CD1d-restricted Natural Killer Research Articles

Aminobisphosphonates inhibit dendritic cell-mediated antigen-specific activation of CD1d-restricted iNKT cells

CD1d-restricted invariant natural killer T (iNKT) cells constitute an important immunoregulatory T cell subset that can be activated by the synthetic glycolipid α-galactosylceramide (α-GalCer) and initiate antitumor immune responses. As cancer patients are frequently treated with aminobisphosphonates (NBP), it is relevant to determine possible effects of NBP on CD1d-restricted glycolipid Ag-presentation to iNKT cells. We report a striking reduction of α-GalCer-induced iNKT cell activation by monocyte derived dendritic cells (moDC) upon their exposure to NBP during maturation. We found that production of apolipoprotein E (apoE), which is a known facilitator of trans-membrane transport of exogenously derived glycolipids, was significantly diminished in moDC exposed to NBP. As the inhibitory effect of NBP on iNKT cell activation was alleviated by exogenous apoE, our data indicate that reduced apoE production by antigen presenting cells (APC) through NBP limits glycolipid-induced iNKT cell activation. This should be taken into account in the design of iNKT cell-based anti-cancer therapies.

Thymus medulla fosters generation of natural Treg cells, invariant γδ T cells, and invariant NKT cells: what we learn from intrathymic migration.

The organization of the thymus into distinct cortical and medullary regions enables it to control the step-wise migration and development of immature T-cell precursors. Such a process provides access to specialized cortical and medullary thymic epithelial cells at defined stages of maturation, ensuring the generation of self-tolerant and MHC-restricted conventional CD4+ and CD8+ αβ T cells. The migratory cues and stromal cell requirements that regulate the development of conventional αβ T cells have been well studied. However, the thymus also fosters the generation of several immunoregulatory T-cell populations that form key components of both innate and adaptive immune responses. These include Foxp3+ natural regulatory T cells, invariant γδ T cells, and CD1d-restricted invariant natural killer T cells (iNKT cells). While less is known about the intrathymic requirements of these nonconventional T cells, recent studies have highlighted the importance of the thymus medulla in their development. Here, we review recent findings on the mechanisms controlling the intrathymic migration of distinct T-cell subsets, and relate this to knowledge of the microenvironmental requirements of these cells.

NKT cell networks in the regulation of tumor immunity.

CD1d-restricted natural killer T (NKT) cells lie at the interface between the innate and adaptive immune systems and are important mediators of immune responses and tumor immunosurveillance. These NKT cells uniquely recognize lipid antigens, and their rapid yet specific reactions influence both innate and adaptive immunity. In tumor immunity, two NKT subsets (type I and type II) have contrasting roles in which they not only cross-regulate one another, but also impact innate immune cell populations, including natural killer, dendritic, and myeloid lineage cells, as well as adaptive populations, especially CD8+ and CD4+ T cells. The extent to which NKT cells promote or suppress surrounding cells affects the host’s ability to prevent neoplasia and is consequently of great interest for therapeutic development. Data have shown the potential for therapeutic use of NKT cell agonists and synergy with immune response modifiers in both pre-clinical studies and preliminary clinical studies. However, there is room to improve treatment efficacy by further elucidating the biological mechanisms underlying NKT cell networks. Here, we discuss the progress made in understanding NKT cell networks, their consequent role in the regulation of tumor immunity, and the potential to exploit that knowledge in a clinical setting.

Adjuvant effects of therapeutic glycolipids administered to a cohort of NKT cell-diverse pigs

CD1d-restricted natural killer T (NKT) cells are a unique lymphocyte population that makes important contributions to host defense against numerous microbial pathogens. The powerful immunomodulatory effects of these cells can be exploited in mice by cognate antigens for multiple therapeutic purposes, including for protection from infectious diseases and as adjuvants to improve vaccines against microbial organisms. These applications have potential to treat and prevent infectious diseases in livestock species that express NKT cells, including pigs. In this study, immune tissues from commercial swine of mixed genetic background were compared for NKT cell frequency, cytokine secretion and subset ratios. Pigs were also injected with the model antigen hen-egg lysozyme (HEL) in conjunction with one of three glycosphingolipids, alpha-galactosylceramide (αGC), OCH and C-glycoside that selectively activate NKT cells, to assess the adjuvant potential of each. There was significant variation between individual pigs for all NKT cell parameters measured. The NKT cell agonists elicited HEL-specific immune responses of different quality, but only αGC increased the systemic concentration of NKT cells. Peripheral blood NKT cell frequency measured prior to treatment was a poor predictor of how individual animals responded to NKT cell therapy. However, our results show that although NKT cells vary considerably between pigs, there exists considerable potential to harness these cells to protect swine from infectious diseases.

