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Background: Upfront docetaxel or novel hormonal agents (NHA) such as abiraterone and enzalutamide have become the standard of care for metastatic hormone sensitive prostate cancer (mHSPC). However, data comparing the efficacy of docetaxel and NHAs in this setting are limited.Patients and Methods: This was a retrospective cohort study of patients with de novo mHSPC treated with upfront docetaxel or an NHA between January 1, 2014 and April 30, 2019 within the Mount Sinai Health System. Clinical data were extracted from the medical record. The primary outcome was failure-free survival (FFS), defined as the time to next treatment. The primary predictor was treatment with docetaxel or NHA. FFS was compared between the two groups using the Kaplan Meier method and multivariable Cox proportional hazards models. We additionally assessed the prognostic value of post-treatment PSA.Results: We identified 94 de novo mHSPC patients; 52 and 42 treated with upfront docetaxel and NHAs, respectively. NHAs were associated with significantly longer FFS compared to docetaxel (20.7 vs. 10.1 months, p=0.023). In a multivariable model adjusting for demographics and clinical factors, docetaxel was independently associated with worse FFS compared to NHAs (HR 1.96, 95% CI 1.12−3.45, p=0.019). High metastasis burden patients had a significantly longer FFS with NHAs than docetaxel (25.12 vs. 9.63 months, p=0.014), while there was no significant difference in FFS among low metastasis burden patients (NHA 20.71 vs. Docetaxel 26.5 months, p=0.9). Regardless of treatment, lower post-treatment PSA levels were associated with improved FFS (58.95 vs. 11.57 vs. 9.4 months for PSA ≤0.2, 0.2-0.4, >0.4ng/ml, respectively; pConclusion: Comparative analysis of real-world data demonstrated longer FFS in de novo mHSPC treated with NHA compared to docetaxel. In addition, the depth of PSA response following combination treatment may hold prognostic value for mHSPC outcomes.

6584 Background: Analysis of real-world data is critical for clear understanding of treatment effectiveness. Here, we explore the clinical utility of next-generation sequencing (NGS) and the impact of mutational landscapes on targeted therapy and outcomes in almost 10,000 patients in real-world setting. Methods: We constructed a data analysis platform that integrated NGS results with machine and manually curated electronic medical records data from a single large healthcare system. We assessed eligibility for a targeted therapy based on the constellation of mutations detected by NGS, investigated whether a matched therapy was given and how this affected patient outcome. Results: The study comprised of 9848 patients with NGS results across 90 cancer types including lung (2208;22%), colorectal (1495;15%), multiple myeloma (1153;12%), breast (783;8%), and prostate (480;5%) and other remaining cancers (3,729; 38%) from 2011 to 2021. Overall, 95% of patients had a positive NGS result, of whom 6364 (65%) had a targetable mutation in 34 genes per any NCCN guidelines in any cancer type. Of these, 2930 (46%) were late-stage (3-4). 820 patients across 31 cancer types received any of 54 targeted therapies based on an NGS result; 605/820 (74%) were stage 3-4; overall 605/2930 (21%) of all tested stage 3-4 patients received targeted therapy. Overall stage 4 patients with a targetable mutation who were treated with a targeted therapy had better 5-year overall survival (OS) than patients who did not (HR = 0.71, 95% CI 0.57-0.89, p = 0.003). We further analyzed important biomarkers in specific cancers. Stage 4 lung cancer patients carrying EGFR, ALK, ROS1 or MET alteration and receiving targeted therapy had better 5-year OS (HR 0.56, 95% CI 0.40-0.80, p = 0.001). We observed that multiple myeloma patients carrying KRAS and NRAS mutation received off-label Trametinib, however it did not improve their OS (HR 1.28, 95% CI 0.76-2.15, p = 0.4). Moreover, there are 665 patients with tumor mutational burden (TMB) across 64 cancer types. TMB has been categorized into high (55, 8%), intermediate (177, 27%) and low (433, 65%). OS was significantly longer in TMB-low patients compared to TMB-high and TMB-intermediate patients across all cancers (HR 0.63, 95% CI 0.43-0.92, p = 0.016). There are 5051 patients with microsatellite instability status across 72 cancers categorized into high (828, 16%), and low (4223, 84%) with no significant survival difference between groups. Conclusions: We show that real-world data offers important insights into clinical practice and patient outcome, with respect to genomic alterations and associated targeted therapies. Moreover, we have built an analytics platform which allows users to ask relevant clinical questions around outcome of patients treated with targeted therapy by genomics biomarker and potential to identify off-label use and clinical response in any cancer type.

