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ObjectiveIdentify autoantibodies in anti-Ro/SS-A negative primary Sjögren’s syndrome (SS).MethodsThis is a proof-of-concept, case-control study of SS, healthy (HC) and other disease (OD) controls. A discovery dataset of plasma samples (n=30 SS, n=15 HC) was tested on human proteome arrays containing 19 500 proteins. A validation dataset of plasma and stimulated parotid saliva from additional SS cases (n=46 anti-Ro+, n=50 anti-Ro–), HC (n=42) and OD (n=54) was tested on custom arrays containing 74 proteins. For each protein, the mean+3 SD of the HC value defined the positivity threshold. Differences from HC were determined by Fisher’s exact test and random forest machine learning using 2/3 of the validation dataset for training and 1/3 for testing. Applicability of the results was explored in an independent rheumatology practice cohort (n=38 Ro+, n=36 Ro–, n=10 HC). Relationships among antigens were explored using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) interactome analysis.ResultsRo+SS parotid saliva contained autoantibodies binding to Ro60, Ro52, La/SS-B and muscarinic receptor 5. SS plasma contained 12 novel autoantibody specificities, 11 of which were detected in both the discovery and validation datasets. Binding to ≥1 of the novel antigens identified 54% of Ro–SS and 37% of Ro+SS cases, with 100% specificity in both groups. Machine learning identified 30 novel specificities showing receiver operating characteristic area under the curve of 0.79 (95% CI 0.64 to 0.93) for identifying Ro–SS. Sera from Ro–cases of an independent cohort bound 17 of the non-canonical antigens. Antigenic targets in both Ro+and Ro–SS were part of leukaemia cell, ubiquitin conjugation and antiviral defence pathways.ConclusionWe identified antigenic targets of the autoantibody response in SS that may be useful for identifying up to half of Ro seronegative SS cases.

Background Women with systemic lupus erythematosus (SLE) are at risk of premature ovarian failure when treated with cyclophosphamide. This risk is increased when autoimmune thyroid disease is present. We undertook this study to determine whether the presence of ovarian autoimmunity also increased the risk of early ovarian failure among women receiving cyclophosphamide. Methods We examined the records of women enrolled in the Lupus Family Registry and Repository, a cross-sectional study of ~3300 SLE subjects, for treatment with cyclophosphamide as well as menopausal status. We defined premature menopause as permanent, spontaneous cessation of menstruation before age 45. We measured anti-ovarian antibodies by enzyme-linked immunosorbent assay using stored sera. Results There were 258 women treated with cyclophosphamide in whom presence of absence or premature menopause could by defined. A total of 169 (65.6%) had premature ovarian failure, while 89 (34.6%) did not. While anti-ovarian antibodies were present in a small percentage of patients, there was no association of premature menopause to either level of these antibodies (16.2 ± 20.3 units vs 17.4 ± 21.7 units, P = NS by Fisher's exact test), or positivity on this testing (11 of 169 [6.5%] positive vs 8 of 89 [8.9%], χ2 = 0.53, P = .46, 95% CI 0.95-1.1). Neither renal disease nor hypothyroidism increased the risk of premature ovarian failure in these women receiving cyclophosphamide. Conclusion Anti-ovarian antibodies among women with SLE are not associated with premature ovarian failure after treatment with cyclophosphamide.

Sjogren's syndrome is in part considered an autoimmune disease because patient sera contain antibodies binding self-structures. In fact, in addition to anti-Ro (or SSA) and anti-La (or SSB), which are included in the classification criteria, there are a wide variety of autoantibodies found among these patients. We reviewed English-language MEDLINE sources. Anti-Ro and anti-La found among healthy individuals, including mothers giving birth to infants with neonatal lupus, predicts future connective tissue disease. Those with Sjogren's syndrome can be divided into two groups; patients with only exocrine gland involvement and those with systemic disease. The presence of anti-Ro/La is associated with systemic, extraglandular disease. Rheumatoid factor is also associated with extraglandular disease while anti-cyclic citrullinated peptide (CCP) is likely associated with inflammatory arthritis and progression to rheumatoid arthritis. Anti-mitochondrial antibodies are uncommon but predict progression to primary biliary cirrhosis. Cryoglobulinemia is found in excess among those with non-Hodgkin's lymphoma. Determination of autoantibodies on the sera of Sjogren's syndrome patients has prognostic implications for Sjogren's syndrome itself as well as associated diseases.

