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Tissue-resident memory T (TRM) cells are non-recirculating cells that exist throughout the body. Although TRM cells 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 TRM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate TRM cell 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- TRM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFβ-responsive CD103+ TRM counterparts. Furthermore, whereas CD103- TRM cells readily modified their phenotype upon relocation, CD103+ TRM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for TRM cell development, tissue adaptation of these cells confers discrete functional properties such that TRM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment.

Preterm infants are more susceptible to severe bacterial and viral infectious diseases than their full-term counterparts. A major contributor to this increased susceptibility may be due to differences in their ability to respond to pathogens. While studies have demonstrated altered bacterial Toll-like receptor (TLR) responses, there is limited data on viral TLR responses in preterm infants. In this study, cord blood mononuclear cells (CBMCs) from 10 moderately preterm (30.4–34.1 wGA), 10 term (37–39.5 wGA) infants, and 5 adults were stimulated with TLR2 (lipoteichoic acid), TLR3 (poly I:C), TLR4 (lipopolysaccharide), TLR7/8 (R848), and TLR9 (CpG-ODN 2216) agonists. Following stimulation, the cellular response was measured by intracellular flow cytometry to detect cell-specific NF-κB (as a marker of the inflammatory response), and multiplex assays were used to measure the cytokine response. This study found that preterm and term infants exhibit very similar baseline TLR expression. In response to both bacterial and viral TLR agonists comparing cell-specific NF-κB activation, preterm infants exhibited increased monocyte activation following LTA stimulation; however, no other differences were observed. Similarly, no difference in cytokine response was observed following stimulation with TLRs. However, a stronger correlation between NF-κB activation and cytokine responses was observed in term infants following poly I:C and R848 stimulation compared to preterm infants. In contrast, despite similar TLR expression, adults produced higher levels of IFN-α following R848 stimulation compared to preterm and term infants. These findings suggest preterm and term infants have a similar capacity to respond to both bacterial and viral TLR agonists. As preterm infants are more likely to develop severe infections, further research is required to determine the immunological factors that may be driving this and develop better interventions for this highly vulnerable group.

Conventional T cells recognise protein-derived antigens in the context of major histocompatibility complex (MHC) class Ia and class II molecules and provide anti-microbial and anti-tumour immunity. Conventional T cells have also been implicated in type IV (also termed delayed-type or T cell-mediated) hypersensitivity reactions in response to protein-derived allergen antigens. In addition to conventional T cells, subsets of unconventional T cells exist, which recognise non-protein antigens in the context of monomorphic MHC class I-like molecules. These include T cells that are restricted to the cluster of differentiation 1 (CD1) family members, known as CD1-restricted T cells, and mucosal-associated invariant T cells (MAIT cells) that are restricted to the MHC-related protein 1 (MR1). Compared with conventional T cells, much less is known about the immune functions of unconventional T cells and their role in hypersensitivities. Here, we review allergen antigen presentation by MHC-I-like molecules, their recognition by unconventional T cells, and the potential role of unconventional T cells in hypersensitivities. We also speculate on possible scenarios of allergen antigen presentation by MHC-I-like molecules to unconventional T cells, the hallmarks of such responses, and the expected frequencies of hypersensitivities within the human population.

BackgroundPreterm infants are highly vulnerable to infectious disease. While many factors are likely to contribute to this enhanced susceptibility, the immature nature of the preterm immune system is postulated as one key factor.MethodsIn our study, we used high-dimensional flow cytometry and cytokine assays to characterise the immune profiles in 25 preterm (range: 30.4-34.1 weeks gestational age) and 25 term infant (range: 37-40 weeks gestational age) cord blood samples.ResultsWe found that preterm infants exhibit reduced frequencies of monocytes, CD56bright NK cells, CD8+ T-cells, γδ T-cells and an increased frequency of intermediate monocytes, CD4+ T-cells, central memory CD4+ and CD8+ T-cells, Tregs and transitional B-cells compared to term infants. Pro-inflammatory cytokines IL-1β, IL-6 and IL-17A were lower in preterm infants in addition to chemokines IL-8, eotaxin, MIP-1α and MIP-1β. However, IL-15 and MCP-1 were higher in preterm infants.ConclusionOverall, we identify key differences in pro-inflammatory immune profiles between preterm and term infants. These findings may help to explain why preterm infants are more susceptible to infectious disease during early life and facilitate the development of targeted interventions to protect this highly vulnerable group.

