CD1-mediated immune responses in mucosal tissues: molecular mechanisms underlying lipid antigen presentation system.

The cluster of differentiation 1 (CD1) molecule differs from major histocompatibility complex class I and II because it presents glycolipid/lipid antigens. Moreover, the CD1-restricted T cells that recognize these self and foreign antigens participate in both innate and adaptive immune responses. CD1s are constitutively expressed by professional and nonprofessional antigen-presenting cells in mucosal tissues, namely, the skin, lung, and intestine. This suggests that CD1-reactive T cells are involved in the immune responses of these tissues. Indeed, evidence suggests that these cells play important roles in diverse diseases, such as inflammation, autoimmune disease, and infection. Recent studies elucidating the molecular mechanisms by which CD1 presents lipid antigens suggest that defects in these mechanisms could contribute to the activities of CD1-reactive T cells. Thus, improving our understanding of these mechanisms could lead to new and effective therapeutic approaches to CD1-associated diseases. In this review, we discuss the CD1-mediated antigen presentation system and its roles in mucosal tissue immunity.

Adenosine A2A receptor activation reduces hepatic ischemia reperfusion injury by inhibiting CD1d-dependent NKT cell activation.

Ischemia reperfusion injury results from tissue damage during ischemia and ongoing inflammation and injury during reperfusion. Liver reperfusion injury is reduced by lymphocyte depletion or activation of adenosine A2A receptors (A2ARs) with the selective agonist 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]- prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL146e). We show that NKT cells are stimulated to produce interferon (IFN)-gamma by 2 h after the initiation of reperfusion, and the use of antibodies to deplete NK1.1-positive cells (NK and NKT) or to block CD1d-mediated glycolipid presentation to NKT cells replicates, but is not additive to, the protection afforded by ATL146e, as assessed by serum alanine aminotransferase elevation, histological necrosis, neutrophil accumulation, and serum IFN-gamma elevation. Reduced reperfusion injury observed in RAG-1 knockout (KO) mice is restored to the wild-type (WT) level by adoptive transfer of NKT cells purified from WT or A2AR KO mice but not IFN-gamma KO mice. Additionally, animals with transferred A2AR-/- NKT cells are not protected from hepatic reperfusion injury by ATL146e. In vitro, ATL146e potently inhibits both anti-CD3 and alpha-galactosylceramide-triggered production of IFN-gamma by NKT cells. These findings suggest that hepatic reperfusion injury is initiated by the CD1d-dependent activation of NKT cells, and the activation of these cells is inhibited by A2AR activation.

Serine/threonine-protein phosphatase 1 α levels are paralleling olfactory memory formation in the CD1 mouse.

Although olfactory discrimination has already been studied in several mouse strains, data on protein levels linked to olfactory memory are limited. Wild mouse strains Mus musculus musculus, Mus musculus domesticus and CD1 laboratory outbred mice were tested in a conditioned odor preference task and trained to discriminate between two odors, Rose and Lemon, by pairing one odor with a sugar reward. Six hours following the final test, mice were sacrificed and olfactory bulbs (OB) were taken for gel-based proteomics analyses and immunoblotting. OB proteins were extracted, separated by 2-DE and quantified using specific software (Proteomweaver). Odor-trained mice showed a preference for the previously rewarded odor suggesting that conditioned odor preference occurred. In CD1 mice levels, one out of 482 protein spots was significantly increased in odor-trained mice as compared with the control group; it was in-gel digested by trypsin and chymotrypsin and analyzed by tandem mass spectrometry (nano-ESI-LC-MS/MS). The spot was unambiguously identified as serine/threonine-protein phosphatase PP1-α catalytic subunit (PP-1A) and differential levels observed in gel-based proteomic studies were verified by immunoblotting. PP-1A is a key signalling element in synaptic plasticity and memory processes and is herein shown to be paralleling olfactory discrimination representing olfactory memory.

Antigen Presentation by CD1 Lipids, T Cells, and NKT Cells in Microbial Immunity.