Objective To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjogren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms. Methods A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding. Results V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens. Conclusion These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.

Background Systemic lupus erythematosus (SLE) is commonly treated with broad immunosuppression, including cyclophosphamide. Use of this agent can result in ovarian toxicity leading to premature menopause. Ovarian failure is associated with concomitant hypothyroidism. We hypothesized that hypothyroidism might be a marker for ovarian autoimmunity because organ-specific autoimmune diseases tend to occur together. We undertook this study to determine whether anti-ovarian antibodies are associated with premature ovarian failure among SLE women who received cyclophosphamide. Methods All SLE women with a history of cyclophosphamide therapy were identified in the Lupus Registry and Repository (LFRR). All subjects were shown to meet the revised ACR classification criteria. Premature menopause was defined as spontaneous lack of menstrual periods prior to age 45. Anti-ovarian antibodies were measured by ELISA (Anti-Ovarian Ab ELISA, IBL, Minneapolis, catalog # IB9184). Data were analyzed by Students T test and chi square testing. The protocol received ethical approval from the University of Oklahoma Health Sciences Center and Oklahoma Medical Research Foundation IRBs. Results Among ~3000 SLE women enrolled in the LFRR who had received cyclophosphamide, we found 169 with menopause before age 45 along with 73 who underwent menopause after age 45 and 16 patients over age 45 who were still having menstrual periods at the time of evaluation. Thus, there were a total of 89 SLE women who did not have premature menopause. Mean anti-ovarian antibodies in the 169 with premature ovarian failure was 16.2 units (SD=20.3), while the mean among those without premature menopause was 17.4 units (SD=21.7). These values were not statistically different between the groups. 11 of 169 (6.5%) prematurely menopausal SLE women had a positive result and 8 of 89 (8.9%) without premature menopausal were positive (2=0.53, p=0.46, OR=1.02 (95% CI 0.95–1.1). Conclusions Anti-ovarian antibodies were present at low levels (~10% positivity) among women with SLE. However, the presence of these antibodies was not related to premature ovarian failure after cyclophosphamide. Funding Source(s): NIH grants AR053483, AI082714, GM104938. VA grant BX0011451

Objective To describe the clinical and serologic manifestations of Sjogren's syndrome (SS) in ethnic groups of the US. Methods This was a cross-sectional study of 648 patients with primary SS: 20 African American (AA), 164 American Indian (AI), 426 European American (EA), and 38 patients of other races evaluated in a multidisciplinary Sjogren's International Collaborative Clinical Alliance research clinic. Results AA subjects comprised 3.1% of the SS cohort, much lower than the percentage of AA in the state of Oklahoma (P = 3.01 × E - 05), the US (P = 2.24E - 13), or a systemic lupus erythematosus (SLE) cohort at the same institution (P = 4.26 × 10E - 27). In contrast, the percentage of AI in the SS cohort (25.3%) was much higher than expected (P = 3.17E - 09 versus SLE cohort, P = 6.36 - 26 versus Oklahoma, and P = 8.14E - 96 versus US population). The SS classification criteria were similar between AA and EA, but subjects of AI ancestry had lower rates of abnormal tear and salivary flow, as well as anti-Ro/SSA and anti-La/SSB antibodies. Paradoxically, AI had higher levels of disease activity (mean ± SD European League Against Rheumatism Sjogren's Syndrome Disease Activity Index score 3.77 ± 4.78) in comparison to EA (2.90 ± 4.12; P = 0.011) and more extraglandular manifestations affecting mainly the articular and glandular domains. Meanwhile, AA patients were characterized by higher rates of hypergammaglobulinemia (odds ratio [OR] 1.39 [95% confidence interval (95% CI) 1.39-8.65]; P = 0.01), elevated erythrocyte sedimentation rate (ESR) (OR 3.95 [95% CI 1.46-9.95]; P = 0.009), and parotid enlargement (OR 4.40 [95% CI 1.49-13.07]; P = 0.02). Conclusion AI are affected at high rates with SS but present with few classical features, potentially preventing timely diagnosis. In contrast to SLE, SS is infrequent and not more severe among AA, but the triad of hypergammaglobulinemia, increased ESR, and parotid enlargement warrants extra vigilance for lymphomagenesis.