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.

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.

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.

Lyme disease is a common multi-system disease caused by infection with a tick-transmitted spirochete, Borrelia burgdorferi and related Borrelia species. The monoglycosylated diacylglycerol known as B. burgdorferi glycolipid II (BbGL-II) is a major target of antibodies in sera from infected individuals. Here we show that CD1b presents BbGL-II to human T cells and that the T cell receptor (TCR) mediates the recognition. However, we did not detect increased frequency of CD1b-BbGL-II binding T cells in the peripheral blood of Lyme disease patients compared to controls. Unexpectedly, mapping the T cell specificity for BbGL-II-like molecules using tetramers and activation assays revealed a concomitant response to CD1b-expressing antigen presenting cells in absence of BbGL-II. Further, among all major classes of self-lipid tested, BbGL-II responsive TCRs show strong cross-reactivity to diacylglycerol, a self-lipid antigen with structural similarities to BbGL-II. Extending prior work on MHC and CD1b, CD1c, and CD1d proteins, this study provides evidence for cross-reactive CD1b-restricted T cell responses to bacterial and self-antigens, and identifies chemically defined targets for future discovery of self and foreign antigen cross-reactive T cells. This article is protected by copyright. All rights reserved.

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.

Mucosal‐associated invariant T (MAIT) cells develop in the thymus and migrate into the periphery to become the largest antigen‐specific αβ T‐cell population in the human immune system. However, the frequency of MAIT cells varies widely between human individuals, and the basis for this is unclear. While MAIT cells are highly conserved through evolution and are phenotypically similar between humans and mice, they represent a much smaller proportion of total T cells in mice. In this review, we discuss how MAIT cells transition through a three‐stage development pathway in both mouse and human thymus, and continue to mature and expand after they leave the thymus. Moreover, we will explore and speculate on how specific factors regulate different stages of this process.

International audience; Objectives. Innate lymphoid cells (ILCs) share many characteristics with CD4(+) T cells, and group 1 ILCs share a requirement for T-bet and the ability to produce IFNc with T helper 1 (Th1) cells. Given this similarity, and the importance of Th1 cells for protection against intracellular protozoan parasites, we aimed to characterise the role of group 1 ILCs during Plasmodium infection. Methods. We quantified group 1 ILCs in peripheral blood collected from subjects infected with with Plasmodium falciparum 3D7 as part of a controlled human malaria infection study, and in the liver and spleens of PcAS-infected mice. We used genetically-modified mouse models, as well as cell-depletion methods in mice to characterise the role of group 1 ILCs during PcAS infection. Results. In a controlled human malaria infection study, we found that the frequencies of circulating ILC1s and NK cells decreased as infection progressed but recovered after volunteers were treated with antiparasitic drug. A similar observation was made for liver and splenic ILC1s in P. chabaudi chabaudi AS (PcAS)-infected mice. The decrease in mouse liver ILC1 frequencies was associated with increased apoptosis. We also identified a population of cells within the liver and spleen that expressed both ILC1 and NK cell markers, indicative of plasticity between these two cell lineages. Studies using genetic and cell-depletion approaches indicated that group 1 ILCs have a limited role in antiparasitic immunity during PcAS infection in mice. Discussion. Our results are consistent with a previous study indicating a limited role for natural killer (NK) cells during Plasmodium chabaudi infection in mice. Additionally, a recent study reported the redundancy of ILCs in humans with competent B and T cells. Nonetheless, our results do not rule out a role for group 1 ILCs in human malaria in endemic settings given that blood stage infection was initiated intravenously in our experimental models, and thus bypassed the liver stage of infection, which may influence the immune response during the blood stage. Conclusion. Our results show that ILC1s are lost early during mouse and human malaria, and this observation may help to explain the limited role for these cells in controlling blood stage infection.

