Summary Background Differentiation and activation of CD4+ T cells is controlled by various cytokines produced by innate immune cells. We have shown that eosinophils (EOS) have the potential to influence Th1 and Th2 cytokine generation by CD4+ cells, but their influence on IL‐17A (IL‐17) has not been established. Objective The purpose of this study is to determine the effect of EOS on IL‐17 production by lymphocytes. Methods Pre‐activated CD4+ T cells were cultured in the presence of either autologous EOS or EOS culture supernatants. Expression of IL‐17 was determined by real‐time quantitative PCR (qPCR) after 5 h and protein level was measured after 48 h. To determine the effect of allergen‐induced airway EOS on IL‐17, subjects with mild allergic asthma underwent bronchoscopic segmental bronchoprovocation with allergen (SBP‐Ag) after a treatment with an anti‐IL‐5 neutralizing antibody (mepolizumab) to reduce airway eosinophilia. IL‐17 mRNA was measured in bronchoalveolar lavage (BAL) cells by qPCR. Results In vitro, EOS significantly increased IL‐17 production by CD4+ T cells. Addition of exogenous IL‐1ß increased expression of IL‐17 mRNA by CD4+ T cells. EOS expressed and released IL‐1ß. Furthermore, levels of IL‐1ß in EOS supernatants highly correlated with their ability to increase IL‐17 expression by CD4+ T cells, and neutralizing antibody to IL‐1ß reduced expression of IL‐17 mRNA. In vivo, reduction of EOS in the airway using mepolizumab was associated with diminished IL‐17 expression after SBP‐Ag. Conclusions and clinical relevance Our data demonstrate that EOS can promote IL‐17 production through the release of IL‐1ß. Enhanced IL‐17 cytokine production is another mechanism by which EOS may participate in pathogenesis of allergic airway inflammation in asthma.

Multi-parameter flow cytometry analysis of T regulatory (Treg) cells is a widely used approach in basic and translational research studies. This approach has been complicated by a lack of specific markers for Treg cells and lack of uniformity in quantification of Treg cells. Given the central role of Treg cells in the inception and perpetuation of diverse immune responses as well as its target as a therapeutic, it is imperative to have established methodologies for Treg cell analysis that are robust and usable for studies with multiple subjects as well as multicenter studies. In this study, we describe an optimized multi-parameter flow cytometry protocol for quantification of human Treg cells from freshly obtained and viably frozen samples and correlations with epigenetic Treg cell analysis (TSDR demethylation). We apply these two methodologies to characterize Treg cell differences between cord blood and adult peripheral blood. In summary, the optimized protocol appears to be robust for Treg cell quantification from freshly isolated or viably frozen cells and the multi-parameter flow cytometry findings are strongly positively correlated with TSDR demethylation thus providing several options for characterization of Treg cell frequency and function in large translational or clinical studies.

Summary Staphylococcal enterotoxin B (SEB) activates T cells via non-canonical signalling through the T cell receptor and is+ GRAIL + T cells were highly suppressive and non-proliferative independent of CD25 expression level and via a glucocorticoid-induced tumour necrosis factor R-related proteinindependent mechanism, whereas naive T regulatory cells were nonsuppressive and partially proliferative with SEB activation in vitro. Lastly, adoptive transfer of conventional T cells revealed that induction of FoxP3 + regulatory cells is not operational in this model system. These data provide a novel paradigm for chronic non-canonical T cell receptor engagement leading to highly suppressive FoxP3 + GRAIL + CD4 + T cells.

GRAIL (gene related to anergy in lymphocytes), is an E3 Anergy describes a functional state of T cells (T)2 that is characterized by failure to proliferate or produce interleukin-2 (IL-2) following presentation of cognate antigen in a known stimulatory fashion. The induction of T cell anergy is an active process dependent upon coordinated up-regulation and degradation of multiple proteins (1-4). Following engagement of the T cell receptor, a distinct biochemical signature has been described in anergic CD4ϩ T cells. In contrast to naïve T cells, anergic T cells demonstrate diminished influx of calcium, impaired PLC-␥ activation, diminished ERK and JNK phosphorylation, and impaired translocation of the transcription factor AP-1 to the nucleus (5, 6). Recent work suggests that T/APC interactions are distinct in anergic CD4ϩ T cells (5, 7). However, the precise mechanisms that regulate this interaction remain poorly understood.Multiple anergy-related E3 ubiquitin ligases, including Cbl-b, Itch, and GRAIL, are up-regulated during, and required for, the induction and maintenance of T cell anergy (5, 8 -11). GRAIL mRNA and protein expression are uniquely up-regulated in anergized CD4ϩ T cells and Foxp3ϩCD25ϩ regulatory T cells (8,12,13). GRAIL expression is necessary for the induction of T cell anergy, and ectopic expression of GRAIL in T cells is sufficient for the induction of anergy and suppressor function (11,14). It is clear that expression of GRAIL in T cells significantly alters proliferative capacity; however, the impact of GRAIL expression on T/APC interactions and signaling events associated with engagement of the TCR has not been fully elucidated. A better understanding of the mechanisms governing T cell activation and T/APC interactions continues to be an area of intense investigation as novel targets for immune control are likely to be realized. In this study, we examine the role of GRAIL expression in modulating T cell signaling and T/APC interactions.

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