Isoelectric focusing (IEF) is an electrophoretic technique that enables the separation of proteins based on their isoelectric points. Until recently, this valuable method was not feasible for single-cell applications, which are necessary to interrogate heterogeneous cell populations. Herein we highlight a recently published method enabling the analysis of single-cell proteomics, which utilizes microfluidics coupled with IEF, photocapture, and immunoprobing of the protein in the same micro-gel, which can be stripped and reprobed multiple times.

Aims Postural tachycardia syndrome (POTS), a common and debilitating cardiovascular disorder, is characterized by an exaggerated heart rate increase during orthostasis and a wide spectrum of adrenergic-related symptoms. To determine the aetiology of POTS, we examined a possible pathophysiological role for autoantibodies against α1-adrenergic (α1AR) and β1/2-adrenergic receptors (β1/2AR). Methods and results Immunoglobulin G (IgG) derived from 17 POTS patients, 7 with recurrent vasovagal syncope (VVS), and 11 normal controls was analysed for its ability to modulate activity and ligand responsiveness of α1AR and β1/2AR in transfected cells and to alter contractility of isolated rat cremaster arterioles in vitro. Immunoglobulin G activation of α1AR and β1/2AR was significantly higher in POTS compared with VVS and controls in cell-based assays. Eight, 11, and 12 of the 17 POTS patients possessed autoantibodies that activated α1AR, β1AR and β2AR, respectively. Pharmacological blockade suppressed IgG-induced activation of α1AR and β1/2AR. Eight of 17 POTS IgG decreased the α1AR responsiveness to phenylephrine and 13 of 17 POTS IgG increased the β1AR responsiveness to isoproterenol irrespective of their ability to directly activate their receptors. Postural tachycardia syndrome IgG contracted rat cremaster arterioles, which was reversed by α1AR blockade. The upright heart rate correlated with IgG-mediated β1AR and α1AR activity but not with β2AR activity. Conclusion These data confirm a strong relationship between adrenergic autoantibodies and POTS. They support the concept that allosteric-mediated shifts in the α1AR and β1AR responsiveness are important in the pathophysiology of postural tachycardia.

Objective: Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) are related by clinical and serologic manifestations as well as genetic risks. Both diseases are more commonly found in women than in men, at a ratio of ~10 to 1. Common X chromosome aneuploidies, 47,XXY and 47,XXX, are enriched among men and women, respectively, in either disease, suggesting a dose effect on the X chromosome. Methods: We examined cohorts of SS and SLE patients by constructing intensity plots of X chromosome single-nucleotide polymorphism alleles, along with determining the karyotype of selected patients. Results: Among ~2,500 women with SLE, we found 3 patients with a triple mosaic, consisting of 45,X/46,XX/47,XXX. Among ~2,100 women with SS, 1 patient had 45,X/46,XX/47,XXX, with a triplication of the distal p arm of the X chromosome in the 47,XXX cells. Neither the triple mosaic nor the partial triplication was found among the controls. In another SS cohort, we found a mother/daughter pair with partial triplication of this same region of the X chromosome. The triple mosaic occurs in ~1 in 25,000–50,000 live female births, while partial triplications are even rarer. Conclusion: Very rare X chromosome abnormalities are present among patients with either SS or SLE and may inform the location of a gene(s) that mediates an X dose effect, as well as critical cell types in which such an effect is operative. © 2017, American College of Rheumatology