A central paradigm in αβ T cell–mediated immunity is the simultaneous co-recognition of antigens and antigen-presenting molecules by the αβ T cell antigen receptor (TCR). CD1a presents a broad repertoire of lipid-based antigens. We found that a prototypical autoreactive TCR bound CD1a when it was presenting a series of permissive endogenous ligands, while other lipid ligands were nonpermissive to TCR binding. The structures of two TCR-CD1a-lipid complexes showed that the TCR docked over the A′ roof of CD1a in a manner that precluded direct contact with permissive ligands. Nonpermissive ligands indirectly inhibited TCR binding by disrupting the TCR-CD1a contact zone. The exclusive recognition of CD1a by the TCR represents a previously unknown mechanism whereby αβ T cells indirectly sense self antigens that are bound to an antigen-presenting molecule.

Rahimpour et al. use MR1 tetramers to characterize the heterogeneous population of mouse MAIT cells and find a close resemblance to their human counterparts. These findings will provide the foundation for further investigation of MAIT cells in health and disease., 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.

Human CMV still remains problematic in immunocompromised patients, particularly after solid organ transplantation. CMV primary disease and reactivation greatly increase the risks associated with incidences of chronic allograft rejection and decreased survival in transplant recipients. But whether this is due to direct viral effects, indirect viral effects including cross-reactive antiviral T cell immunopathology, or a combination of both remains undetermined. In this article, we report the novel TCR signature of cross-reactive HLA-A*02:01 (A2) CMV (NLVPMVATV [NLV])–specific CD8+ T cells recognizing a specific array of HLA-B27 alleles using technical advancements that combine both IFN-γ secretion and multiplex nested RT-PCR for determining paired CDR3α/β sequences from a single cell. This study represents the first evidence, to our knowledge, of the same A2-restricted cross-reactive NLV-specific TCR-α/β signature (TRAV3TRAJ31_TRBV12-4TRBJ1-1) in two genetically distinct individuals. Longitudinal posttransplant monitoring of a lung transplant recipient (A2, CMV seropositive) who received a HLA-B27 bilateral lung allograft showed a dynamic expansion of the cross-reactive NLV-specific TCR repertoire before CMV reactivation. After resolution of the active viral infection, the frequency of cross-reactive NLV-specific CD8+ T cells reduced to previremia levels, thereby demonstrating immune modulation of the T cell repertoire due to antigenic pressure. The dynamic changes in TCR repertoire, at a time when CMV reactivation was subclinical, illustrates that prospective monitoring in susceptible patients can reveal nuances in immune profiles that may be clinically relevant.

Godfrey, Rossjohn, and colleagues define a population of T cells in healthy humans that express T cell receptors (TCRs) comprised of δ variable gene segments fused to α joining and constant domains and paired with a variety of TCR-β chains. Functional and structural analyses reveal how components of αβ and γδ TCR gene loci combine to create T cells with unique patterns of antigen recognition., 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.

The T cell repertoire comprises αβ and γδ T cell lineages. Although it is established how αβ T cell antigen receptors (TCRs) interact with antigen presented by antigen-presenting molecules, this is unknown for γδ TCRs. We describe a population of human V δ 1 + γδ T cells that exhibit autoreactivity to CD1d and provide a molecular basis for how a γδ TCR binds CD1d-α- galactosylceramide (α-GalCer). The γδ TCR docked orthogonally, over the A′ pocket of CD1d, in which the V δ 1-chain, and in particular the germ line-encoded CDR1δ loop, dominated interactions with CD1d. The TCR γ-chain sat peripherally to the interface, with the CDR3γ loop representing the principal determinant for α-GalCer specificity. Accordingly, we provide insight into how a γδ TCR binds specifically to a lipid-loaded antigen-presenting molecule.

