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Tissue-resident memory T (TRM) cells are non-recirculating cells that exist throughout the body. Although TRMcells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze TRMcell heterogeneity between organs and find that the different environments in which these cells differentiate dictate TRMcell function, durability and malleability. We find that unequal responsiveness to TGFβ is a major driver of this diversity. Notably, dampened TGFβ signaling results in CD103−TRMcells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFβ-responsive CD103+TRMcounterparts. Furthermore, whereas CD103−TRMcells readily modified their phenotype upon relocation, CD103+TRMcells were comparatively resistant to transdifferentiation. Thus, despite common requirements for TRMcell development, tissue adaptation of these cells confers discrete functional properties such that TRMcells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment.

Respiratory syncytial virus (RSV) is the most common viral pathogen associated with acute lower respiratory tract infection (LRTI) in children under 5 years of age. Severe RSV disease is associated with the development of chronic respiratory complications such as recurrent wheezing and asthma. A common risk factor for developing severe RSV disease is premature gestation and this is largely due to an immature innate immune system. This increases susceptibility to RSV since the innate immune system is less able to protect against pathogens at a time when adaptive immunity has not fully developed. This review focuses on comparing different aspects of innate immunity between preterm and term infants to better understand why preterm infants are more susceptible to severe RSV disease. Identifying early life innate immune biomarkers associated with the development of severe RSV disease, and understanding how these compare between preterm and term infants, remains a critically important question that would aid the development of interventions to reduce the burden of disease in this vulnerable population.

T cell lineages are defined by specialized functions and differential expression of surface antigens, cytokines and transcription factors. Conventional CD4+and CD8+T cells are the best studied of the T cell subsets, but ‘unconventional’ T cells have emerged as being more abundant and influential than has previously been appreciated. Key subsets of unconventional T cells include natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells and ?d T cells; collectively, these make up ~10% of circulating T cells, and often they are the majority of T cells in tissues such as the liver and gut mucosa. Defects and deficiencies in unconventional T cells are associated with autoimmunity, chronic inflammation and cancer, so it is important to understand how their development is regulated. In this Review, we describe the thymic development of NKT cells, MAIT cells and ?d T cells and highlight some of the key differences between conventional and unconventional T cell development.

Mucosal-associated invariant T (MAIT) cells, natural killer T (NKT) cells, and γδT cells are collectively referred to as ‘unconventional T cells due to their recognition of non-peptide antigens and restriction to non-classical MHC-I-like molecules. Though the frequencies of MAIT, NKT, and γδT cells and the distribution of their functional subsets vary widely between organs and individuals, the factors controlling these differences are poorly understood. Here, we show that MAIT cells are increased in mice deficient in either NKT or γδT cells and even more so in mice deficient in both NKT and γδT cells. In addition, characterization of the TCRα repertoire within γδT cell-deficient thymocytes revealed an altered Travgene segment usage relative to that of wild-type thymocytes, highlighting a retention of the Tcra-Tcrdlocus from 129 strain embryonic stem cells used to generate Tcrd−/−mice. Accordingly, this resulted in a moderate increase in distal Travsegment usage such as Trav1, potentially contributing to increased generation of Trav1-Traj33+MAIT cells in the Tcrd−/−thymus. However, peripheral MAIT cells also underwent increased homeostatic proliferation in the absence of NKT and γδT cells and adoptively transferred MAIT cell subsets exhibited tissue-specific homing patterns. Collectively, our data reveal a shared niche for MAIT, NKT, and γδT cells, suggesting a competition for common factors that could be exploited for treating disease. Importantly, these data highlight that careful assessment of studies using NKT or γδT cell-deficient mouse models is required when investigating the role of unconventional T cells in disease.

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.

Mucosal-associated invariant T (MAIT) cells detect microbial infection via recognition of riboflavin-based antigens presented by the major histocompatibility complex class I (MHC-I)–related protein 1 (MR1). Most MAIT cells in human peripheral blood express CD8αα or CD8αβ coreceptors, and the binding site for CD8 on MHC-I molecules is relatively conserved in MR1. Yet, there is no direct evidence of CD8 interacting with MR1 or the functional consequences thereof. Similarly, the role of CD8αα in lymphocyte function remains ill-defined. Here, using newly developed MR1 tetramers, mutated at the CD8 binding site, and by determining the crystal structure of MR1–CD8αα, we show that CD8 engaged MR1, analogous to how it engages MHC-I molecules. CD8αα and CD8αβ enhanced MR1 binding and cytokine production by MAIT cells. Moreover, the CD8–MR1 interaction was critical for the recognition of folate-derived antigens by other MR1-reactive T cells. Together, our findings suggest that both CD8αα and CD8αβ act as functional coreceptors for MAIT and other MR1-reactive T cells.