Background. Sjogren's syndrome and systemic lupus erythematosus (SLE) are related by clinical and serological manifestations as well as genetic risks. Both diseases are more commonly found in women compared to men at a ratio of about 10 to 1. Common X chromosome aneuploidies, 47,XXY and 47,XXX, are enriched among men and women, respectively, in either disease suggesting a dose effect on the X chromosome. Methods. We examined cohorts of Sjogren's syndrome or SLE patients with intensity plots of X chromosome single nucleotide polymorphism (SNP) alleles along with karyotype of selected subjects. Results. Among ∼2500 women with SLE we found three patients with a triple mosaic consisting of 45,X/46,XX/47,XXX. Among ∼2100 women with Sjogren's syndrome, one patient had 45,X/46,XX/47,XXX with a triplication of the distal p arm of the X chromosome in the 47,XXX cells. Neither the triple mosaic nor the partial triplication were found among controls. In another Sjogren's cohort, we found a mother-daughter pair with partial triplication of this same region of the X chromosome. The triple mosaic occurs in approximately 1 in 25,000 to 50,000 live female births, while partial triplications such are even rarer. Conclusions. Very rare X chromosome abnormalities are present among patients with either Sjogren's or SLE, and may inform the location of a gene(s) that mediate an X dose effect as well as critical cell types in which such effect is operative. This article is protected by copyright. All rights reserved.

Objective Primary Sjogren's syndrome (SS) is a systemic autoimmune disease with incompletely understood etiology. This study was undertaken to investigate the role of epigenetic dysregulation in the pathogenesis of primary SS. Methods A genome-wide DNA methylation study was performed in naive CD4+ T cells from 11 patients with primary SS compared to age-, sex-, and ethnicity-matched healthy controls. Cytosine methylation was quantified using the Illumina Infinium HumanMethylation450 BeadChip array, and the data were validated using bisulfite sequencing. Results Genome-wide analyses identified 553 hypomethylated CpG sites and 200 hypermethylated CpG sites in naive CD4+ T cells from patients with primary SS as compared to healthy controls, representing 311 hypomethylated and 115 hypermethylated gene regions. The hypomethylated genes in patients with primary SS included LTA (encoding lymphotoxin α). Other relevant genes, such as CD247, TNFRSF25, PTPRC, GSTM1, and PDCD1, were also hypomethylated. The interferon signature pathway was represented by hypomethylation of STAT1, IFI44L, USP18, and IFITM1. A group of genes encoding members of the solute carrier proteins were differentially methylated. In addition, the transcription factor gene RUNX1 was hypermethylated in patients with primary SS, suggesting a possible connection to lymphoma predisposition. Gene ontology (GO) analysis of hypomethylated genes demonstrated enrichment of genes involved in lymphocyte activation and immune response. GO terms for hypermethylated genes included antigen processing and presentation. Conclusion This is the first epigenome-wide DNA methylation study in patients with primary SS. These findings highlight a role for DNA methylation in primary SS and identify disease-associated DNA methylation changes in several genes and pathways in naive CD4+ T cells from patients with primary SS that may be involved in the pathogenesis of this disease.

Primary Sjögren's syndrome (pSS) has a strong female bias. We evaluated an X chromosome dose effect by analyzing 47,XXY (Klinefelter's syndrome, 1 in 500 live male births) among subjects with pSS. 47,XXY was determined by examination of fluorescence intensity of single nucleotide polymorphisms from the X and Y chromosomes. Among 136 pSS men there were 4 with 47,XXY. This was significantly different from healthy controls (1 of 1254 had 47,XXY, p=0.0012 by Fisher's exact test) as well men with rheumatoid arthritis (0 of 363 with 47,XXY), but not different compared to men with systemic lupus erythematosus (SLE) (4 of 136 versus 8 of 306, Fisher's exact test p=NS). These results are consistent with the hypothesis that the number of X chromosomes is critical for the female bias of pSS, a property that may be shared with SLE but not RA.

To characterise the serological and clinical findings in primary Sjögren's syndrome in which anti-La was found without anti-Ro. We hypothesised that a significant portion of these are falsely negative for anti-Ro60.Twenty-nine sera from primary Sjögren's syndrome patients were tested for antibodies directed against La and Ro. Anti-La was detected using bovine La treated with or without DNAase and RNAase to identify potential false positivity. Anti-Ro60 antibodies were detected using HEp-2000 substrate (in which cells are transfected with human Ro60) and HEp-2 substrate. Anti-Ro60 and Ro-52 were also tested by in vitro transcription/translation followed by immunoprecipitation assay.All 29 sera bound La, even after treatment with DNAase and RNAase. Of the 29 sera, 25 were unequivocally negative on HEp-2000 (1:40 dilution). Four samples were anti-Ro60 positive with a speckled pattern, three of the four at 1:320 dilution. Thus, false negative anti-Ro60 exists in a small fraction (14%) of the Ro-negative/La-positive primary Sjögren's patients. However, all the samples were negative for Ro60 and Ro52 by in vitro immunoprecipitation assay. Clinically these patients tended not to have salivary gland pathology characteristic of Sjögren's syndrome.We found only a small fraction of Ro negative/La positive sera to show positive HEp-2000 pattern. These subjects did not have characteristic findings on pathological examination of minor salivary glands, suggesting these subjects have a process distinct from Sjögren's syndrome.

