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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 γδ 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 γδ T cells and highlight some of the key differences between conventional and unconventional T cell development.

S1PR5 impairs TRM cell differentiation by limiting T cell entry and promoting T cell egress from peripheral tissues. Local TGF-β signaling coordinates suppression of the T-bet–ZEB2–S1PR5 emigration axis, thus enforcing tissue residency., 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., Graphical Abstract

Mucosal‐associated invariant T (MAIT) cells are unconventional T cells that recognize antigens derived from riboflavin biosynthesis. In addition to anti‐microbial functions, human MAIT cells are associated with cancers, autoimmunity, allergies and inflammatory disorders, although their role is poorly understood. Activated MAIT cells are well known for their rapid release of Th1 and Th17 cytokines, but we have discovered that chronic stimulation can also lead to potent interleukin (IL)‐13 expression. We used RNA‐seq and qRT‐PCR to demonstrate high expression of the IL‐13 gene in chronically stimulated MAIT cells, and directly identify IL‐13 using intracellular flow cytometry and multiplex bead analysis of MAIT cell cultures. This unexpected finding has important implications for IL‐13‐dependent diseases, such as colorectal cancer (CRC), that occur in mucosal areas where MAIT cells are abundant. We identify MAIT cells near CRC tumors and show that these areas and precancerous polyps express high levels of the IL‐13 receptor, which promotes tumor progression and metastasis. Our data suggest that MAIT cells have a more complicated role in CRC than currently realized and that they represent a promising new target for immunotherapies where IL‐13 can be a critical factor., Mucosal‐associated invariant T (MAIT) cells are regarded as proinflammatory lymphocytes with a Th1/Th17 cytokine response. This study shows human MAIT cells can also be prominent IL‐13‐producing cells. The findings shed new light on the potential role of MAIT cells in tumor immunity and their potential as new targets for immunotherapies in colorectal cancer and other IL‐13‐dependent diseases.

The plakin family of cytoskeletal proteins play an important role in cancer progression yet are under-studied in cancer, especially ovarian cancer. These large cytoskeletal proteins have primary roles in the maintenance of cytoskeletal integrity but are also associated with scaffolds of intermediate filaments and hemidesmosomal adhesion complexes mediating signalling pathways that regulate cellular growth, migration, invasion and differentiation as well as stress response. Abnormalities of plakins, and the closely related spectraplakins, result in diseases of the skin, striated muscle and nervous tissue. Their prevalence in epithelial cells suggests that plakins may play a role in epithelial ovarian cancer progression and recurrence. In this review article, we explore the roles of plakins, particularly plectin, periplakin and envoplakin in disease-states and cancers with emphasis on ovarian cancer. We discuss the potential role the plakin family of proteins play in regulating cancer cell growth, survival, migration, invasion and drug resistance. We highlight potential relationships between plakins, epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) and discuss how interaction of these processes may affect ovarian cancer progression, chemoresistance and ultimately recurrence. We propose that molecular changes in the expression of plakins leads to the transition of benign ovarian tumours to carcinomas, as well as floating cellular aggregates (commonly known as spheroids) in the ascites microenvironment, which may contribute to the sustenance and progression of the disease. In this review, attempts have been made to understand the crucial changes in plakin expression in relation to progression and recurrence of ovarian cancer. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12964-021-00726-x.

Summary: 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, CD26hiCD94lo Vδ2+ cells exhibit substantial similarities to CD26hi mucosal-associated invariant T (MAIT) cells, although CD26− Vδ2+ cells exhibit cytotoxic, effector-like profiles. At birth, the Vδ2+Vγ9+ population is dominated by CD26hiCD94lo cells; during adolescence and adulthood, Vδ2+ cells acquire CD94/NKG2A expression and the relative frequency of the CD26hiCD94lo subset declines. Critically, exposure of the CD26hiCD94lo subset 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 CD26hiCD94lo Vδ2+ cells, combined with their adenosine-binding capacity, may make them ideal targets for immunotherapeutic expansion and adoptive transfer.

Special AT‐rich binding protein‐1 (SATB1) is a global chromatin organizer capable of activating or repressing gene transcription in mice and humans. The role of SATB1 is pivotal for T‐cell development, with SATB1‐knockout mice being neonatally lethal, although the exact mechanism is unknown. Moreover, SATB1 is dysregulated in T‐cell lymphoma and proposed to suppress transcription of the Pdcd1 gene, encoding the immune checkpoint programmed cell death protein 1 (PD‐1). Thus, SATB1 expression in T‐cell subsets across different tissue compartments in humans is of potential importance for targeting PD‐1. Here, we comprehensively analyzed SATB1 expression across different human tissues and immune compartments by flow cytometry and correlated this with PD‐1 expression. We investigated SATB1 protein levels in pediatric and adult donors and assessed expression dynamics of this chromatin organizer across different immune cell subsets in human organs, as well as in antigen‐specific T cells directed against acute and chronic viral infections. Our data demonstrate that SATB1 expression in humans is the highest in T‐cell progenitors in the thymus, and then becomes downregulated in mature T cells in the periphery. Importantly, SATB1 expression in peripheral mature T cells is not static and follows fine‐tuned expression dynamics, which appear to be tissue‐ and antigen‐dependent. Furthermore, SATB1 expression negatively correlates with PD‐1 expression in virus‐specific CD8+ T cells. Our study has implications for understanding the role of SATB1 in human health and disease and suggests an approach for modulating PD‐1 in T cells, highly relevant to human malignancies or chronic viral infections.

