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Item description: Chapter 7 in book 'MAIT Cells' (2020), ed H Kaipe and I Magalhaes. Methods in Molecular Biology, volume 2098. Mucosal-associated invariant T (MAIT) cells are an abundant innate-like T cell subset in humans, enriched in mucosal tissues and the liver. MAIT cells express a semi-invariant T cell receptor (TCR) and recognize microbial-derived riboflavin metabolites presented on the MHC Class I-like molecule MR1. In addition to activation via the TCR, MAIT cells can also be activated in response to cytokines such as IL-12 and IL-18, in contrast to conventional T cells. Here we describe TCR-dependent and -independent methods for MAIT cell activation. The TCR-dependent approaches include stimulation with microbead- or plate-bound anti-CD3/anti-CD28 antibodies, and with 5-OP-RU or paraformaldehyde (PFA)-fixed E. coli in the presence of antigen-presenting cells (APCs). The latter method includes a combination of TCR- and cytokine-mediated stimulation. The TCR-independent methods include direct stimulation with the recombinant cytokines IL-12 and IL-18, and indirect stimulation with TLR-4/TLR-8 agonists or influenza A virus in the presence of APCs. Finally, we outline a protocol to analyze activated MAIT cells using flow cytometry.

Background: Severe asthma with persistent airway eosinophilia despite high-intensity inhaled corticosteroids is a phenotype associated with high exacerbation risk. A major barrier to progress is poor understanding of the pathogenic mechanisms and cell types contributing to the disease. Aims and objectives: To determine the function of type-2 cytokine producing CD8+ T cells in severe eosinophilic asthma, and investigate their contribution to pathogenesis of the disease. Methods: Profiles of type-2 CD8+ T cells (Tc2) and type-2 T helper (Th2), in different asthma phenotypes were analysed with flow cytometry. Tc2 cells were isolated and cultured in vitro. Effects of prostaglandin D2 (PGD2) and leukotriene E4 (LTE4) on Tc2 cells were defined using chemotaxis assays, Luminex, quantitative RT-PCR, PrimeFlow RNA assay and microarray. Effects of Tc2 supernatants were determined on eosinophil shape change and apoptosis, and on eosinophil chemokine production from airway epithelial cells. Results: In two independent patient cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ Tc2 cells are enriched in blood and airways, and concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways in severe eosinophilic asthma. In vitro PGD2 and LTE4 function synergistically to enhance Tc2 cell recruitment and activation. They regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines which could contribute to eosinophilia. Conclusions: These finding are consistent with an important role for human Tc2 cells in severe eosinophilic asthma.

Human type-2 CD8+ T cells are a cell population with potentially important roles in allergic disease. We investigated this in the context of severe asthma with persistent airway eosinophilia—a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines, which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

The functions and in vivo roles of type-2 CD8+ T cells in humans have not been well defined and this cell type has been largely overlooked in models of disease. We investigated this in the context of severe asthma with persistent airway eosinophilia - a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.