Objective More than 80% of autoimmune disease predominantly affects females, but the mechanism for this female bias is poorly understood. We suspected that an X chromosome dose effect accounts for this, and we undertook this study to test our hypothesis that trisomy X (47,XXX; occurring in ∼1 in 1,000 live female births) would be increased in patients with female-predominant diseases (systemic lupus erythematosus [SLE], primary Sjogren's syndrome [SS], primary biliary cirrhosis, and rheumatoid arthritis [RA]) compared to patients with diseases without female predominance (sarcoidosis) and compared to controls. Methods All subjects in this study were female. We identified subjects with 47,XXX using aggregate data from single-nucleotide polymorphism arrays, and, when possible, we confirmed the presence of 47,XXX using fluorescence in situ hybridization or quantitative polymerase chain reaction. Results We found 47,XXX in 7 of 2,826 SLE patients and in 3 of 1,033 SS patients, but in only 2 of 7,074 controls (odds ratio in the SLE and primary SS groups 8.78 [95% confidence interval 1.67–86.79], P = 0.003 and odds ratio 10.29 [95% confidence interval 1.18–123.47], P = 0.02, respectively). One in 404 women with SLE and 1 in 344 women with SS had 47,XXX. There was an excess of 47,XXX among SLE and SS patients. Conclusion The estimated prevalence of SLE and SS in women with 47,XXX was ∼2.5 and ∼2.9 times higher, respectively, than that in women with 46,XX and ∼25 and ∼41 times higher, respectively, than that in men with 46,XY. No statistically significant increase of 47,XXX was observed in other female-biased diseases (primary biliary cirrhosis or RA), supporting the idea of multiple pathways to sex bias in autoimmunity.

Primary Sjögren's syndrome (pSS) has a strong female bias. We evaluated an X chromosome dose effect by analyzing 47,XXY (Klinefelter's syndrome, 1 in 500 live male births) among subjects with pSS. 47,XXY was determined by examination of fluorescence intensity of single nucleotide polymorphisms from the X and Y chromosomes. Among 136 pSS men there were 4 with 47,XXY. This was significantly different from healthy controls (1 of 1254 had 47,XXY, p = 0.0012 by Fisher's exact test) as well men with rheumatoid arthritis (0 of 363 with 47,XXY), but not different compared to men with systemic lupus erythematosus (SLE) (4 of 136 versus 8 of 306, Fisher's exact test p = NS). These results are consistent with the hypothesis that the number of X chromosomes is critical for the female bias of pSS, a property that may be shared with SLE but not RA. © 2016.

Self-reactive B cells not controlled by receptor editing or clonal deletion may become anergic. We report that fully mature human B cells negative for surface IgM and retaining only IgD are autoreactive and functionally attenuated (referred to as naive IgD+IgM− B cells [BND]). These BND cells typically make up 2.5% of B cells in the peripheral blood, have antibody variable region genes in germline (unmutated) configuration, and, by all current measures, are fully mature. Analysis of 95 recombinant antibodies expressed from the variable genes of single BND cells demonstrated that they are predominantly autoreactive, binding to HEp-2 cell antigens and DNA. Upon B cell receptor cross-linkage, BND cells have a reduced capacity to mobilize intracellular calcium or phosphorylate tyrosines, demonstrating that they are anergic. However, intense stimulation causes BND cells to fully respond, suggesting that these cells could be the precursors of autoantibody secreting plasma cells in autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. This is the first identification of a distinct mature human B cell subset that is naturally autoreactive and controlled by the tolerizing mechanism of functional anergy.

