Medlindoherty0488

Innate lymphoid cells (ILCs) are important for both tissue immunity and tissue homeostasis. They are classified into three groups Group 1 ILCs include NK cells, which are important in eliciting immunity against intracellular pathogens; group 2 ILCs protect against parasitic helminths; and group 3 ILCs protect against extracellular pathogens. The role of ILCs in cancer immunity remains unclear. In this chapter, we discuss methods for isolating and characterizing tumor-infiltrating ILC subsets within the tumor microenvironment in an experimental murine model of B16 melanoma. The chapter also highlights the expression of PD-1 on the various ILC subsets within the tumor microenvironment.Intestinal intraepithelial lymphocytes (IEL) comprise distinct groups of innate-like and memory T cells that collectively form one of the largest T cell compartments in the body. IEL are located within the intestinal epithelium and are the first immune cells in the gut to interact with the food, microbiota, and pathogens that the gut is continually exposed to. IEL can respond rapidly to external insults to protect the small intestinal epithelium but are also considered regulatory cells that are important to maintain the homeostasis of the gut. However, the mechanisms of IEL activation and their interactions within the epithelium remain largely elusive. selleckchem Indeed, IEL are not commonly evaluated even in studies of gut immunology, potentially because they are perceived as being difficult to isolate and study. In this protocol, we present a simplified method to isolate IEL from the murine small intestine and provide representative data for flow cytometric analyses of the different IEL subsets. We also outline two procedures for culturing IEL, which can permit functional studies and coculture with epithelial cells. These strategies should make studies of this large but enigmatic T cell compartment more accessible and open up understanding of homeostatic mechanisms in the intestine, and tissue-associated immunity.Innate lymphoid cells (ILCs) are important regulators of the early responses to infection at mucosal barriers, including the intestine. Recently, we have shown that specific ILC3 subsets protect against enteric bacterial pathogens. Here, we describe a mouse model of oral infection by Yersinia enterocolitica (Y. enterocolitica) and several different methodologies to assess the severity of the infection. We also detail how ILC3 subsets can be isolated from the mouse small intestine and transferred into recipient immune deficient mice to study the function of these ILCs in the small intestine.Innate lymphoid cells (ILCs) are lymphocytes with critical roles in homeostasis, inflammation, and immunity to pathogens. ILCs are rare relative to other immune cell populations and are primarily defined by lack of expression of markers associated with other immune cell lineages and are predominantly found in mucosal tissues like the gut, lung and skin. They are classified into distinct subsets, ILC1, ILC2, and ILC3, which mirror subsets of CD4+ helper T cells. ILC subsets have distinct cytokine and transcription factor profiles which align with their biological functions, although recently it has emerged that ILC subsets are not phenotypically fixed and exhibit considerable heterogeneity and plasticity in different contexts. Here, we describe protocols for the maintenance, expansion, and induction of plasticity in mouse and human ILC2s. The resulting cells can be used for molecular interrogation of ILC function and biology, both in vivo and in vitro.ILC2s are a rare innate cell population capable of rapidly producing type 2 cytokines prior to the recruitment and expansion of adaptive type 2 T helper cells. As a result, they are implicated in the pathogenesis of many type-2 immune-mediated diseases, including allergic airway inflammation. Here we describe methods for interrogating and analyzing ILC2 biology in the context of allergic airway inflammation, such as flow cytometric analysis of mouse and human ILC2s as well as live imaging of pulmonary ILC2s.Innate lymphoid cells (ILCs) are a recently identified family of immune cells mostly present at barrier surfaces. They play an important role in the induction, regulation, and resolution of inflammatory responses. Environmental signals play an important role in development and function of ILCs. G-protein coupled receptors (GPCRs) sense and mediate cellular responses to the environmental signals. ILCs express several G-protein coupled receptors, which play a critical role in migration of these cells to appropriate sites. Here, we describe a method to test the migration of ILCs toward 7α,25-hydroxycholesterol, which is mediated by cell surface-expressed GPR183. A similar strategy can be employed to test the role of other GPCRs in mediating the migration of ILCs toward other chemotactic ligands.Neuroendocrine hormones are recognized as important mediators of inflammation that participate in the regulation of the magnitude and length of the immune response. It was demonstrated that endogenous glucocorticoids control the function of innate lymphoid cells (ILCs), and this regulatory mechanism is both cell type- and tissue-specific and is required for host protection during infections. We describe here how to analyze in vitro the effects of corticosterone on murine ILCs, using flow cytometry. The protocols described allow for the identification of the specific combination of stimuli with which glucocorticoids cooperate to regulate the function of ILCs. These methods are instrumental to understanding the molecular mechanisms downstream of glucocorticoid receptor activation and can explain the tissue specificity of ILC response to glucocorticoids.Group 2 innate lymphoid cells are important innate effectors and regulators of adaptive immunity in response to parasitic infections and allergic inflammation. Their low frequency in vivo during steady state condition may complicate research on the cells. During type 2 biased immune responses they are activated, increase in frequency and release cytokines as well as regulate T cell functions through direct interactions including MHC class II-T cell receptor interactions. Importantly, coreceptors significantly influence the ILC2-T cell cross talk and shape the adaptive immune response.Here, we provide an experimental framework to study the function of coreceptors expressed on tissue ILC2. In brief, we describe flow cytometric analysis of the coreceptor of interest, the isolation and culture of mouse pulmonary ILC2 and splenic T cells, as well as approaches to manipulate their coculture. Finally, downstream readout options are outlined.