Improving Mycobacterium bovis bacillus Calmette-Guèrin as a vaccine delivery vector for viral antigens by incorporation of glycolipid activators of NKT cells.

Recombinant Mycobacterium bovis bacillus Calmette-Guèrin (rBCG) has been explored as a vector for vaccines against HIV because of its ability to induce long lasting humoral and cell mediated immune responses. To maximize the potential for rBCG vaccines to induce effective immunity against HIV, various strategies are being employed to improve its ability to prime CD8+ T cells, which play an important role in the control of HIV infections. In this study we adopted a previously described approach of incorporating glycolipids that activate CD1d-restricted natural killer T (NKT) cells to enhance priming of CD8+ T cells by rBCG strains expressing an SIV Gag antigen (rBCG-SIV gag). We found that the incorporation of the synthetic NKT activating glycolipid α-galactosylceramide (α-GC) into rBCG-SIV gag significantly enhanced CD8+ T cell responses against an immunodominant Gag epitope, compared to responses primed by unmodified rBCG-SIV gag. The abilities of structural analogues of α-GC to enhance CD8+ T cell responses to rBCG were compared in both wild type and partially humanized mice that express human CD1d molecules in place of mouse CD1d. These studies identified an α-GC analogue known as 7DW8-5, which has previously been used successfully as an adjuvant in non-human primates, as a promising compound for enhancing immunogenicity of antigens delivered by rBCG.vectors. Our findings support the incorporation of synthetic glycolipid activators of NKT cells as a novel approach to enhance the immunogenicity of rBCG-vectored antigens for induction of CD8+ T cell responses. The glycolipid adjuvant 7DW8-5 may be a promising candidate for advancing to non-human primate and human clinical studies for the development of HIV vaccines based on rBCG vectors.

Circulating CD56dim natural killer cells and CD56+ T cells that produce interferon-γ or interleukin-10 are expanded in asymptomatic, E antigen-negative patients with persistent hepatitis B virus infection.

Infection with hepatitis B virus (HBV) can result in spontaneous resolution or chronic infection, which can remain asymptomatic or can progress to cirrhosis and/or hepatocellular carcinoma. The host immune response is thought to be a major determinant of the outcome of HBV infection and virus-specific cytotoxic T lymphocytes (CTL) can mediate immunity against the virus and cause liver pathology. Antigen-nonspecific innate lymphocytes may also contribute to HBV infection and liver disease, therefore, we examined the frequencies, phenotypes, cytolytic activities and cytokine profiles of circulating natural killer (NK) cells, CD1d-restricted invariant natural killer T (iNKT) cells and CD56(+) T cells in 102 asymptomatic HBV-infected patients and compared them with those in 66 uninfected control subjects. NK cells expressing low levels of CD56 (CD56(dim)) and CD56(+) T cells were significantly expanded in the circulation of HBV-infected patients compared with control subjects. CD1d expression and iNKT cell frequencies were similar in both groups. Despite these expansions, we did not detect augmented natural or cytokine-induced cytotoxicity in the HBV-infected subjects. All lymphocyte populations studied produced interferon-γ (IFN-γ) significantly more frequently when taken from HBV-infected patients compared with when taken from healthy controls. Additionally, NK cells from the patients more frequently produced interleukin-10. As our HBV-infected cohort consisted of asymptomatic patients with low viral loads, we propose that CD56(dim) NK cells and CD56(+) T cells control HBV infection by noncytolytic mechanisms.

Activation of CD1d-restricted natural killer T cells can inhibit cancer cell proliferation during chemotherapy by promoting the immune responses in murine mesothelioma.

