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1.  PhIP-Seq characterization of autoantibodies from patients with multiple sclerosis, type 1 diabetes and rheumatoid arthritis 
Journal of autoimmunity  2013;43:1-9.
Autoimmune disease results from a loss of tolerance to self-antigens in genetically susceptible individuals. Completely understanding this process requires that targeted antigens be identified, and so a number of techniques have been developed to determine immune receptor specificities. We previously reported the construction of a phage-displayed synthetic human peptidome and a proof-of-principle analysis of antibodies from three patients with neurological autoimmunity. Here we present data from a large-scale screen of 298 independent antibody repertoires, including those from 73 healthy sera, using phage immunoprecipitation sequencing. The resulting database of peptide-antibody interactions characterizes each individual’s unique autoantibody fingerprint, and includes specificities found to occur frequently in the general population as well as those associated with disease. Screening type 1 diabetes (T1D) patients revealed a prematurely polyautoreactive phenotype compared with their matched controls. A collection of cerebrospinal fluids and sera from 63 multiple sclerosis patients uncovered novel, as well as previously reported antibody-peptide interactions. Finally, a screen of synovial fluids and sera from 64 rheumatoid arthritis patients revealed novel disease-associated antibody specificities that were independent of seropositivity status. This work demonstrates the utility of performing PhIP-Seq screens on large numbers of individuals and is another step toward defining the full complement of autoimmunoreactivities in health and disease.
doi:10.1016/j.jaut.2013.01.013
PMCID: PMC3677742  PMID: 23497938
autoantigen discovery; high throughput screening; PhIP-Seq; proteomics
2.  Fine-Mapping the Genetic Association of the Major Histocompatibility Complex in Multiple Sclerosis: HLA and Non-HLA Effects 
PLoS Genetics  2013;9(11):e1003926.
The major histocompatibility complex (MHC) region is strongly associated with multiple sclerosis (MS) susceptibility. HLA-DRB1*15:01 has the strongest effect, and several other alleles have been reported at different levels of validation. Using SNP data from genome-wide studies, we imputed and tested classical alleles and amino acid polymorphisms in 8 classical human leukocyte antigen (HLA) genes in 5,091 cases and 9,595 controls. We identified 11 statistically independent effects overall: 6 HLA-DRB1 and one DPB1 alleles in class II, one HLA-A and two B alleles in class I, and one signal in a region spanning from MICB to LST1. This genomic segment does not contain any HLA class I or II genes and provides robust evidence for the involvement of a non-HLA risk allele within the MHC. Interestingly, this region contains the TNF gene, the cognate ligand of the well-validated TNFRSF1A MS susceptibility gene. The classical HLA effects can be explained to some extent by polymorphic amino acid positions in the peptide-binding grooves. This study dissects the independent effects in the MHC, a critical region for MS susceptibility that harbors multiple risk alleles.
Author Summary
Multiple sclerosis (MS) is an inflammatory and neurodegenerative disease with a heritable component. Although it has been known for a long time that the strongest MS risk factor maps to the major histocompatibility complex (MHC) on chromosome 6, there are still many unresolved questions as to the identity and the nature of the risk variants within the MHC. Because the MHC has a complex structure, systematic investigation across this region has been challenging. In this study, we used state-of-the-art imputation methods coupled to statistical regression to query variants in the human leukocyte antigen (HLA) class I and II genes for a role in MS risk. Starting from available SNP genotype data, we replicated the strongest risk factor, the HLA-DRB1*15:01 allele, and were able to identify 11 independent effects in total. Functional studies are now needed to understand their mechanism in MS etiology.