The discovery of molecules capable of presenting lipid antigens, the CD1 family, and of the T cells that recognize them, has opened a new dimensionin our understanding of cell-mediated immunity against infection. Like MHC Class I molecules, CD1 isoforms (CD1a, b, c and d) are assembled in the ER and sent to the cell surface. However, in contrast to MHC molecules, CD1 complexes are then re-internalized into specific endocytic compartments where they can bind lipid antigens. These include a broad scope of both self and foreign molecules that range from simple fatty acids or phospholipids, to more complex glycolipids, isoprenoids, mycolates and lipopeptides. Lipid-loaded CD1 molecules are then delivered to the cell surface and can be surveyed by CD1-restricted T cells expressing alphabeta or gammadelta T Cell Receptors (TCR). It has become clear that T cell-mediated lipid antigen recognition plays an important role in detection and clearance of pathogens. CD1a, b and c-restricted T cells have been found to recognize a number of lipid antigens from M. tuberculosis. CD1d-restricted T cells are the only CD1-restricted T cell subset present in mice, which lack the genes encoding CD1a, b and c. Evidence from experiments in CD1d-restricted T cell-deficient mice indicates that these cells play an important role in the immune response against awide range of pathogens including several bacteria, viruses and parasites. One subset of CD1d-restricted T cells in particular, invariant Natural Killer T (iNKT) cells, has been extensively studied. iNKT cells are characterized by the expression of a semi-invariant TCR composed of a strictly conserved alpha chain paired with a limited repertoire of beta chains. During infection, iNKT cells are rapidly elicited. Activated iNKT cells can produce a vast array of cytokines that profoundly affect both the innate and the adaptive arms of the immune response. In this review, we describe the pathways and mechanisms of lipid antigen binding and presentation by CD1 in detail, as well as the diverse roles played by CD1-restricted T cells in the context of microbial infection.

Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity.

Using natural killer T (NKT) cell-deficient mice, we show here that allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma, does not develop in the absence of V(alpha)14i NKT cells. The failure of NKT cell-deficient mice to develop AHR is not due to an inability of these mice to produce type 2 T-helper (Th2) responses because NKT cell-deficient mice that are immunized subcutaneously at non-mucosal sites produce normal Th2-biased responses. The failure to develop AHR can be reversed by the adoptive transfer of tetramer-purified NKT cells producing interleukin (IL)-4 and IL-13 to Ja281(-/-) mice, which lack the invariant T-cell receptor (TCR) of NKT cells, or by the administration to Cd1d(-/-) mice of recombinant IL-13, which directly affects airway smooth muscle cells. Thus, pulmonary V(alpha)14i NKT cells crucially regulate the development of asthma and Th2-biased respiratory immunity against nominal exogenous antigens. Therapies that target V(alpha)14i NKT cells may be clinically effective in limiting the development of AHR and asthma.

CD1d function is regulated by microsomal triglyceride transfer protein.

CD1d is a major histocompatibility complex (MHC) class I-related molecule that functions in glycolipid antigen presentation to distinct subsets of T cells that express natural killer receptors and an invariant T-cell receptor-alpha chain (invariant NKT cells). The acquisition of glycolipid antigens by CD1d occurs, in part, in endosomes through the function of resident lipid transfer proteins, namely saposins. Here we show that microsomal triglyceride transfer protein (MTP), a protein that resides in the endoplasmic reticulum of hepatocytes and intestinal epithelial cells (IECs) and is essential for lipidation of apolipoprotein B, associates with CD1d in hepatocytes. Hepatocytes from animals in which Mttp (the gene encoding MTP) has been conditionally deleted, and IECs in which Mttp gene products have been silenced, are unable to activate invariant NKT cells. Conditional deletion of the Mttp gene in hepatocytes is associated with a redistribution of CD1d expression, and Mttp-deleted mice are resistant to immunopathologies associated with invariant NKT cell-mediated hepatitis and colitis. These studies indicate that the CD1d-regulating function of MTP in the endoplasmic reticulum is complementary to that of the saposins in endosomes in vivo.

CD1a function in human skin disease.