We studied the impact of natural killer T (NKT) cell activation by alpha-galactocysylceramide (α-GalCer, α-GC) on cancer cell repopulation during chemotherapy in murine mesothelioma. The number of NKT cells was found to be increased during the development of murine mesothelioma. NKT cells specifically recognize α-GC through CD1d resulting in their activation and expansion. Tumor-bearing mice were treated with chemotherapy once weekly, and α-GC was followed after each cycle of chemotherapy. Anti-tumor effect was evaluated on wild-type (WT) and CD1d knockout (CD1dKO) mice. Cancer cell proliferation and apoptosis were evaluated by Ki67 and TUNEL immunohistochemistry. CD4(+) and CD8(+) T cell proportion and activation in tumor, spleen, draining lymph node and peripheral blood were determined by flow cytometry, and gene expression of activated T cell-related cytokines was quantified by reverse transcription PCR. NKT cells were identified by CD1d-α-GC-tetramer staining. In WT mice, tumor growth delay was achieved by cisplatin (Cis), and this effect was improved in combination with α-GC, but α-GC alone had little effect. Cancer cell proliferation during chemotherapy was significantly inhibited by α-GC, while cancer cell death was significantly upregulated. α-GC following chemotherapy resulted in NKT cell expansion and an increase of interferon-γ production in the draining lymph node, blood and spleen. Gene expression of immune-associated cytokines was upregulated. Strikingly, the percentage of inducible T cell co-stimulator(+)CD4 T cells, Th17/Tc17 cells increased in splenocytes. In CD1d KO mice, however, Cis alone was less effective and Cis+α-GC provided no additional benefit over Cis alone. α-GC alone had minimal effect in both mice. NKT activation between cycles of chemotherapy could improve the outcome of mesothelioma treatment.

Vγ9Vδ2-T cells as antigen presenting cells for iNKT cell based cancer immunotherapy

CD1d-restricted invariant natural killer T cells (iNKT) constitute an important immunoregulatory T-cell subset involved in the induction of antitumor immune responses. Here, we provide a view on the recent observation that Vγ9Vδ2-T cells, through trogocytosis of CD1d-containing membrane fragments, have the capacity to act as antigen presenting cells for iNKT.

INKT-cell help to B cells: a cooperative job between innate and adaptive immune responses.

T-cell help to B lymphocytes is one of the most important events in adaptive immune responses in health and disease. It is generally delivered by cognate CD4(+) T follicular helper (T(FH)) cells via both cell-to-cell contacts and soluble mediators, and it is essential for both the clonal expansion of antibody (Ab)-secreting B cells and memory B-cell formation. CD1d-restricted invariant natural killer T (iNKT) cells are a subset of innate-like T lymphocytes that rapidly respond to stimulation with specific lipid antigens (Ags) that are derived from infectious pathogens or stressed host cells. Activated iNKT cells produce a wide range of cytokines and upregulate costimulatory molecules that can promote activation of dendritic cells (DCs), natural killer (NK) cells, and T cells. A decade ago, we discovered that iNKT cells can help B cells to proliferate and to produce IgG Abs in vitro and in vivo. This adjuvant-like function of Ag-activated iNKT cells provides a flexible set of helper mechanisms that expand the current paradigm of T-cell-B-cell interaction and highlights the potential of iNKT-cell targeting vaccine formulations.

Biology of CD1- and MR1-Restricted T Cells

Over the past 15 years, investigators have shown that T lymphocytes can recognize not only peptides in the context of MHC class I and class II molecules but also foreign and self-lipids in association with the nonclassical MHC class I-like molecules, CD1 proteins. In this review, we describe the most recent events in the field, with particular emphasis on (a) structural and functional aspects of lipid presentation by CD1 molecules, (b) the development of CD1d-restricted invariant natural killer T (iNKT) cells and transcription factors required for their differentiation, (c) the ability of iNKT cells to modulate innate and adaptive immune responses through their cross talk with lymphoid and myeloid cells, and (d) MR1-restricted and group I (CD1a, CD1b, and CD1c)-restricted T cells.

Enhancing T Cell Immune Responses by B Cell-based Therapeutic Vaccine Against Chronic Virus Infection.

