A divergent transcriptional landscape underpins the development and functional branching of MAIT cells.

MR1-restricted mucosal-associated invariant T (MAIT) cells play a unique role in the immune system. These cells develop intrathymically through a three-stage process, but the events that regulate this are largely unknown. Here, using bulk and single-cell RNA sequencing-based transcriptomic analysis in mice and humans, we studied the changing transcriptional landscape that accompanies transition through each stage. Many transcripts were sharply modulated during MAIT cell development, including SLAM (signaling lymphocytic activation molecule) family members, chemokine receptors, and transcription factors. We also demonstrate that stage 3 "mature" MAIT cells comprise distinct subpopulations including newly arrived transitional stage 3 cells, interferon-γ-producing MAIT1 cells and interleukin-17-producing MAIT17 cells. Moreover, the validity and importance of several transcripts detected in this study are directly demonstrated using specific mutant mice. For example, MAIT cell intrathymic maturation was found to be halted in SLAM-associated protein (SAP)-deficient and CXCR6-deficient mouse models, providing clear evidence for their role in modulating MAIT cell development. These data underpin a model that maps the changing transcriptional landscape and identifies key factors that regulate the process of MAIT cell differentiation, with many parallels between mice and humans.

Mucosal-associated invariant T-cell activation and accumulation after in vivo infection depends on microbial riboflavin synthesis and co-stimulatory signals.

Despite recent breakthroughs in identifying mucosal-associated invariant T (MAIT) cell antigens (Ags), the precise requirements for in vivo MAIT cell responses to infection remain unclear. Using major histocompatibility complex-related protein 1 (MR1) tetramers, the MAIT cell response was investigated in a model of bacterial lung infection employing riboflavin gene-competent and -deficient bacteria. MAIT cells were rapidly enriched in the lungs of C57BL/6 mice infected with Salmonella Typhimurium, comprising up to 50% of αß-T cells after 1 week. MAIT cell accumulation was MR1-dependent, required Ag derived from the microbial riboflavin synthesis pathway, and did not occur in response to synthetic Ag, unless accompanied by a Toll-like receptor agonist or by co-infection with riboflavin pathway-deficient S. Typhimurium. The MAIT cell response was associated with their long-term accumulation in the lungs, draining lymph nodes and spleen. Lung MAIT cells from infected mice displayed an activated/memory phenotype, and most expressed the transcription factor retinoic acid-related orphan receptor γt. T-bet expression increased following infection. The majority produced interleukin-17 while smaller subsets produced interferon-γ or tumor necrosis factor, detected directly ex vivo. Thus the activation and expansion of MAIT cells coupled with their pro-inflammatory cytokine production occurred in response to Ags derived from microbial riboflavin synthesis and was augmented by co-stimulatory signals.

CD3bright signals on γδ T cells identify IL-17A-producing Vγ6Vδ1+ T cells.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine that has an important role at mucosal sites in a wide range of immune responses including infection, allergy and auto-immunity. γδ T cells are recognized as IL-17 producers, but based on the level of CD3 expression, we now define the remarkable ability of a CD3(bright) γδ T-cell subset with an effector memory phenotype to rapidly produce IL-17A, but not interferon-γ. CD3(bright) γδ T cells uniformly express the canonical germline encoded Vγ6/Vδ1(+) T-cell receptor. They are widely distributed with a preferential representation in the lungs and skin are negatively impacted in the absence of retinoic acid receptor-related orphan receptor gammat expression or endogenous flora. This population responded rapidly to various stimuli in a mechanism involving IL-23 and NOD-like receptor family, pyrin domain containing 3 (NLRP3)-inflammasome-dependent IL-1ß. Finally, we demonstrated that IL-17-producing CD3(bright) γδ T cells responded promptly and strongly to pneumococcal infection and during skin inflammation. Here, we propose a new way to specifically analyze IL-17-producing Vγ6/Vδ1(+) T cells based on the level of CD3 signals. Using this gating strategy, our data reinforce the crucial role of this γδ T-cell subset in respiratory and skin disorders.

IL-17-producing NKT cells depend exclusively on IL-7 for homeostasis and survival.

