Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.     

CD8+ T cells rapidly acquire NK1.1 and NK cell-associated molecules upon stimulation in vitro and in vivo

NKT cells express both NK cell-associated markers and TCR. Classically, these NK1.1+TCRalphabeta+ cells have been described as being either CD4+CD8- or CD4-CD8-. Most NKT cells interact with the nonclassical MHC class I molecule CD1 through a largely invariant Valpha14-Jalpha281 TCR chain in conjunction with either a Vbeta2, -7, or -8 TCR chain. In the present study, we describe the presence of significant numbers of NK1.1+TCRalphabeta+ cells within lymphokine-activated killer cell cultures from wild-type C57BL/6, CD1d1-/-, and Jalpha281-/- mice that lack classical NKT cells. Unlike classical NKT cells, 50-60% of these NK1.1+TCRalphabeta+ cells express CD8 and have a diverse TCR Vbeta repertoire. Purified NK1.1-CD8alpha+ T cells from the spleens of B6 mice, upon stimulation with IL-2, IL-4, or IL-15 in vitro, rapidly acquire surface expression of NK1.1. Many NK1.1+CD8+ T cells had also acquired expression of Ly-49 receptors and other NK cell-associated molecules. The acquisition of NK1.1 expression on CD8+ T cells was a particular property of the IL-2Rbeta+ subpopulation of the CD8+ T cells. Efficient NK1.1 expression on CD8+ T cells required Lck but not Fyn. The induction of NK1.1 on CD8+ T cells was not just an in vitro phenomenon as we observed a 5-fold increase of NK1.1+CD8+ T cells in the lungs of influenza virus-infected mice. These data suggest that CD8+ T cells can acquire NK1.1 and other NK cell-associated molecules upon appropriate stimulation in vitro and in vivo.     

NKT cell ligand recognition logic: molecular basis for a synaptic duet and transmission of inflammatory effectors

NKT cells that express the semi-invariant TCR are innate-like lymphocytes whose functions are regulated by self and foreign glycolipid ligands presented by the Ag-presenting, MHC class I-like molecule CD1d. Activation of NKT cells in vivo results in rapid release of copious amounts of effector cytokines and chemokines with which they regulate innate and adaptive immune responses to pathogens, certain types of cancers, and self-antigens. The nature of CD1d-restricted ligands, the manner in which they are recognized, and the unique effector functions of NKT cells suggest an immunoregulatory role for this T cell subset. Their ability to respond fast and our ability to steer NKT cell cytokine response to altered lipid ligands make them an important target for vaccine design and immunotherapies against autoimmune diseases. This review summarizes our current understanding of CD1d-restricted ligand recognition by NKT cells and how these innate-like lymphocytes regulate inflammation.     

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.     

Interplay of cytokines and microbial signals in regulation of CD1d expression and NKT cell activation

In this study we show that like MHC class I and class II molecules, cell surface CD1d expression on APC is regulated and affects T cell activation under physiological conditions. Although IFN-gamma alone is sufficient for optimum expression of MHC, CD1d requires two signals, one provided by IFN-gamma and a second mediated by microbial products or by the proinflammatory cytokine TNF. IFN-gamma-dependent CD1d up-regulation occurs on macrophages following infection with live bacteria or exposure to microbial products in vitro and in vivo. APC expressing higher CD1d levels more efficiently activate NKT cell hybridomas and primary NKT cells independently of whether the CD1d-restricted TCR recognizes foreign or self-lipid Ags. Our findings support a model in which CD1d induction regulates NKT cell activation.     

Prevention of diabetes in nonobese diabetic mice mediated by CD1d-restricted nonclassical NKT cells

A role for regulatory lymphocytes has been demonstrated in the pathogenesis of type 1 diabetes in the NOD mouse but the nature of these cells is debated. CD1d-restricted NKT lymphocytes have been implicated in this process. Previous reports of reduced diabetes incidence in NOD mice in which the numbers of NKT cells are artificially increased have been attributed to the enhanced production of IL-4 by these cells and a role for classical NKT cells, using the Valpha14-Jalpha18 rearrangement. We now show that overexpression in NOD mice of CD1d-restricted TCR Valpha3.2(+)Vbeta9(+) NKT cells producing high levels of IFN-gamma but low amounts of IL-4 leads to prevention of type 1 diabetes, demonstrating a role for nonclassical CD1d-restricted NKT cells in the regulation of autoimmune diabetes.     