Familial hypercholesterolemia (FH) is a monogenic disease characterized by a lifelong exposure to high LDL-C levels that can lead to early onset coronary heart disease (CHD). The main causes of FH identified to date include loss-of-function mutations in

Purpose To develop a comprehensive genetic test for female and male infertility in support of medical decisions during assisted reproductive technology (ART) protocols. Methods We developed a next-generation sequencing (NGS) gene panel consisting of 87 genes including promoters, 5′ and 3′ untranslated regions, exons, and selected introns. In addition, sex chromosome aneuploidies and Y chromosome microdeletions were analyzed concomitantly using the same panel. Results The NGS panel was analytically validated by retrospective analysis of 118 genomic DNA samples with known variants in loci representative of female and male infertility. Our results showed analytical accuracy of > 99%, with > 98% sensitivity for single-nucleotide variants (SNVs) and > 91% sensitivity for insertions/deletions (indels). Clinical sensitivity was assessed with samples containing variants representative of male and female infertility, and it was 100% for SNVs/indels, CFTR IVS8-5T variants, sex chromosome aneuploidies, and copy number variants (CNVs) and > 93% for Y chromosome microdeletions. Cost analysis shows potential savings when comparing this single NGS assay with the standard approach, which includes multiple assays. Conclusions A single, comprehensive, NGS panel can simplify the ordering process for healthcare providers, reduce turnaround time, and lower the overall cost of testing for genetic assessment of infertility in females and males, while maintaining accuracy. Electronic supplementary material The online version of this article (10.1007/s10815-018-1204-7) contains supplementary material, which is available to authorized users.

e17047 Background: The addition of docetaxel or a novel hormonal agent (NHA), such as abiraterone acetate or enzalutamide, has become standard of care for mHSPC, but prospective data for comparative efficacy remains limited. Clinical variables abstracted through natural language processing of free text from patient charts can provide real-world data to answer important clinical questions. Methods: Using an innovative data abstraction process, we retrospectively identified men with de novo mHSPC within deidentified charts from the Mount Sinai Health System treated with either docetaxel or a NHA between January 1, 2014 and April 30, 2019. Dates were chosen to reflect the timeline for which these therapeutic agents received regulatory approval for mHSPC, but also to allow sufficient clinical follow up after treatment initiation. Primary outcome of failure-free survival (FFS), defined as the time to next treatment, was assessed using the Kaplan-Meier method and multivariable Cox proportional hazards models. We additionally performed multivariable analysis to evaluate the prognostic significance of post-treatment PSA nadir on FFS. Results: A total of 94 de novo mHSPC patients that received either docetaxel or an NHA were identified using proprietary Sema4 data abstraction process: 52 received docetaxel, 42 received an NHA. Use of an upfront NHA in mHSPC was associated with a significantly longer FFS compared to docetaxel (20.7 vs. 10.1 months, p = 0.023). While NHAs were associated with a significantly longer FFS than docetaxel in patients with high metastatic burden of disease (25.12 vs. 9.63 months, p = 0.014), this was not observed in low-volume disease (20.71 vs. 26.5 months, p = 0.9). In multivariable model analysis adjusting for age, baseline PSA, and metastasis burden, docetaxel remains independently associated with worse FFS compared to NHA (HR 1.96, 95% CI 1.12−3.45, p = 0.019). Irrespective of docetaxel or NHA use, lower post-treatment PSA nadir levels were associated with improved FFS (58.95 vs. 11.57 vs. 9.4 months for PSA ≤0.2, 0.2-0.4, > 0.4ng/ml, respectively; p < 0.001). Conclusions: Clinical variables abstracted from deidentified clinical documentation can efficiently provide relevant and reliable data upon which clinical research can be based. Comparative analysis of real-world data demonstrates superior FFS in de novo mHSPC treated with a NHA as compared to docetaxel. The depth of PSA response holds prognostic value for mHSPC outcomes, regardless of treatment used.