doi:10.1371/journal.pgen.1003926
PMCID: PMC3836799  PMID: 24278027
3.  Prostaglandin E2 affects T cell responses through modulation of CD46 expression1 
The ubiquitous protein CD46, a regulator of complement activity, promotes T cell activation and differentiation towards a regulatory Tr1-like phenotype. CD46-mediated differentiation pathway is defective in several chronic inflammatory diseases, underlying the importance of CD46 in controlling T cell function and the need to understand its regulatory mechanisms. Using an RNAi-based screening approach in primary T cells, we have identified that two members of the G-protein coupled receptor (GPCR) kinases were involved in regulating CD46 expression at the surface of activated cells. We have investigated the role of prostaglandin E2 (PGE2), which binds to the E-prostanoid family of GPCRs through four subtypes of receptors called EP1-4, in the regulation of CD46 expression and function. Conflicting roles of PGE2 in T cell functions have been reported, and the reasons for these apparent discrepancies are not well understood. We show that addition of PGE2 strongly downregulates CD46 expression in activated T cells. Moreover, PGE2 differentially affects T cell activation, cytokine production and phenotype depending on the activation signals received by the T cells. This was correlated with a distinct pattern of the PGE2 receptors induced, with EP4 being preferentially induced by CD46 activation. Indeed, addition of an EP4 antagonist could reverse the effects observed on cytokine production observed following CD46 costimulation. These data demonstrate a novel role of the PGE2-EP4-GRK axis in CD46 functions, which might at least partly explain the diverse roles of PGE2 in T cell functions.
doi:10.4049/jimmunol.1103090
PMCID: PMC3758685  PMID: 22544928
4.  An RNA Profile Identifies Two Subsets of Multiple Sclerosis Patients Differing in Disease Activity 
Science translational medicine  2012;4(153):153ra131.
The multiple sclerosis (MS) patient population is highly heterogeneous in terms of disease course and treatment response. We used a transcriptional profile generated from peripheral blood mononuclear cells to define the structure of an MS patient population. Two subsets of MS subjects (MSA and MSB) were found among 141 untreated subjects. We replicated this structure in two additional groups of MS subjects treated with one of the two first-line disease-modifying treatments in MS: glatiramer acetate (GA) (n = 94) and interferon-β (IFN-β) (n = 128). One of the two subsets of subjects (MSA) was distinguished by higher expression of molecules involved in lymphocyte signaling pathways. Further, subjects in this MSA subset were more likely to have a new inflammatory event while on treatment with either GA or IFN-β (P = 0.0077). We thus report a transcriptional signature that differentiates subjects with MS into two classes with different levels of disease activity.
doi:10.1126/scitranslmed.3004186
PMCID: PMC3753678  PMID: 23019656
5.  Sodium Chloride Drives Autoimmune Disease by the Induction of Pathogenic Th17 Cells 
Nature  2013;496(7446):518-522.
There has been a marked increase in the incidence of autoimmune diseases in the last half-century. While the underlying genetic basis of this class of diseases has recently been elucidated implicating predominantly immune response genes1, changes in environmental factors must ultimately be driving this increase. The newly identified population of interleukin (IL)-17 producing CD4+ helper T cells (Th17 cells) plays a pivotal role in autoimmune diseases2. Pathogenic IL-23 dependent Th17 cells have been shown to be critical for the development of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), and genetic risk factors associated with MS are related to the IL23/Th17 pathway1, 2. However, little is known regarding the environmental factors that directly influence Th17 cells. Here we show that increased salt (sodium chloride; NaCl) concentrations found locally under physiological conditions in vivo dramatically boost the induction of murine and human Th17 cells. High-salt conditions activate the p38/MAPK pathway involving the tonicity-responsive enhancer binding protein (TonEBP/NFAT5) and the serum/glucocorticoid-regulated kinase 1 (SGK1) during cytokine-induced Th17 polarization. Gene silencing or chemical inhibition of p38/MAPK, NFAT5 or SGK1 abrogates the high-salt induced Th17 cell development. The Th17 cells generated under high-salt display a highly pathogenic and stable phenotype characterized by the up-regulation of the pro-inflammatory cytokines GM-CSF, TNFα and IL-2. Moreover, mice fed with a high-salt diet develop a more severe form of EAE, in line with augmented central nervous system infiltrating and peripherally induced antigen specific Th17 cells. Thus, increased dietary salt intake might represent an environmental risk factor for the development of autoimmune diseases through the induction of pathogenic Th17 cells.
