decapeptides. Cross-reactive antibodies were isolated by column absorption of EF-binding antibodies from LF immune sera and by column absorption of LF-binding antibodies from EF immune sera. The resulting fractions were subjected to the same assays. Major cross-reactive epitopes were identified as EF amino acids (aa) 257 to 268 and LF aa 265 to 274. Whole LF and EF immune sera neutralized LeTx and EdTx, respectively. However, LF sera did not neutralize EdTx, nor did EF sera neutralize LeTx. Purified cross-reactive immunoglobulin G also failed to cross-neutralize. Cross-reactive B-cell epitopes in the PA-binding domains of whole rLF and rEF occur and have been identified; however, the major anthrax toxin-neutralizing humoral responses to these antigens are constituted by non-cross-reactive epitopes. This work increases understanding of the immunogenicity of EF and LF and offers perspective for the development of new strategies for vaccination against anthrax.

FVB/NJ mice frequently are used as transgenic hosts, but the suitability of this genetic background for transgenic and congenic models of systemic autoimmunity have not been reported. In this study, FVB/NJ mice were evaluated for the presence of serum autoantibodies and autoimmune kidney pathology. Previously unreported albuminuria was observed in aged female FVB/NJ mice; however, serum autoantibody testing, light microscopic evaluation of differentially stained renal sections, and evaluation of renal sections for immunoglobulin deposits revealed that the albuminuria was not of autoimmune etiology. Anecdotally, multiple characteristics of the FVB/NJ strain, including albuminuria, cholesterolemia, mild podocyte foot process effacement in aged female FVB/NJ kidneys and predisposition to enhanced Th2 immune responses, is reminiscent of human minimal change nephrotic syndrome (MCNS). We propose that mapping of genetic polymorphisms that are responsible for these traits in FVB/NJ mice may lead to increased understanding of mild nephrotic syndromes including MCNS and other proteinurias.

As the immediate precursors to mature follicular B cells in splenic development, immature transitional cells are an essential component for understanding late B cell differentiation. It has been shown that T2 cells can give rise to mature B cells; however, whether T3 B cells represent a normal stage of B cell development, which has been widely assumed, has not been fully resolved. In this study, we demonstrate both in vitro and in vivo that T3 B cells do not give rise to mature B cells and are instead selected away from the T1→T2→mature B cell developmental pathway and are hyporesponsive to stimulation through the BCR. Significantly reduced numbers of T3 B cells in young lupus-prone mice further suggest that the specificity of this subset holds clues to understanding autoimmunity.

Several diseases are characterized by the presence of point mutations, which are amenable to molecular detection using a number of methods such as PCR. However, certain mutations are particularly difficult to detect due to factors such as low abundance and the presence of special (e.g., oligonucleotide repeat) sequences. The mutation 7A in the oligoA sequence of exon 7 of the gene encoding the La autoantigen is difficult to detect at the DNA level, and even at the RNA level, due to both its estimated low abundance and its differentiation from the wild-type 8A sequence. This article describes a technique in which amplification of the excess wild-type 8A La sequence is suppressed by a peptide nucleic acid (PNA) during a nested PCR step. Detection of the amplified 7A mutant form was then performed by simple electrophoresis following a final primer extension step with an infrared dye-labeled primer. This technique allowed us to detect the mutation in 3 of 7 individuals harboring serum immunoglobulin G (IgG) antibodies reactive with a neo-B cell epitope in the 7A mutant protein product. We propose that this method is a viable screening test for mutations in regions containing simple polynucleotide repeats.

In previous studies, we detected a frame shift mutation in the gene encoding the autoantigen La of a patient with systemic lupus erythematosus. The mutant La mRNA contains a premature termination codon. mRNAs that prematurely terminate translation should be eliminated by RNA quality control mechanisms. As we find Abs specific for the mutant La form in ∼30% of sera from anti-La-positive patients, we expected that mutant La mRNAs circumvent RNA control and the expression of mutant La protein could become harmful. Indeed, real-time PCR, immunostaining, and immunoblotting data of mice transgenic for the mutant La form show that mutant La mRNAs are not repressed in these animals and are translated to mutant La protein. In addition to the mutant La protein, we detected a minor portion of native human La in the mutant La-transgenic mice. Therefore, ribosomal frame shifting may allow the mutant La mRNA to escape from RNA control. Interestingly, expression of the mutant La mRNA results in a lupus-like disease in the experimental mice. Consequently, escape of mutant La mRNA from RNA control can have two effects: it 1) results in the expression of an immunogenic (neo)epitope, and 2) predisposes to autoimmunity.

In the lipid raft model, cell membranes contain detergent-resistant membrane domains, or rafts, that function in cell signaling. Recent findings have shown rafts are assembled to form macrodomains that are micrometers in diameter, one example of which is the immune synapse in stimulated lymphocytes. Because immune synapses are necessary for lymphocyte activation, deciphering the mechanism of their assembly has been an active topic of research. A current model, in which macrodomains are formed and maintained through association of rafts with the actin cytoskeleton, is discussed here. Recent evidence that either supports or challenges this model is presented, as well as data suggesting that macrodomain assembly might act as a gatekeeper to lymphocyte signaling and functioning.

