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1.  Conservation of Pathogenic TCR Homology Across Class II Restrictions in Anti-RNP Autoimmunity: Extended Efficacy of T Cell Vaccine Therapy1 
T cells have been shown to mediate aspects of anti-RNP autoimmunity, and are a potential target of therapy in lupus and related diseases. In this study, we assessed the relevance of a conserved class of anti-RNP T cells to autoimmune disease expression and therapy. Our data show that anti-RNP T cell selection induced a limited set of homologous CDR3 motifs at high frequency. Homologous CDR3 motifs have been reported in other autoimmune diseases. Vaccination with irradiated anti-RNP (but not anti-Tetanus Toxoid) CD4+ cells induced remission of anti-RNP-associated nephritis in at least 80% of treated mice, even with donor/recipient MHC Class II mismatch, and in both induced and spontaneous autoimmunity. Vaccine responder sera inhibited anti-70k T cell proliferation and bound hybridomas expressing the conserved CDR3 motifs. Our data indicate that a limited set of TCR CDR3 motifs may be important for the pathogenesis of anti-RNP lupus and other autoimmune diseases. The ability to target a consistent set of pathogenic T cells between individuals and across Class II restrictions may allow for the more practical development of a standardized anti-RNP T cell vaccine preparation useful for multiple patients.
doi:10.4049/jimmunol.1203197
PMCID: PMC4001251  PMID: 24670800
Systemic Lupus Erythematosus; T Cells; Vaccination; Rodent
2.  Differential immunoglobulin class-mediated responses to components of the U1 small nuclear ribonucleoprotein in Systemic Lupus Erythematosus and Mixed Connective Tissue Disease 
Lupus  2013;22(13):10.1177/0961203313508444.
Summary
Objective
To determine whether patients with Systemic Lupus Erythematosus (SLE) and Mixed Connective Tissue Disease (MCTD) possess differential IgM-and IgG-specific reactivity against peptides from the U1 small nuclear ribonucleoprotein particle (U1 snRNP).
Methods
The IgM- and IgG-mediated responses against 15 peptides from subunits of the U1 snRNP were assessed by indirect ELISAs in sera from patients with SLE and MCTD and healthy individuals (n = 81, 41 and 31, respectively). Additionally, 42 laboratory tests and 40 clinical symptoms were evaluated to uncover potential differences. Binomial logistic regression analyses (BLR) were performed to construct models to support the independent nature of SLE and MCTD. Receiver Operating Characteristic (ROC) curves corroborated the classification power of the models.
Results
We analyzed IgM and IgG anti-U1 snRNP titers to classify SLE and MCTD patients. IgG anti-U1 snRNP reactivity segregates SLE and MCTD from non-disease controls with an accuracy of 94.1% while IgM-specific anti-U1 snRNP responses distinguish SLE from MCTD patients with an accuracy of 71.3%. Comparison of the IgG and IgM anti-U1 snRNP approach with clinical tests used for diagnosing SLE and MCTD revealed that our method is the best classification tool of those analyzed (p ≤ 0.0001).
Conclusions
Our IgM anti-U1 snRNP system along with lab tests and symptoms provide additional molecular and clinical evidence to support the hypothesis that SLE and MCTD may be distinct syndromes.
doi:10.1177/0961203313508444
PMCID: PMC3875166  PMID: 24158973
Systemic Lupus Erythematosus (SLE); Mixed Connective Tissue Disease (MCTD); immunoglobulin M (IgM); U1 small nuclear ribonucleoprotein particle (U1 snRNP); auto-immune disorders; classification criteria
3.  Ultraviolet Radiation Damages Self Noncoding RNA And Is Detected By TLR3 
Nature medicine  2012;18(8):10.1038/nm.2861.
Exposure to ultraviolet B (UVB) radiation from the sun can result in sunburn, premature aging and carcinogenesis, but the mechanism responsible for acute inflammation of the skin is not well understood. Here we show that RNA is released from keratinocytes after UVB exposure and that this stimulates production of the inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) from nonirradiated keratinocytes and peripheral blood mononuclear cells (PBMCs). Whole-transcriptome sequencing revealed that UVB irradiation of keratinocytes induced alterations in the double-stranded domains of some noncoding RNAs. We found that this UVB-damaged RNA was sufficient to induce cytokine production from nonirradiated cells, as UVB irradiation of a purified noncoding RNA (U1 RNA) reproduced the same response as the one we observed to UVB-damaged keratinocytes. The responses to both UVB-damaged self-RNAs and UVB-damaged keratinocytes were dependent on Toll-like receptor 3 (TLR3) and Toll-like receptor adaptor molecule 1 (TRIF). In response to UVB exposure, Tlr3−/− mice did not upregulate TNF-α in the skin. Moreover, TLR3 was also necessary for UVB-radiation–induced immune suppression. These findings establish that UVB damage is detected by TLR3 and that self-RNA is a damage-associated molecular pattern that serves as an endogenous signal of solar injury.