The gut microbiome plays an important role in immune function and has been implicated in several autoimmune disorders. Here we use 16S rRNA sequencing to investigate the gut microbiome in subjects with multiple sclerosis (MS, n=60) and healthy controls (n=43). Microbiome alterations in MS include increases in Methanobrevibacter and Akkermansia and decreases in Butyricimonas, and correlate with variations in the expression of genes involved in dendritic cell maturation, interferon signalling and NF-kB signalling pathways in circulating T cells and monocytes. Patients on disease-modifying treatment show increased abundances of Prevotella and Sutterella, and decreased Sarcina, compared with untreated patients. MS patients of a second cohort show elevated breath methane compared with controls, consistent with our observation of increased gut Methanobrevibacter in MS in the first cohort. Further study is required to assess whether the observed alterations in the gut microbiome play a role in, or are a consequence of, MS pathogenesis., The gut microbiome has been implicated in several autoimmune disorders. Here, the authors study the gut microbiome of patients with multiple sclerosis, and find correlations between altered abundance of certain gut microorganisms and changes in expression of immune defence genes.

After upregulation of AHR in astrocytes by type I interferons, commensal-microbe-derived metabolites of dietary tryptophan act on astrocytes to suppress CNS inflammation.

Familial hypercholesterolemia (FH) is a monogenic disease characterized by a lifelong exposure to high LDL-C levels that can lead to early onset coronary heart disease (CHD). The main causes of FH identified to date include loss-of-function mutations in LDLR or APOB, or gain-of-function mutations in PCSK9. Early diagnosis and genetic testing of FH suspects is critical for improved prognosis of affected individuals as lipid lowering treatments are effective in preventing CHD related morbidity and mortality. In the present manuscript, we developed a comprehensive next generation sequencing (NGS) panel which we applied on two different resources of FH in the Icelandic population: 62 subjects from 23 FH families with known or unknown culprit mutations, and a population-based sampling of 315 subjects selected for total cholesterol levels above the 95th percentile cut-point. The application of the NGS panel revealed significant diagnostic yields in identifying pathogenic LDLR mutations in both family and population-based genetic testing.

Our understanding of the pathways that regulate lymphocyte metabolism, as well as the effects of metabolism and its products on the immune response, is still limited. We report that a metabolic program controlled by the transcription factors hypoxia inducible factor-1α (HIF1-α) and aryl hydrocarbon receptor (AHR) supports the differentiation of type 1 regulatory T cell (Tr1) cells. HIF1-α controls the early metabolic reprograming of Tr1 cells. At later time points, AHR promotes HIF1-α degradation and takes control of Tr1 cell metabolism. Extracellular ATP (eATP) and hypoxia, linked to inflammation, trigger AHR inactivation by HIF1-α and inhibit Tr1 cell differentiation. Conversely, CD39 promotes Tr1 cell differentiation by depleting eATP. CD39 also contributes to Tr1 suppressive activity by generating adenosine in cooperation with CD73 expressed by responder T cells and antigen-presenting cells. These results suggest that HIF1-α and AHR integrate immunological, metabolic and environmental signals to regulate the immune response.

ObjectiveTo develop a comprehensive genetic test for female and male infertility in support of medical decisions during assisted reproductive technology (ART) protocols.DesignRetrospective analysis of results from 118 DNA samples with known variants in loci representative of female and male infertility.Interventions(s)NoneMain Outcome Measure(s)Next-Generation Sequencing (NGS) of 87 genes including promoters, 5’ and 3’ untranslated regions, exons and selected introns. In addition, sex chromosome aneuploidies and Y chromosome microdeletions are analyzed concomitantly using the same panel.ResultsAnalytical accuracy was >99%, with >98% sensitivity for Single Nucleotide Variants (SNVs) and >91% sensitivity for insertions/deletions (indels). Clinical sensitivity was assessed with samples containing variants representative of male and female infertility, and it was 100% for SNVs/indels, CFTR IVS8-5T variants, sex chromosome aneuploidies and Copy Number Variants (CNVs), and >93% for Y chromosome microdeletions. Cost analysis comparing the NGS assay with standard, multiple analysis approach, shows potential savings of $2723 per case. Conclusion: A single, comprehensive, NGS panel can simplify the ordering process for healthcare providers, reduce turnaround time, and lower the overall cost of testing for genetic assessment of infertility in females and males, while maintaining accuracy.