Contains fulltext : 184433.pdf (Publisher’s version ) (Open Access) In this study, phylogenetically conserved structural features of the Ro RNP associated Y RNAs were investigated. The human, iguana, and frog Y3 and Y4 RNA sequences have been determined previously and the respective RNAs were subjected to enzymatic and chemical probing to obtain structural information. For all of the analyzed RNAs, the probing data were used to compose secondary structures, which partly deviate from previously predicted structures. Our results confirm the existence of two stem structures, which are also found at similar positions in hY1 and hY5 RNA. For the remaining parts of hY3 and hY4 RNA the secondary structures differ from those previously proposed based upon computer predictions. What might be more important is that certain parts of the RNAs appear to be flexible, i.e., to adopt several conformations. Another striking feature is that a characteristic pyrimidine-rich region, present in every Y RNA known, is single-stranded in all secondary structures. This may suggest that this region is readily available for base pairing inter-actions with other cellular nucleic acids, which might be important for the as yet unknown function of the RNAs.

Ultraviolet radiation plays an important role in the induction of lesions in many patients with cutaneous lupus. In the photosensitive subset of lupus, subacute cutaneous lupus, the effects of ultraviolet radiation likely act in concert with specific autoantibodies, particularly anti-Ro-related autoantibodies, to produce lesions. Potential effects of ultraviolet radiation on the induction of cutaneous lupus, and the potential interplay of specific autoantibodies with ultraviolet radiation are discussed. The steps involved in the induction of cutaneous lupus lesions by ultraviolet radiation have not been fully elucidated. Recent advances in phototesting and analysis of the genetics of lupus should clarify the events leading to photosensitive cutaneous lupus lesions.

TLR7 is a single-stranded RNA-recognizing innate immune receptor with roles in anti-viral immunity and systemic lupus erythematosus (SLE) in humans and SLE-like autoimmunity in mice. TLR7 gene duplication drives inflammation and autoimmunity in the BXSB mouse model and other Y autoimmune accelerator (Yaa)-derived models of murine lupus. Additionally, TLR7 SNPs associate with SLE in Eastern Asian populations. In this study, we investigated effects of TLR7 overexpression on CD4+ T cell subsets. In unmanipulated, 5-6 month old TLR7 BAC transgenic mice, we observed amplified activated:naïve T cell ratios and preferential expansion of the Th1 subset compared to wild type controls. We also evaluated the phenotype of TCR transgenic T cells specific for the RNA-binding autoantigen, human La, following adoptive transfer into TLR7 BAC transgenic mice. These human La-specific T cells demonstrated enhanced differentiation to Th1 and PSGL1lowCXCR4+ extrafollicular helper phenotypes after recovery from TLR7-overexpressing human La transgenic mice compared to wild type human La transgenic controls seven days after transfer. Moreover, TLR7 overexpression in recipient mice resulted in significantly reduced expansion of the CD4+ Foxp3+ phenotype among transferred, human La-specific cells. These data indicate that TLR7 overexpression alters CD4+ T cell populations in a manner that potentially contributes to autoinflammatory disease in these animals.

Abstract 3694 Background: Current treatments for Thrombotic Thrombocytopenic Purpura (TTP) fail to successfully control this life-threatening syndrome in up to 20% of patients, and of those patients who survive the first episode, a substantial fraction experience at least one relapse. Genetic mutation or autoantibody inhibition of the ultra-large von-Willebrand factor protease, A Disintegrin And Metalloproteinase with a Thrombospondin Type 1 Motif (ADAMTS13), reduces its enzymatic activity and is an important factor that promotes TTP development. Case reports of acute TTP episodes following infections or Type I interferon (IFN) treatment, a phenomenon also observed in systemic lupus erythematosus (SLE), suggest that inflammatory dysregulation could be important in this disease. SLE patients have elevated serum Type I IFN activity and a peripheral blood Type I IFN gene signature, which are associated with the presence of autoantibodies specific for RNA-binding nuclear antigens. Purpose: This study was undertaken to determine whether, in the absence of SLE, Type I IFN is elevated in acquired TTP patients, and/or is associated with autoantibodies to RNA-binding proteins. Study Participants: We obtained blood and serum samples from patients with acquired ADAMTS13-deficient TTP and matched healthy controls. Patient Inclusion Criteria: 1) ADAMTS13 activity Methods and Results: Blood samples were collected from 38 eligible TTP subjects during remission and from 38 age- (±5 years), race- and sex-matched healthy controls. While anti-nuclear autoantibody prevalence (titer ≥1:120) did not significantly differ between TTP patients and controls, specific autoantibodies against one or more of the following RNA-binding antigens: Ro, La, Sm or nRNP, were more frequent in TTP patients (31.6%) compared to controls (5.3%) (McNemar c2 p=0.0063). Using previously-banked serum samples from acute phase(s) of the disease obtained from the same patients, we observed no differences in the prevalence of these autoantibodies among TTP subjects in the acute phase of disease compared to remission. Serum Type I IFN activity was measured by the capacity of serum samples to induce expression of Type I IFN genes in the WISH epithelial cell line and was significantly increased in remission TTP samples compared to controls (McNemar c2 p=0.0313). Finally, global gene expression was examined (Illumina WG6 version 3 chips) in globin RNA-cleared total RNA taken from whole blood samples of the TTP patients in remission and controls. In initial analysis, 17 genes were found to be ≥ 1.5× differentially expressed in the peripheral blood of TTP patients compared to controls. Among the 7 genes exhibiting the greatest expression difference, 6 are recognized interferon-regulated genes. The relative expression of IFI44 effectively partitioned 13 of the TTP samples (34.2%) into a subset bearing a Type I interferon gene signature similar to that observed in SLE, compared to occurrence of a Type I interferon gene signature observed in 3 of the healthy controls (7.9%) (McNemar c2 p=0.0020). The Type I IFN gene signature was significantly associated with autoantibodies against specific RNA-binding nuclear antigens, defined as the presence of detectable IgG antibodies to one or more of the Ro, La, Sm, or nRNP antigens (Fisher p=0.0086) and correlated with serum Type I interferon activity in the WISH assay (r=.47, p=.0031). However, the Type I interferon gene signature did not associate with tendency of a patient to relapse after a first episode. Conclusion: From these studies, we propose a new subset of acquired, ADAMTS13–deficient TTP patients characterized by elevated serum Type I interferon activity, Type I interferon gene signature and IgG antibodies directed to RNA-binding proteins. Disclosures: Merrill: MedImmune: Consultancy, Honoraria; Genentech: Consultancy, Honoraria.

