Vivian H. Gersuk

Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25– T cells

CD4+CD25+ regulatory T (TR) cells have been described in both humans and mice. In mice, TR are thymically derived, and lack of TR leads to organ-specific autoimmunity. Recently, the forkhead/winged helix transcription factor, FoxP3, has been shown to be important for the function of TR cells in mice. In this study, human TR cells were examined and, in results similar to those of studies done in mice, expression of FoxP3 was found exclusively in CD4+CD25+ T cells and correlated with the suppressive activity of these cells. In contrast to the mouse studies, activation of human CD4+CD25- T cells led to expression of FoxP3. Expression of FoxP3 in activated human CD4+CD25+ cells also correlated with suppression of proliferation by these cells in freshly isolated CD4+CD25- T cells from the same donor. This suppression was cell-contact dependent and cytokine independent. Thus, in humans, during activation of CD4+CD25- T cells in an immune response, two populations of cells may arise, effector CD4+CD25+ and regulatory CD4+CD25+ T cells, with expression of FoxP3 correlated with regulatory activity. These data also raise the possibility that a failure to generate peripheral TR cells properly may contribute to autoimmune disease and suggest a possible therapeutic role for FoxP3 in the treatment of such diseases.

MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data

Single-cell transcriptomics reveals gene expression heterogeneity but suffers from stochastic dropout and characteristic bimodal expression distributions in which expression is either strongly non-zero or non-detectable. We propose a two-part, generalized linear model for such bimodal data that parameterizes both of these features. We argue that the cellular detection rate, the fraction of genes expressed in a cell, should be adjusted for as a source of nuisance variation. Our model provides gene set enrichment analysis tailored to single-cell data. It provides insights into how networks of co-expressed genes evolve across an experimental treatment. MAST is available at https://github.com/RGLab/MAST.

Modular transcriptional repertoire analyses of adults with systemic lupus erythematosus reveal distinct type I and type II interferon signatures.

The role of interferon‐α (IFNα) in the pathogenesis of systemic lupus erythematosus (SLE) is strongly supported by gene expression studies. The aim of this study was to improve characterization of the blood IFN signature in adult SLE patients.

A prospective approach to investigating the natural history of preclinical rheumatoid arthritis (RA) using first-degree relatives of probands with RA

OBJECTIVE To describe a large, multicenter prospective cohort study of first-degree relatives (FDRs) of probands with rheumatoid arthritis (RA), and outline the use of such a study in investigating the natural history of RA development. METHODS A total of 1,058 FDRs, none of whom met the American College of Rheumatology criteria for RA, were enrolled in a prospective study investigating genetic and environmental influences on the development of RA-related autoimmunity. Demographic, epidemiologic, genetic, autoantibody, and physical examination data from the initial study enrollment visit were described for these FDRs, and the relationship was examined between genetic factors, autoantibodies, inflammation, and joint disease. RESULTS Fifty-five percent of the FDRs had > or =1 copy of the shared epitope, 20% had > or =1 copy of the PTPN22 polymorphism, and approximately 16% were positive for rheumatoid factor (RF; including isotypes) and/or anti-cyclic citrullinated peptide antibody. IgM-RF positivity is associated with > or =1 tender joint on examination (odds ratio [OR] 2.50, 95% confidence interval [95% CI] 1.27-4.89; P < 0.01) and elevated C-reactive protein (CRP) levels (OR 5.31, 95% CI 1.45-19.52; P = 0.01). CONCLUSION FDRs without RA demonstrate high prevalences of genetic risk factors and RA-related autoantibodies. Additionally, an RF association with tender joints and elevated CRP levels suggests that autoantibodies are a valid intermediate marker of RA-related autoimmunity in this cohort. This prospective FDR cohort will be a valuable resource for evaluating the relationship between genetic and epidemiologic factors and the development of RA-related autoimmunity.

Intermolecular Antigen Spreading Occurs During the Preclinical Period of Human Type 1 Diabetes