doi:10.1038/nm.2861
PMCID: PMC3812946  PMID: 22772463
4.  T cell vaccination therapy in an induced model of anti-RNP autoimmune glomerulonephritis 
Clinical immunology (Orlando, Fla.)  2010;137(2):281-287.
To establish the relevance of targeting disease-associated T cells in anti-RNP-associated glomerulonephritis, mice developing nephritis following immunization with U1-70-kd small nuclear ribonucleoprotein (snRNP) were treated with a single dose of irradiated antigen-selected T cell vaccine. T cell receptor usage in nephritic kidneys revealed oligoclonal use of T Cell Receptor V Beta (TRBV) genes as previously found in spleens and lungs of immunized mice with pulmonary disease. The CDR3 regions from T cell isolates showed sequence homology to those in humans with anti-RNP autoimmunity. Following T cell vaccination, urinalysis returned to normal in 5/7 treated mice (71% response rate) whereas all mock treated mice continued to have an active urinary sediment (Fisher’s Exact p=0.02). An oligoclonal population of T cells homologous to those identified in humans with anti-RNP autoimmunity is implicated in disease pathogenesis, and T cell vaccination is associated with a high rate of clinical improvement in established nephritis.
doi:10.1016/j.clim.2010.07.013
PMCID: PMC2950256  PMID: 20797908
5.  Tissue Targeting of Anti-RNP Autoimmunity: Effects of T Cells and Myeloid Dendritic Cells 
Arthritis and rheumatism  2009;60(2):534-542.
Using adoptive transfer techniques, we explored the immune cells implicated in a model of anti-RNP autoimmunity that presents either with pneumonitis or glomerulonephritis. Unfractionated splenocytes from donors without renal disease induced predominantly lung disease (8/14 (57%) lung versus 2/14 (14%) renal, p = 0.046). However, the CD4+ cells taken from these splenocytes induced renal disease more frequently than lung disease (7/10 (70%) renal versus 2/10 (20%) lung, p = 0.01). Adoptive transfer of RNP+ CD4+ T cells from short-term culture gave results similar to those with donor splenic CD4+ cells (8/11 (73%) of recipients with renal disease versus 3/11 (27%) with lung disease). Co-transfer of myeloid dendritic cells (MDCs) from the spleens of immunized mice along with CD4+ cells from immunized donors prevented the induction of renal disease (0/5 mice, p = 0.026 versus recipients of fresh CD4+ cells), though lung disease was still seen in 1/5 mice. Transfer of MDCs alone from immunized donors induced lung disease in 3/5 (60%) of recipients, with no nephritis. Co-transfer of splenocytes from mice with nephritis along with splenocytes from mice without nephritis led to renal disease in 4/5 recipients, with lung disease in 0/5 recipients. These findings indicate that RNP+ CD4+ T cells are sufficient to induce anti-RNP autoimmunity, that the tissue targeting of anti-RNP autoimmunity can be deviated to either a renal or pulmonary phenotype depending upon the presence of accessory cells including MDCs, and that dendritic cell subsets can play roles in both propagation of autoimmunity and end organ targeting.