Evidence for two subgroups of CD4-CD8- NKT cells with distinct TCR alpha beta repertoires and differential distribution in lymphoid tissues

NKT cells are a subset of T lymphocytes that is mainly restricted by the nonclassical MHC class I molecule, CD1d, and that includes several subpopulations, in particular CD4+ and CD4-CD8- (DN) cells. In the mouse, differential distribution of these subpopulations as well as heterogeneity in the expression of various markers as a function of tissue localization have been reported. We have thus undertaken a detailed study of the DN NKT cell subpopulation. With a highly sensitive semiquantitative RT-PCR technique, its TCR repertoire was characterized in various tissues. We found that mouse DN NKT cells are a variable mixture of two subgroups, one bearing the invariant Valpha14 chain paired to rearranged Vbeta2, Vbeta7, Vbeta8.1, Vbeta8.2, or Vbeta8.3 beta-chains and the other exhibiting unskewed alpha- and beta-chains. The proportion of these subgroups varies from about 100:0 in thymus, 80:20 in liver, and 50:50 in spleen to 20:80% in bone marrow, respectively. Finally, further heterogeneity in the tissue-derived DN NKT cells was discovered by sequencing extensively Vbeta8.2-Jbeta2.5 rearrangements in individual mice. Despite a few recurrences in TCR sequences, we found that each population exhibits its own and broad TCRbeta diversity.     

Cross-regulation between distinct natural killer T cell subsets influences immune response to self and foreign antigens

Natural killer T (NKT) cells generally recognize lipid-antigens presented in the context of the MHC class I-like molecule CD1d. CD1d-restricted NKT cells consist of two broad subsets: Type I, which express an invariant T cell receptor (TCR) and type II, which utilize diverse TCR gene segments. A major type II NKT subset has been shown to recognize a self-glycolipid, sulfatide. Both subsets play important roles in autoimmune diseases, tumor surveillance, and infectious diseases. While type I NKT cells protect from tumor growth by enhancing tumor surveillance, type II NKT cells may suppress anti-tumor immune responses. In a murine autoimmune hepatitis model, type I NKT cells contribute to pathogenesis, whereas activation of sulfatide-reactive type II NKT cells protects from disease. Sulfatide-mediated activation of type II NKT cells results in modification of dendritic cells and induction of anergy in type I NKT cells. Elucidation of this novel pathway of cross-regulation among NKT cell subsets will provide tools for intervention in autoimmune diseases and for designing strategies for effective anti-tumor immunity.     

CD1d-specific NK1.1+ T cells with a transgenic variant TCR

The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Valpha14-Jalpha281 rearrangement and a limited set of TCR Vbeta segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Valpha3. 2/Vbeta9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-gamma, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the "non-Valpha 14" diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics.     

Activation of nonclassical CD1d-restricted NK T cells induces airway hyperreactivity in beta 2-microglobulin-deficient mice

Allergic asthma is characterized by Th2-driven eosinophilic airway inflammation and by a central feature called airway hyperreactivity (AHR), development of which requires the presence of classical type I invariant NK T (iNKT) cells. Allergen-induced AHR, however, develops in beta(2)-microglobulin (beta(2)m)(-/-) mice, which lack classical iNKT cells, suggesting that in some situations iNKT cells may be dispensable for the development of AHR. In contrast, our studies now suggest that a CD1d-restricted, NK1.1(+) noninvariant TCR NKT cell population is present in beta(2)m(-/-) mice and is responsible for the development of AHR but not for Th2 responses. Furthermore, treatment of beta(2)m(-/-) mice with anti-CD1d mAb or anti-NK1.1 mAb unexpectedly abolished allergen-induced AHR. The CD1-restricted NKT cells in these mice, which failed to respond to alpha-galactosylceramide and which therefore were not classical type I iNKT cells, appear to represent an NKT cell subset restricted by a beta(2)m-independent form of CD1d. These results indicate that, although classical type I iNKT cells are normally required for the development of AHR, under different circumstances other NKT cell subsets, including nonclassical NKT cells, may substitute for classical iNKT cells and induce AHR.     

A subset of NKT cells that lacks the NK1.1 marker, expresses CD1d molecules, and autopresents the alpha-galactosylceramide antigen