Astrocytes have complex roles in health and disease, thus it is important to study the pathways that regulate their function. Here we report that lactosylceramide (LacCer) synthesized by β-1,4-galactosyltransferase 6 (B4GALT6) is upregulated in the central nervous system (CNS) of mice during chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). LacCer acts in an autocrine manner to control astrocyte transcriptional programs that promote neurodegeneration. In addition, LacCer in astrocytes controls the recruitment and activation of microglia and CNS-infiltrating monocytes in a non-cell autonomous manner by regulating production of the chemokine CCL2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. We also detected high B4GALT6 gene expression and LacCer concentrations in CNS MS lesions. Inhibition of LacCer synthesis in mice suppressed local CNS innate immunity and neurodegeneration in EAE and interfered with the activation of human astrocytes in vitro. Thus, B4GALT6 regulates astrocyte activation and is a potential therapeutic target for MS and other neuroinflammatory disorders.

Yeste, Ada et al., Type 1 diabetes (T1D) is a T cell-dependent autoimmune disease that is characterized by the destruction of insulin-producing β cells in the pancreas. The administration to patients of ex vivo-differentiated FoxP3 regulatory T (T) cells or tolerogenic dendritic cells (DCs) that promote T cell differentiation is considered a potential therapy for T1D; however, cell-based therapies cannot be easily translated into clinical practice. We engineered nanoparticles (NPs) to deliver both a tolerogenic molecule, the aryl hydrocarbon receptor (AhR) ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and the β cell antigen proinsulin (NP) to induce a tolerogenic phenotype in DCs and promote T cell generation in vivo. NP administration to 8-week-old nonobese diabetic mice suppressed autoimmune diabetes. NP induced a tolerogenic phenotype in DCs, which was characterized by a decreased ability to activate inflammatory effector T cells and was concomitant with the increased differentiation of FoxP3 T cells. The induction of a tolerogenic phenotype in DCs by NPs was mediated by the AhR-dependent induction of Socs2, which resulted in inhibition of nuclear factor κB activation and proinflammatory cytokine production (properties of tolerogenic DCs). Together, these data suggest that NPs constitute a potential tool to reestablish tolerance in T1D and potentially other autoimmune disorders., This work was supported by grants ES025530, AI075285, and AI093903 from the NIH, 17-2011-371 from the Juvenile Diabetes Research Foundation, the Boston Area Diabetes Endocrinology Research Center (to F.J.Q.), and PI14-1600 from the Spanish Ministry of Economy and Competitiveness (ISCIII) with FEDER cofunding (to D.P.). M.C.T. received support from an educational grant from Mallinckrodt Pharmaceuticals (A113307) and from a postdoctoral fellowship (0571-15-6) from Coordination for the Improvement of Higher Education Personnel, Brazil. I.D.M. received support from an educational grant from Questcor (A219074) and from a postdoctoral fellowship (FG 2036-A1/1) from the National Multiple Sclerosis Society. S.C.K. received support from The Helmsley Charitable Trust.

Background Fingolimod (FTY720), the first oral treatment for multiple sclerosis (MS), blocks immune cell trafficking and prevents disease relapses by downregulation of sphingosine-1-phosphate receptor. We determined the effect of FTY720 on human T cell activation and effector function. Methods T cells from MS patients and healthy controls were isolated to measure gene expression profiles in the presence or absence of FTY720 using nanostring and quantitative real-time polymerase chain reaction (qPCR). Cytokine protein expression was measured using luminex assay and flow cytometry analysis. Lentivirus vector carrying short hairpin RNA (shRNA) was used to knock down the expression of specific genes in CD4+ T cells. Chromatin immunoprecipitation was performed to assess T cell factor 1 (TCF-1) binding to promoter regions. Luciferase assays were performed to test the direct regulation of interferon gamma (IFN-γ) and granzyme B (GZMB) by TCF-1. Western blot analysis was used to assess the phosphorylation status of Akt and GSK3β. Results We showed that FTY720 treatment not only affects T cell trafficking but also T cell activation. Patients treated with FTY720 showed a significant reduction in circulating CD4 T cells. Activation of T cells in presence of FTY720 showed a less inflammatory phenotype with reduced production of IFN-γ and GZMB. This decreased effector phenotype of FTY720-treated T cells was dependent on the upregulation of TCF-1. FTY720-induced TCF-1 downregulated the pathogenic cytokines IFN-γ and GZMB by binding to their promoter/enhancer regions and mediating epigenetic modifications. Furthermore, we observed that TCF-1 expression was lower in T cells from multiple sclerosis patients than in those from healthy individuals, and FTY720 treatment increased TCF-1 expression in multiple sclerosis patients. Conclusions These results reveal a previously unknown mechanism of the effect of FTY720 on human CD4+ T cell modulation in multiple sclerosis and demonstrate the role of TCF-1 in human T cell activation and effector function. Electronic supplementary material The online version of this article (doi:10.1186/s12974-015-0460-z) contains supplementary material, which is available to authorized users.