The high expression of CD1a on Langerhans cells in normal human skin suggests a central role for this lipid antigen presenting molecule in skin homeostasis and immunity. Although the lipid antigen presenting function of CD1a has been known for years, the physiological and pathological functions of the CD1a system in human skin remain incompletely understood. This review provides an overview of this active area of investigation, and discusses recent insights into the functions of CD1a, CD1a-restricted T cells, and lipid antigens in inflammatory and allergic skin disease. We include recent publications and work presented at the biennial CD1-MR1 EMBO workshop held in 2019 in Oxford, regarding lipids that increase and those that decrease T cell responses to CD1a.

Animal models for human group 1 CD1 protein function.

The CD1 antigen presenting system is evolutionary conserved and found in mammals, birds and reptiles. Humans express five isoforms, of which CD1a, CD1b and CD1c represent the group 1 CD1-molecules. They are recognized by T cells that express diverse αß-T cell receptors. Investigation of the role of group 1 CD1 function has been hampered by the fact that CD1a, CD1b and CD1c are not expressed by mice. However, other animals, such as guinea pigs or cattle, serve as alternative models and have established basic aspects of CD1-dependent, antimicrobial immune functions. Group 1 CD1 transgenic mouse models became available about ten years ago. In a series of seminal studies these mouse models coined the mechanistical understanding of the role of the corresponding CD1 restricted T cell responses. This review gives a short overview of available animal studies and the lessons that have been and still can be learned.

Natural killer T cell ligand alpha-galactosylceramide enhances protective immunity induced by malaria vaccines.

The important role played by CD8(+) T lymphocytes in the control of parasitic and viral infections, as well as tumor development, has raised the need for the development of adjuvants capable of enhancing cell-mediated immunity. It is well established that protective immunity against liver stages of malaria parasites is primarily mediated by CD8(+) T cells in mice. Activation of natural killer T (NKT) cells by the glycolipid ligand, alpha-galactosylceramide (alpha-GalCer), causes bystander activation of NK, B, CD4(+), and CD8(+) T cells. Our study shows that coadministration of alpha-GalCer with suboptimal doses of irradiated sporozoites or recombinant viruses expressing a malaria antigen greatly enhances the level of protective anti-malaria immunity in mice. We also show that coadministration of alpha-GalCer with various different immunogens strongly enhances antigen-specific CD8(+) T cell responses, and to a lesser degree, Th1-type responses. The adjuvant effects of alpha-GalCer require CD1d molecules, Valpha14 NKT cells, and interferon gamma. As alpha-GalCer stimulates both human and murine NKT cells, these findings should contribute to the design of more effective vaccines against malaria and other intracellular pathogens, as well as tumors.

Distinct functional lineages of human V(alpha)24 natural killer T cells.

CD1d-restricted autoreactive natural killer (NK)T cells have been reported to regulate a range of disease conditions, including type I diabetes and immune rejection of cancer, through the secretion of either T helper (Th)2 or Th1 cytokines. However, mechanisms underlying Th2 versus Th1 cytokine secretion by these cells are not well understood. Since most healthy subjects express <1 NKT cell per 1,000 peripheral blood lymphocytes (PBLs), we devised a new method based on the combined used of T cell receptor (TCR)-specific reagents alpha-galactosylceramide (alphaGalCer) loaded CD1d-tetramers and anti-V(alpha)24 monoclonal antibody, to specifically identify and characterize these rare cells in fresh PBLs. We report here that CD4(+) and CD4(-)CD8(-) (double negative [DN]) NKT cell subsets represent functionally distinct lineages with marked differences in their profile of cytokine secretion and pattern of expression of chemokine receptors, integrins, and NK receptors. CD4(+) NKT cells were the exclusive producers of interleukin (IL)-4 and IL-13 upon primary stimulation, whereas DN NKT cells had a strict Th1 profile and prominently expressed several NK lineage receptors. These findings may explain how NKT cells could promote Th2 responses in some conditions and Th1 in others, and should be taken into consideration for intervention in relevant diseases.

CD1d-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells.

