Clinical relevance of RORγ positive and negative subsets of CD161+CD4+ T cells in primary Sjögren's syndrome.

OBJECTIVE: The relevance of the Th17 pathway in primary SS (pSS) is unclear. Published studies have relied on restimulating circulating CD161+ T cells in vitro for quantitation of IL-17-producing cells. While CD161 marks all IL-17+ T cells, it is also expressed by other Th subsets. The aim of this study was to directly analyse retinoic acid receptor-related orphan nuclear receptor (ROR)-γ expressing and non-expressing subsets of CD161+ T cells to determine the relevance of the Th17 pathway in pSS. METHODS: We quantitated the frequencies of both CD161- and RORγ-expressing T cells by comparative flow cytometry in peripheral blood mononuclear cells from a well-stratified cohort of pSS patients and control subjects. We also analysed the expression of antigen D-related HLA (HLA-DR) and CD161 in labial salivary glands from nine subjects undergoing a diagnostic biopsy. RESULTS: While the frequencies of both RORγ+ and RORγ- subsets of CD161+ CD4+ T cells were increased in peripheral blood from pSS patients, the increase in the RORγ+ subset positively correlated with humoral manifestations of the disease (anti-SSA/SSB autoantibodies and hypergammaglobulinaemia), but not with disease activity, and vice versa for the RORγ- subset. An increased frequency of HLA-DR+ CD161+CD4+ T cells was observed in labial salivary gland biopsies from pSS patients, suggesting chronic activation of CD161+CD4+ T cells in the target tissue of the disease. CONCLUSION: In addition to pointing to CD161 as a marker of a pathogenic subset of CD4+ T cells in pSS patients, our data indicate that even though the RORγ+ (Th17) CD161+ subset might contribute to humoral manifestations of the disease, the RORγ- (non-Th17) CD161+ subset is the one associated with disease activity in pSS patients.

Discovery of biaryls as RORγ inverse agonists by using structure-based design.

RORγ plays a critical role in controlling a pro-inflammatory gene expression program in several lymphocyte lineages including T cells, γδ T cells, and innate lymphoid cells. RORγ-mediated inflammation has been linked to susceptibility to Crohn's disease, arthritis, and psoriasis. Thus inverse agonists of RORγ have the potential of modulating inflammation. Our goal was to optimize two RORγ inverse agonists: T0901317 from literature and 1 that we obtained from internal screening. We used information from internal X-ray structures to design two libraries that led to a new biaryl series.

Small molecule mediated inhibition of RORγ-dependent gene expression and autoimmune disease pathology in vivo.

Retinoic acid receptor-related orphan nuclear receptor γ (RORγ) orchestrates a pro-inflammatory gene expression programme in multiple lymphocyte lineages including T helper type 17 (Th17) cells, γδ T cells, innate lymphoid cells and lymphoid tissue inducer cells. There is compelling evidence that RORγ-expressing cells are relevant targets for therapeutic intervention in the treatment of autoimmune and inflammatory diseases. Unlike Th17 cells, where RORγ expression is induced under specific pro-inflammatory conditions, γδ T cells and other innate-like immune cells express RORγ in the steady state. Small molecule mediated disruption of RORγ function in cells with pre-existing RORγ transcriptional complexes represents a significant and challenging pharmacological hurdle. We present data demonstrating that a novel, selective and potent small molecule RORγ inhibitor can block the RORγ-dependent gene expression programme in both Th17 cells and RORγ-expressing γδ T cells as well as a disease-relevant subset of human RORγ-expressing memory T cells. Importantly, systemic administration of this inhibitor in vivo limits pathology in an innate lymphocyte-driven mouse model of psoriasis.

Discovery of novel pyrazole-containing benzamides as potent RORγ inverse agonists.