Mucosal‐associated invariant T (MAIT) cells represent up to 10% of circulating human T cells. They are usually defined using combinations of non‐lineage‐specific (surrogate) markers such as anti‐TRAV1‐2, CD161, IL‐18Rα and CD26. The development of MR1‐Ag tetramers now permits the specific identification of MAIT cells based on T‐cell receptor specificity. Here, we compare these approaches for identifying MAIT cells and show that surrogate markers are not always accurate in identifying these cells, particularly the CD4+ fraction. Moreover, while all MAIT cell subsets produced comparable levels of IFNγ, TNF and IL‐17A, the CD4+ population produced more IL‐2 than the other subsets. In a human ontogeny study, we show that the frequencies of most MR1 tetramer+ MAIT cells, with the exception of CD4+ MAIT cells, increased from birth to about 25 years of age and declined thereafter. We also demonstrate a positive association between the frequency of MAIT cells and other unconventional T cells including Natural Killer T (NKT) cells and Vδ2+ γδ T cells. Accordingly, this study demonstrates that MAIT cells are phenotypically and functionally diverse, that surrogate markers may not reliably identify all of these cells, and that their numbers are regulated in an age‐dependent manner and correlate with NKT and Vδ2+ γδ T cells.

Vδ2

Langerhans cell histiocytosis (LCH) lesions contain myeloid lineage 'LCH' cells. Regulatory T cells (Tregs) are also enriched within lesions, although their role in LCH pathogenesis is unknown. LCH cells are thought to produce the transforming growth factor beta (TGF-β) within lesions, however whether Tregs contribute is unestablished. Using flow cytometry, we analyzed relative frequencies of live Tregs from LCH patients and identified CD56 expression and TGF-β production by lesion Tregs. While CD56

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 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.

Langerhans cell histiocytosis (LCH) lesions contain myeloid lineage 'LCH' cells. Regulatory T cells (Tregs) are also enriched within lesions, although their role in LCH pathogenesis is unknown. LCH cells are thought to produce the transforming growth factor beta (TGF-β) within lesions, however whether Tregs contribute is unestablished. Using flow cytometry, we analyzed relative frequencies of live Tregs from LCH patients and identified CD56 expression and TGF-β production by lesion Tregs. While CD56+ Tregs were enriched in lesions, overall CD56+ T cells were reduced in the blood from active LCH patients compared to non-active disease patients, and there was a negative correlation between CD8+CD56+ T cells and Tregs. We propose that inducing a Treg phenotype in T cells such as CD56+ T cells may be a mechanism by which LCH cells divert inflammatory T cell responses. Thus, Tregs within LCH lesions are likely an important component in LCH pathogenesis.

Langerhans cell histiocytosis (LCH) lesions are defined by the presence of CD1a+/CD207+ myeloid cells, but many other immune cells are present including unconventional T cells, which have powerful immunoregulatory functions. Unconventional T cell lineages include mucosal-associated invariant T (MAIT) cells, type I natural killer T (NKT) cells and gamma-delta (γδ) T cells, which are associated with many inflammatory conditions, although their importance has not been studied in LCH. We characterized their phenotype and function in blood and lesions from patients with LCH, and identified a deficiency in MAIT cell frequency and abnormalities in the subset distributions of γδ T cells and NKT cells. Such abnormalities are associated with immune dysregulation in other disease settings and are therefore potentially important in LCH. Our study is the first to recognize alterations to MAIT cell proportions in patients with LCH. This finding along with other abnormalities identified amongst unconventional T cells could potentially influence the onset and progression of LCH, thereby highlighting potential targets for new immune based therapies.

Langerhans cell histiocytosis is characterized by lesions containing inflammatory immune cells, including myeloid cells and T cells. Patient mortality remains unacceptably high and new treatment options are required. Several LCH studies have identified aberrant frequencies of T cell subsets with potential immune regulatory properties. High numbers of Foxp3+ regulatory T cells and gamma-delta T cells have been reported in patients with LCH, although, the cause of their presence or their significance is not yet clear. This review describes the current understanding of how LCH develops and progresses, focusing on the growing evidence that regulatory T cell subsets may be important and discussing the exciting potential for harnessing these cells to treat LCH using immune based therapies.

The last few years has seen the burgeoning of a new category of therapeutics for cancer targeting immune regulatory pathways. Antibodies that block the PD-1/PD-L1 interaction are perhaps the most prominent of these new anti-cancer therapies, but several other inhibitory receptor ligand interactions have also shown promise as targets in clinical trials, including CTLA-4/CD80 and Lag-3/MHC class II. Related to this is a rapidly improving knowledge of ‘regulatory’ lymphocyte lineages, including NKT cells, MAIT cells, B regulatory cells and others. These cells have potent cytokine responses that can influence the functioning of other immune cells and many researchers believe that they could be effective targets for therapies designed to enhance immune responses to cancer. This review will outline our current understanding of FOXP3 + ‘Tregs’, NKT cells, MAIT cells and B regulatory cells immune regulatory cell populations in cancer, with a particular focus on chronic lymphocytic leukaemia (CLL). We will discuss evidence linking CLL with immune regulatory dysfunction and the potential for new therapies targeting regulatory cells.