SummaryThe antigen receptor for natural killer T cells (NKT TCR) binds CD1d-restricted microbial and self-lipid antigens, although the molecular basis of self-CD1d recognition is unclear. Here, we have characterized NKT TCR recognition of CD1d molecules loaded with natural self-antigens (Ags) and report the 2.3 Å resolution structure of an autoreactive NKT TCR-phosphatidylinositol-CD1d complex. NKT TCR recognition of self- and foreign antigens was underpinned by a similar mode of germline-encoded recognition of CD1d. However, NKT TCR autoreactivity is mediated by unique sequences within the non-germline-encoded CDR3β loop encoding for a hydrophobic motif that promotes self-association with CD1d. Accordingly, NKT cell autoreactivity may arise from the inherent affinity of the interaction between CD1d and the NKT TCR, resulting in the recognition of a broad range of CD1d-restricted self-antigens. This demonstrates that multiple self-antigens can be recognized in a similar manner by autoreactive NKT TCRs.

SummaryNatural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t1/2 life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells.

alphabeta T cell receptors (TCRs) have traditionally been viewed as receptors for peptide antigens presented by either Major Histocompatibility Complex (MHC) class I (for CD8 T cells) or MHC class II (for CD4 T cells) antigen-presenting molecules. However, it is now clear that some T cell lineages express TCRs that are specialized for recognition of lipid-based antigens presented by the MHC class I-like CD1 family. Recently, the molecular basis for the TCR recognition of glycolipid antigens presented by CD1d has revealed an evolutionarily conserved-docking mode that is distinct from that of peptide-based recognition. T cells carrying these receptors follow a unique developmental pathway that results not only in unconventional antigen specificity, but also seemingly exaggerated functional capabilities, which makes these cells and their antigens highly attractive targets for immunotherapeutic manipulation.

Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.

Invariant NKT cells play a critical role in controlling the strength and character of adaptive immune responses. We have previously reported deficiencies in the numbers and function of NKT cells in the NOD mouse strain, which is a well-validated model of type 1 diabetes and systemic lupus erythematosus. Genetic control of thymic NKT cell numbers was mapped to two linkage regions: Nkt1 on distal chromosome 1 and Nkt2 on chromosome 2. In this study, we report the production and characterization of a NOD.Nkrp1b.Nkt1b congenic mouse strain, apply microarray expression analyses to limit candidate genes within the 95% confidence region, identify Slamf1 (encoding signaling lymphocyte activation molecule) and Slamf6 (encoding Ly108) as potential candidates, and demonstrate retarded signaling lymphocyte activation molecule expression during T cell development of NOD mice, resulting in reduced expression at the CD4+CD8+ stage, which is consistent with decreased NKT cell production and deranged tolerance induction in NOD mice.

NKT cells recognize lipid-based Ags presented by CD1d. Type I NKT cells are often referred to as invariant owing to their mostly invariant TCR α-chain usage (Vα14-Jα18 in mice, Vα24-Jα18 in humans). However, these cells have diverse TCR β-chains, including Vβ8, Vβ7, and Vβ2 in mice and Vβ11 in humans, joined to a range of TCR Dβ and Jβ genes. In this study, we demonstrate that TCR β-chain composition can dramatically influence lipid Ag recognition in an Ag-dependent manner. Namely, the glycolipids α-glucosylceramide and isoglobotrihexosylceramide were preferentially recognized by Vβ7+ NKT cells from mice, whereas the α-galactosylceramide analog OCH, with a truncated sphingosine chain, was preferentially recognized by Vβ8+ NKT cells from mice. We show that the influence of the TCR β-chain is due to a combination of Vβ-, Jβ-, and CDR3β-encoded residues and that these TCRs can recapitulate the selective Ag reactivity in TCR-transduced cell lines. Similar observations were made with human NKT cells where different CDR3β-encoded residues determined Ag preference. These findings indicate that NKT TCR β-chain diversity results in differential and nonhierarchical Ag recognition by these cells, which implies that some Ags can preferentially activate type I NKT cell subsets.