doi:10.1038/nature11868
PMCID: PMC3746493  PMID: 23467095
6.  RNAi screen in primary human T cells reveals Flt3 as a modulator of IL-10 levels1 
Functional studies of human primary immune cells have been hampered by the lack of tools to silence gene functions. Here we report the application of a lentiviral RNAi library in primary human T cells. Using a subgenomic shRNA library targeting ~1,000 signaling genes, we identified novel genes that control the levels of IL-10 produced. IL-10 is a potent anti-inflammatory cytokine secreted by several cell types, including Tr1 cells, a subset of Tregs that exert their suppressive activity through IL-10 secretion. FLT3, a known hematopoeitic growth factor, was found to be a negative regulator of IL-10 levels in activated T cells. This was based on several observations. First, FLT3 and its ligand, FL, were both induced by T cell activation. Second, silencing of FLT3 led to increased IL-10 levels while addition of FL suppressed IL-10 secretion and increased FLT3 surface levels. Third, engagement of CD46, a known inducer of Tr1 regulatory T cells, upregulated surface FLT3 and secreted FL, which then inhibited IL-10 production in T cells. Hence, FL and FLT3 form a novel regulatory feedback loop that limits IL-10 production in T cells. Our results identified Flt3 as a new regulator of T cell function and offer a strategy to genetically dissect specific pathways in T cells.
doi:10.4049/jimmunol.0902443
PMCID: PMC3746748  PMID: 20018615
7.  Microbial Reprogramming Inhibits Western Diet-Associated Obesity 
PLoS ONE  2013;8(7):e68596.
A recent epidemiological study showed that eating ‘fast food’ items such as potato chips increased likelihood of obesity, whereas eating yogurt prevented age-associated weight gain in humans. It was demonstrated previously in animal models of obesity that the immune system plays a critical role in this process. Here we examined human subjects and mouse models consuming Westernized ‘fast food’ diet, and found CD4+ T helper (Th)17-biased immunity and changes in microbial communities and abdominal fat with obesity after eating the Western chow. In striking contrast, eating probiotic yogurt together with Western chow inhibited age-associated weight gain. We went on to test whether a bacteria found in yogurt may serve to lessen fat pathology by using purified Lactobacillus reuteri ATCC 6475 in drinking water. Surprisingly, we discovered that oral L. reuteri therapy alone was sufficient to change the pro-inflammatory immune cell profile and prevent abdominal fat pathology and age-associated weight gain in mice regardless of their baseline diet. These beneficial microbe effects were transferable into naïve recipient animals by purified CD4+ T cells alone. Specifically, bacterial effects depended upon active immune tolerance by induction of Foxp3+ regulatory T cells (Treg) and interleukin (Il)-10, without significantly changing the gut microbial ecology or reducing ad libitum caloric intake. Our finding that microbial targeting restored CD4+ T cell balance and yielded significantly leaner animals regardless of their dietary ‘fast food’ indiscretions suggests population-based approaches for weight management and enhancing public health in industrialized societies.
doi:10.1371/journal.pone.0068596
PMCID: PMC3707834  PMID: 23874682
8.  Identification of T helper type 1–like, Foxp3+ regulatory T cells in human autoimmune disease 
Nature medicine  2011;17(6):673-675.
CD4+CD25highCD127low/− forkhead box p3 (Foxp3)+ regulatory T cells (Treg cells) possess functional plasticity. Here we describe a higher frequency of T helper type 1 (TH1)-like, interferon-γ (IFN-γ)-secreting Foxp3+ T cells in untreated subjects with relapsing remitting multiple sclerosis (RRMS) as compared to healthy control individuals. In subjects treated with IFN-β, the frequency of IFN-γ+Foxp3+ T cells is similar to that in healthy control subjects. In vitro, human Treg cells from healthy subjects acquire a TH1-like phenotype when cultured in the presence of interleukin-12 (IL-12). TH1-like Treg cells show reduced suppressive activity in vitro, which can partially be reversed by IFN-γ–specific antibodies or by removal of IL-12.
doi:10.1038/nm.2389
PMCID: PMC3675886  PMID: 21540856
9.  Specific peripheral B cell tolerance defects in patients with multiple sclerosis 
The Journal of Clinical Investigation  2013;123(6):2737-2741.