Seasonal changes in disease activity have been observed in multiple sclerosis, an autoimmune disorder that affects the CNS. These epidemiological observations suggest that environmental factors influence the disease course. Here, we report that melatonin levels, whose production is modulated by seasonal variations in night length, negatively correlate with multiple sclerosis activity in humans. Treatment with melatonin ameliorates disease in an experimental model of multiple sclerosis and directly interferes with the differentiation of human and mouse T cells. Melatonin induces the expression of the repressor transcription factor Nfil3, blocking the differentiation of pathogenic Th17 cells and boosts the generation of protective Tr1 cells via Erk1/2 and the transactivation of the IL-10 promoter by ROR-α. These results suggest that melatonin is another example of how environmental-driven cues can impact T cell differentiation and have implications for autoimmune disorders such as multiple sclerosis. Fil: Farez, Mauricio. Fundacion para la Lucha contra las Enfermedades Neurologicas Infancia. Instituto de Investigaciones Neurologicas "Raul Carrea"; Argentina Fil: Mascanfroni, Ivan Darío. Harvard Medical School; Estados Unidos Fil: Mendez Huergo, Santiago Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Yeste, Ada. Harvard Medical School; Estados Unidos Fil: Murugaiyan, Gopal. Harvard Medical School; Estados Unidos Fil: Garo, Lucien P.. Harvard Medical School; Estados Unidos Fil: Balbuena Aguirre, María E.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Fundacion para la Lucha contra las Enfermedades Neurologicas Infancia. Instituto de Investigaciones Neurologicas "Raul Carrea"; Argentina Fil: Patel, Bonny. Harvard Medical School; Estados Unidos Fil: Ysrraelit, María C.. Fundacion para la Lucha contra las Enfermedades Neurologicas Infancia. Instituto de Investigaciones Neurologicas "Raul Carrea"; Argentina Fil: Zhu, Chen . Harvard Medical School; Estados Unidos Fil: Kuchroo, Vjay K.. Harvard Medical School; Estados Unidos Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: Quintana, Francisco. Harvard Medical School; Estados Unidos Fil: Correale, Jorge. Fundacion para la Lucha contra las Enfermedades Neurologicas Infancia. Instituto de Investigaciones Neurologicas "Raul Carrea"; Argentina

We have previously reported the molecular signature of murine pathogenic TH17 cells that induce experimental autoimmune encephalomyelitis (EAE) in animals. Here we show that human peripheral blood IFN-γ+IL-17+ (TH1/17) and IFN-γ−IL-17+ (TH17) CD4+ T cells display distinct transcriptional profiles in high-throughput transcription analyses. Compared to TH17 cells, TH1/17 cells have gene signatures with marked similarity to mouse pathogenic TH17 cells. Assessing 15 representative signature genes in patients with multiple sclerosis, we find that TH1/17 cells have elevated expression of CXCR3 and reduced expression of IFNG, CCL3, CLL4, GZMB, and IL10 compared to healthy controls. Moreover, higher expression of IL10 in TH17 cells is found in clinically stable vs. active patients. Our results define the molecular signature of human pro-inflammatory TH17 cells, which can be used to both identify pathogenic TH17 cells and to measure the effect of treatment on TH17 cells in human autoimmune diseases., CD4+ T cells secreting interleukin-17 (TH17) have diverse functions in modulating autoimmune diseases. Here the authors show via transcriptome analyses that a subset of human TH 17 co-expressing interferon-γ (TH1/17) has a molecular signature similar to “pathogenic” mouse TH 17 but distinct from “non-pathogenic” mouse TH 17.