Tissue-resident memory T (Trm) cells contribute to local immune protection in non-lymphoid tissues such as skin and mucosa, but little is known about their transcriptional regulation. Here we showed that CD8(+)CD103(+) Trm cells, independent of circulating memory T cells, were sufficient for protection against infection and described molecular elements that were crucial for their development in skin and lung. We demonstrated that the T-box transcription factors (TFs) Eomes and T-bet combined to control CD8(+)CD103(+) Trm cell formation, such that their coordinate downregulation was crucial for TGF-β cytokine signaling. TGF-β signaling, in turn, resulted in reciprocal T-box TF downregulation. However, whereas extinguishment of Eomes was necessary for CD8(+)CD103(+) Trm cell development, residual T-bet expression maintained cell surface interleukin-15 (IL-15) receptor β-chain (CD122) expression and thus IL-15 responsiveness. These findings indicate that the T-box TFs control the two cytokines, TGF-β and IL-15, which are pivotal for CD8(+)CD103(+) Trm cell development and survival.

A characteristic of mucosal-associated invariant T (MAIT) cells is the expression of TRAV1-2(+) T cell receptors (TCRs) that are activated by riboflavin metabolite-based antigens (Ag) presented by the MHC-I related molecule, MR1. Whether the MR1-restricted T cell repertoire and associated Ag responsiveness extends beyond these cells remains unclear. Here, we describe MR1 autoreactivity and folate-derivative reactivity in a discrete subset of TRAV1-2(+) MAIT cells. This recognition was attributable to CDR3β loop-mediated effects within a consensus TRAV1-2(+) TCR-MR1-Ag footprint. Furthermore, we have demonstrated differential folate- and riboflavin-derivative reactivity by a diverse population of "atypical" TRAV1-2(-) MR1-restricted T cells. We have shown that TRAV1-2(-) T cells are phenotypically heterogeneous and largely distinct from TRAV1-2(+) MAIT cells. A TRAV1-2(-) TCR docks more centrally on MR1, thereby adopting a markedly different molecular footprint to the TRAV1-2(+) TCR. Accordingly, diversity within the MR1-restricted T cell repertoire leads to differing MR1-restricted Ag specificity.

Glycolipid ligands for invariant natural killer T cells (iNKT cells) are loaded onto CD1d molecules in the late endosome/lysosome. Accumulation of glycosphingolipids (GSLs) in lysosomal storage diseases could potentially influence endogenous and exogenous lipid loading and/or presentation and, thus, affect iNKT cell selection or function. The percentages and frequency of iNKT cells were reduced in multiple mouse models of lysosomal GSL storage disease, irrespective of the specific genetic defect or lipid species stored. Reduced numbers of iNKT cells resulted in the absence of cytokine production in response to alpha-galactosylceramide (alpha-GalCer) and reduced iNKT cell-mediated lysis of wild-type targets loaded with alpha-GalCer. The reduction in iNKT cells did not result from defective expression of CD1d or a lack of antigen-presenting cells. Although H-2 restricted CD4(+) T cell responses were generally unaffected, processing of a lysosome-dependent analogue of alpha-GalCer was impaired in all the strains of mice tested. These data suggest that GSL storage may result in alterations in thymic selection of iNKT cells caused by impaired presentation of selecting ligands.

Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.

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.

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.

The development of CD1d-dependent natural killer T (NKT) cells is poorly understood. We have used both CD1d/alpha-galactosylceramide (CD1d/alphaGC) 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/alphaGC 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/alphaGC 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.

Natural killer T cells (NKT cells) recognize lipid-based antigens presented by CD1d. The mammalian glycolipid β-glucosylceramide, a ubiquitous self antigen for NKT cells, is upregulated by microbial danger signals, which leads to activation of NKT cells in the absence of foreign glycolipid antigen.