Multiple sclerosis (MS) is a genetically mediated autoimmune disease of the central nervous system. B cells have recently emerged as major contributors to disease pathogenesis, but the mechanisms responsible for the loss of B cell tolerance in patients with MS are largely unknown. In healthy individuals, developing autoreactive B cells are removed from the repertoire at 2 tolerance checkpoints during early B cell development. Both of these central and peripheral B cell tolerance checkpoints are defective in patients with rheumatoid arthritis (RA) and type 1 diabetes (T1D). Here, we found that only the peripheral, but not the central, B cell tolerance checkpoint is defective in patients with MS. We show that this specific defect is accompanied by increased activation and homeostatic proliferation of mature naive B cells. Interestingly, all of these MS features parallel defects observed in FOXP3-deficient IPEX patients, who harbor nonfunctional Tregs. We demonstrate that in contrast to patients with RA or T1D, bone marrow central B cell selection in MS appears normal in most patients. In contrast, patients with MS suffer from a specific peripheral B cell tolerance defect that is potentially attributable to impaired Treg function and that leads to the accumulation of autoreactive B cell clones in their blood.
doi:10.1172/JCI68775
PMCID: PMC3668812  PMID: 23676463
10.  The TIGIT/CD226 axis regulates human T cell function 
TIGIT is a newly identified receptor expressed on T cells that binds to CD155 on the dendritic cell surface driving them to a more tolerogenic phenotype. Given that TIGIT contains an ITIM motif in its intracellular domain and considering the potential importance of the TIGIT/CD226 pathway in human autoimmune disease, we investigated the specific role of TIGIT in human CD4+ T cells. Using an agonistic anti-TIGIT mAb, we demonstrate a direct inhibitory effect on T cell proliferation with a decrease in expression of T-bet, GATA3, IRF4 and RORc with inhibition of cytokine production, predominately IFNγ. Knockdown of TIGIT expression by shRNA resulted in an increase of both T-bet and IFNγ mRNA and protein expression with concomitant decrease in IL-10 expression. Increases in IFNγ with TIGIT knockdown could be overcome by blocking CD226 signaling indicating that TIGIT exerts immunosuppressive effects by competing with CD226 for the same CD155 ligand. These data demonstrate that TIGIT can inhibit T cell functions by competing with CD226 and can also directly inhibit T cells in a T cell intrinsic manner. Our results provide evidence for a novel role of this alternative co-stimulatory pathway in regulating human T cell responses associated with autoimmune disease.
doi:10.4049/jimmunol.1103627
PMCID: PMC3324669  PMID: 22427644
11.  Induction and molecular signature of pathogenic TH17 cells 
Nature immunology  2012;13(10):991-999.
Interleukin 17 (IL-17)-producing TH17 cells are often present at the sites of tissue inflammation in autoimmune diseases, which has lead to the conclusion that TH17 are main drivers of autoimmune tissue injury. However, not all TH17 cells are pathogenic, in fact TH17 generated with TGF-β1 and IL-6 produce IL-17 but do not readily induce autoimmune disease without further exposure to IL-23. Here we show that TGF-β3, produced by developing TH17 cells, is dependent on IL-23, which together with IL-6 induces highly pathogenic TH17 cells. Moreover, TGF-β3-induced TH17 cells are functionally and molecularly distinct from TGF-β1-induced TH17 cells and possess a molecular signature that defines pathogenic effector TH17 cells in autoimmune disease.