Glucocorticoids (GCs) are powerful inhibitors of inflammation and immunity. Although glucocorticoid-induced cell death (GICD) is an important part of GCs actions, the cell types and molecular mechanisms involved are not well understood. Untranslated exon 1A3 of the human glucocorticoid receptor (GR) gene is a major determinant of GICD in GICD-sensitive human cancer cell lines, operating to dynamically upregulate GR levels in response to GCs. We measured the GICD sensitivity of freshly isolated peripheral blood mononuclear cells and thymocytes to dexamethasone in vitro, relating this to GR exon 1A3 expression. A clear GICD sensitivity hierarchy was detected: B cells>thymocytes/natural killer (NK) cells>peripheral T cells. Within thymocyte populations, GICD sensitivity decreased with maturation. Interestingly, NK cell subsets were differentially sensitive to GICD, with CD16(+)CD56(int) (cytotoxic) NK cells being highly resistant to GICD, whereas CD16(-)CD56(hi) (cytokine producing) NK cells were highly sensitive (similar to B cells). B-cell GICD was rescued by co-culture with interleukin-4. Strikingly, although no significant increases in GR protein were observed during 48 h of culture of GICD-sensitive and -resistant cells alike, GR 1A3 expression was increased over pre-culture levels in a manner directly proportional to the GICD sensitivity of each cell type. Accordingly, this is the first evidence that the GR exon 1A3 promoter is differentially regulated during thymic development and maturation of human T cells. Furthermore, human peripheral blood B cells are exquisitely GICD-sensitive in vitro, giving new insight into how GCs may downregulate immunity. Collectively, these data show that GR 1A3 expression is tied with GICD sensitivity in human lymphocytes, underscoring the potential for GR 1A3 expression to be used as a biomarker for sensitivity to GICD.

Summary 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.

The role of specific members of the NF-κB family of transcription factors in CD8 T-cell selection and development is largely unknown. Here, we show that mice lacking NF-κB1 develop a unique population of conventional CD8 single-positive (SP) thymocytes with memory T cell-like properties that populate peripheral immune organs. Development of this memory-like population is not due to PLZF+ thymocytes and instead coincides with changes in CD8 T-cell selection. These include a reduction in the efficiency of negative selection and a dependence on MHC class Ia or Ib expressed by haematopoietic cells. These findings indicate that NF-κB1 regulates multiple events in the thymus that collectively inhibit the excess development of CD8+ thymocytes with memory cell characteristics.

Introduction: Langerhans cell histiocytosis (LCH) is a rare disease involving inflammatory lesions that can occur in essentially any organ of the body. LCH lesions are defined by the presence of CD1a+/CD207+ myeloid lineage cells (LCH cells), which often have mutations within the RAS/RAF/MEK/ERK pathway and constitutive activation of ERK. A range of other immune cells are also present within lesions including an enrichment of Foxp3 regulatory T cells (Tregs). The immune suppressive cytokine transforming growth factor beta (TGF-β), which is commonly produced by Foxp3 Tregs has also been detected within lesions and blood from patients with active LCH disease. Groups have suggested that LCH cells are a source of TGF-β and that TGF-β is one of the drivers of the LCH cell phenotype. Given the enrichment of Foxp3 Tregs and the presence of TGF-β within LCH lesions it is conceivable that Foxp3 Tregs too are a source of TGF-β production. Historically the identification of Foxp3 Tregs has not allowed for functional studies to be conducted because staining for the Foxp3 transcription factor requires cell permeabilization. A newer surrogate gating strategy to detect CD3+CD4+CD25+CD127low lymphocytes allows for downstream functional assays on the Foxp3 Treg population. Our study aimed to better define the role of Tregs in LCH pathogenesis using this surrogate staining method. Results: A surrogate Treg identification method was employed to gate on CD3+CD4+CD25+CD127low lymphocytes in the blood from healthy donors and patients with LCH and in lesions from patients with LCH. Using this gating strategy we identified similarly to previous studies that the proportion of lesional Tregs (median = 12.85 %, IQR = 7.85-26.72 %, n = 6) was significantly (p < 0.0001) increased in the total T cell population when compared to those in the blood from healthy donors (median = 1.02 %, IQR = 0.84-1.20 %, n = 19). The proportion of Tregs in blood from patients with active LCH (median = 1.73 %, IQR = 1.58-3.80 %, n = 8) was also significantly (p = 0.232) increased when compared to those in the blood from healthy donors. We confirmed Foxp3 expression in Tregs in lesions from three independent LCH donors by staining for the transcription factor, and we confirmed that other lesional CD4+ T cells were mostly negative for Foxp3. Due to our interest in unconventional T cells we analysed specimens for CD56 expression in conjunction with investigating Tregs and we unexpectedly found CD56 expression on a considerable proportion of the Treg population in LCH lesions (median = 36.48 %, IQR = 24.94-55.14 %, n = 6). The proportion of Tregs that displayed CD56 expression on their cell surface was significantly higher in the lesions from patients with LCH compared to the blood from healthy donors (median = 2.29 %, IQR = 0.61-4.10 %, n = 15, p = 0.0092) and patients with active LCH (median = 0.57 %, IQR = 0.11-0.85 %, n = 8, p < 0.0001). Additionally, we found that the proportion of CD56+ Tregs in total T cells from LCH lesions was directly correlated to the proportion of total Tregs in overall lesional T cells (r = 1, p = 0.0028). Importantly, we confirmed Foxp3 expression by both the CD56+ and CD56- Treg populations in lesions from three independent LCH donors. We purified and then stimulated Tregs from healthy donors, and CD56+ and CD56- Treg populations from lesions from patients with LCH. Using supernatants from this assay we found that Tregs from lesions from patients with LCH produced active TGF-β when challenged with PMA and ionomycin for 16 hours (CD56+ Tregs n = 3, CD56- Tregs n = 3). Conclusion: We have confirmed that both CD56+ and CD56- Treg populations from the lesions from patients with LCH are able to produce TGF-β. Given their cytokine potential, the functional status of Tregs in LCH lesions is therefore most likely to be suppressive in nature. This means that Tregs are possibly responsible for a component of the well documented expression of TGF-β within lesions. Disclosures No relevant conflicts of interest to declare.