Natural killer T (NKT) cells are innate-like T cells that rapidly recognize pathogens and produce cytokines that shape the ensuing immune response. IL-17-producing NKT cells are enriched in barrier tissues, such as the lung, skin, and peripheral lymph nodes, and the factors that maintain this population in the periphery have not been elucidated. Here we show that NKT17 cells deviate from other NKT cells in their survival requirements. In contrast to conventional NKT cells that are maintained by IL-15, RORγt(+) NKT cells are IL-15 independent and instead rely completely on IL-7. IL-7 initiates a T-cell receptor-independent (TCR-independent) expansion of NKT17 cells, thus supporting their homeostasis. Without IL-7, survival is dramatically impaired, yet residual cells remain lineage committed with no downregulation of RORγt evident. Their preferential response to IL-7 does not reflect enhanced signaling through STAT proteins, but instead is modulated via the PI3K/AKT/mTOR signaling pathway. The ability to compete for IL-7 is dependent on high-density IL-7 receptor expression, which would promote uptake of low levels of IL-7 produced in the non-lymphoid sites of lung and skin. This dependence on IL-7 is also reported for RORγt(+) innate lymphoid cells and CD4(+) Th17 cells, and suggests common survival requirements for functionally similar cells.

Natural killer T cell defects in multiple myeloma and the impact of lenalidomide therapy.

The causes of multiple myeloma (MM) remain obscure and there are few known risk factors; however, natural killer T (NKT) cell abnormalities have been reported in patients with MM, and therapeutic targeting of NKT cells is promoted as a potential treatment. We characterized NKT cell defects in treated and untreated patients with MM and determined the impact of lenalidomide therapy on the NKT cell pool. Lenalidomide is an immunomodulatory drug with co-stimulatory effects on NKT cells in vitro and is an approved treatment for MM, although its mode of action in that context is not well defined. We find that patients with relapsed/progressive MM had a marked deficiency in NKT cell numbers. In contrast, newly diagnosed patients had relatively normal NKT cell frequency and function prior to treatment, although a specific NKT cell deficiency emerged after high-dose melphalan and autologous stem cell transplantation (ASCT) regimen. This also impacted NK cells and conventional T cells, but the recovery of NKT cells was considerably delayed, resulting in a prolonged, treatment-induced NKT cell deficit. Longitudinal analysis of individual patients revealed that lenalidomide therapy had no in-vivo impact on NKT cell numbers or cytokine production, either as induction therapy, or as maintenance therapy following ASCT, indicating that its clinical benefits in this setting are independent of NKT cell modulation.

In-vivo stimulation of macaque natural killer T cells with α-galactosylceramide.

Natural killer T cells are a potent mediator of anti-viral immunity in mice, but little is known about the effects of manipulating NKT cells in non-human primates. We evaluated the delivery of the NKT cell ligand, α-galactosylceramide (α-GalCer), in 27 macaques by studying the effects of different dosing (1-100 µg), and delivery modes [directly intravenously (i.v.) or pulsed onto blood or peripheral blood mononuclear cells]. We found that peripheral NKT cells were depleted transiently from the periphery following α-GalCer administration across all delivery modes, particularly in doses of ≥10 µg. Furthermore, NKT cell numbers frequently remained depressed at i.v. α-GalCer doses of >10 µg. Levels of cytokine expression were also not enhanced after α-GalCer delivery to macaques. To evaluate the effects of α-GalCer administration on anti-viral immunity, we administered α-GalCer either together with live attenuated influenza virus infection or prior to simian immunodeficiency virus (SIV) infection of two macaques. There was no clear enhancement of influenza-specific T or B cell immunity following α-GalCer delivery. Further, there was no modulation of pathogenic SIVmac251 infection following α-GalCer delivery to a further two macaques in a pilot study. Accordingly, although macaque peripheral NKT cells are modulated by α-GalCer in vivo, at least for the dosing regimens tested in this study, this does not appear to have a significant impact on anti-viral immunity in macaque models.

Ex-vivo analysis of human natural killer T cells demonstrates heterogeneity between tissues and within established CD4(+) and CD4(-) subsets.