doi:10.1038/ni.2416
PMCID: PMC3459594  PMID: 22961052
12.  Models of Somatic Hypermutation Targeting and Substitution Based on Synonymous Mutations from High-Throughput Immunoglobulin Sequencing Data 
Analyses of somatic hypermutation (SHM) patterns in B cell immunoglobulin (Ig) sequences contribute to our basic understanding of adaptive immunity, and have broad applications not only for understanding the immune response to pathogens, but also to determining the role of SHM in autoimmunity and B cell cancers. Although stochastic, SHM displays intrinsic biases that can confound statistical analysis, especially when combined with the particular codon usage and base composition in Ig sequences. Analysis of B cell clonal expansion, diversification, and selection processes thus critically depends on an accurate background model for SHM micro-sequence targeting (i.e., hot/cold-spots) and nucleotide substitution. Existing models are based on small numbers of sequences/mutations, in part because they depend on data from non-coding regions or non-functional sequences to remove the confounding influences of selection. Here, we combine high-throughput Ig sequencing with new computational analysis methods to produce improved models of SHM targeting and substitution that are based only on synonymous mutations, and are thus independent of selection. The resulting “S5F” models are based on 806,860 Synonymous mutations in 5-mer motifs from 1,145,182 Functional sequences and account for dependencies on the adjacent four nucleotides (two bases upstream and downstream of the mutation). The estimated profiles can explain almost half of the variance in observed mutation patterns, and clearly show that both mutation targeting and substitution are significantly influenced by neighboring bases. While mutability and substitution profiles were highly conserved across individuals, the variability across motifs was found to be much larger than previously estimated. The model and method source code are made available at http://clip.med.yale.edu/SHM
doi:10.3389/fimmu.2013.00358
PMCID: PMC3828525  PMID: 24298272
immunoglobulin; B cell; somatic hypermutation; mutability; substitution; targeting; AID; affinity maturation
13.  Multiple sclerosis 
The Journal of Clinical Investigation  2012;122(4):1180-1188.
Multiple sclerosis (MS) is a multifocal demyelinating disease with progressive neurodegeneration caused by an autoimmune response to self-antigens in a genetically susceptible individual. While the formation and persistence of meningeal lymphoid follicles suggest persistence of antigens to drive the continuing inflammatory and humoral response, the identity of an antigen or infectious agent leading to the oligoclonal expansion of B and T cells is unknown. In this review we examine new paradigms for understanding the immunopathology of MS, present recent data defining the common genetic variants underlying disease susceptibility, and explore how improved understanding of immune pathway disruption can inform MS prognosis and treatment decisions.
doi:10.1172/JCI58649
PMCID: PMC3314452  PMID: 22466660
14.  Interrogating the complex role of chromosome 16p13.13 in multiple sclerosis susceptibility: independent genetic signals in the CIITA–CLEC16A–SOCS1 gene complex 
Human Molecular Genetics  2011;20(17):3517-3524.
Multiple sclerosis (MS) is a neurodegenerative, autoimmune disease of the central nervous system, and numerous studies have shown that MS has a strong genetic component. Independent studies to identify MS-associated genes have often indicated multiple signals in physically close genomic regions, although by their proximity it is not always clear if these data indicate redundant or truly independent genetic signals. Recently, three MS study samples were genotyped in parallel using an Illumina Custom BeadChip. These revealed multiple significantly associated single-nucleotide polymorphisms within a 600 kb stretch on chromosome 16p13. Here we present a detailed analysis of variants in this region that clarifies the independent nature of these signals. The linkage disequilibrium patterns in the region and logistic regression analysis of the associations suggest that this region likely harbors three independent MS disease loci. Further, we examined cis-expression QTLs, histone modifications and CCCTC-binding factor (CTCF) binding data in the region. We also tested for correlated expression of the genes from the region using whole-genome expression array data from lymphoblastoid cell lines. Three of the genes show expression correlations across loci. Furthermore, in the GM12878 lymphoblastoid cell line, these three genes are in a continuous region devoid of H3K27 methylation, suggesting an open chromatin configuration. This region likely only contributes minimal risk to MS; however, investigation of this region will undoubtedly provide insight into the functional mechanisms of these genes. These data highlight the importance of taking a closer look at the expression and function of chromosome 16p13 in the pathogenesis of MS.
doi:10.1093/hmg/ddr250
PMCID: PMC3153306  PMID: 21653641
15.  Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein 
Nature medicine  2007;13(2):211-217.