Chronic virus infection leads to the functional impairment of dendritic cells (DCs) as well as T cells, limiting the clinical usefulness of DC-based therapeutic vaccine against chronic virus infection. Meanwhile, B cells have been known to maintain the ability to differentiate plasma cells producing antibodies even during chronic virus infection. Previously, α-galactosylceramide (αGC) and cognate peptide-loaded B cells were comparable to DCs in priming peptide-specific CD8+ T cells as antigen presenting cells (APCs). Here, we investigated whether B cells activated by αGC can improve virus-specific T cell immune responses instead of DCs during chronic virus infection. We found that comparable to B cells isolated from naïve mice, chronic B cells isolated from chronically infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) after αGC-loading could activate CD1d-restricted invariant natural killer T (iNKT) cells to produce effector cytokines and upregulate co-stimulatory molecules in both naïve and chronically infected mice. Similar to naïve B cells, chronic B cells efficiently primed LCMV glycoprotein (GP) 33-41-specific P14 CD8+ T cells in vivo, thereby allowing the proliferation of functional CD8+ T cells. Importantly, when αGC and cognate epitope-loaded chronic B cells were transferred into chronically infected mice, the mice showed a significant increase in the population of epitope-specific CD8+ T cells and the accelerated control of viremia. Therefore, our studies demonstrate that reciprocal activation between αGC-loaded chronic B cells and iNKT cells can strengthen virus-specific T cell immune responses, providing an effective regimen of autologous B cell-based therapeutic vaccine to treat chronic virus infection.

Proliferatory defect of invariant population and accumulation of non-invariant CD1d-restricted natural killer T cells in the joints of RA patients

Objectives. While numerical and functional defects of invariant NKT cells have been demonstrated in rheumatoid arthritis (RA), the detailed characterization of proliferative and secretory responses following CD1d-mediated presentation is lacking; the presence of non-invariant populations has never been assessed in human autoimmunity. We have evaluated both invariant and non-invariant populations in the blood and synovial fluid from patients to assess feasibility of NKT cell-directed manipulations in RA.Methods. NKT cell populations were quantified by anti-CD4/anti-Vα24 staining and/or CD1d tetramers. Proliferation was measured in cultures of mononuclear cells following stimulations with αGalCer and cytokine secretion determined by multi-bead assay.Results. We have confirmed a proliferative defect of iNKT cells in both peripheral blood and synovial fluid from RA patients, but no changes in baseline frequencies. Moreover, we have detected an enlargement of non-invariant cell pool in synovial fluid samples. In addition, we noted an evident Th2 shift following exposure to αGalCer and pronounced IL-6 secretion.Conclusions. While RA patients suffer from defective proliferative responses of invariant NKT cells, non-invariant cells accumulate at the site of inflammation. While stimulation with αGalCer results in reduced TNF-α and increased suppressive IL-10, abundantly produced IL-6 could potentially contribute to the induction of Th17 cells in the joints.

Blockade of invariant TCR-CD1d interaction specifically inhibits antibody production against blood group A carbohydrates

AbstractPreviously, we detected B cells expressing receptors for blood group A carbohydrates in the CD11b+CD5+ B-1a subpopulation in mice, similar to that in blood group O or B in humans. In the present study, we demonstrate that CD1d-restricted natural killer T (NKT) cells are required to produce anti-A antibodies (Abs), probably through collaboration with B-1a cells. After immunization of wild-type (WT) mice with human blood group A red blood cells (A-RBCs), interleukin (IL)-5 exclusively and transiently increased and the anti-A Abs were elevated in sera. However, these reactions were not observed in CD1d−/− mice, which lack NKT cells. Administration of anti-mouse CD1d blocking monoclonal Abs (mAb) prior to immunization abolished IL-5 production by NKT cells and anti-A Ab production in WT mice. Administration of anti-IL-5 neutralizing mAb also diminished anti-A Ab production in WT mice, suggesting that IL-5 secreted from NKT cells critically regulates anti-A Ab production by B-1a cells. In nonobese diabetic/severe combined immunodeficient (NOD/SCID/γcnull) mice, into which peripheral blood mononuclear cells from type O human volunteers were engrafted, administration of anti-human CD1d mAb prior to A-RBC immunization completely inhibited anti-A Ab production. Thus, anti-CD1d treatment might constitute a novel approach that could help in evading Ab-mediated rejection in ABO-incompatible transplant recipients.