Natural killer T (NKT) cells are a unique immunoregulatory T cell population that is positively selected by CD1d-expressing thymocytes. Previous studies have shown that NKT cells exhibit autoreactivity, which raises the question of whether they are subject to negative selection. Here, we report that the addition of agonist glycolipid alpha-galactosylceramide (alpha-GalCer) to a fetal thymic organ culture (FTOC) induces a dose-dependent disappearance of NKT cells, suggesting that NKT cells are susceptible to negative selection. Overexpression of CD1d in transgenic (Tg) mice results in reduced numbers of NKT cells, and the residual NKT cells in CD1d-Tg mice exhibit both an altered Vbeta usage and a reduced sensitivity to antigen. Furthermore, bone marrow (BM) chimeras between Tg and WT mice reveal that CD1d-expressing BM-derived dendritic cells, but not thymic epithelial cells, mediate the efficient negative selection of NKT cells. Thus, our data suggest that NKT cells developmentally undergo negative selection when engaged by high-avidity antigen or abundant self-antigen.

Cross-presentation of disialoganglioside GD3 to natural killer T cells.

GD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2- CD1-) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of approximately 1:2000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.

A pathway of costimulation that prevents anergy in CD28- T cells: B7-independent costimulation of CD1-restricted T cells.

A class of molecules that is expressed on antigen presenting cells, exemplified by CD80 (B7), has been found to provide a necessary costimulatory signal for T cell activation and proliferation. CD28 and CTLA4 are the B7 counterreceptors and are expressed on the majority of human CD4+ T cells and many CD8+ T cells. The signal these molecules mediate is distinguished from other costimulatory signals by the finding that T cell recognition of antigen results in a prolonged state of T cell unresponsiveness or anergy, unless these costimulatory molecules are engaged. However, nearly half of the CD8+ and CD4-CD8- T cells lack CD28, and the costimulatory signals required for the activation of such cells are unknown. To understand the pathways of activation used by CD28- T cells, we have examined the costimulatory requirements of antigen-specific CD4-CD8- TCR(+)-alpha/beta circulating T cells that lack the expression of CD28. We have characterized two T cell lines, DN1 and DN6, that recognize a mycobacterial antigen, and are restricted not by major histocompatibility complex class I or II, but by CD1b or CD1c, two members of a family of major histocompatibility complex-related molecules that have been recently implicated in a distinct pathway for antigen presentation. Comparison of antigen-specific cytolytic responses of the DN1 and DN6 T cell lines against antigen-pulsed CD1+ monocytes or CD1+ B lymphoblastoid cell lines (B-LCL) demonstrated that these T cells recognized antigen presented by both types of cells. However, T cell proliferation occurred only when antigen was presented by CD1+ monocytes, indicating that the CD1+ monocytes expressed a costimulatory molecule that the B-LCL transfectants lacked. This hypothesis was confirmed by demonstrating that the T cells became anergic when incubated with the CD1(+)-transfected B-LCL in the presence of antigen, but not in the absence of antigen. The required costimulatory signal occurred by a CD28-independent mechanism since both the CD1+ monocytes and CD1+ B-LCL transfectants expressed B7-1 and B7-2, and DN1 and DN6 lacked surface expression of CD28. We propose that these data define a previously unrecognized pathway of costimulation for T cells distinct from that involving CD28 and its counterreceptors. We suggest that this B7-independent pathway plays a crucial role in the activation and maintenance of tolerance of at least a subset of CD28- T cells.

CD1d-mediated recognition of an alpha-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution.

Natural killer (NK) T cells are a lymphocyte subset with a distinct surface phenotype, an invariant T cell receptor (TCR), and reactivity to CD1. Here we show that mouse NK T cells can recognize human CD1d as well as mouse CD1, and human NK T cells also recognize both CD1 homologues. The unprecedented degree of conservation of this T cell recognition system suggests that it is fundamentally important. Mouse or human CD1 molecules can present the glycolipid alpha-galactosylceramide (alpha-GalCer) to NK T cells from either species. Human T cells, preselected for invariant Valpha24 TCR expression, uniformly recognize alpha-GalCer presented by either human CD1d or mouse CD1. In addition, culture of human peripheral blood cells with alpha-GalCer led to the dramatic expansion of NK T cells with an invariant (Valpha24(+)) TCR and the release of large amounts of cytokines. Because invariant Valpha14(+) and Valpha24(+) NK T cells have been implicated both in the control of autoimmune disease and the response to tumors, our data suggest that alpha-GalCer could be a useful agent for modulating human immune responses by activation of the highly conserved NK T cell subset.

CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells.

A conserved subset of mature circulating T cells in humans expresses an invariant Valpha24-JalphaQ T cell receptor (TCR)-alpha chain rearrangement and several natural killer (NK) locus-encoded C-type lectins. These human T cells appear to be precise homologues of the subset of NK1.1(+) TCR-alpha/beta+ T cells, often referred to as NK T cells, which was initially identified in mice. Here we show that human NK T cell clones are strongly and specifically activated by the same synthetic glycolipid antigens as have been shown recently to stimulate murine NK T cells. Responses of human NK T cells to these synthetic glycolipids, consisting of certain alpha-anomeric sugars conjugated to an acylated phytosphingosine base, required presentation by antigen-presenting cells expressing the major histocompatibility complex class I-like CD1d protein. Presentation of synthetic glycolipid antigens to human NK T cells required internalization of the glycolipids by the antigen-presenting cell and normal endosomal targeting of CD1d. Recognition of these compounds by human NK T cells triggered proliferation, cytokine release, and cytotoxic activity. These results demonstrate a striking parallel in the specificity of NK T cells in humans and mice, thus providing further insight into the potential mechanisms of immune recognition by NK T cells and the immunological function of this unique T cell subset.

Macrophage inflammatory protein 3alpha is involved in the constitutive trafficking of epidermal langerhans cells.

Certain types of dendritic cells (DCs) appear in inflammatory lesions of various etiologies, whereas other DCs, e.g., Langerhans cells (LCs), populate peripheral organs constitutively. Until now, the molecular mechanism behind such differential behavior has not been elucidated. Here, we show that CD1a(+) LC precursors respond selectively and specifically to the CC chemokine macrophage inflammatory protein (MIP)-3alpha. In contrast, CD14(+) precursors of DC and monocytes are not attracted by MIP-3alpha. LCs lose the migratory responsiveness to MIP-3alpha during their maturation, and non-LC DCs do not acquire MIP-3alpha sensitivity. The notion that MIP-3alpha may be responsible for selective LC recruitment into the epidermis is further supported by the following observations: (a) MIP-3alpha is expressed by keratinocytes and venular endothelial cells in clinically normal appearing human skin; (b) LCs express CC chemokine receptor (CCR)6, the sole MIP-3alpha receptor both in situ and in vitro; and (c) non-LC DCs that are not found in normal epidermis lack CCR6. The mature forms of LCs and non-LC DCs display comparable sensitivity for MIP-3beta, a CCR7 ligand, suggesting that DC subtype-specific chemokine responses are restricted to the committed precursor stage. Although LC precursors express primarily CCR6, non-LC DC precursors display a broad chemokine receptor repertoire. These findings reflect a scenario where the differential expression of chemokine receptors by two different subpopulations of DCs determines their functional behavior. One type, the LC, responds to MIP-3alpha and enters skin to screen the epidermis constitutively, whereas the other type, the "inflammatory" DC, migrates in response to a wide array of different chemokines and is involved in the amplification and modulation of the inflammatory tissue response.

Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens.

CD1b and CD1c are antigen-presenting molecules that mediate recognition of bacterial lipids by T cells, but it is currently not known whether these two molecules are redundant or are specialized to perform different immunological functions. Here, we show that the distribution of CD1c in human dendritic cells was characterized by a high ratio of cell surface to intracellular molecules, whereas CD1b showed a reciprocal pattern of distribution. In contrast to the accumulation of CD1b in lysosomal major histocompatibility complex class II compartments, intracellular CD1c molecules accumulated in other endocytic compartments, most likely early and late endosomes. Deletion of the cytoplasmic tail of CD1c, containing a tyrosine-based internalization motif, abolished most of its intracellular localization. Functional studies using T cells specific for defined lipid antigens revealed that in contrast to CD1b-mediated antigen presentation, antigen presentation by CD1c was resistant to drugs inhibiting endosomal acidification and was independent of endosomal localization of CD1c. Taken together, these results support the hypothesis that CD1b and CD1c are specialized to survey the lipid content of different intracellular compartments.

Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining.