The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS.
doi:10.1038/nm1488
PMCID: PMC3429369  PMID: 17237795
16.  Increased Frequencies of MOG/MHC Class II binding CD4 cells in Patients with Multiple Sclerosis 
Multiple sclerosis (MS) is an autoimmune disease characterized by infiltration of pathogenic immune cells in the central nervous system resulting in destruction of the myelin sheath and surrounding axons. We and others have previously measured the frequency of human myelin reactive T cells in peripheral blood was observed. Using T cell cloning techniques, a modest increase in the frequency of myelin reactive T cells in patients as compared to control subjects. Here, we investigated whether MOG-specific T cells could be detected and their frequency measured using DRB1*0401/MOG97–109(107E-S) tetramers in MS subjects and healthy controls expressing HLA Class II DRB1*0401. We defined the optimal culture conditions for expansion of MOG reactive T cells upon MOG peptide stimulation of PMBC. MOG97–109 reactive CD4+ T cells, isolated with DRB1*0401/MOG97–109 tetramers, after a short-term culture of PMBC with MOG97–109 peptides were detected more frequently from patients with MS as compared to healthy controls. T cell clones from single cell cloning of DRB1*0401/MOG97–109(107E-S) tetramer positive cells confirmed that these T cell clones were responsive to both the native and the substituted MOG peptide. These data indicate that autoantigen-specific T cells can be detected and enumerated from the blood of subjects utilizing Class II tetramers and the frequency of MOG97–109 reactive T cells is greater in patients with MS as compared to healthy controls.
doi:10.4049/jimmunol.1001543
PMCID: PMC3131477  PMID: 21653833
Human; Class II tetramers; myelin; autoimmunity; T lymphocytes
17.  Related B cell clones that populate the CSF and CNS of patients with multiple sclerosis produce CSF immunoglobulin 
Journal of neuroimmunology  2011;233(1-2):245-248.
We investigated the overlap shared between the immunoglobulin (Ig) proteome of the cerebrospinal fluid (CSF) and the B cell Ig-transcriptome of CSF and the central nervous system (CNS) tissue of three patients with multiple sclerosis. We determined the IgG-proteomes of CSF by mass spectrometry, and compared them to the IgG-transcriptomes from CSF and brain lesions, which were analyzed by cDNA cloning. Characteristic peptides that were identified in the CSF-proteome could also be detected in the transcriptomes of both, brain lesions and CSF, providing evidence for a strong overlap of the IgG repertoires in brain lesions and in the CSF.
doi:10.1016/j.jneuroim.2011.01.010
PMCID: PMC3090654  PMID: 21353315
multiple sclerosis; B cells; cerebrospinal fluid; central nervous system; oligoclonal bands
18.  Related B cell clones populate the meninges and parenchyma of patients with multiple sclerosis 
Brain  2011;134(2):534-541.
In the central nervous system of patients with multiple sclerosis, B cell aggregates populate the meninges, raising the central question as to whether these structures relate to the B cell infiltrates found in parenchymal lesions or instead, represent a separate central nervous system immune compartment. We characterized the repertoires derived from meningeal B cell aggregates and the corresponding parenchymal infiltrates from brain tissue derived primarily from patients with progressive multiple sclerosis. The majority of expanded antigen-experienced B cell clones derived from meningeal aggregates were also present in the parenchyma. We extended this investigation to include 20 grey matter specimens containing meninges, 26 inflammatory plaques, 19 areas of normal appearing white matter and cerebral spinal fluid. Analysis of 1833 B cell receptor heavy chain variable region sequences demonstrated that antigen-experienced clones were consistently shared among these distinct compartments. This study establishes a relationship between extraparenchymal lymphoid tissue and parenchymal infiltrates and defines the arrangement of B cell clones that populate the central nervous system of patients with multiple sclerosis.
doi:10.1093/brain/awq350
PMCID: PMC3030766  PMID: 21216828
multiple sclerosis; B cells; clonal expansion; antigen experience; central nervous system
19.  Class II MHC Self-Antigen Presentation in Human B and T Lymphocytes 
PLoS ONE  2012;7(1):e29805.
Human CD4+ T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4+ T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.
doi:10.1371/journal.pone.0029805
PMCID: PMC3267721  PMID: 22299025
20.  A knowledge-driven interaction analysis reveals potential neurodegenerative mechanism of multiple sclerosis susceptibility 
Genes and immunity  2011;12(5):335-340.