Objectives Commercial curcumin (CU), derived from food spice turmeric (TU), has been widely studied as a potential therapeutic for a variety of oncological and inflammatory conditions. Lack of solubility/bioavailability has hindered curcumin9s therapeutic efficacy in human diseases. We have solubilised curcumin in water applying heat/pressure, obtaining up to 35-fold increase in solubility (ultrasoluble curcumin (UsC)). We hypothesised that UsC or ultrasoluble turmeric (UsT) will ameliorate systemic lupus erythematosus (SLE) and Sjogren9s syndrome (SS)-like disease in MRL-lpr/lpr mice. Methods Eighteen female MRL-lpr/lpr (6 weeks old) and 18 female MRL-MpJ mice (6 weeks old) were used. Female MRL-lpr/lpr mice develop lupus-like disease at the 10th week and die at an average age of 17 weeks. MRL-MpJ mice develop lupus-like disease around 47 weeks and typically die at 73 weeks. Six mice of each strain received autoclaved water only (lpr-water or MpJ-water group), UsC (lpr-CU or MpJ-CU group) or UsT (lpr-TU or MpJ-TU group) in the water bottle. Results UsC or UsT ameliorates SLE in the MRL-lpr/lpr mice by significantly reducing lymphoproliferation, proteinuria, lesions (tail) and autoantibodies. lpr-CU group had a 20% survival advantage over lpr-water group. However, lpr-TU group lived an average of 16 days shorter than lpr-water group due to complications unrelated to lupus-like illness. CU/TU treatment inhibited lymphadenopathy significantly compared with lpr-water group (p=0.03 and p=0.02, respectively) by induction of apoptosis. Average lymph node weights were 2606±1147, 742±331 and 385±68 mg, respectively, for lpr-water, lpr-CU and lpr-TU mice. Transferase dUTP nick end labelling assay showed that lymphocytes in lymph nodes of lpr-CU and lpr-TU mice underwent apoptosis. Significantly reduced cellular infiltration of the salivary glands in the lpr-TU group compared with the lpr-water group, and a trend towards reduced kidney damage was observed in the lpr-CU and lpr-TU groups. Conclusions These studies show that UsC/UsT could prove useful as a therapeutic intervention in SLE/SS.

The B-cell ELISPOT assay is a sensitive tool that can be utilized to measure total immunoglobulin (Ig) and antigen-specific antibody-secreting cells. Typically, membrane-bound antigen enables binding of antibody secreted by B-cells. Bound antibody is then detected by using an anti-Ig antibody and a colorimetric substrate, resulting in colored spots on the membrane that can be easily enumerated. Here we have described a method to quantitate antigen-specific antibody-secreting cells from the spleen or bone marrow of a vaccinated mouse.

The ELISPOT is a powerful functional assay used to detect biological activity and immunological secretions from immune cells. In this chapter, we specifically discuss T cell ELISPOT methods for the detection of secreted cytokines. A detailed protocol is given enabling the detection of interferon gamma-secreting CD8(+) T cells and/or peripheral blood mononuclear cells following their isolation and polyclonal activation. Included is a brief discussion on choosing the activation method for your T cell ELISPOT assay, as well as additional instructions for the adaptation of this protocol for the study of memory and antigen-specific T cell responses.

Objective. Systemic lupus erythematosus (SLE) and Sjögren’s syndrome (SS) share clinical and immunogenetic features and may occur together. We undertook this study to determine the risk of primary SS among SLE-unaffected relatives of SLE patients and whether or not primary and secondary SS tended to occur in the same families.Methods. We collected clinical and serological data on 2694 SLE patients, 7390 SLE-unaffected relatives of the SLE patients, and 1470 matched controls.Results. Of the 2694 subjects with SLE, 548 had secondary SS, while 71 of their 7390 SLE-unaffected relatives had primary SS. None of the 1470 controls had SS as defined herein (p=5×10-5compared to SLE-unaffected relatives). Of the 71 SLE-unaffected relatives with primary SS, 18 (25.3%) had an SLE-affected family member with secondary SS, while only 530 of the 7319 (7.2%) SLE-unaffected relatives without SS did so (p=1×10-8).Conclusion. Among families identified for the presence of SLE, primary and secondary SS tend to occur within the same families. These results highlight the commonalities between these two forms of SS, which in fact correspond to the same disease.