In the present report, we characterize a novel T cell subset that shares with the NKT cell lineage both CD1d-restriction and high reactivity in vivo and in vitro to the alpha-galactosylceramide (alpha-GalCer) glycolipid. These cells preferentially use the canonical Valpha14-Jalpha281 TCR-alpha-chain and Vbeta8 TCR-beta segments, and are stimulated by alpha-GalCer in a CD1d-dependent fashion. However, in contrast to classical NKT cells, they lack the NK1.1 marker and express high surface levels of CD1d molecules. In addition, this NK1.1(-) CD1d(high) T subset, further referred to as CD1d(high) NKT cells, can be distinguished by its unique functional features. Although NK1.1(+) NKT cells require exogenous CD1d-presenting cells to make them responsive to alpha-GalCer, CD1d(high) NKT cells can engage their own surface CD1d in an autocrine and/or paracrine manner. Furthermore, in response to alpha-GalCer, CD1d(high) NKT cells produce high amounts of IL-4 and moderate amounts of IFN-gamma, a cytokine profile more consistent with a Th2-like phenotype rather than the Th0-like phenotype typical of NK1.1(+) NKT cells. Our work reveals a far greater level of complexity within the NKT cell population than previously recognized and provides the first evidence for T cells that can be activated upon TCR ligation by CD1d-restricted recognition of their ligand in the absence of conventional APCs.     

Regulation of antitumour immunity by CD1d-restricted NKT cells

An understanding of the complex interactions occurring between tumours and the immune system is a prerequisite for the rational design of effective cancer immunotherapies. To date, attention has focused mainly on the role the adaptive immune system plays in controlling tumourigenesis, with conventional T cells, which recognize peptide antigens presented by classical MHC molecules, coming under close scrutiny. Accumulating reports now suggest that an additional T-cell subset, known as CD1d-restricted natural killer T (NKT) cells, also plays a pivotal role in modulating antitumour responses. Found in both humans and mice, CD1d-restricted NKT cells are a highly specialized cell type that, in contrast to conventional T cells, recognize lipid/glycolipid antigens presented by the non-classical MHC molecule CD1d. Several features of NKT cells, including their ability to rapidly produce large quantities of cytokines upon primary stimulation, make them ideal targets for developing anticancer immunotherapies. This intriguing cell type is the focus of this review.     

Human NKT cells express granulysin and exhibit antimycobacterial activity

Human NKT cells are a unique subset of T cells that express an invariant V alpha 24 TCR that recognizes the nonclassical Ag-presenting molecule CD1d. Activation of NKT cells is greatly augmented by the marine sponge-derived glycolipid alpha-galactosylceramide (alpha GalCer). Because human monocyte-derived cells express CD1d and can harbor the intracellular pathogen Mycobacterium tuberculosis, we asked whether the addition of alpha GalCer could be used to induce effector functions of NKT cells against infected monocytes, macrophages, and monocyte-derived dendritic cells. NKT cells secreted IFN-gamma, proliferated, and exerted lytic activity in response to alpha GalCer-pulsed monocyte-derived cells. Importantly, alpha GalCer-activated NKT cells restricted the growth of intracellular M. tuberculosis in a CD1d-dependent manner. NKT cells that exhibited antimycobacterial activity also expressed granulysin, an antimicrobial peptide shown to mediate an antimycobacterial activity through perturbation of the mycobacterial surface. Degranulation of NKT cells resulted in depletion of granulysin and abrogation of antimycobacterial activity. The detection of CD1d in granulomas of tuberculosis patients supports the potential interaction of NKT cells with CD1d-expressing cells at the site of disease activity. These studies provide evidence that alpha Gal Cer-activated CD1d-restricted T cells can participate in human host defense against M. tuberculosis infection.     

Cutting edge: NKT cell development is selectively impaired in Fyn- deficient mice.

Most NK1.1+ T (NKT) cells express a biased TCRalphabeta repertoire that is positively selected by the monomorphic MHC class I-like molecule CD1d. The development of CD1d-dependent NKT cells is thymus dependent but, in contrast to conventional T cells, requires positive selection by cells of hemopoietic origin. Here, we show that the Src protein tyrosine kinase Fyn is required for development of CD1d-dependent NKT cells but not for the development of conventional T cells. In contrast, another Src kinase, Lck, is required for the development of both NKT and T cells. Impaired NKT cell development in Fyn-deficient mice cannot be rescued by transgenic expression of CD8, which is believed to increase the avidity of CD1d recognition by NKT cells. Taken together, our data reveal a selective and nonredundant role for Fyn in NKT cell development.     