doi:10.1002/art.24256
PMCID: PMC3059848  PMID: 19180485
Lupus Erythematosus; Systemic; Mixed Connective Tissue Disease; Lung Diseases; Interstitial; Ribonucleoproteins; small nuclear; Models; animal
6.  CD4+ T Cells Target Epitopes Residing within the RNA-Binding Domain of the U1-70-kDa Small Nuclear Ribonucleoprotein Autoantigen and Have Restricted TCR Diversity in an HLA-DR4-Transgenic Murine Model of Mixed Connective Tissue Disease1 
Mixed connective tissue disease (MCTD) is a systemic autoimmune disease with significant morbidity and premature mortality of unknown pathogenesis. In the present study, we characterized U1-70-kDa small nuclear ribonucleoprotein (70-kDa) autoantigen-specific T cells in a new murine model of MCTD. These studies defined 70-kDa-reactive T cell Ag fine specificities and TCR gene usage in this model. Similar to patients with MCTD, CD4+ T cells can be readily identified from 70-kDa/U1-RNA-immunized HLA-DR4-transgenic mice. Using both freshly isolated CD4+ T cells from spleen and lung, and T cell lines, we found that the majority of these T cells were directed against antigenic peptides residing within the RNA-binding domain of 70 kDa. We also found that TCR-β (TRB) V usage was highly restricted among 70-kDa-reactive T cells, which selectively used TRBV subgroups 1, 2, 6, 8.1, 8.2, and 8.3, and that the TRB CDR3 had conserved sequence motifs which were shared across different TRBV subgroups. Finally, we found that the TRBV and CDR3 regions used by both murine and human 70-kDa-specific CD4+ T cells were homologous. Thus, T cell recognition of the 70-kDa autoantigen by HLA-DR4-transgenic mice is focused on a limited number of T cell epitopes residing primarily within the RBD of the molecule, using a restricted number of TRBV and CDR3 motifs that are homologous to T cells isolated from MCTD patients.
PMCID: PMC2919051  PMID: 18523312
7.  Clinical and Immunologic Manifestations of Mixed Connective Tissue Disease in a Miami Population Compared to a Midwestern US Caucasian Population 
The Journal of rheumatology  2008;35(3):429-437.
Objective
A cross-sectional study of mixed connective tissue disease (MCTD) was performed to determine if there were identifiable differences in the clinical expression of MCTD associated with race or ethnicity.
Methods
Miami, Florida, and Midwestern US (Missouri) Caucasian MCTD cohorts were studied. Clinical and laboratory features of the 2 MCTD cohorts were compared. A concurrently collected cohort of Sm-positive patients with systemic lupus erythematosus (SLE) was studied as a control. Disease activity and severity and functional status were measured. CD4+CD25high-expressing T-regulatory cells were enumerated and serum soluble L selectin was measured as biomarkers of disease activity.
Results
The Miami and Missouri Caucasian MCTD groups, while differing from the SLE group, were largely similar; however, gastroesophageal reflux, sclerodactyly, and malar rash were significantly more frequent in the Missouri MCTD group and alopecia was more frequent in the Miami MCTD group. Significant clinical and laboratory differences were found between the Miami MCTD and Miami SLE groups despite similar disease duration, activity, severity and functional status. Raynaud's phenomenon (RP), hand swelling, synovitis, myositis, and sclerodactyly were all significantly more common in RNP-positive MCTD versus Sm-positive SLE subjects.
Conclusion
Ethnic differences were observed in the frequency of end-organ involvement in the Miami MCTD versus the Missouri Caucasian MCTD groups. Clinical and laboratory features of all MCTD groups were clearly different from the SLE group, despite similar disease activity, disease severity, and functional status. Disease activity measures appeared to behave similarly as valid measures of disease activity in SLE and MCTD.
PMCID: PMC2919224  PMID: 18260175
Mixed Connective Tissue Disease; Systemic Lupus Erythematosus; L-Selectin; T-Regulatory Cells; Hispanic
8.  Endosomal Toll-like receptors in autoimmunity: mechanisms for clinical diversity 
Therapy  2009;6(3):433-442.
The endosomal Toll-like receptors (TLR3, TLR7 and TLR9) have been implicated in the pathogenesis of autoimmune diseases. Their signaling pathways show remarkable similarities and yet the outcomes following activation of each of these TLRs lead to clinically distinct autoimmune disease phenotypes. This review discusses how differences may arise at a molecular and cellular level to account for this diversity of responses. Understanding the roles of individual TLR pathways and the relationships between them and non-TLR innate immune pathways in the pathogenesis of diseases such as systemic lupus erythematosis highlights potential treatment targets for this spectrum of autoimmune diseases.
doi:10.2217/thy.09.2
PMCID: PMC2779546  PMID: 20161373
dendritic cells; innate immunity; interferon regulatory factors; kidney; lupus; Toll-like receptors
9.  Recent Advances and Opportunities in Research on Lupus: Environmental Influences and Mechanisms of Disease 
Environmental Health Perspectives  2008;116(6):695-702.
Objectives
In this review we summarize research on mechanisms through which environmental agents may affect the pathogenesis of lupus, discuss three exposures that have been the focus of research in this area, and propose recommendations for new research initiatives.