The nuclear receptor RORγ plays a central role in controlling a pro-inflammatory gene expression program in several lymphocyte lineages including TH17 cells. RORγ-dependent inflammation has been implicated in the pathogenesis of several major autoimmune diseases and thus RORγ is an attractive target for therapeutic intervention in these diseases. Starting from a lead biaryl compound 4a, replacement of the head phenyl moiety with a substituted aminopyrazole group resulted in a series with improved physical properties. Further SAR exploration led to analogues (e.g., 4j and 5m) as potent RORγ inverse agonists.

Discovery of biaryl carboxylamides as potent RORγ inverse agonists.

RORγt is a pivotal regulator of a pro-inflammatory gene expression program implicated in the pathology of several major human immune-mediated diseases. Evidence from mouse models demonstrates that genetic or pharmacological inhibition of RORγ activity can block the production of pathogenic cytokines, including IL-17, and convey therapeutic benefit. We have identified and developed a biaryl-carboxylamide series of RORγ inverse agonists via a structure based design approach. Co-crystal structures of compounds 16 and 48 supported the design approach and confirmed the key interactions with RORγ protein; the hydrogen bonding with His479 was key to the significant improvement in inverse agonist effect. The results have shown this is a class of potent and selective RORγ inverse agonists, with demonstrated oral bioavailability in rodents.

An alternative process to treat boiler feed water for reuse.

A bench-scale process to treat boiler feed water for reuse in steam generation was developed. Industrial water samples from a steam-assisted gravity drainage plant in northern Alberta, Canada, were obtained and samples characterized. The technology, which consists of coagulation-settling to remove oil/grease and particulates followed by an advanced oxidative treatment, led to clean water samples with negligible organic carbon. Coagulation followed by settling removed most particulates and some insoluble organics. The advanced oxidative treatment removed any remaining color in the samples, decreased the organic content to near-zero, and provided water ready for reuse.

Lentiviral gene transfer method to study integrin function in T lymphocytes.

Integrins play critical roles in adhesion and migration of T cells during an immune response and inflammation. It is of great importance to understand the molecular pathways that regulate integrin function in T cells. Lentiviral vector-based gene transfer method has emerged in the past decade as an efficient means of transferring genes into both resting and activated hard-to-transfect cells, including T cells to knockdown gene expression. Therefore, this technology could be utilized effectively to study different aspects of integrin function or even to perform genome-wide RNAi screens to look globally for regulators of integrin function in T cells. In this chapter, we provide the simplest protocol to infect activated CD4(+) human T cells with high efficiency.

c-Rel-dependent priming of naive T cells by inflammatory cytokines.

The intrinsic refractoriness of naive T cells for cytokine production is counteracted by cells of the innate immune system. Upon sensing danger via Toll-like receptors, these cells upregulate T cell costimulatory molecules and secrete cytokines that enhance T cell activation. We show that cytokine-mediated priming of naive T cells requires the NF-kappaB family member c-Rel. In resting naive cells c-Rel is associated primarily with IkappaBbeta, an inhibitory molecule that is not effectively degraded by TCR signals. Exposure of T cells to proinflammatory cytokines, TNF-alpha and IL-1beta, shifts c-Rel to IkappaBalpha-associated complexes that are readily targeted by the TCR. As a consequence, IL-2 and IFN-gamma mRNA are produced more quickly, and at higher levels, in cytokine-primed T cells. This mechanism does not operate in effector T cells where cytokine gene expression is c-Rel-independent. We propose that c-Rel plays a crucial role as a target of innate signals in T cells.

Deletion of beta-catenin impairs T cell development.

T cells encounter two main checkpoints during development in the thymus. These checkpoints are critically dependent on signals derived from the thymic microenvironment as well as from the pre-T cell receptor (pre-TCR) and the alphabeta TCR. Here we show that T cell-specific deletion of beta-catenin impaired T cell development at the beta-selection checkpoint, leading to a substantial decrease in splenic T cells. In addition, beta-catenin also seemed to be a target of TCR-CD3 signals in thymocytes and mature T cells. These data indicate that beta-catenin-mediated signals are required for normal T cell development.