Antitumor Impact of Interferon-γ Producing CD1d-restricted NKT Cells in Murine Malignant Mesothelioma

CD1d-restricted natural killer T (iNKT) cells have been shown to provide adjuvant activity against cancer by producing interferon (IFN)-γ. However, the role of invariant NKT (iNKT) cells in the tumor microenvironment has not yet been fully addressed. Our aim is to elucidate the antitumor effect of iNKT cells in the tumor microenvironment by using an intrathoracic murine malignant pleural mesothelioma model that we had previously developed and to provide pleural effusion as a good surrogate of the tumor microenvironment. We found that the number of iNKT cells increased dramatically in the pleural effusion after intrathoracic tumor cell injection at an earlier phase compared with accumulation of CD8 T cells. These iNKT cells showed increased expression of CD25 and increased ratio of cells positive for IFN-γ intracellular staining. iNKT cells sorted from pleural effusion of tumor burden mice produced larger amount of IFN-γ compared with naive mice. Mice pretreated in vivo with anti-CD1d-blocking Ab showed increased amount of pleural effusion and decreased ratio of total and effector-type CD8 T cells as well as decreased intracellular IFN-γ expression of CD8T-cell in the pleural effusion. In vivo administration of α-galactosylceramide (α-GalCer) showed prolonged survival associated with less pleural effusion and increased ratio of IFN-γ-positive iNKT cells and CD8 T cells in the pleural effusion. Therefore, this study suggests that iNKT cells accumulating in the tumor microenvironment play an antitumor effect by producing IFN-γ and enhancing subsequent CD8 T-cell response. Furthermore, in vivo administration of α-GalCer could suppress mesothelioma growth by activating iNKT cells.

CD1d-restricted NKT cells modulate placental and uterine leukocyte populations during chlamydial infection in mice

Invariant CD1d-restricted natural killer T cells play an important immunoregulatory role and can influence a broad spectrum of immunological responses including against bacterial infections. They are present at the fetal–maternal interface and although it has been reported that experimental systemic iNKT cell activation can induce mouse abortion, their role during pregnancy remain poorly understood. In the present work, using a physiological Chlamydia muridarum infection model, we have shown that, in vaginally infected pregnant mice, C. muridarum is cleared similarly in C57BL/6 wild type (WT) and CD1d−/− mice. We have also shown that infected- as well as uninfected-CD1d−/− mice have the same litter size as WT counterparts. Thus, CD1d-restricted cells are required neither for the resolution of chlamydial infection of the lower-genital tract, nor for the maintenance of reproductive capacity. However, unexpected differences in T cell populations were observed in uninfected pregnant females, as CD1d−/− placentas contained significantly higher percentages of CD4+ and CD8+ T cells than WT counterparts. However, infection triggered a significant decrease in the percentages of CD4+ T cells in CD1d−/− mice. In infected WT pregnant mice, the numbers of uterine CD4+ and CD8+ T cells, monocytes and granulocytes were greatly increased, changes not observed in infected CD1d−/− mice. An increase in the percentage of CD8+ T cells seems independent of CD1d-restricted cells as it occurred in both WT and CD1d−/− mice. Thus, in the steady state, the lack of CD1d-restricted NKT cells affects leukocyte populations only in the placenta. In Chlamydia-infected pregnant mice, the immune response against Chlamydia is dampened in the uterus. Our results suggest that CD1d-restricted NKT cells play a role in the recruitment or homeostasis of leukocyte populations at the maternal–fetal interface in the presence or absence of Chlamydia infection.

Production of α-Galactosylceramide by a Prominent Member of the Human Gut Microbiota

Author SummaryWhile human gut bacteria are thought to produce diffusible molecules that influence host biology, few of these molecules have been identified. Species of Bacteroides, a Gram-negative bacterial genus whose members often comprise >50% of the gut community, are unusual in that they produce sphingolipids, signaling molecules that play a key role in modulating the host immune response. Sphingolipid production is ubiquitous among eukaryotes but present in only a few bacterial genera. We set out to construct a Bacteroides strain that is incapable of producing sphingolipids, knocking out a gene predicted to encode the first enzymatic step in the Bacteroides sphingolipid biosynthetic pathway. The resulting mutant is indeed deficient in sphingolipid production, and we purified and solved the structures of three sphingolipids that are present in the wild-type strain but absent in the mutant. To our surprise, one of these molecules is a close chemical relative of a sponge sphingolipid that is the prototypical ligand for a host receptor that controls the activity of natural killer T cells. Like the sponge sphingolipid, the Bacteroides sphingolipid can modulate natural killer T cell activity, suggesting a novel mechanism by which Bacteroides in the gut might influence the host immune response.