e13105 Background: Diagnosis of hereditary cancer syndromes involves time-consuming comprehensive clinical and laboratory work-up, however, timely and accurate diagnosis is pivotal to the clinical management of cancer patients. Germline genetic testing has shown to facilitate the diagnostic process, allowing for identification and management of individuals at risk for inherited cancers. However, the laboratory diagnostics process requires not only development and validation of comprehensive gene panels to improve diagnostic yields, but a quality driven workflow including an end-to-end bioinformatics pipeline, and a robust process for variant classification. We will present a gene panel for the evaluation of hereditary cancer syndromes, conducted utilizing our novel end-to-end workflow, and validated in the CLIA-approved environment. Methods: A targeted Next-Generation Sequencing (NGS) panel consisting of 130 genes, including exons, promoters, 5’-UTRs, 3’-UTRs and selected introns, was designed to include genes associated with hereditary cancers. The assay was validated using samples from the 1000 genomes project and samples with known pathogenic variants. Elements software was utilized for end-to-end bioinformatic process ensuring adherence with the CLIA quality standards, and supporting manual curation of sequence variants. Results: Preliminary data from our current panel of genes associated with hereditary cancer syndromes revealed high sensitivity, specificity, and positive predictive value. Accuracy was confirmed by analysis of known SNVs, indels, and CNVs using 1000 Genomes and samples carrying pathogenic variants. The bioinformatics software allowed for an end-to-end quality controlled process of handling and analyzing of the NGS data, showing applicability for a clinical laboratory workflow. Conclusions: We have developed a comprehensive and accurate genetic testing process based on an automated and quality driven bioinformatics workflow that can be used to identify clinically important variants in genes associated with hereditary cancers. It's performance allows for implementation in the clinical laboratory setting.

Mice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific “humanized” mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. To address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHC II) and instead expresses human leukocyte antigen DR1 (HLA-DR1). These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T-cell reconstitution, delayed-type hypersensitivity responses, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, associated with aberrant FOXP3 function, mice developed a lethal inflammatory disorder with multiorgan involvement and autoantibody production mimicking the pathology seen in affected humans. This humanized mouse model permits in vivo evaluation of immune responses associated with genetically altered HSCs, including primary immunodeficiencies, and should facilitate the study of human immune pathobiology and the development of targeted therapeutics.

Objectives: For most adults with initial clinical presentation of multiple sclerosis (MS), biological disease was likely initiated many years prior. Pediatric-onset MS provides an opportunity to study early disease processes. Methods: Using antigen microarrays, including CNS-related proteins, lipids, and other autoantigens, we studied early immunologic events involved in clinical onset of pediatric MS. Serum samples were collected at the time of incident acquired CNS demyelinating syndromes (ADS) in children who, in subsequent prospective follow-up, were ascertained to have either pediatric MS (ADS-MS) or a monophasic illness (ADS-mono). Samples were obtained both at the time of ADS presentation and 3 months into follow-up. We used an initial training set of samples to implicate antibody signatures associated with each group, and then a test set. An additional set of follow-up samples (stability set) was used as a form of internal validation. Results: Children with ADS-MS tended to have distinguishable serum antibody patterns both at the time of ADS presentation and 3 months into follow-up. At the time of ADS, serum samples from patients with ADS-MS or ADS-mono reacted against similar numbers of CNS antigens, although CNS antigens implicated in adult MS were more often targeted in children with ADS-MS. The follow-up ADS-MS samples reacted against a broader panel of CNS antigens, while corresponding ADS-mono samples exhibited a contraction of the initial antibody response. Conclusions: Our findings in this prospective cohort of pediatric-onset CNS demyelinating diseases point to an active process of epitope spreading during early stages of MS, not seen in monophasic CNS inflammatory conditions.

Interleukin (IL)-22 produced by innate lymphoid cells (ILCs) and CD4+ T cells plays an important role in host defence and mucosal homeostasis, thus it is important to investigate the mechanisms that regulate IL-22 production. We investigated the regulation IL-22 production by CD4+ T cells. Here we show that IL-21 triggers IL-22, but not IL-17 production by CD4+ T cells. STAT3, activated by IL-21, controls the epigenetic status of the il22 promoter and its interaction with the aryl hydrocarbon receptor (AhR). Moreover, IL-21 and AhR signalling in T cells control IL-22 production and the development of dextran sodium sulphate-induced colitis in ILC-deficient mice. Thus, we have identified IL-21 as an inducer of IL-22 production in CD4+ T cells in vitro and in vivo.