Phenotypic and functional differences between NKT cells colonizing splanchnic and peripheral lymph nodes

NKT cells are considered unconventional T cells. First, they are restricted by a nonclassical MHC class I molecule, CD1d, which presents glycolipids; second, their TCR repertoire is very limited. After stimulation by their TCR, NKT cells rapidly release large amounts of cytokines, such as IL-4 and IFN-gamma. Little is known about NKT cells present in lymph nodes. In the present report we show that NKT cells are differently distributed in various lymph nodes and are, for instance, abundant in pancreatic and mesenteric lymph nodes of C57BL/6 mice and nonobese diabetic mice. The high frequency of NKT cells in splanchnic lymph nodes is not simply a consequence of inflammatory signals, as draining lymph nodes still contain low frequencies of NKT cells after IFA or CFA injections. NKT cells from splanchnic lymph nodes harbor a Vbeta repertoire similar to that of splenic and liver NKT cells, in contrast to peripheral NKT cells that are not biased toward Vbeta8 segments. Analysis of cytokine production by NKT cells from splanchnic lymph nodes reveals that they produce at least as much IL-4 as IFN-gamma, in contrast to NKT cells from other organs (spleen, liver, and peripheral lymph nodes), which produce much more IFN-gamma than IL-4. These specific features of NKT cells from splanchnic lymph nodes might explain their protective action against the development of pathogenic Th1 cells in type 1 diabetes.     

Proliferation of activated CD1d-restricted NKT cells is down-modulated by lymphocyte activation gene-3 signaling via cell cycle arrest in S phase

Upon antigenic stimulation, CD1d-restricted NKT cells quickly secrete large amounts of cytokines. This prompt response demonstrates that CD1d-restricted NKT cells may potentially prove to be useful therapeutic agents for the treatment of many diseases. Despite the clinical importance of CD1d-restricted NKT cells, the regulating mechanisms of this unique T cell population remain to be defined. We found murine LAG-3 is inducible on CD1d-restricted NKT cells as the result of a variety of stimulants such as concanavalin A (con A) and anti-CD3. Also, antigen-specific CD1d stimulation can elicit LAG-3 in CD1d-restricted NKT cells. Moreover, ectopic LAG-3 expression on CD1d-restricted NKT cells results in cell cycle arrest in the S phase. These results show that LAG-3 signaling on activated CD1d-restricted NKT cells may down-modulate NKT cell proliferation.     

Monte Carlo simulations of voltage-driven translocation of a signal sequence

CD1d-deficient (CD1d-/-) mouse lymphocytes were analyzed to classify the natural killer T (NKT) cells without reactivity to CD1d. The cells bearing a V(alpha)19.1-J(alpha)26 (AV19-AJ33) invariant TCR alpha chain, originally found in the peripheral blood lymphocytes, were demonstrated to be abundant in the NK1.1+ but not NK1.1- T cell population isolated from CD1d-/- mice. Moreover, more than half (11/21) of the hybrid cell lines established from CD1d-/- NKT cells expressed the V(alpha)19.1-J(alpha)26 invariant TCR alpha chain. The expression of the invariant V(alpha)19.1-J(alpha)26 mRNA was absent in beta2-microglobulin-deficient mice. Collectively, the present findings suggest the presence of a second NKT cell repertoire characterized by an invariant TCR alpha chain (V(alpha)19.1-J(alpha)26) that is selected by an MHC class I-like molecule other than CD1d.     

Injury-induced suppression of effector T cell immunity requires CD1d-positive APCs and CD1d-restricted NKT cells

Overwhelming infection remains the leading cause of death from serious burn injury despite recent advances in the care of burn patients and a better understanding of immune and inflammatory consequences of injury. In this study, we report a critical requirement for CD1d-restricted NKT cells and CD1d expression by APCs in the immune dysfunction that occurs early after burn injury. Using a well-established murine scald injury model with BALB/c and BALB/c CD1d knockout mice, we investigated whether peripheral T cell immunity was affected by the presence or absence of CD1d-restricted NKT cells in the early stages after injury. Using Ag-specific delayed-type hypersensitivity, T cell proliferation, and cytokine production as indices of immune responsiveness, we observed that both CD1d expression by APCs and CD1d-restricted NKT cells are required for immune suppression after injury. Via adoptive transfer of splenocytes from injured mice to uninjured recipients, we found injury-induced suppression of immunity to be Ag specific, long lasting, and critically dependent on cell surface expression of CD1d by APCs. Together, our results suggest that the defects in T cell responsiveness that occur subsequent to severe burn injury are not merely the result of global or passive suppression, but instead represent an active form of CD1d/NKT cell-dependent immunologic tolerance.     

CD1d-independent NKT cells in beta 2-microglobulin-deficient mice have hybrid phenotype and function of NK and T cells

Unlike CD1d-restricted NK1.1(+)TCRalphabeta(+) (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in beta(2)-microglobulin (beta(2)m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in beta(2)m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-independent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed beta(2)m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by D(b) killed RMA-S, but not RMA, cells. They also killed RMA (H-2(b)) cells that were preincubated with the HY peptide. NKT cells from beta(2)m-deficient mice, upon CD3 cross-linking, secreted IFN-gamma and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.