Autoimmune lymphocytic infiltration of the salivary glands, termed sialadenitis, is a pathologic feature of Sjögren’s syndrome (SjS) that is also prominent in nonobese diabetic (NOD) mice. Genetic factors regulate sialadenitis, and a previous (NOD × NZW)F2 study detected linkage to murine chromosome (Chr) 7. The locus, subsequently annotated as Ssial3, maps to the distal end of Chr7 and overlaps a region associated with type 1 diabetes susceptibility in NOD mice. To examine whether Ssial3 could contribute to both diseases, or was specific for SjS, we generated a congenic mouse strain that harbored an NZW-derived Chr7 interval on the NOD genetic background. This congenic strain exhibited reduced sialadenitis compared with NOD mice and confirmed Ssial3. This reduction, however, did not ameliorate saliva abnormalities associated with SjS-like disease in NOD mice, nor were congenic mice protected against insulitis (lymphocytic infiltration of the pancreatic islets) or diabetes onset. Thus, the Ssial3 locus appears to have a tissue-specific effect for which the NZW allele is unable to prevent other autoimmune traits in the NOD mouse. Anomalous increases for antinuclear Ab production and frequency of marginal-zone B cells were also identified in congenic mice, indicating that the NZW-derived Chr7 interval has a complex effect on the NOD immune system.

Autoimmune regulator gene (Aire)-deficient mice develop an array of autoimmune lesions that reflect failures of immune tolerance. Negative selection is clearly compromised in these mice, but there is evidence to suggest that other mechanisms of tolerance might also be affected, including a possible impairment of regulatory T cell (Treg) development. Studies to date have failed to demonstrate any significant impact on the development or function of the FOXP31 Treg compartment, but NKT cells represent a distinct regulatory cell lineage that also develop in the thymus and which are known to influence self-tolerance. Aire-related defects coincide with NKT cell deficiencies in a number of animal models, but the direct consequence of Aire-deficiency on NKT cell development has not been established. In this study, we demonstrate that the frequency, distribution and cytokine production of NKT cells and their subsets is principally normal in Aire-deficient mice. We conclude that Aire has little or no effect on regulatory T cell development in general and NKT cells in particular. Refereed/Peer-reviewed

NKT cell subsets can be divided based on CD4 and NK1.1 expression and tissue of origin, but the developmental and functional relationships between the different subsets still are poorly understood. A comprehensive study of 19 cytokines across different NKT cell subsets revealed that no two NKT subpopulations exhibited the same cytokine profile, and, remarkably, the amounts of each cytokine produced varied by up to 100-fold or more among subsets. This study also revealed the existence of a population of CD4 − NK1.1 − NKT cells that produce high levels of the proinflammatory cytokine IL-17 within 2–3 h of activation. On intrathymic transfer these cells develop into mature CD4 − NK1.1 + but not into CD4 + NK1.1 + NKT cells, indicating that CD4 − NK1.1 − NKT cells include an IL-17–producing subpopulation, and also mark the elusive branch point for CD4 + and CD4 − NKT cell sublineages.

CD1d-dependent natural killer T (NKT) cells are a unique T-cell subset with the ability to regulate the immune system in response to a broad range of diseases. That low NKT-cell numbers are associated with many different disease states in mice and humans, combined with the fact that NKT-cell numbers vary widely between individuals, makes it crucial to understand how these cells develop and how their numbers are maintained. Here, we review the current state of knowledge of NKT-cell development and attempt to highlight the most important questions in this field.

T cell development in the thymus involves a series of TCR-mediated control points including TCR-β selection and positive and negative selection. Approximately half of the thymic sojourn is spent in the medulla, where thymocytes undergo final maturation before emigrating to the periphery. Although it is acknowledged that thymic emigration is an active process, relatively little is known about how this is regulated, why it takes so long, and whether TCR-mediated signaling can influence this step. Using wild-type and TCR transgenic mice, we found that Ag injected i.v. or intrathymically led to a striking reduction in the number of recent thymic emigrants (RTE) in the periphery. This was caused by inhibition of T cell export rather than peripheral deletion, because a cohort of RTE that was already released before in vivo Ag challenge was not depleted, and similar results were observed in Bim-deficient mice, which have impaired T cell deletion. Within the thymus, the loss of RTE was associated with retention of medullary thymocytes rather than increased negative selection. In addition to Ag-specific inhibition of export, some TCR-independent suppression of emigration was also observed that appeared to be partly the result of the inflammatory cytokine TNF. Thus, in addition to its accepted role in intrathymic selection events, TCR signaling can also play an important role in the regulation of thymic emigration.