Intra- and intermolecular spreading of T cell responses to autoantigens has been implicated in the pathogenesis of autoimmune diseases. Therefore, we questioned whether T cell responses from subjects identified as at-risk (positive for autoantibody reactivity to islet proteins) for the development of type 1 diabetes, a cell-mediated autoimmune disease, would demonstrate intermolecular Ag spreading of T cell responses to islet cell proteins. Previously, we have demonstrated that by the time subjects develop type 1 diabetes, they have T cell responses to numerous islet proteins, whereas T cells from normal controls respond to a limited number of islet proteins. Initial testing of PBMC responses from 25 nondiabetic at-risk subjects demonstrated that 16 of the 25 subjects have PBMC responses to islet proteins similar to controls. Fourteen of these 16 subjects were available for follow-up. Eleven of the 14 developed T cell responses to increasing numbers of islet proteins, and 6 of these subjects developed type 1 diabetes. In the nine subjects who already demonstrated T cell Ag spreading at the initial visit, four were available for follow-up. Of these four, two had increases in T cell reactivity to islet proteins, while two maintained their initial levels of T cell reactivity. We also observed Ag spreading in autoantibody reactivity to islet proteins in nine of the 18 at-risk subjects available for follow-up. Our data strongly support the conclusion that intermolecular spreading of T cell and Ab responses to islet proteins occurs during the preclinical period of type 1 diabetes.

Prevalence of narcolepsy in King County, Washington, USA

BACKGROUND Relatively few epidemiologic studies have focused on narcolepsy, a disabling sleep disorder with a strong association with HLA-DQB1 *0602. METHODS We sought to estimate the prevalence of narcolepsy using multiple overlapping techniques to identify residents of King County, WA who were 18 years or older with physician-diagnosed narcolepsy. Patients were entered into a registry and recruited into an epidemiologic study entailing interview and buccal scrapings to determine HLA-DQB1 *0602 status. Missing values were imputed to allow prevalence to be estimated based on all 425 patients entered into the registry between 2001 and 2005, whether they were recruited into the epidemiologic study (n=279) or not (n=146). RESULTS As of July 01, 2001, estimated prevalence per 100,000 of physician-diagnosed narcolepsy with cataplexy was 21.8 (95% confidence interval (CI): 18.8-24.8), similar to prior studies. The median age of onset was 14 (interquartile range: 10-18). For narcolepsy with HLA-DQB1 *0602, prevalence was 15.3 (95% CI: 12.8-17.9). Estimated prevalence was higher in women than men and in African-Americans than other racial groups. CONCLUSIONS These differences could reflect problems in identification and recruitment or may provide etiologic clues about narcolepsy. This study illustrates the challenges in performing population-based studies of narcolepsy.

A phenotypically and functionally distinct human TH2 cell subpopulation is associated with allergic disorders

A unique T helper cell signature in allergic patients isolates the pathogenic cells and provides a target for disease intervention. Defining damaging cells Although T helper type 2 (TH2) cells provide necessary protection from certain types of pathogens, they are also implicated in allergy pathogenesis. Until now, methods to distinguish pathogenic cells that are reactive to allergens from the rest of the TH2 population were very limited. Wambre et al. characterized a population of memory TH2 cells, termed TH2A, that were only found in allergic individuals. They were able to do so without the use of antigen-specific tetramers. These cells decreased in patients that benefited from allergen immunotherapy, indicating that targeting TH2A cells could disrupt allergic responses. Allergen-specific type 2 helper T (TH2) cells play a central role in initiating and orchestrating the allergic and asthmatic inflammatory response pathways. One major factor limiting the use of such atopic disease–causing T cells as both therapeutic targets and clinically useful biomarkers is the lack of an accepted methodology to identify and differentiate these cells from overall nonpathogenic TH2 cell types. We have described a subset of human memory TH2 cells confined to atopic individuals that includes all allergen-specific TH2 cells. These cells are terminally differentiated CD4+ T cells (CD27− and CD45RB−) characterized by coexpression of CRTH2, CD49d, and CD161 and exhibit numerous functional attributes distinct from conventional TH2 cells. Hence, we have denoted these cells with this stable allergic disease–related phenotype as the TH2A cell subset. Transcriptome analysis further revealed a distinct pathway in the initiation of pathogenic responses to allergen, and elimination of these cells is indicative of clinical responses induced by immunotherapy. Together, these findings identify a human TH2 cell signature in allergic diseases that could be used for response-monitoring and designing appropriate immunomodulatory strategies.

Vitamin D deficiency is associated with increased risk of non-alcoholic steatohepatitis in adults with non-alcoholic fatty liver disease: Possible role for MAPK and NF-κB?