Gene-gene interactions are proposed as one important component of the genetic architecture of complex diseases, and are just beginning to be evaluated in the context of genome wide association studies (GWAS). In addition to detecting epistasis, a benefit to interaction analysis is that it also increases power to detect weak main effects. We conducted a knowledge-driven interaction analysis of a GWAS of 931 multiple sclerosis trios to discover gene-gene interactions within established biological contexts. We identify heterogeneous signals, including a gene-gene interaction between CHRM3 and MYLK (joint p = 0.0002), an interaction between two phospholipase-β isoforms, PLCβ1 & PLCβ4 (joint p = 0.0098), and a modest interaction between ACTN1 and MYH9 (joint p = 0.0326), all localized to calcium-signaled cytoskeletal regulation. Furthermore, we discover a main effect (joint p = 5.2E-5) previously unidentified by single-locus analysis within another related gene, SCIN, a calcium-binding cytoskeleton regulatory protein. This work illustrates that knowledge-driven interaction analysis of GWAS data is a feasible approach to identify new genetic effects. The results of this study are among the first gene-gene interactions and non-immune susceptibility loci for multiple sclerosis. Further, the implicated genes cluster within inter-related biological mechanisms that suggest a neurodegenerative component to multiple sclerosis.
doi:10.1038/gene.2011.3
PMCID: PMC3136581  PMID: 21346779
21.  Human Regulatory T Cells in Autoimmune Diseases 
Current opinion in immunology  2010;22(6):753-760.
Summary of Recent Advances
Human regulatory T cells (Tregs) play a critical role in preventing autoimmunity, and their failure contributes to autoimmune diseases. In recent years, our understanding of human Tregs has been greatly enhanced by improvements in the definition and isolation of pure human Tregs, as well as by the discovery of phenotypically and functionally distinct human Treg subsets. This progress has also yielded a better understanding of the mechanisms of human Treg suppression and the role of human Tregs in autoimmune diseases. An unexpected discovery is that human Tregs have considerable plasticity that allows them to produce the pro-inflammatory cytokine IL-17 under certain conditions. These recent advances highlight the importance of studying the roles of both mouse and human Tregs in autoimmunity.
doi:10.1016/j.coi.2010.08.012
PMCID: PMC2997859  PMID: 20869862
22.  Evidence for CRHR1 in multiple sclerosis using supervised machine learning and meta-analysis in 12 566 individuals 
Human Molecular Genetics  2010;19(21):4286-4295.
The primary genetic risk factor in multiple sclerosis (MS) is the HLA-DRB1*1501 allele; however, much of the remaining genetic contribution to MS has yet to be elucidated. Several lines of evidence support a role for neuroendocrine system involvement in autoimmunity which may, in part, be genetically determined. Here, we comprehensively investigated variation within eight candidate hypothalamic–pituitary–adrenal (HPA) axis genes and susceptibility to MS. A total of 326 SNPs were investigated in a discovery dataset of 1343 MS cases and 1379 healthy controls of European ancestry using a multi-analytical strategy. Random Forests, a supervised machine-learning algorithm, identified eight intronic SNPs within the corticotrophin-releasing hormone receptor 1 or CRHR1 locus on 17q21.31 as important predictors of MS. On the basis of univariate analyses, six CRHR1 variants were associated with decreased risk for disease following a conservative correction for multiple tests. Independent replication was observed for CRHR1 in a large meta-analysis comprising 2624 MS cases and 7220 healthy controls of European ancestry. Results from a combined meta-analysis of all 3967 MS cases and 8599 controls provide strong evidence for the involvement of CRHR1 in MS. The strongest association was observed for rs242936 (OR = 0.82, 95% CI = 0.74–0.90, P = 9.7 × 10−5). Replicated CRHR1 variants appear to exist on a single associated haplotype. Further investigation of mechanisms involved in HPA axis regulation and response to stress in MS pathogenesis is warranted.
doi:10.1093/hmg/ddq328
PMCID: PMC2951862  PMID: 20699326
23.  Genome-Wide Assessment for Genetic Variants Associated with Ventricular Dysfunction after Primary Coronary Artery Bypass Graft Surgery 
PLoS ONE  2011;6(9):e24593.