We showed previously that NKT cell–deficient TCR Jα18−/− mice are more susceptible to methylcholanthrene (MCA)-induced sarcomas, and that normal tumor surveillance can be restored by adoptive transfer of WT liver-derived NKT cells. Liver-derived NKT cells were used in these studies because of their relative abundance in this organ, and it was assumed that they were representative of NKT cells from other sites. We compared NKT cells from liver, thymus, and spleen for their ability to mediate rejection of the sarcoma cell line (MCA-1) in vivo, and found that this was a specialized function of liver-derived NKT cells. Furthermore, when CD4+ and CD4− liver-derived NKT cells were administered separately, MCA-1 rejection was mediated primarily by the CD4− fraction. Very similar results were achieved using the B16F10 melanoma metastasis model, which requires NKT cell stimulation with α-galactosylceramide. The impaired ability of thymus-derived NKT cells was due, in part, to their production of IL-4, because tumor immunity was clearly enhanced after transfer of IL-4–deficient thymus-derived NKT cells. This is the first study to demonstrate the existence of functionally distinct NKT cell subsets in vivo and may shed light on the long-appreciated paradox that NKT cells function as immunosuppressive cells in some disease models, whereas they promote cell-mediated immunity in others.

NKT cells are typically defined as CD1d-dependent T cells that carry an invariant TCR α-chain and produce high levels of cytokines. Traditionally, these cells were defined as NK1.1+ T cells, although only a few mouse strains express the NK1.1 molecule. A popular alternative marker for NKT cells has been DX5, an Ab that detects the CD49b integrin, expressed by most NK cells and a subset of T cells that resemble NKT cells. Interpretation of studies using DX5 as an NKT cell marker depends on how well DX5 defines NKT cells. Using a range of DX5 and other anti-CD49b Abs, we reveal major differences in reactivity depending on which Ab and which fluorochrome are used. The brightest, PE-conjugated reagents revealed that while most CD1d-dependent NKT cells expressed CD49b, they represented only a minority of CD49b+ T cells. Furthermore, CD49b+ T cell numbers were near normal in CD1d−/− mice that are completely deficient for NKT cells. CD1d tetramer− CD49b+ T cells differ from NKT cells by their activation and memory marker expression, tissue distribution, and CD4/CD8 coreceptor profile. Interestingly, both NKT cells and CD1d tetramer− CD49b+ T cells produce cytokines, but the latter are clearly biased toward Th1-type cytokines, in contrast to NKT cells that produce both Th1 and Th2 cytokines. Finally, we demonstrate that expression of CD49b by NKT cells does not dramatically alter with age, contrasting with earlier reports proposing DX5 as a maturation marker for NKT cells. In summary, our data demonstrate that DX5/CD49b is a poor marker for identifying CD1d-dependent NKT cells.

After being positively selected on CD1d-expressing thymocytes, NKT cells undergo a series of developmental changes that can take place inside or outside the thymus. We asked whether CD1d continues to play a role in late-stage NKT cell development and, in particular, during the functionally significant acquisition of NK1.1 that is indicative of NKT cell maturity. We report that CD1d is indeed crucial for this step, because immature NK1.1− NKT cells fail to fully mature when transferred to a CD1d-deficient environment. Surprisingly, however, the lack of CD1d did not greatly affect the long-term survival of NKT cells, and they continued to express CD69 and slowly proliferate. This directly contradicts the currently held view that these phenomena are caused by autoreactivity directed against CD1d/TCR-restricted self-Ags. Our findings demonstrate an ongoing role for TCR-mediated signaling throughout NKT cell development, but the characteristic semiactivated basal state of NKT cells is controlled by CD1d-independent factors or is intrinsic to the cells themselves.

Our understanding of NKT cells has been rapidly advancing over recent years, with many research groups studying how these cells behave and how they can be manipulated to prevent disease. Although good progress has been made, a difficulty is the lack of a clear consensus about how to assay, or even identify, NKT cells. The different approaches have been an ongoing source of uncertainty about the biological behaviour and function of NKT cells and have complicated efforts to define their role in immunity. An important step towards reaching agreement on the behaviour of NKT cells is to have a clear appreciation of the advantages and disadvantages of the different approaches that are employed in this field of study. This should help determine the most appropriate ways to investigate NKT cell function, thus bringing us closer to successfully exploiting these cells in the treatment of human diseases.