Current views emphasize TCR diversity as a key feature that differentiates the group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 systems. Whereas TCR sequence motifs define CD1d-reactive NKT cells, the available data do not allow a TCR-based organization of the group 1 CD1 repertoire. The observed TCR diversity might result from donor-to-donor differences in TCR repertoire, as seen for MHC-restricted T cells. Alternatively, diversity might result from differing CD1 isoforms, Ags, and methods used to identify TCRs. Using CD1b tetramers to isolate clones recognizing the same glycolipid, we identified a previously unknown pattern of V gene usage (TRAV17, TRBV4-1) among unrelated human subjects. These TCRs are distinct from those present on NKT cells and germline-encoded mycolyl lipid–reactive T cells. Instead, they resemble the TCR of LDN5, one of the first known CD1b-reactive clones that was previously thought to illustrate the diversity of the TCR repertoire. Interdonor TCR conservation was observed in vitro and ex vivo, identifying LDN5-like T cells as a distinct T cell type. These data support TCR-based organization of the CD1b repertoire, which consists of at least two compartments that differ in TCR sequence motifs, affinity, and coreceptor expression.

CD1d-restricted Vα14-Jα281 invariant αβTCR+ (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 αβTCR+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/α-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.

Natural killer T cells (NKT cells) are divided into type I and type II subsets on the basis of differences in their T cell antigen receptor (TCR) repertoire and CD1d-antigen specificity. Although the mode by which type I NKT cell TCRs recognize CD1d-antigen has been established, how type II NKT cell TCRs engage CD1d-antigen is unknown. Here we provide a basis for how a type II NKT cell TCR, XV19, recognized CD1d-sulfatide. The XV19 TCR bound orthogonally above the A' pocket of CD1d, in contrast to the parallel docking of type I NKT cell TCRs over the F' pocket of CD1d. At the XV19 TCR-CD1d-sulfatide interface, the TCRα and TCRβ chains sat centrally on CD1d, where the malleable CDR3 loops dominated interactions with CD1d-sulfatide. Accordingly, we highlight the diverse mechanisms by which NKT cell TCRs can bind CD1d and account for the distinct antigen specificity of type II NKT cells.

The most potent foreign antigens for natural killer T cells (NKT cells) are α-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct β-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated β-linked agonists β-galactosylceramide (β-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the β-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the β-linked self ligands to resemble the conformation of foreign α-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to β-linked antigens.

Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(α)14-J(α)18) T cell antigen receptor (TCR) α-chain and recognition of the glycolipid α-galactosylceramide (α-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of α-GalCer-reactive NKT cells that expressed a canonical V(α)10-J(α)50 TCR α-chain, which showed a preference for α-glucosylceramide (α-GlcCer) and bacterial α-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCRα sequence identity, the V(α)10 TCR-CD1d-α-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-α-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.

NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag α-galactosylceramide (α-GalCer). A modified analog of α-GalCer with a carbon-based glycosidic linkage (α-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to α-C-GalCer, and related C-glycoside ligands, is weaker than that of α-GalCer. Furthermore, the Vβ8.2 and Vβ7 NKT TCR affinity for CD1d–α-C-GalCer, and some related analogs, is ∼10-fold lower than that for the NKT TCR–CD1d–α-GalCer interaction. Nevertheless, the crystal structure of the Vβ8.2 NKT TCR–CD1d–α-C-GalCer complex is similar to that of the corresponding NKT TCR–CD1d–α-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR–CD1d–α-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies., We thank the synchrotron staff at the MX2 beamline of the Australian synchrotron for assistance with data collection and David Taylor and the animal house staff at the University of Melbourne, Department of Microbiology and Immunology, for animal husbandry assistance. We also thank the National Institutes of Health tetramer facility for providing us with a-C-GalCer and Maria Sandoval for technical assistance.