Dendritic cells (DCs) control the balance between effector T cells and regulatory T cells in vivo. Hence, the study of DCs might identify mechanisms of disease pathogenesis and guide new therapeutic approaches for disorders mediated by the immune system. We found that interleukin 27 (IL-27) signaling in mouse DCs limited the generation of effector cells of the TH1 and TH17 subsets of helper T cells and the development of experimental autoimmune encephalomyelitis (EAE). The effects of IL-27 were mediated at least in part through induction of the immunoregulatory molecule CD39 in DCs. IL-27-induced CD39 decreased the extracellular concentration of ATP and downregulated nucleotide-dependent activation of the NLRP3 inflammasome. Finally, therapeutic vaccination with IL-27-conditioned DCs suppressed established relapsing-remitting EAE. Thus, IL-27 signaling in DCs limited pathogenic T cell responses and the development of autoimmunity.

Immune sequencing allows for the study of complex immunological diseases by sequencing millions of V(D)J combinations from B-cell antibody and T-cell receptors. The popularity of this technique has increased due to recent throughput and read length improvements in next-generation sequencing technologies. However, structural and sequence complexities of antibody genes have made reliable targeting approaches challenging. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. The method uses a unique barcoding scheme specifically designed to tag every mRNA molecule with a unique identifier (UID) so that all PCR copies of each mRNA fragment can be collapsed into a single consensus sequence. This makes the assay extremely accurate, by resolving PCR bias and sequencing errors as well as allowing quantitative digital molecule counting. Immune sequencing libraries were generated from total RNA extracted from Peripheral Blood Mononuclear Cells in duplicate from a single patient. The use of UIDs enabled absolute quantification of starting RNA molecules present in the original sample and therefore accurate ranking of the antibody clone abundance, by avoiding the bias incorporated by PCR or sequencing when total reads only were measured. Using the same sequencing method, tumor samples were analyzed for abundance of expanded clones via grouping clones by V gene, J gene and CDR3 similarity and ranking by mRNA abundance. Additionally, the use of isotype-specific primers (IgM, IgD, IgG, IgA and IgE) enabled measurement of the heavy chain isotype proportions within the samples. Further, alignment of full-length heavy chain antibody sequences generated using this method to germline genes from reference databases enabled quantitation of the mutation level of each antibody sequence, thereby providing information on the overall maturity and mutational profile of the sample repertoire. Citation Format: Fiona J. Stewart, Mehmet Karaca, Kris Adams, Chris Clouser, Bonny Patel, Sonia Timberlake, William Donahue, Lynne Apone, Salvatore Russello, Eileen T. Dimalanta, Theodore B. Davis, Francois Vigneault. Sequencing the B-cell and T-cell repertoire. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3998.

Background Cytokine producing effector T helper cells are the prime mediators of inflammation during multiple sclerosis (MS) and other autoimmune diseases. The basic immune mechanisms associated with increased pro-inflammatory effector T cell function in MS are not understood. TCF-1 (T cell factor 1, also known, as TCF-7) is a transcription factor that plays an important role in T cell biology in mice. Methods T cells from MS patients and healthy controls were isolated to measure gene expression profiles using nanostring and qPCR. Cytokine protein expression was measured using luminex assay and flow cytometry analysis. Lentivirus vector carrying shRNA was used to knock down the expression of specific genes in CD4+ T cells. Chromatin immunoprecipitation was performed to assess TCF-1 binding to promoter regions. Luciferase assays were performed to test the regulation of IFN-γ and granzyme B by TCF-1. Western blot analysis was used to assess the phosphorylation status of Akt and GSK3β. Results We found that TCF-1 expression is decreased in MS T cells, and FTY720 increased the expression of TCF-1 in treated T cells. We found that FTY720 treated T cell have decreased effector phenotype as measured by decreased T cell proliferation, decreased cytokine and granzyme B production. The decreased effector phenotype of the T cells is dependent upon regulatory transcription factor, TCF-1. TCF-1 blocked T cell effector function by directly binding and negatively regulating the expression of cytokines and granzyme B. Thus, our study for the first time showed that TCF-1 regulates human effector T cell function and its decreased expression could be related to altered T cell function in MS.

