Hematopoietic prostaglandin D synthase defines a proeosinophilic pathogenic effector human T(H)2 cell subpopulation with enhanced function.

BACKGROUND: IL-5(+) pathogenic effector T(H)2 (peT(H)2) cells are a T(H)2 cell subpopulation with enhanced proinflammatory function that has largely been characterized in murine models of allergic inflammation. OBJECTIVE: We sought to identify phenotype markers for human peT(H)2 cells and characterize their function in patients with allergic eosinophilic inflammatory diseases. METHODS: Patients with eosinophilic gastrointestinal disease (EGID), patients with atopic dermatitis (AD), and nonatopic healthy control (NA) subjects were enrolled. peT(H)2 and conventional T(H)2 (cT(H)2) cell phenotype, function, and cytokine production were analyzed by using flow cytometry. Confirmatory gene expression was measured by using quantitative RT-PCR. Prostaglandin D2 levels were measured with ELISA. Gut T(H)2 cells were obtained by means of esophagogastroduodenoscopy. RESULTS: peT(H)2 cells were identified as chemoattractant receptor-homologous molecule expressed on T(H)2 cells-positive (CRTH2(+)), hematopoietic prostaglandin D synthase-positive CD161(hi) CD4 T cells. peT(H)2 cells expressed significantly greater IL-5 and IL-13 than did hematopoietic prostaglandin D synthase-negative and CD161(-) cT(H)2 cells. peT(H)2 cells were highly correlated with blood eosinophilia (r = 0.78-0.98) and were present in 30- to 40-fold greater numbers in subjects with EGID and those with AD versus NA subjects. Relative to cT(H)2 cells, peT(H)2 cells preferentially expressed receptors for thymic stromal lymphopoietin, IL-25, and IL-33 and demonstrated greater responsiveness to these innate pro-TH2 cytokines. peT(H)2 but not cT(H)2 cells produced prostaglandin D2. In patients with EGID and those with AD, peT(H)2 cells expressed gut- and skin-homing receptors, respectively. There were significantly greater numbers of peT(H)2 cells in gut tissue from patients with EGID versus NA subjects. CONCLUSION: peT(H)2 cells are the primary functional proinflammatory human T(H)2 cell subpopulation underlying allergic eosinophilic inflammation. The unambiguous phenotypic identification of human peT(H)2 cells provides a powerful tool to track these cells in future pathogenesis studies and clinical trials.

Functional and physical interaction between the mismatch repair and FA-BRCA pathways.

Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure and an increased risk for leukemia and cancer. Fifteen proteins thought to function in the repair of DNA interstrand crosslinks (ICLs) comprise what is known as the FA-BRCA pathway. Activation of this pathway leads to the monoubiquitylation and chromatin localization of FANCD2 and FANCI. It has previously been shown that FANCJ interacts with the mismatch repair (MMR) complex MutLα. Here we show that FANCD2 interacts with the MMR proteins MSH2 and MLH1. FANCD2 monoubiquitylation, foci formation and chromatin loading are greatly diminished in MSH2-deficient cells. Human or mouse cells lacking MSH2 or MLH1 display increased sensitivity and radial formation in response to treatment with DNA crosslinking agents. Studies in human cell lines and Drosophila mutants suggest an epistatic relationship between FANCD2, MSH2 and MLH1 with regard to ICL repair. Surprisingly, the interaction between MSH2 and MLH1 is compromised in multiple FA cell lines, and FA cell lines exhibit deficient MMR. These results suggest a significant role for MMR proteins in the activation of the FA pathway and repair of ICLs. In addition, we provide the first evidence for a defect in MMR in FA cell lines.

Pathogenic Effector Th2 Cells in Allergic Eosinophilic Inflammatory Disease.

There is an absolute requirement for Th2 cells in the pathogenesis of allergen-driven eosinophil-rich type 2 inflammation. Although Th2 cells are generally regarded as a homogeneous population, in the past decade there has been increasing evidence for a minority subpopulation of IL-5+ Th2 cells that have enhanced effector function. This IL-5+ Th2 subpopulation has been termed pathogenic effector Th2 (peTh2), as it exhibits greater effector function and disease association than conventional Th2 cells. peTh2 cells have a different expression profile, differentially express transcription factors, and preferentially use specific signaling pathways. As such, peTh2 cells are a potential target in the treatment of allergic eosinophilic inflammation. This review examines peTh2 cells, both in mouse models and human disease, with an emphasis on their role in the pathogenesis of allergic eosinophilic inflammation.

Detection of Intracellular Cytokines by Flow Cytometry.

Intracellular cytokine staining (ICCS), employing fluorescently labeled MAbs detected by flow cytometry, has emerged as the premier technique for studying cytokine expression at the single-cell level. Advances in polychromatic flow cytometry have dramatically enhanced the sophistication of ICCS investigations. ICCS can simultaneously measure multiple cytokines within a single cell, allowing the detection of complex cytokine phenotypes. Additionally, cytokines can be measured with a variety of other analytes, including transcription factors, proliferation dilution dyes, activation markers, and viability dyes. This capability, combined with the high throughput inherent in the instrumentation, gives ICCS an enormous advantage over other single-cell techniques such as ELISPOT, limiting dilution, and T cell cloning. The unit describes intracellular staining of cells that have already been stimulated in vitro and fixed. Methods for in vitro activation by PMA and ionomycin or antigens, fixation of cell suspensions, and cell surface staining are also described.