Data sources and synthesis
We examined studies pertaining to key mechanistic events and specific exposures. Apoptosis leading to increased production or decreased clearance of immunogenic intracellular self-antigens and defective apoptosis of autoreactive immune cells both have been implicated in the loss of self-tolerance. The adjuvant or bystander effect is also needed to produce a sustained autoimmune response. Activation of toll-like receptors is one mechanism through which these effects may occur. Abnormal DNA methylation may also contribute to the pathogenesis of lupus. Each of the specific exposures we examined—Epstein-Barr virus, silica, and trichloroethylene—has been shown, in humans or in mice, to act upon one or more of these pathogenic steps. Specific recommendations for the continued advancement of our understanding of environmental influences on lupus and other autoimmune diseases include the development and use of mouse models with varying degrees of penetrance and manifestations of disease, identification of molecular or physiologic targets of specific exposures, development and use of improved exposure assessment methodologies, and multisite collaborations designed to examine understudied environmental exposures in humans.
Conclusions
The advances made in the past decade concerning our understanding of mechanisms involved in the development of lupus and the influence of environmental agents on this process provide a strong foundation for further developments in this field.
doi:10.1289/ehp.11092
PMCID: PMC2430222  PMID: 18560522
adjuvant effect; apoptosis; autoimmune diseases; bystander effect; demethylation; epigenetics; Epstein-Barr virus; silica; systemic lupus erythematosus; trichloroethylene
10.  U1 RNA Induces Innate Immunity Signaling 
Arthritis and rheumatism  2004;50(9):2891-2896.
Objective
The U1–70-kd RNP is a prominent target of autoimmunity in connective tissue diseases. In this study, we explored whether its endogenous ligand, U1 RNA, mediates a proimmune signal and may be immunogenic.
Methods
We assayed the proliferation of control and MyD88-knockout splenocytes in response to in vitro–synthesized U1 RNA, and measured interleukin-6 (IL-6) and IL-8 secretion induced by U1 RNA in a human cell line competent for signaling through Toll-like receptor 3 (TLR-3) and TLR-5.
Results
Treatment with U1 RNA or with poly(I-C), a known agonist of TLR-3, induced approximately twice as much control splenocyte proliferation as did treatment with RNase-digested U1 RNA. Proliferation in response to either poly(I-C) or U1 RNA by MyD88-knockout splenocytes was similarly attenuated. Similar to poly(I-C), U1 RNA induced significant secretion of both IL-6 and IL-8 from a TLR-3–expressing human cell line; in contrast, the TLR-5 agonist flagellin induced predominantly IL-8 secretion. Pretreatment of U1 RNA with RNase abolished IL-6 and IL-8 secretion.
Conclusion
U1 RNA is capable of inducing manifestations consistent with TLR-3 activation. The ability of U1 RNA (which has a substantial double-stranded secondary structure) to activate TLR-3 may contribute to the immunogenicity of the U1–70-kd autoantigen. Stimulation of innate immunity by native RNA molecules with a double-stranded secondary structure may help explain the high prevalence of autoimmunity to RNA binding proteins.
doi:10.1002/art.20428
PMCID: PMC1475509  PMID: 15457457
11.  Lack of autoantibody production associated with cytomegalovirus infection 
Arthritis Research  2002;4(5):R6.
To confirm an association between cytomegalovirus (CMV) infection and the presence of antibodies to Smith (Sm), to ribonucleoprotein (RNP), and to a component of the U1 ribonucleoproteins (U1-70 kD), we measured antibodies to these protein antigens using an enzyme immunoassay and an immunoblot. The antibodies were measured in the sera of 80 healthy subjects, one-half of whom were naturally CMV seropositive and one-half were CMV seronegative, and in eight subjects immunized with a live attenuated strain of CMV. None of the vaccinees developed antibodies to Sm, to RNP, or to U1-70 kD at either 4 or 12 months after immunization. Additionally, there was no statistically significant association between levels of antibodies to Sm or to RNP and between sera obtained from vaccinees, natural CMV seropositive individuals, and CMV seronegative individuals. One CMV seropositive serum and one CMV seronegative serum tested positive for antibodies to U1-70 kD. These data indicate that neither wild-type infection nor the live-attenuated Towne vaccine frequently induce autoantibody production.
doi:10.1186/ar429
PMCID: PMC125300  PMID: 12223109
autoantibodies; cytomegalovirus; RNP antigen; Sm antigen; U1-70kD

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