SummaryAire promotes the tolerization of thymocytes by inducing the expression of a battery of peripheral-tissue antigens in thymic medullary epithelial cells. We demonstrate that the cellular mechanism by which Aire exerts its tolerance-promoting function is not primarily positive selection of regulatory T cells, but rather negative selection of T effector cells. Surprisingly, supplementing its influence on the transcription of genes encoding peripheral-tissue antigens, Aire somehow enhances the antigen-presentation capability of medullary epithelial cells. Thus, this transcriptional control element promotes central tolerance both by furnishing a specific thymic stromal cell type with a repertoire of self antigens and by better arming such cells to present these antigens to differentiating thymocytes. In Aire’s absence, autoimmunity and ultimately overt autoimmune disease develops.

In the diabetes-prone NOD mouse, there is a proven association between a systemic deficiency of NKT cells and the onset of type 1 diabetes. Numerous reports of similar defects within the NKT cell compartment of human type 1 diabetes patients suggested NKT cell levels might be a valuable predictor of susceptibility and could provide a target for therapeutic intervention. Two recent studies, however, found no association between type 1 diabetes and blood NKT cell levels in humans and consequently rejected a link between the onset of diabetes and NKT cell deficiency. This cast considerable doubts on the potential for NKT cell-based clinical applications and challenged the validity of the NOD mouse as a model of human type 1 diabetes. We now report that NKT cell levels in blood are a poor representation of those in other organs. Strikingly, systemic NKT cell deficiencies were identified in NOD mice with normal, or even raised, blood levels. This re-establishes the correlation between NKT cell deficiency and type 1 diabetes and raises important questions regarding the assaying of NKT cell levels in humans.

CD1d-restricted invariant natural killer T cell (iNKT cells) have a limited T cell receptor (TCR) repertoire and share characteristics common to T cells and natural killer cells. While intrathymic selection facilitates the production of T cells carrying self major histocompatibility complex-restricted TCRs, natural killer cells carry an appropriate repertoire of self major histocompatibility complex-recognizing receptors to avoid self-reactivity. Here we show that chronic exposure to specific glycolipid antigen resulted in iNKT cell disappearance and thymus-dependent repopulation of iNKT cells with increased expression of inhibitory Ly-49 molecules that resulted in impaired responsiveness. Thymic selection of peripheral Ly-49-expressing iNKT cell repertoire inhibited cytokine production and other functions in vivo. These observations emphasize the acquisition of self-recognizing inhibitory receptors on NKT cells as a previously unknown mechanism of thymic tolerance after chronic antigen exposure.

The molecular basis of thymocyte negative selection, which plays a critical role in establishing and maintaining immunological tolerance, is not yet resolved. In particular, the importance of the death receptor subgroup of the tumor necrosis factor (TNF)-family has been the subject of many investigations, with equivocal results. A recent report suggested that TRAIL was a critical factor in this process, a result that does not fit well with previous studies that excluded a role for the FADD-caspase 8 pathway, which is essential for TRAIL and Fas ligand (FasL) signaling, in negative selection. We have investigated intrathymic negative selection of TRAIL-deficient thymocytes, using four well-established models, including antibody-mediated TCR/CD3 ligation in vitro, stimulation with endogenous superantigen in vitro and in vivo, and treatment with exogenous superantigen in vitro. We were unable to demonstrate a role for TRAIL signaling in any of these models, suggesting that this pathway is not a critical factor for thymocyte negative selection.

Given the importance of the NOD mouse as a model of type 1 diabetes, there is a surprising lack of published information on the overall composition of the thymic and peripheral T-cell compartments. In this study, we revisited some earlier reports of T-cell abnormalities in this strain and examined a number of additional parameters to provide a global view of T-cells in prediabetic NOD mice. In some cases, we concur with past conclusions, but in other important areas, we find that NOD mice closely resemble nonautoimmune strains. Specifically, and contrary to published reports, the thymocyte subset distribution, the rate and composition of thymic export, and the composition of the peripheral T-cell pool, including the proportion of CD25+CD4+ T-cells, are essentially normal in prediabetic NOD mice. These factors are therefore unlikely to be involved in the loss of tolerance that leads to autoimmunity within this strain.

Recent studies in mice and humans show that the importance of the thymus extends well beyond the initial seeding of the peripheral T-cell pool. Although peripheral homeostasis can maintain T-cell numbers, the thymus is the major, if not the exclusive, source of new T-cell specificities. With age, thymus atrophy dramatically reduces the export of new T cells and predisposes an individual to impaired T-cell function, reduced T-cell immunity, and increased autoimmunity. Thymus atrophy is also the primary obstacle to restoration of the T-cell pool in the aftermath of HIV treatment or lymphoablative therapies. Here, we review thymus T-cell production, with particular attention to the factors that influence thymocyte export, and examine the impact that recent thymic emigrants have on the peripheral pool. In the future, thymic regeneration might become a feasible and potentially powerful approach to rejuvenating a depleted peripheral T-cell pool.

Natural killer T (NKT) cells are credited with regulatory roles in immunity against cancers, autoimmune diseases, allergies, and bacterial and viral infections. Studies in mice and observational research in patient groups have suggested that NKT cell-based therapies could be used to prevent or treat these diseases, yet the translation into clinical settings has been disappointing. We support the view that NKT cells have regulatory characteristics that could be exploited in clinical settings, but there are doubts about the natural roles of NKT cells in vivo and whether NKT cell defects are fundamental drivers of disease in humans. In this Opinion article, we discuss the uncertainties and opportunities regarding NKT cells in humans, and the potential for NKT cells to be manipulated to prevent or treat disease.