The ELISPOT is a powerful functional assay used to detect biological activity and immunological secretions from immune cells. In this chapter, we specifically discuss T cell ELISPOT methods for the detection of secreted cytokines. A detailed protocol is given enabling the detection of interferon gamma-secreting CD8(+) T cells and/or peripheral blood mononuclear cells following their isolation and polyclonal activation. Included is a brief discussion on choosing the activation method for your T cell ELISPOT assay, as well as additional instructions for the adaptation of this protocol for the study of memory and antigen-specific T cell responses.

The B-cell ELISPOT assay is a sensitive tool that can be utilized to measure total immunoglobulin (Ig) and antigen-specific antibody-secreting cells. Typically, membrane-bound antigen enables binding of antibody secreted by B-cells. Bound antibody is then detected by using an anti-Ig antibody and a colorimetric substrate, resulting in colored spots on the membrane that can be easily enumerated. Here we have described a method to quantitate antigen-specific antibody-secreting cells from the spleen or bone marrow of a vaccinated mouse.

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.

Systemic lupus erythematosus is an autoimmune disease characterized by multi-system involvement and autoantibody production. Abnormal T cell DNA methylation and type-I interferon play an important role in the pathogenesis of lupus. We performed a genome-wide DNA methylation study in two independent sets of lupus patients and matched healthy controls to characterize the DNA methylome in naive CD4+ T cells in lupus. DNA methylation was quantified for over 485,000 methylation sites across the genome, and differentially methylated sites between lupus patients and controls were identified and then independently replicated. Gene expression analysis was also performed from the same cells to investigate the relationship between the DNA methylation changes observed and mRNA expression levels. We identified and replicated 86 differentially methylated CG sites between patients and controls in 47 genes, with the majority being hypomethylated. We observed significant hypomethylation in interferon-regulated genes in naive CD4+ T cells from lupus patients, including IFIT1, IFIT3, MX1, STAT1, IFI44L, USP18, TRIM22 and BST2, suggesting epigenetic transcriptional accessibility in these genetic loci. Indeed, the majority of the hypomethylated genes (21 out of 35 hypomethylated genes) are regulated by type I interferon. The hypomethylation in interferon-regulated genes was not related to lupus disease activity. Gene expression analysis showed overexpression of these genes in total but not naive CD4+ T cells from lupus patients. Our data suggest epigenetic "poising" of interferon-regulated genes in lupus naive CD4+ T cells, argue for a novel pathogenic implication for abnormal T cell DNA methylation in lupus, and suggest a mechanism for type-I interferon hyper-responsiveness in lupus T cells.

There are many Green Fluorescent Proteins (GFPs) originating from diverse species that are invaluable to cell biologists today because of their ability to provide experimental visualization of protein expression. Since their initial discovery, they have been modified and improved to provide more stable variants with emission ranges spanning a wide array of colors. Due to their ease of expression both in-vitro and in-vivo, they are an attractive choice for use as markers in molecular biology. GFPs are generally assumed to have negligible effects on the cells to which they have been introduced. However, a growing number of reports indicate that this is not always the case. Consequently, because of GFP's ubiquitous use, it is important to document the nature and extent of unintended effects. In this report, we find that GFP affects T cell activation, leading to defects in clustering, upregulation of the activation marker CD25 and IL-2 cytokine production following stimulation in human primary T cells that also express TurboGFP. We utilized a reporter assay which has been routinely used to assay the NF-κB pathway and found reduced NF-κB activitation in stimulated HEK293 and HeLa cells that were co-transfected with TurboGFP, suggesting that GFP interferes with signaling through the NF-κB pathway. These findings indicate that the utilization of GFP-tagged vectors may negatively impact in vitro experiments in T cells, emphasizing the critical importance of controls to identify any GFP-induced effects.