CD1d-restricted natural killer (NK)T cells are known to potently secrete T helper (Th)1 and Th2 cytokines and to mediate cytolysis, but it is unclear how these contrasting functional activities are regulated. Using lipid antigen-loaded CD1d tetramers, we have distinguished two subsets of CD1d-restricted T cells in fresh peripheral blood that differ in cytokine production and cytotoxic activation. One subset, which was CD4(-), selectively produced the Th1 cytokines interferon gamma and tumor necrosis factor alpha, and expressed NKG2d, a marker associated with cytolysis of microbially infected and neoplastic cells. This subset up-regulated perforin after exposure to interleukin (IL)-2 or IL-12. In contrast, CD4(+) CD1d-restricted NKT cells potently produced both Th1 and Th2 cytokines, up-regulated perforin in response to stimulation by phorbol myristate acetate and ionomycin but not IL-2 or IL-12, and could be induced to express CD95L. Further, for both CD1d-restricted NKT cell subsets, we found that antigenic stimulation induced cytokine production but not perforin expression, whereas exposure to inflammatory factors enhanced perforin expression but did not stimulate cytokine production. These results show that the various activities of CD1d-restricted T cells in tumor rejection, autoimmune disease, and microbial infections could result from activation of functionally distinct subsets, and that inflammatory and antigenic stimuli may influence different effector functions.

Cutaneous immunization rapidly activates liver invariant Valpha14 NKT cells stimulating B-1 B cells to initiate T cell recruitment for elicitation of contact sensitivity.

T cell recruitment to elicit contact sensitivity (CS) requires a CS-initiating process mediated by B-1 cells that produce IgM, which activates complement to promote T cell passage into the tissues. We now show that Valpha14i NKT cells induce B-1 cell activation likely by releasing IL-4 early postimmunization. The CS initiation process is absent in Jalpha18-/- and CD1d-/- NKT cell-deficient mice and is reconstituted by populations enriched for Valpha14i NKT cells. Transfers are not effective if cells are derived from IL-4-/- mice. Staining with specific tetramers directly showed that hepatic Valpha14i NKT cells increase by 30 min and nearly double by 2 h postimmunization. Transfer of immune B-1 cells also reconstitutes CS responses in NKT cell-deficient mice. The B-1 cells act downstream of the Valpha14i NKT cells to restore CS initiation. In addition, IL-4 given systemically to Jalpha18-/- or CD1d-/- NKT cell-deficient mice reconstitutes elicitation of CS. Further, splenocytes from immune Jalpha18-/- mice produce less antigen (Ag)-specific IgM antibodies compared with sensitized WT mice. Together these findings indicate that very early after skin immunization Valpha14i NKT cells are stimulated to produce IL-4, which activates B-1 cells to produce Ag-specific IgM, subsequently needed to recruit effector T cells for elicitation of CS responses.

Ligand-dependent inhibition of CD1d-restricted NKT cell development in mice transgenic for the activating receptor Ly49D.

In addition to their CD1d-restricted T cell receptor (TCR), natural killer T (NKT) cells express various receptors normally associated with NK cells thought to act, in part, as modulators of TCR signaling. Immunoreceptor-tyrosine activation (ITAM) and inhibition (ITIM) motifs associated with NK receptors may augment or attenuate perceived TCR signals respectively, potentially influencing NKT cell development and function. ITIM-containing Ly49 family receptors expressed by NKT cells are proposed to play a role in their development and function. We have produced mice transgenic for the ITAM-associated Ly49D and ITIM-containing Ly49A receptors and their common ligand H2-Dd to determine the importance of these signaling interplays in NKT cell development. Ly49D/H2-Dd transgenic mice had selectively and severely reduced numbers of thymic and peripheral NKT cells, whereas both ligand and Ly49D transgenics had normal numbers of NKT cells. CD1d tetramer staining revealed a blockade of NKT cell development at an early precursor stage. Coexpression of a Ly49A transgene partially rescued NKT cell development in Ly49D/H2-Dd transgenics, presumably due to attenuation of ITAM signaling. Thus, Ly49D-induced ITAM signaling is incompatible with the early development of cells expressing semi-invariant CD1d-restricted TCRs and appropriately harmonized ITIM-ITAM signaling is likely to play an important role in the developmental program of NKT cells.