Background
Postoperative ventricular dysfunction (VnD) occurs in 9–20% of coronary artery bypass graft (CABG) surgical patients and is associated with increased postoperative morbidity and mortality. Understanding genetic causes of postoperative VnD should enhance patient risk stratification and improve treatment and prevention strategies. We aimed to determine if genetic variants associate with occurrence of in-hospital VnD after CABG surgery.
Methods
A genome-wide association study identified single nucleotide polymorphisms (SNPs) associated with postoperative VnD in male subjects of European ancestry undergoing isolated primary CABG surgery with cardiopulmonary bypass. VnD was defined as the need for ≥2 inotropes or mechanical ventricular support after CABG surgery. Validated SNPs were assessed further in two replication CABG cohorts and meta-analysis was performed.
Results
Over 100 SNPs were associated with VnD (P<10−4), with one SNP (rs17691914) encoded at 3p22.3 reaching genome-wide significance (Padditive model = 2.14×10−8). Meta-analysis of validation and replication study data for 17 SNPs identified three SNPs associated with increased risk for developing postoperative VnD after adjusting for clinical risk factors. These SNPs are located at 3p22.3 (rs17691914, ORadditive model = 2.01, P = 0.0002), 3p14.2 (rs17061085, ORadditive model = 1.70, P = 0.0001) and 11q23.2 (rs12279572, ORrecessive model = 2.19, P = 0.001).
Conclusions
No SNPs were consistently associated with strong risk (ORadditive model>2.1) of developing in-hospital VnD after CABG surgery. However, three genetic loci identified by meta-analysis were more modestly associated with development of postoperative VnD. Studies of larger cohorts to assess these loci as well as to define other genetic mechanisms and related biology that link genetic variants to postoperative ventricular dysfunction are warranted.
doi:10.1371/journal.pone.0024593
PMCID: PMC3184087  PMID: 21980348
24.  Pervasive Sharing of Genetic Effects in Autoimmune Disease 
PLoS Genetics  2011;7(8):e1002254.
Genome-wide association (GWA) studies have identified numerous, replicable, genetic associations between common single nucleotide polymorphisms (SNPs) and risk of common autoimmune and inflammatory (immune-mediated) diseases, some of which are shared between two diseases. Along with epidemiological and clinical evidence, this suggests that some genetic risk factors may be shared across diseases—as is the case with alleles in the Major Histocompatibility Locus. In this work we evaluate the extent of this sharing for 107 immune disease-risk SNPs in seven diseases: celiac disease, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes. We have developed a novel statistic for Cross Phenotype Meta-Analysis (CPMA) which detects association of a SNP to multiple, but not necessarily all, phenotypes. With it, we find evidence that 47/107 (44%) immune-mediated disease risk SNPs are associated to multiple—but not all—immune-mediated diseases (SNP-wise PCPMA<0.01). We also show that distinct groups of interacting proteins are encoded near SNPs which predispose to the same subsets of diseases; we propose these as the mechanistic basis of shared disease risk. We are thus able to leverage genetic data across diseases to construct biological hypotheses about the underlying mechanism of pathogenesis.
Author Summary
Over the last five years we have found over 100 genetic variants predisposing to common diseases affecting the immune system. In this study we analyze 107 such variants across seven diseases and find that almost half are shared across diseases. We also find that the patterns of sharing across diseases cluster these variants into groups; proteins encoded near variants in the same group tend to interact. This suggests that genetic variation may influence entire pathways to create risk to multiple diseases.
doi:10.1371/journal.pgen.1002254
PMCID: PMC3154137  PMID: 21852963
25.  Droplet-based microfluidic platforms for single T cell secretion analysis of IL-10 cytokine 
Biosensors & bioelectronics  2010;26(5):2707-2710.
Here we present a microfluidic method for the analysis of single cell secretions. The method co-encapsulates cells with microspheres conjugated with capture antibodies and detection fluorescence-labeled antibodies. The secreted substance captured on the microsphere surface and detected via detection antibodies generating a localized fluorescent signal on a microsphere surface. Using this method, CD4+CD25+ regulatory T cells were encapsulated and assayed to detect IL-10 secreting cell in population.
doi:10.1016/j.bios.2010.09.006
PMCID: PMC3141325  PMID: 20888750
Microfluidic; Droplets; Single cell; Cell secretion; Microsphere

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