CD1d presents lipid-based antigens (Ag) that are recognised by the semi-invariant T cell receptor (TCR) expressed on Natural Killer T (NKT) cells. While the TCR α-chain is typically invariant, the TCR β-chain expression is more diverse, particularly in mice where at least three different Vβ chains are commonly expressed. We report the structures of Vα14-Vβ8.2 and Vα14-Vβ7 NKT TCRs in complex with CD1d-α-galactosylceramide (α-GalCer), as well as a 2.5 Å structure of the human NKT TCR-CD1d-α-GalCer complex. Both Vβ8.2 and Vβ7 NKT TCRs, as well as the human NKT TCR, ligated CD1d-α-GalCer in a broadly similar manner, thereby highlighting the evolutionarily-conserved nature of this interaction. However, differences within the Vβ domains of the Vβ8.2 and Vβ7 NKT TCR-CD1d complexes not only resulted in altered TCR-β-CD1d-mediated contacts, but also surprisingly modulated recognition mediated by the invariant α-chain. Mutagenesis studies revealed the differing contributions of Vβ8.2 and Vβ7 residues within the CDR2β loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR Vβ usage in NKT cells.

One interesting aspect of NKT cell development is that although they are thymus dependent, the pivotal transition from NK1.1− to NK1.1+ can often take place after immature NK1.1− NKT cells are exported to the periphery. NK1.1− NKT cells in general are regarded as immature precursors of NK1.1+ NKT cells, meaning that peripheral NK1.1− NKT cells are regarded as a transient, semimature population of recent thymic emigrant NKT cells. In this study, we report the unexpected finding that most NK1.1− NKT cells in the periphery of naive mice are actually part of a stable, mature and functionally distinct NKT cell population. Using adult thymectomy, we show that the size of the peripheral NK1.1− NKT cell pool is maintained independently of thymic export and is not the result of NK1.1 down-regulation by mature cells. We also demonstrate that most peripheral NK1.1− NKT cells are functionally distinct from their immature thymic counterparts, and from NK1.1+ NKT cells in the periphery. We conclude that the vast majority of peripheral NK1.1− NKT cells are part of a previously unrecognized, mature NKT cell subset.

Activation of NKT cells using the glycolipid α-galactosylceramide (α-GalCer4) has availed many investigations into their immunoregulatory and therapeutic potential. However, it remains unclear how NKT cells respond to stimulation in vivo, which co-stimulatory pathways are important, and what factors (eg. antigen availability and activation-induced cell death) limit their response. We have explored these questions in the context of anin vivo model of NKT cell dynamics spanning activation, population expansion and subsequent contraction. Neither the B7/CD28 nor the CD40/CD40-L co-stimulatory pathways were necessary for cytokine production by activated NKT cells, either early (2 hours) or late (3 days) following initial stimulation, but both pathways were necessary for normal proliferative expansion of NKT cells in vivo. The pro-apoptotic Bcl-2 family member Bim was necessary for normal contraction of the NKT cell population between days 3-9 after stimulation, suggesting the pool size is regulated by apoptotic cell death in a manner similar to that of conventional T cells. Antigen availability was not the limiting factor for NKT cell expansion in vivo, and a second injection of α-GalCer induced a very blunted response, whereby cytokine production was reduced and further expansion did not occur. This appeared to be a form of anergy that was intrinsic to the NKT cells and not associated with up-regulation of inhibitory NK cell receptors such as NKG2A or Ly49 family members. Furthermore, NKT cells from mice pre-challenged with α-GalCer in vivoshowed little cytokine production and reduced proliferation in vitro. In summary, this study significantly enhances our understanding of how NKT cells respond to α-GalCer in vivo, revealing that the full primary response depends on costimulation via the CD28 and CD40 pathways, with subsequent Bim-dependent contraction. After contraction, the NKT cells are hypo-responsive to further antigenic induced expansion.