Our understanding of human type 1 natural killer T (NKT) cells has been heavily dependent on studies of cells from peripheral blood. These have identified two functionally distinct subsets defined by expression of CD4, although it is widely believed that this underestimates the true number of subsets. Two recent studies supporting this view have provided more detail about diversity of the human NKT cells, but relied on analysis of NKT cells from human blood that had been expanded in vitro prior to analysis. In this study we extend those findings by assessing the heterogeneity of CD4(+) and CD4(-) human NKT cell subsets from peripheral blood, cord blood, thymus and spleen without prior expansion ex vivo, and identifying for the first time cytokines expressed by human NKT cells from spleen and thymus. Our comparative analysis reveals highly heterogeneous expression of surface antigens by CD4(+) and CD4(-) NKT cell subsets and identifies several antigens whose differential expression correlates with the cytokine response. Collectively, our findings reveal that the common classification of NKT cells into CD4(+) and CD4(-) subsets fails to reflect the diversity of this lineage, and that more studies are needed to establish the functional significance of the antigen expression patterns and tissue residency of human NKT cells.

Testing the NKT cell hypothesis in lenalidomide-treated myelodysplastic syndrome patients.

Myelodysplastic syndrome (MDS) comprises a group of clonal bone marrow disorders characterized by ineffective hematopoiesis and increased predisposition to acute myeloid leukemia. The causes of MDS remain poorly defined, but several studies have reported the NKT cell compartment of patients with MDS is deficient in number and functionally defective. In support of a central role for NKT cells, a pilot clinical study reported that lenalidomide (an approved treatment for MDS) increased NKT cell numbers in patients with MDS, and several in vitro studies showed lenalidomide specifically promoted NKT cell proliferation and cytokine production. We tested this in a much larger study and confirm a moderate in vitro augmentation of some NKT cell functions by lenalidomide, but find no impact on the NKT cell compartment of patients treated with lenalidomide, despite a consistently positive clinical response. We further show that the frequency and cytokine production of NKT cells is normal in patients with MDS before treatment and remains stable throughout 10 months of lenalidomide therapy. Collectively, our data challenge the concept that NKT cell defects contribute to the development of MDS, and show that a clinical response to lenalidomide is not dependent on modulation of NKT cell frequency or function.

Immune characterization of an individual with an exceptionally high natural killer T cell frequency and her immediate family.

Natural killer T cells (NKT) are a regulatory subset of T lymphocytes whose frequency in peripheral blood is highly variable within the human population. Lower than normal NKT frequencies are associated with increased predisposition to a number of diseases, including type 1 diabetes and some forms of cancer, raising the possibility that an increased frequency may be protective. However, there is little or no understanding of how high NKT frequencies arise or, most importantly, whether the potential exists to boost and maintain NKT levels for therapeutic advantage. Here, we provide a detailed functional and phenotypic characterization of the NKT compartment of a human donor with NKT levels approximately 50 times greater than normal, including an analysis of NKT in her immediate family members. The study focuses upon the characteristics of this donor and her family, but demonstrates more broadly that the size and flexibility of the NKT niche is far greater than envisioned previously. This has important implications for understanding how the human NKT compartment is regulated, and supports the concept that the human NKT compartment might be expanded successfully for therapeutic benefit.

CD1-restricted T cells and tumor immunity.

CD1d-restricted T cells (NKT cells) are potent regulators of a broad range of immune responses. In particular, an abundance of research has focussed on the role of NKT cells in tumor immunity. This field of research has been greatly facilitated by the finding of agonist ligands capable of potently stimulating NKT cells and also animal models where NKT cells have been shown to play a natural role in the surveillance of tumors. Herein, we review the capability of NKT cells to promote the rejection of tumors and the mechanisms by which this occurs. We also highlight a growing field of research that has found that NKT cells are capable of suppressing anti-tumor immunity and discuss the progress to date for the immunotherapeutic use of NKT cells.

NKT cells: potential targets for autoimmune disease therapy?

NKT cells represent a unique T cell lineage that recognize glycolipid antigens in the context of the non-classical MHC class I molecule CD1d. NKT cells are potent producers of immunoregulatory cytokines, and have been implicated in several different autoimmune diseases in mice and humans, including Type 1 diabetes, experimental autoimmune encephalomyelitis--a mouse model for multiple sclerosis, systemic lupus erythematosus, and scleroderma. This review will cover the evidence for an involvement for NKT cells in these autoimmune diseases, and discuss the potential for therapeutic manipulation of these cells as a means of preventing autoimmune disease in the clinic.

The effect of antigen stimulation on the migration of mature T cells from the peripheral lymphoid tissues to the thymus.