SP0086 Regulatory B cells in healthy and in patients with SLE

Recent advances have demonstrated the existence of a B cell subset with regulatory function named (Bregs). Bregs have been shown to restrain the severity of several autoimmune disease and contribute...

The Functions of Type I and Type II Natural Killer T Cells in Inflammatory Bowel Diseases

CD1d-restricted natural killer T (NKT) cells are a distinct subset of T cells that rapidly produce an array of cytokines on activation and play a critical role in regulating various immune responses. NKT cells are classified into 2 groups based on differences in T-cell receptor usage. Type I NKT cells have an invariant T-cell receptor α-chain and are readily detectable by α-galactosylceramide (α-GalCer)-loaded CD1d tetramers. Type II NKT cells have a more diverse T-cell receptor repertoire and cannot be directly identified. Both types of NKT cells and multiple CD1d-expressing cell types are present in the intestine, and their interactions are likely to be modulated by pathogenic and commensal microbes, which in turn contribute to the intestinal immune responses in health and disease. Indeed, in several animal models of inflammatory bowel disease, type I NKT cells have been shown to make both protective and pathogenic contributions to disease. In contrast, in patients with ulcerative colitis, and a mouse model in which both CD1d expression and the frequency of type II NKT cells are increased, type II NKT cells seem to promote intestinal inflammation. In this review, we summarize the present knowledge on the antigen recognition, activation, and function of NKT cells with a particular focus on their role in inflammatory bowel disease and discuss factors that may influence the functional outcome of NKT cell responses in intestinal inflammation.

CD1d-restricted invariant natural killer T cells mediate airway inflammation through IL-25 and IL-18. (P6257)

Abstract CD1d-restricted iNKT cells are characterized by their expression of an invariant Vα14-Jα18 antigen receptor in mice and of an invariant Vα24-Jα18 in humans that recognizes glycolipid antigen presented by MHC class I-like molecule CD1d. Another distinguishing feature of iNKT cells is their promiscuous production of Th1, Th2, Th9 and Th17 cytokines. Here we identified iNKT cells consist from distinct functional subtypes based on the expression of IL-17 receptor B (IL-17RB), a receptor for IL-25: IL-17RB+ iNKT cells produce robust IL-13 and IL-9 with moderate IL-17A in response to IL-25, which is phenotypically and functionally distinct from classical IL-12-reactive IFN-γ-producing iNKT cells bearing CD122 without IL-17RB. In the in vivo experimental settings, IL-17RB+ iNKT cells play a crucial role in the induction of airway inflammation both of OVA and of RSV model. Through the elucidation of the mechanisms in the activation of IL-17RB+ iNKT cells, IL-18 instead of IL-25 is responsible for the activation of these cells in RSV model, resulting in an acute and robust production of Th2 and Th17 cytokines. Our findings reveal that IL-17RB+ iNKT cells and leading effector cells as target cells of IL-25 and IL-18 in the pathogenesis of airway diseases, illustrating the clinical potential of severe exacerbation of developing asthma by IL-17RB+ iNKT cell-dependent cellular cascade targeted therapy in humans.

Human CD1d knock-in mouse model demonstrates potent antitumor potential of human CD1d-restricted invariant natural killer T cells

Despite a high degree of conservation, subtle but important differences exist between the CD1d antigen presentation pathways of humans and mice. These differences may account for the minimal success of natural killer T (NKT) cell-based antitumor therapies in human clinical trials, which contrast strongly with the powerful antitumor effects in conventional mouse models. To develop an accurate model for in vivo human CD1d (hCD1d) antigen presentation, we have generated a hCD1d knock-in (hCD1d-KI) mouse. In these mice, hCD1d is expressed in a native tissue distribution pattern and supports NKT cell development. Reduced numbers of invariant NKT (iNKT) cells were observed, but at an abundance comparable to that in most normal humans. These iNKT cells predominantly expressed mouse Vβ8, the homolog of human Vβ11, and phenotypically resembled human iNKT cells in their reduced expression of CD4. Importantly, iNKT cells in hCD1d knock-in mice exert a potent antitumor function in a melanoma challenge model. Our results show that replacement of mCD1d by hCD1d can select a population of functional iNKT cells closely resembling human iNKT cells. These hCD1d knock-in mice will allow more accurate in vivo modeling of human iNKT cell responses and will facilitate the preclinical assessment of iNKT cell-targeted antitumor therapies.