Telomeres are specialized nucleoprotein structures that protect chromosomal ends from degradation. These structures progressively shorten during cellular division and can signal replicative senescence below a critical length. Telomere length is predominantly maintained by the enzyme telomerase. Significant decreases in telomere length and telomerase activity are associated with a host of chronic diseases; conversely their maintenance underpins the optimal function of the adaptive immune system. Habitual physical activity is associated with longer leukocyte telomere length; however, the precise mechanisms are unclear. Potential hypotheses include regulation of telomeric gene transcription and/or microRNAs (miRNAs). We investigated the acute exercise-induced response of telomeric genes and miRNAs in twenty-two healthy males (mean age = 24.1±1.55 years). Participants undertook 30 minutes of treadmill running at 80% of peak oxygen uptake. Blood samples were taken before exercise, immediately post-exercise and 60 minutes post-exercise. Total RNA from white blood cells was submitted to miRNA arrays and telomere extension mRNA array. Results were individually validated in white blood cells and sorted T cell lymphocyte subsets using quantitative real-time PCR (qPCR). Telomerase reverse transcriptase (TERT) mRNA (P = 0.001) and sirtuin-6 (SIRT6) (P

Natural killer T (NKT) cells are a subset of CD1d-restricted, glycolipid-reactive T cells that express a semi-invariant T cell receptor alpha (TCRa) chain (Va14–Ja18 in mice, Va24–Ja18 in humans) paired with Vb8.2, Vb7 or Vb2 in mice and Vb11 in humans. NKT cells are thought to have an immunoregulatory role mediated by their rapid production of Th1, Th2 and Th17 type cytokines, with NKT cell dysfunction and/or deficiency associated with many diverse diseases.1,2 These defects can emerge as a consequence of problems associated with NKT cell development, making it crucially important to understand this process in order to identify targets for treating NKT cell-associated human diseases. NKT cells develop from CD4+CD8+ double-positive (DP) thymocytes that have randomly produced (through TCR gene rearrangement) the semi-invariant NKT cell TCR. These cells are selected by CD1d (probably in combination with self-glycolipid antigens) expressed by other DP thymocytes. Although several different factors that regulate NKT cell development have been identified (for example, SLAM/SAP, PLZF, c-myc, Egr2),2 in most cases, they regulate the maturation of selected NKT cells, rather than the initial selection event that generates this lineage. A new study by D’Cruz et al. in Nature Immunology3 identifies the E-protein HeLa E-box binding protein (HEB) as a key factor in the earliest stages of NKT cell selection. HEB is a member of a family of basic helix–loop–helix transcription factors, collectively called E proteins (also comprising E12 and E47 (E2A), and E2-2), that regulate key steps in lymphocyte development. E proteins function by forming homodimers and heterodimers that bind to E-box regions on DNA to regulate transcriptional activation and repression.4 They are negatively regulated by inhibitor of DNA binding (Id) factors, which dimerize with E proteins to prevent DNA binding.5 In the thymus, E proteins control TCRa and TCRb gene rearrangements and expression, regulate thymocyte survival and proliferation, and enforce TCR-dependent selection checkpoints, including TCRb selection and positive selection.4 Severe T cell deficiencies are observed in HEB/E2A double-knockout mice and dominant-negative HEB mutants, whereas only mild defects in T cell development are found in E2A and HEB single-gene knockouts, demonstrating functional redundancy within the E protein family.6–9 A role for E proteins has not previously been described for NKT cell development; however, there is evidence that the E protein antagonist Id2 may control NKT cell homeostasis,10 with Id2-deficient NKT cells developing normally in the thymus, but having reduced expression of factors important for survival (Bcl-2 and Bcl-xL) and normal accumulation in the liver (CXCR6).10 The study by D’Cruz et al.3 has now demonstrated severely impaired NKT cell development (but seemingly normal T cell development) in mice with a T cell-specific HEB deletion (HEB-TKO), but not in mice with E2Adeficient thymocytes. NKT cells follow a maturational pathway in the thymus that begins at stage 0 (CD24+) and proceeds through stage 1 (CD24 CD44loNK1.1 ), stage 2 (CD24 CD44hiNK1.1 ) and stage 3 (CD24 CD44hiNK1.1+) (Figure 1), with the transition from stage 2 to stage 3 occurring either within the thymus or after export of stage 2 cells to the periphery.11 In many instances, defects in NKT cell development manifest between stage 1 and stage 3.11 HEB deletion appeared to impact heavily on the NKT cell numbers even at stage 0, suggesting an effect at the earliest selection stage and resulting in a dramatic reduction of NKT cells in the thymus, spleen and liver.3 The NKT cell developmental deficiency was not restored in the HEB-deficient thymocyte component of WT/HEB-TKO mixed BM chimeras, demonstrating that HEB deletion impacted directly on NKT cells, rather than affecting NKT cell selection by antigenpresenting cells. HEB deletion directly impacted on the survival and proliferation of DP thymocytes and was associated with lower expression of RORgt and BclxL. Rearrangement of Va to Ja segments occurs at the DP thymocyte stage, and this typically involves proximally located Ja genes in the initial (primary) rearrangements. DP cells that are not positively selected continue to rearrange TCR-Va genes with progressively more distal TCR-Ja genes (secondary rearrangements) until they are either positively selected or die through neglect.12 The TCR Ja18 gene, essential for formation of the NKT cell invariant TCRa chain, is distally located and typically rearranged late in the DP thymocyte lifespan as a secondary rearrangement. Thus, NKT cell development, which is critically dependent on the TCR Va14-Ja18 rearrangement, can be impaired in mice with a shortened DP lifespan, as observed in RORgt / mice.13,14 Indeed, HEB-TKO mutants had a skewed TCR repertoire with an abnormally low frequency of Va to distal Ja rearrangements. Moreover, the NKT cell defect in these mice was almost fully rescued by expression of a Va14–Ja18 TCR transgene, or by retroviral BclxL expression that prolongs DP thymocyte survival to allow more distal TCR rearrangements to occur.3 Taken together, these findings indicate that the defect in NKT cell development observed in HEB-deficient thymocytes was primarily because of an impaired ability to generate the NKT semi-invariant, Va14–Ja18 TCR. However, a further role for HEB in NKT cell maturation was suggested by two addiAC Chan, SP Berzins and DI Godfrey are at the Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail: godfrey@unimelb.edu.au Immunology and Cell Biology (2010) 88, 507–509 & 2010 Australasian Society for Immunology Inc. All rights reserved 0818-9641/10 $32.00