Genetic polymorphism in MECP2/IRAK1 on chromosome Xq28 is a confirmed and replicated susceptibility locus for lupus. High linkage disequilibrium in this locus suggests that both MECP2 and IRAK1 are candidate genes for the disease. DNA methylation changes in lupus T cells play a central role in the pathogenesis of lupus, and MeCp-2 (encoded by MECP2) is a master regulator of gene expression and is also known to recruit DNA methyltransferase 1 (DNMT1) during DNA synthesis. Using human T cells from normal individuals with either the lupus risk or the lupus protective haplotype in MECP2/IRAK1, we demonstrate that polymorphism in this locus increases MECP2 isoform 2 mRNA expression in stimulated but not unstimulated T cells. By assessing DNA methylation levels across over 485,000 methylation sites across the entire genome, we further demonstrate that the lupus risk variant in this locus is associated with significant DNA methylation changes, including in the HLA-DR and HLA-DQ loci, as well as interferon-related genes such as IFI6, IRF6, and BST2. Further, using a human MECP2 transgenic mouse, we show that overexpression of MECP2 alters gene expression in stimulated T cells. This includes overexpression of Eif2c2 that regulates the expression of multiple microRNAs (such as miR-21), and the histone demethylase Jhdm1d. In addition, we show that MECP2 transgenic mice develop antinuclear antibodies. Our data suggest that the lupus-associated variant in the MECP2/IRAK1 locus has the potential to affect all 3 epigenetic mechanisms: DNA methylation, microRNA expression, and histone modification. Importantly, these data support the notion that variants within the MECP2 gene can alter DNA methylation in other genetic loci including the HLA and interferon-regulated genes, thereby providing evidence for genetic-epigenetic interaction in lupus.

Determining the origin and fate of autoreactive B cells is critical to understanding and treating autoimmune diseases. We report that compared to antibodies from naïve and IgG memory B cells those from cells that have class switched to IgD via genetic recombination (Cδ-CS) are highly reactive with self-antigens, despite being derived from healthy people. Half of the antibodies from Cδ-CS B cells bind various autoantigens, and they are frequently polyreactive. Many also bind HEp-2 antigens. Intriguingly, some Cδ-CS B cells have accumulated residues in the variable regions that mediate anti-DNA reactivity via somatic hypermutation and selection, suggesting an immunological origin for anti-DNA antibodies in healthy people, while other Cδ-CS B cells are naturally autoreactive. We interpret these findings to indicate that autoreactive B cells can either be induced to class switch to IgD or autoreactive B cell clones that use IgD as the B cell receptor are not effectively deleted. Determining the mechanism by which the majority of Cδ-CS B cells are autoreactive may be important in understanding peripheral tolerance mechanisms and may provide insight to the enigmatic function of the secreted IgD antibody.

Even though only 1.5% of the human genome is translated into proteins, recent reports indicate that most of it is transcribed into non-coding RNAs (ncRNAs), which are becoming the subject of increased scientific interest. We hypothesized that examining how different classes of ncRNAs co-localized with annotated epigenomic elements could help understand the functions, regulatory mechanisms, and relationships among ncRNA families. We examined 15 different ncRNA classes for statistically significant genomic co-localizations with cell type-specific chromatin segmentation states, transcription factor binding sites (TFBSs), and histone modification marks using GenomeRunner ( http://www.genomerunner.org ). P-values were obtained using a Chi-square test and corrected for multiple testing using the Benjamini-Hochberg procedure. We clustered and visualized the ncRNA classes by the strength of their statistical enrichments and depletions. We found piwi-interacting RNAs (piRNAs) to be depleted in regions containing activating histone modification marks, such as H3K4 mono-, di- and trimethylation, H3K27 acetylation, as well as certain TFBSs. piRNAs were further depleted in active promoters, weak transcription, and transcription elongation regions, and enriched in repressed and heterochromatic regions. Conversely, transfer RNAs (tRNAs) were depleted in heterochromatin regions and strongly enriched in regions containing activating H3K4 di- and trimethylation marks, H2az histone variant, and a variety of TFBSs. Interestingly, regions containing CTCF insulator protein binding sites were associated with tRNAs. tRNAs were also enriched in the active, weak and poised promoters and, surprisingly, in regions with repetitive/copy number variations. Searching for statistically significant associations between ncRNA classes and epigenomic elements permits detection of potential functional and/or regulatory relationships among ncRNA classes, and suggests cell type-specific biological roles of ncRNAs.