Although the maturation and export of T cells from the thymus has been extensively studied, the movement of cells in the opposite direction has been less well documented. In particular, the question of whether T cells which have been activated by antigen in the periphery are more likely to return to the thymus had been raised but not clearly answered. We examined this issue by activating T cells present in the periphery with their cognate antigen, and assessing migration to the thymus. TCR-transgenic cells from OT-I mice (Thy1.2+), which recognise the ovalbumin peptide OVA257-264 in the context of H-2Kb, were transferred into otherwise unmanipulated Thy1.1+ C57BL/6 mice. Recipient mice were injected i.v. with 5 microg peptide (SIINFEKL) approximately 24 hours later. The numbers of donor-derived (Thy1.2+) cells in the thymus and peripheral lymphoid tissue were determined. The results clearly show increased numbers of transgenic cells in the thymus 3 days after antigenic stimulation. However, since numbers of transgenic cells increased in the spleen and LN in about the same proportion, the data do not support the notion that there is highly increased selective migration of activated T cells to the thymus. Rather, they suggest that a sample of peripheral cells enters the thymus each day, and that the mature immigrants detected in the thymus merely reflect the contents of the peripheral T cell pool.

Lymphotoxin controls alphaEbeta7-integrin expression by peripheral CD8+ T cells.

Lymphotoxin (LT)-alpha, a member of the TNF family, is recognized as an important mediator in different aspects of lymphoid organ development. Targeted disruption of this molecule resulted in a substantial reduction in the proportion of alphaEbeta7-integrin(high) CD8+ T cells detectable in peripheral lymphoid organs. This defect, however, was not observed on mature CD4-CD8+ thymocytes. To determine whether this was due to downregulation of beta7-integrin expression by peripheral CD8+ T cells or a failure of thymic emigration of CD8+ beta7-integrin(high) T cells, beta7-integrin was examined on recent thymic emigrants (RTE). When analysed within 16 h after leaving the thymus CD4-CD8+ RTE in both LT-alpha-/- and wild type (wt) mice remained beta7-integrin(high) and were indistinguishable. However, within 3-5 days, emigration loss of beta7-integrin became evident in LT-alpha-/- mice. Despite this loss, the proportion of thymically derived alphabetaTCR+ T-cell populations in the intestinal epithelium, an important target tissue of CD8+ alphaEbeta7-integrin(high) T cells, was increased in the absence of LT-alpha. In contrast, B cells were detectable only rarely in the intestinal tissue of LT-alpha-/- mice. The expression of E-Cadherin remained unchanged. These results indicate that a LT-alpha-dependent process maintains a high level of alphaEbeta7-integrin expression by peripheral CD8+ T cells, and with this control mechanism LT-alpha may help to regulate CD8+ T-cell numbers in the tissues.

CD1d-restricted NKT cells: an interstrain comparison.

CD1d-restricted Valpha14-Jalpha281 invariant alphabetaTCR(+) (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as alphabetaTCR(+)CD4(-)CD8(-) and DX5(+)CD3(+) have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1.1-congenic BALB/c, and NK1.1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1.1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/alpha-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5(+)CD3(+)) actually excludes these cells. Finally, our results support the concept that NK1.1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1.1-negative, especially within the CD4(+) subset and particularly in NK1.1-congenic BALB/c mice.

Cytometric and functional analyses of NK and NKT cell deficiencies in NOD mice.

Defects in NK and NKT cell activities have been implicated in the etiology of type 1 (autoimmune) diabetes in NOD mice on the basis of experiments performed using surrogate phenotypes for the identification of these lymphocyte subsets. Here, we have generated a congenic line of NOD mice (NOD.b-Nkrp1(b)) which express the allelic NK1.1 marker, enabling the direct study of NK and NKT cells in NOD mice. Major deficiencies in both populations were identified when NOD.b-Nkrp1(b) mice were compared with C57BL/6 and BALB.B6-Cmv1(r) mice by flow cytometry. The decrease in numbers of peripheral NK cells was associated with an increase in their numbers in the bone marrow, suggesting that a defect in NK cell export may be involved. In contrast, the most severe deficiency of NKT cells found was in the thymus, indicating that defects in thymic production were probably responsible. The deficiencies in NK cell activity in NOD mice could only partly be accounted for by the reduced numbers of NK cells, and fewer NKT cells from NOD mice produced IL-4 following stimulation, suggesting that NK and NKT cells from NOD mice shared functional deficiencies in addition to their numerical deficiencies. Despite the relative lack of IL-4 production by NOD NKT cells, adoptive transfer of alpha beta TCR(+)NK1.1(+) syngeneic NKT cells into 3-week-old NOD recipients successfully prevented the onset of spontaneous diabetes. As both NK and NKT cells play roles in regulating immune responses, we postulate that the synergistic defects reported here contribute to the susceptibility of NOD mice to autoimmune disease.