Studies on the biology of mucosal-associated invariant T cells (MAIT cells) in mice have been hampered by a lack of specific reagents. Using MR1-antigen (Ag) tetramers that specifically bind to the MR1-restricted MAIT T cell receptors (TCRs), we demonstrate that MAIT cells are detectable in a broad range of tissues in C57BL/6 and BALB/c mice. These cells include CD4−CD8−, CD4−CD8+, and CD4+CD8− subsets, and their frequency varies in a tissue- and strain-specific manner. Mouse MAIT cells have a CD44hiCD62Llo memory phenotype and produce high levels of IL-17A, whereas other cytokines, including IFN-γ, IL-4, IL-10, IL-13, and GM-CSF, are produced at low to moderate levels. Consistent with high IL-17A production, most MAIT cells express high levels of retinoic acid–related orphan receptor γt (RORγt), whereas RORγtlo MAIT cells predominantly express T-bet and produce IFN-γ. Most MAIT cells express the promyelocytic leukemia zinc finger (PLZF) transcription factor, and their development is largely PLZF dependent. These observations contrast with previous reports that MAIT cells from Vα19 TCR transgenic mice are PLZF− and express a naive CD44lo phenotype. Accordingly, MAIT cells from normal mice more closely resemble human MAIT cells than previously appreciated, and this provides the foundation for further investigations of these cells in health and disease.

αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1+ human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity.

Tissue-resident memory T (TRM) cells provide long-lasting immune protection. One of the key events controlling TRM cell development is the local retention of TRM cell precursors coupled to downregulation of molecules necessary for tissue exit. Sphingosine-1-phosphate receptor 5 (S1PR5) is a migratory receptor with an uncharted function in T cells. Here, we show that S1PR5 plays a critical role in T cell infiltration and emigration from peripheral organs, as well as being specifically downregulated in TRM cells. Consequentially, TRM cell development was selectively impaired upon ectopic expression of S1pr5, whereas loss of S1pr5 enhanced skin TRM cell formation by promoting peripheral T cell sequestration. Importantly, we found that T-bet and ZEB2 were required for S1pr5 induction and that local TGF-β signaling was necessary to promote coordinated Tbx21, Zeb2, and S1pr5 downregulation. Moreover, S1PR5-mediated control of tissue residency was conserved across innate and adaptive immune compartments. Together, these results identify the T-bet–ZEB2–S1PR5 axis as a previously unappreciated mechanism modulating the generation of tissue-resident lymphocytes.

The development of CD1d-dependent natural killer T (NKT) cells is poorly understood. We have used both CD1d/α-galactosylceramide (CD1d/αGC) tetramers and anti-NK1.1 to investigate NKT cell development in vitro and in vivo. Confirming the thymus-dependence of these cells, we show that CD1d/αGC tetramer-binding NKT cells, including NK1.1+ and NK1.1− subsets, develop in fetal thymus organ culture (FTOC) and are completely absent in nude mice. Ontogenically, CD1d/αGC tetramer-binding NKT cells first appear in the thymus, at day 5 after birth, as CD4+CD8−NK1.1−cells. NK1.1+ NKT cells, including CD4+ and CD4−CD8− subsets, appeared at days 7–8 but remained a minor subset until at least 3 wk of age. Using intrathymic transfer experiments, CD4+NK1.1− NKT cells gave rise to NK1.1+ NKT cells (including CD4+ and CD4− subsets), but not vice-versa. This maturation step was not required for NKT cells to migrate to other tissues, as NK1.1− NKT cells were detected in liver and spleen as early as day 8 after birth, and the majority of NKT cells among recent thymic emigrants (RTE) were NK1.1−. Further elucidation of this NKT cell developmental pathway should prove to be invaluable for studying the mechanisms that regulate the development of these cells.

The antimetastatic effect of the CD1d-binding glycolipid, α-galactosylceramide (α-GalCer), is mediated by NK1.1+T (NKT) cells; however, the mechanisms behind this process are poorly defined. Although it has been shown to involve NK cells and interferon-γ (IFN-γ) production, the way these factors collaborate to mediate effective tumor rejection and the importance of other factors characteristic of NKT cell and NK cell activation are unknown. Using gene-targeted mice and antibody treatments, the critical need for interleukin 12 (IL-12), IFN-γ, and NK cells has been shown in the antimetastatic activity of α-GalCer in the lungs and the liver. By contrast, in lung and liver metastasis models, cytotoxic molecules expressed by NK cells and NKT cells (perforin, Fas ligand, and tumor necrosis factor-related apoptosis-inducing ligand) and an NKT cell-secreted cytokine, IL-4, were not necessary for the antitumor activity of α-GalCer. Like IL-12, IL-18 was required for optimal serum IFN-γ induction and control of lung metastases by α-GalCer. IL-18 was unnecessary for α-GalCer–related suppression of liver metastases. Most importantly, after adoptive transfer of α-GalCer–reactive NKT cells or NK cells into NKT cell-deficient, IFN-γ–deficient, or RAG-1–deficient mice, it was demonstrated that the sequential production of IFN-γ by NKT cells and NK cells was absolutely required to reconstitute the antimetastatic activity of α-GalCer.