NKT cells are a CD1d-restricted T cell subset with strong immunoregulatory properties. Human NKT deficiencies are associated with autoimmune diseases such as type 1 diabetes and several types of cancer, yet there is little understanding of how the human NKT cell pool develops or is maintained. In this study, we present the first detailed analysis of human NKT cells from donor-matched postnatal thymus and blood samples. In mice, NKT cells are a thymus-dependent population that migrates to the periphery at an immature stage. Our data show that human NKT cells also undergo early stages of development in the thymus, forming a CD4(+)CD161(-/low) population that predominates neonatal thymic and blood NKT cell pools. CD4(-) and CD161(+) NKT cells accumulate with age in the blood, but not thymus, to the point that they dominate the NKT cell compartment in adult blood. This is consistent with the post-thymic maturation of NKT cells exported from the thymus at the putatively immature CD4(+)CD161(-/low) stage. Interestingly, while thymus and peripheral NKT cell frequencies vary widely between patients and are relatively stable between age groups, there is no clear relationship between the NKT cell frequency in thymus and blood.

NKT cells are emerging as an extremely influential regulatory subset of T lymphocytes that are functionally and developmentally distinct from their mainstream counterparts. Like other T cells, NKT cells are thymus-dependent but their apparently unique pathway of differentiation is poorly characterized. Given the strong association between NKT cell deficiency and increased incidences of autoimmunity and cancer it is imperative that the mechanisms by which NKT cells are generated becomes better understood. This review examines what is known about NKT cell development in the thymus and highlights elements of the pathway that differ significantly from mainstream T-cell development. It is here that NKT cell-specific disorders may originate and may best be addressed.

NKT cell development takes place in the thymus, beginning when these cells branch away from CD4+CD8+ mainstream thymocytes upon expression of the Valpha14Jalpha18 T cell receptor (TCR) and recognition of the CD1d molecule. Although NKT cells express an invariant TCR alpha chain, the diverse TCR beta expression leaves open the possibility that the development of these cells is shaped by glycolipid antigen recognition in the context of CD1d. Here, we show that the presence of an agonist glycolipid ligand, alpha-galactosylceramide, while NKT cells are developing in vitro or in vivo, specifically ablates their development. In contrast, the delayed introduction of this compound in vitro or in vivo, after NKT cells have developed, does not deplete these cells. These data indicate that NKT cells pass through a developmental window where they are susceptible to TCR-mediated negative selection, and suggest that NKT cells with a potentially high level of self reactivity can be removed from the NKT cell repertoire before they exit the thymus.

The T lymphocyte membrane glycoprotein CD8 enhances antigen recognition by class I-restricted T cells. There are two naturally occurring forms of CD8, an alphabeta heterodimer expressed by the majority of CD8(+) T cells, and a less abundant alphaalpha homodimer found on specialised T cell subsets. An expression strategy was developed for production of soluble CD8alphaalpha and CD8alphabeta extracellular domains for use in ligand binding studies. Mouse CD8alpha was expressed autonomously as a homodimer at 10 mg/l in mammalian fibroblasts, but CD8beta was not expressed at significant levels in the absence of CD8alpha. Co-expression with CD8alpha led to significant enhancement in the level of CD8beta expression, which was secreted as a non-covalent heterodimer at 3 mg/l with CD8alpha. Despite the marked increase of CD8beta expression in the presence of CD8alpha, an excess of soluble CD8alphaalpha homodimer was also present in the supernatant of co-expressing cell clones. In order to resolve the CD8alphaalpha homodimer from the CD8alphabeta heterodimer, affinity chromatographic techniques specific for the CD8beta subunit were employed. Purification procedures requiring elution from affinity matrices at low pH led to substantial losses in the total antigenic activity and partial subunit dissociation of the soluble CD8alphabeta heterodimer. The inclusion of a hexahistidine tag at the C-terminus of CD8beta enabled affinity purification of soluble CD8alphabeta (and sCD8alphaalpha) under neutral conditions, yielding recombinant protein with the correct stoichiometry and full antigenic activity. This method may prove useful for production of other soluble recombinant heterodimeric receptor proteins whose antigenicity is affected by denaturation during immunoaffinity purification.