NK cells and NKT cells collaborate in host protection from methylcholanthrene-induced fibrosarcoma.

NK1.1(+) V(alpha)14J(alpha)281(+) (NKT) cells can be induced by IL-12 therapy to mediate tumor rejection; however, methylcholanthrene (MCA)-induced fibrosarcoma is the only tumor model described where NKT cells play a natural role in controlling tumor initiation. From our previous study in C57BL/6 mice it remained unclear whether NK cells were also involved in this natural response. Herein, to discriminate the function of NK and NKT cells, we have evaluated fibrosarcoma development in mice deficient in NKT cells, but not NK cells, and mice deficient in NK cells, but not NKT cells. The results indicate that both NK cells and NKT cells are essential and collaborate in natural host immunity against MCA-induced sarcoma. In contrast, sarcoma incidence and growth rate were reduced using IL-12 therapy, this effect was mediated in the absence of T cells (including NKT cells), but not NK cells.

A fresh look at tumor immunosurveillance and immunotherapy.

Despite major advances in our understanding of adaptive immunity and dendritic cells, consistent and durable responses to cancer vaccines remain elusive and active immunotherapy is still not an established treatment modality. The key to developing an effective anti-tumor response is understanding why, initially, the immune system is unable to detect transformed cells and is subsequently tolerant of tumor growth and metastasis. Ineffective antigen presentation limits the adaptive immune response; however, we are now learning that the host's innate immune system may first fail to recognize the tumor as posing a danger. Recent descriptions of stress-induced ligands on tumor cells recognized by innate effector cells, new subsets of T cells that regulate tumor tolerance and the development of spontaneous tumors in mice that lack immune effector molecules, beckon a reflection on our current perspectives on the interaction of transformed cells with the immune system and offer new hope of stimulating therapeutic immunity to cancer.

GRKO mice express an aberrant dexamethasone-binding glucocorticoid receptor, but are profoundly glucocorticoid resistant.

The introduction of a targeted insertion mutation into exon 2 of the gene coding for the glucocorticoid receptor (GR) enabled production of glucocorticoid receptor knock-out (GRKO) mice. GRKO mice on a C57BL/6/129sv mixed genetic background show a variable phenotype, with 90% of -/- mice dying at birth with respiratory insufficiency but 10% of mutant mice surviving to maturity. To investigate the possibility of residual GR expression in surviving GRKO mice we have measured binding of the synthetic glucocorticoid dexamethasone in tissue extracts from adrenalectomized mice. High affinity binding of dexamethasone in protein extracts of liver, kidney, lung and brain from adult GRKO mice is found at levels 30-60% those in wild-type mice, with heterozygotes (+/-) having intermediate levels. PCR and ribonuclease protection analysis showed comparable levels of GR mRNA on the 3' side of the gene-targeted insertional mutation in exon 2 of the GR gene, with almost no GR mRNA detected from exons 1 and 2 on the 5' side of the gene-targeted insertional mutation. Western blot analysis using a C-terminal specific GR antibody detects a 39 kDa GR fragment in extracts from adult GRKO mice. Despite the evidence for expression of a ligand-binding domain fragment of the glucocorticoid receptor these mice are profoundly glucocorticoid resistant, with elevated levels of plasma ACTH and corticosterone. Thymocytes from adult and fetal GRKO mice are resistant to dexamethasone-induced apoptosis and cultured fetal hepatocytes from GRKO mice are completely refractory to glucocorticoid induction of the gluconeogenic enzyme glucose-6-phosphatase. Thus although the surviving adult homozygous GRKO mice express a dexamethasone-binding GR fragment, their classic target tissues remain profoundly glucocorticoid insensitive.

Stress-free T-cell development: glucocorticoids are not obligatory.

A role for glucocorticoids in thymopoiesis has been suggested by studies using glucocorticoid receptor (GR) anti-sense transgenic mice, glucocorticoid synthesis inhibitors and GR antagonists. Unfortunately, no consensus has been reached on exactly how glucocorticoids influence T-cell development. The most recent approach, using GR knockout (GR(-/-)) mice, indicates that GR signaling is, in fact, dispensable in this entire process.