Objectives:The objective of this study was to determine the relationship of serum vitamin D deficiency (VDD) to histologic features of non-alcoholic fatty liver disease (NAFLD), and associated demographic, clinical, laboratory, and transcriptomic data in the well-characterized Non-alcoholic Steatohepatitis Clinical Research Network (NASH CRN) cohort.Methods:Serum vitamin D 25(OH)D (VD) was quantified by liquid chromatography-tandem mass spectrometry in 190 adults (>18 years) with biopsy-proven NAFLD. Subjects were categorized according to their level of VD as either sufficient (>30 ng/ml), insufficient (≥20≤30 ng/ml), or deficient (VDD; <20 ng/ml). Multivariable logistic regression was used to investigate the association of VDD and the presence of definite NASH and individual histological features of NAFLD after adjusting for age, sex, race, body mass index, alanine aminotransferase, and diabetes status. Hepatic transcriptomic data was compared between VDD and non-VDD subjects.Results:VDD was present in 55% of subjects and was independently associated with definitive NASH (odds ratio (OR) 3.15, 95% confidence interval (CI), 1.62–6.15, P=0.001), increased lobular inflammation (OR=1.98, 95% CI, 1.08–3.61, P=0.026), more ballooning (OR=2.38, 95% CI, 1.32–4.30, P=0.004), and the presence of fibrosis (OR=2.32, 95% CI, 1.13–4.77, P=0.022). There was a significant inverse relationship between lower levels of serum resistin and increased VD level category (P=0.013). The KRT10, SEMA3B, SNORD3C, ARSD, and IGKV4-1 genes were differentially expressed (false discovery rate <0.05) between VDD and non-VDD subjects. Gene ontology and pathway analysis suggest activation of the mitogen-activated protein kinase and nuclear factor-κB pathways in VDD NAFLD subjects.Conclusions:VDD is prevalent among US adult NAFLD patients and is independently associated with a definitive diagnosis of NASH and increased histological severity. Novel associations in proinflammatory pathways were identified, which suggest the mechanism for VDD in the pathogenesis of NASH and support dietary and/or lifestyle modifications to increase vitamin D levels in these patients.

The Toll-Like Receptor Signaling Molecule Myd88 Contributes to Pancreatic Beta-Cell Homeostasis in Response to Injury

Commensal flora and pathogenic microbes influence the incidence of diabetes in animal models yet little is known about the mechanistic basis of these interactions. We hypothesized that Myd88, an adaptor molecule in the Toll-like-receptor (TLR) pathway, regulates pancreatic β-cell function and homeostasis. We first examined β-cells histologically and found that Myd88−/− mice have smaller islets in comparison to C57Bl/6 controls. Myd88−/− mice were nonetheless normoglycemic both at rest and after an intra-peritoneal glucose tolerance test (IPGTT). In contrast, after low-dose streptozotocin (STZ) challenge, Myd88−/−mice had an abnormal IPGTT relative to WT controls. Furthermore, Myd88−/− mice suffer enhanced β-cell apoptosis and have enhanced hepatic damage with delayed recovery upon low-dose STZ treatment. Finally, we treated WT mice with broad-spectrum oral antibiotics to deplete their commensal flora. In WT mice, low dose oral lipopolysaccharide, but not lipotichoic acid or antibiotics alone, strongly promoted enhanced glycemic control. These data suggest that Myd88 signaling and certain TLR ligands mediate a homeostatic effect on β-cells primarily in the setting of injury.

Allelic Exclusion and Peripheral Reconstitution by TCR Transgenic T Cells Arising From Transduced Human Hematopoietic Stem/Progenitor Cells

Transduction and transplantation of human hematopoietic stem/progenitor cells (HSPC) with the genes for a T-cell receptor (TCR) that recognizes a tumor-associated antigen may lead to sustained long-term production of T cells expressing the TCR and confer specific antitumor activity. We evaluated this using a lentiviral vector (CCLc-MND-F5) carrying cDNA for a human TCR specific for an HLA-A*0201-restricted peptide of Melanoma Antigen Recognized by T cells (MART-1). CD34(+) HSPC were transduced with the F5 TCR lentiviral vector or mock transduced and transplanted into neonatal NSG mice or NSG mice transgenic for human HLA-A*0201 (NSG-A2). Human CD8(+) and CD4(+) T cells expressing the human F5 TCR were present in the thymus, spleen, and peripheral blood after 4-5 months. Expression of human HLA-A*0201 in NSG-A2 recipient mice led to significantly increased numbers of human CD8(+) and CD4(+) T cells expressing the F5 TCR, compared with control NSG recipients. Transduction of the human CD34(+) HSPC by the F5 TCR transgene caused a high degree of allelic exclusion, potently suppressing rearrangement of endogenous human TCR-β genes during thymopoiesis. In summary, we demonstrated the feasibility of engineering human HSPC to express a tumor-specific TCR to serve as a long-term source of tumor-targeted mature T cells for immunotherapy of melanoma.