The antimetastatic effect of the CD1d-binding glycolipid, α-galactosylceramide (α-GalCer), is mediated by NK1.1+T (NKT) cells; however, the mechanisms behind this process are poorly defined. Although it has been shown to involve NK cells and interferon-γ (IFN-γ) production, the way these factors collaborate to mediate effective tumor rejection and the importance of other factors characteristic of NKT cell and NK cell activation are unknown. Using gene-targeted mice and antibody treatments, the critical need for interleukin 12 (IL-12), IFN-γ, and NK cells has been shown in the antimetastatic activity of α-GalCer in the lungs and the liver. By contrast, in lung and liver metastasis models, cytotoxic molecules expressed by NK cells and NKT cells (perforin, Fas ligand, and tumor necrosis factor-related apoptosis-inducing ligand) and an NKT cell-secreted cytokine, IL-4, were not necessary for the antitumor activity of α-GalCer. Like IL-12, IL-18 was required for optimal serum IFN-γ induction and control of lung metastases by α-GalCer. IL-18 was unnecessary for α-GalCer–related suppression of liver metastases. Most importantly, after adoptive transfer of α-GalCer–reactive NKT cells or NK cells into NKT cell-deficient, IFN-γ–deficient, or RAG-1–deficient mice, it was demonstrated that the sequential production of IFN-γ by NKT cells and NK cells was absolutely required to reconstitute the antimetastatic activity of α-GalCer.

Vδ2+T cells play a critical role in immunity to micro-organisms and cancer but exhibit substantial heterogeneity in humans. Here, we demonstrate that CD26 and CD94 define transcriptionally, phenotypically, and functionally distinct Vδ2+T cell subsets. Despite distinct antigen specificities, CD26hiCD94loVδ2+cells exhibit substantial similarities to CD26himucosal-associated invariant T (MAIT) cells, although CD26−Vδ2+cells exhibit cytotoxic, effector-like profiles. At birth, the Vδ2+Vγ9+population is dominated by CD26hiCD94locells; during adolescence and adulthood, Vδ2+cells acquire CD94/NKG2A expression and the relative frequency of the CD26hiCD94losubset declines. Critically, exposure of the CD26hiCD94losubset to phosphoantigen in the context of interleukin-23 (IL-23) and CD26 engagement drives the acquisition of a cytotoxic program and concurrent loss of the MAIT cell-like phenotype. The ability to modulate the cytotoxic potential of CD26hiCD94loVδ2+cells, combined with their adenosine-binding capacity, may make them ideal targets for immunotherapeutic expansion and adoptive transfer.

Mucosal‐associated invariant T (MAIT) cells are a subset of unconventional T cells restricted by the major histocompatibility complex (MHC) class I–like molecule MHC‐related protein 1 (MR1). MAIT cells are found throughout the body, especially in human blood and liver. Unlike conventional T cells, which are stimulated by peptide antigens presented by MHC molecules, MAIT cells recognize metabolite antigens derived from an intermediate in the microbial biosynthesis of riboflavin. MAIT cells mediate protective immunity to infections by riboflavin‐producing microbes via the production of cytokines and cytotoxicity. The discovery of stimulating MAIT cell antigens allowed for the development of an analytical tool, the MR1 tetramer, that binds specifically to the MAIT T cell receptor (TCR) and is becoming the gold standard for identification of MAIT cells by flow cytometry. This article describes protocols to characterize the phenotype of human MAIT cells in blood and tissues by flow cytometry using fluorescently labeled human MR1 tetramers alongside antibodies specific for MAIT cell markers. © 2019 by John Wiley & Sons, Inc. The main protocols include: Basic Protocol 1: Determining the frequency and steady‐state surface phenotype of human MAIT cells Basic Protocol 2: Determining the activation phenotype of human MAIT cells in blood Basic Protocol 3: Characterizing MAIT cell TCRs using TCR‐positive reporter cell lines Alternate protocols are provided for determining the absolute number, transcription factor phenotype, and TCR usage of human MAIT cells; and determining activation phenotype by staining for intracellular markers, measuring secreted cytokines, and measuring fluorescent dye dilution due to proliferation. Additional methods are provided for determining the capacity of MAIT cells to produce cytokine independently of antigen using plate‐bound or bead‐immobilized CD3/CD28 stimulation; and determining the MR1‐Ag dependence of MAIT cell activation using MR1‐blocking antibody or competitive inhibition. For TCR‐positive reporter cell lines, methods are also provided for evaluating the MAIT TCR‐mediated MR1‐Ag response, determining the capacity of the reporter lines to produce cytokine independently of antigen, determining the MR1‐Ag dependence of the reporter lines, and evaluating the MR1‐Ag response of the reporter lines using IL‐2 secretion. Support Protocols describe the preparation of PBMCs from human blood, the preparation of single‐cell suspensions from tissue, the isolation of MAIT cells by FACS and MACS, cloning MAIT TCRα and β chain genes and MR1 genes for transduction, generating stably and transiently transfected cells lines, generating a stable MR1 knockout antigen‐presenting cell line, and generating monocyte‐derived dendritic cells.

No relevant conflicts of interest to declare.

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