MicroRNAs (miRNAs) are single-stranded, small noncoding RNAs that regulate gene expression. Because they are stable in serum, they are being developed as biomarkers for cancer and other diseases. In multiple sclerosis (MS), miRNAs have been studied in cell populations but not in the circulation. In MS, a major challenge is to develop immune biomarkers to monitor disease. We asked whether circulating miRNAs could be identified in MS and whether they are linked to disease stage and/or disability.A total of 368 miRNAs were measured in ethylenediaminetetraacetic acid plasma in 10 relapsing-remitting MS (RRMS) patients, 9 secondary progressive MS (SPMS) patients, and 9 healthy controls (HCs) using miRCURY LNA™ Universal RT microRNA polymerase chain reaction panels. Nineteen miRNAs from this discovery set were validated using qPCR on an independent set of 50 RRMS patients, 51 SPMS patients, and 32 HCs.We found that circulating miRNAs are differentially expressed in RRMS and SPMS versus HCs and in RRMS versus SPMS. We also found miRNAs to be linked to Expanded Disability Status Scale (EDSS). hsa-miR-92a-1* was identified in the largest number of comparisons. It was different in RRMS versus SPMS, and RRMS versus HCs, and showed an association with EDSS and disease duration. miR-92 has target genes involved in cell cycle regulation and cell signaling. The let-7 family of miRNAs differentiated SPMS from HCs and RRMS from SPMS. let-7 miRNAs regulate stem cell differentiation and T cell activation, activate Toll-like receptor 7, and are linked to neurodegeneration. hsa-miR-454 differentiated RRMS from SPMS, and hsa-miR-145 differentiated RRMS from HCs and RRMS from SPMS. Interestingly, the same circulating miRNAs (let-7 and miR-92) that were differentially expressed in RRMS versus SPMS also differentiated amyotrophic lateral sclerosis (ALS) from RRMS subjects, but were not different between SPMS and ALS, suggesting that similar processes may occur in SPMS and ALS.Our results establish circulating miRNAs as a readily accessible blood biomarker to monitor disease in MS.

Objective: To determine whether peripheral immune responses as measured by serum antigen arrays are linked to cerebral MRI measures of disease severity in multiple sclerosis (MS). Methods: In this cross-sectional study, serum samples were obtained from patients with relapsing-remitting MS (n = 21) and assayed using antigen arrays that contained 420 antigens including CNS-related autoantigens, lipids, and heat shock proteins. Normalized compartment-specific global brain volumes were obtained from 3-tesla MRI as surrogates of atrophy, including gray matter fraction (GMF), white matter fraction (WMF), and total brain parenchymal fraction (BPF). Total brain T2 hyperintense lesion volume (T2LV) was quantified from fluid-attenuated inversion recovery images. Results: We found serum antibody patterns uniquely correlated with BPF, GMF, WMF, and T2LV. Furthermore, we identified immune signatures linked to MRI markers of neurodegeneration (BPF, GMF, WMF) that differentiated those linked to T2LV. Each MRI measure was correlated with a specific set of antibodies. Strikingly, immunoglobulin G (IgG) antibodies to lipids were linked to brain MRI measures. Based on the association between IgG antibody reactivity and each unique MRI measure, we developed a lipid index. This comprised the reactivity directed against all of the lipids associated with each specific MRI measure. We validated these findings in an additional independent set of patients with MS (n = 14) and detected a similar trend for the correlations between BPF, GMF, and T2LV vs their respective lipid indexes. Conclusions: We propose serum antibody repertoires that are associated with MRI measures of cerebral MS involvement. Such antibodies may serve as biomarkers for monitoring disease pathology and progression.

SummaryFoxp3+ T regulatory (Treg) cells regulate immune responses and maintain self-tolerance. Recent work shows that Treg cells are comprised of many subpopulations with specialized regulatory functions. Here we identified Foxp3+ T cells expressing the coinhibitory molecule TIGIT as a distinct Treg cell subset that specifically suppresses proinflammatory T helper 1 (Th1) and Th17 cell, but not Th2 cell responses. Transcriptional profiling characterized TIGIT+ Treg cells as an activated Treg cell subset with high expression of Treg signature genes. Ligation of TIGIT on Treg cells induced expression of the effector molecule fibrinogen-like protein 2 (Fgl2), which promoted Treg-cell-mediated suppression of T effector cell proliferation. In addition, Fgl2 was necessary to prevent suppression of Th2 cytokine production in a model of allergic airway inflammation. TIGIT expression therefore identifies a Treg cell subset that demonstrates selectivity for suppression of Th1 and Th17 cell but not Th2 cell responses.