Thymic shared Ag-2 (TSA-2) is a 28-kDa, glycophosphatidylinitosol-linked cell surface molecule expressed on various T cell and thymic stromal cell subsets. It is expressed on most CD3−CD4−CD8−, CD4+CD8+, and CD3highCD4−CD8+ thymocytes but is down-regulated on ∼40% of CD3highCD4+CD8− thymocytes. Expression on peripheral TCR-αβ+ T cells is similar to that of CD3+ thymocytes, although a transient down-regulation occurs with cell activation. Consistent with the recent hypothesis that emigration from the thymus is an active process, recent thymic emigrants are primarily TSA-2−/low. TSA-2 expression reveals heterogeneity among subpopulations of CD3highCD4+CD8− thymocytes and TCR-γδ+ T cell previously regarded as homogenous. The functional importance of TSA-2 was illustrated by the severe block in T cell differentiation caused by adding purified anti-TSA-2 mAb to reconstituted fetal thymic organ culture. While each CD25/CD44-defined triple-negative subset was present, differentiation beyond the TN stage was essentially absent, and cell numbers of all subsets were significantly below those of control cultures. Cross-linking TSA-2 on thymocytes caused a significant Ca2+ influx but no increase in apoptosis, unless anti-TSA-2 was used in conjunction with suboptimal anti-CD3 mAb. Similar treatment of mature TSA-2+ T cells had no effect on cell survival or proliferation. This study reveals TSA-2 to be a functionally important molecule in T cell development and a novel indicator of heterogeneity among a variety of developing and mature T cell populations.

The thymus is essential for the initial seeding of T cells to the periphery, but its role in maintaining the adult T cell pool remains poorly defined. We investigated whether changes to the rate of T cell export could form part of the mechanism(s) controlling the homeostatic regulation of the size and composition of the peripheral T cell pool. Using neonatal thymi grafted under the kidney capsule, we found that irrespective of whether the pool was oversupplied (by thymic grafts) or undersupplied (due to neonatal thymectomy), the thymic export rate was constant from both the host and graft thymus, and the periphery remained constant in size. Recent thymic emigrants (RTE) were also tracked to determine the extent of their acceptance into the T cell pool of a normal mouse. As a population, RTE are phenotypically mature, but were distinct from resident T cells in the periphery, being released in a CD4/CD8 ratio approximately twice that of established peripheral T cells. This export ratio is similar to that of T cells in the mature thymic compartment, but soon after entry into the periphery, the ratio falls, indicating separate thymic and peripheral regulation of the CD4/CD8 ratio. RTE may also be preferentially incorporated into the periphery, causing displacement of resident T cells, thus maintaining the size of the peripheral pool. Although not vital for the maintenance of a functional T cell pool, the acceptance of RTE in a “full” peripheral pool would ensure that the T cell receptor repertoire is kept diverse and that the T cell population encompasses a broad range of naive as well as memory T cells.

Whether natural killer T cells recognize antigens derived from dangerous pathogens remains unclear. New data demonstrate that a glycolipid from Borrelia burgdorferi, the causative agent of Lyme disease, directly stimulates mouse and human natural killer T cells.

Initially identified as a nuclear factor in B cells, the family of NFκB transcription factors have since been found to operate in almost all cell types, regulating the transcription of a wide range of target genes. The NFκB signalling pathway is of particular importance to T lymphocytes, playing a prominent role in both T cell development and function. This review will focus on the current understanding of the roles of NFκB during thymic T cell development, with an emphasis on some of the emerging roles for NFκB signalling in regulating the development of non-conventional thymocyte lineages. We will also evaluate the function of NFκB signalling in the polarization of T-helper subsets in the periphery, and how NFκB intersects with other T cell-intrinsic pathways through mechanisms of signalling crosstalk. Dysregulated NFκB signalling is implicated in numerous disease states, and a thorough understanding of NFκB function during different phases of T cell development and function will be vital for optimal targeting in a therapeutic setting.