The release of Bacillus anthracis spores can cause significant morbidity and mortality and an effective vaccine as well as directed immunotherapeutics are needed. The currently licensed vaccine requires six primary vaccinations, annual boosters, and has been associated with serious adverse events. Thus there is increasing interest in developing an improved vaccination strategy which can generate lasting protective immunity with reduced vaccinations. This study seeks to identify the major humoral targets of B. anthracis that provide protection. To this end, plasma was obtained from two hundred vaccinated individuals and thirty unvaccinated controls. Ninety-five percent of all vaccinated individuals had anti-protective antigen antibodies and these antibodies correlated with in vitro protection against lethal toxin. Utilizing overlapping decapeptides, we identified common antigenic regions within those individuals with high toxin neutralizing activity. Epitope-specific antibodies directed against three of these regions were isolated and were able to mediate protection in vitro and in a mouse model of in vivo lethal toxin challenge. Identification of common, protective, peptide-specific humoral immune responses provide key information for further immunotherapeutic and vaccine development. NIAID: 5U19AI062629

The currently licensed anthrax vaccine (AVA) has been designed to elicit primarily an anti-protective antigen response. However, animal studies have clearly demonstrated that antibodies to the lethal factor component of the toxin can provide protection against challenge. This study was designed to identify AVA vaccinated individuals that generate anti-LF responses and to determine if these anti-LF antibodies can mediate protection. Plasma was obtained from one thousand vaccinated individuals and thirty unvaccinated controls. Only 69 individuals (6.9%) had significant levels of anti-LF antibodies. The presence of these antibodies did not correlate with in vitro protection against lethal toxin. To determine if antibodies against specific regions of LF could mediate protection, we used overlapping decapeptides to identify six common antigenic regions within those individuals with high levels of anti-LF antibodies and high toxin neutralizing activity. Epitope-specific antibodies directed against three of these common antigenic regions were isolated and select antibodies were able to protect in both in vitro tissue culture and in vivo mouse toxin challenge models. Findings from these studies have important implications for vaccine design and immunotherapeutic development. NIAID: 5U19AI062629

BACKGROUND: Anthrax Lethal and Edema Toxins (LTx and ETx) are formed by binding of Lethal Factor (LF) or Edema Factor (EF) to the pore-forming moiety, Protective Antigen (PA). Immunity to LF and EF neutralize anthrax toxins. AIM: LF and EF immune sera were used to identify cross-reactive B cell epitopes in their conserved PA-binding domains and to determine the relative contribution of such antibodies to LTx and ETx neutralization. METHODS: A/J mice were immunized with recombinant (r) LF or EF. EF-binding antibodies were purified from LF immune sera, and LF-binding antibodies from EF immune sera by column absorption. Whole immune sera and purified cross-reactive antibodies were tested for IgG to rLF or rEF by ELISA, in vitro neutralization of LTx and ETx, and binding to solid phase, overlapping LF and EF decapeptides. RESULTS: Cross-reactive epitopes were identified as EF aa 98-257 and 257-268 and LF aa 265-274. Whole LF and EF immune sera neutralized LTx and ETx, respectively. However, LF sera did not neutralize ETx nor did EF sera neutralize LTx. Purified cross-reactive IgG also failed to cross-neutralize. CONCLUSIONS: Cross-reactive B cell epitopes in the PA-binding domains of whole rLF and rEF occur and have been identified; however, the major anthrax toxin-neutralizing humoral responses to these antigens are constituted by non-cross-reactive epitopes that likely lie outside of their PA-binding sites.