Engineering allorejection-resistant CAR-NKT cells from hematopoietic stem cells for off-the-shelf cancer immunotherapy.

The clinical potential of current FDA-approved chimeric antigen receptor (CAR)-engineered T (CAR-T) cell therapy is encumbered by its autologous nature, which presents notable challenges related to manufacturing complexities, heightened costs, and limitations in patient selection. Therefore, there is a growing demand for off-the-shelf universal cell therapies. In this study, we have generated universal CAR-engineered NKT (UCAR-NKT) cells by integrating iNKT TCR engineering and HLA gene editing on hematopoietic stem cells (HSCs), along with an ex vivo, feeder-free HSC differentiation culture. The UCAR-NKT cells are produced with high yield, purity, and robustness, and they display a stable HLA-ablated phenotype that enables resistance to host cell-mediated allorejection. These UCAR-NKT cells exhibit potent antitumor efficacy to blood cancers and solid tumors, both in vitro and in vivo, employing a multifaceted array of tumor-targeting mechanisms. These cells are further capable of altering the tumor microenvironment by selectively depleting immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells. In addition, UCAR-NKT cells demonstrate a favorable safety profile with low risks of graft-versus-host disease and cytokine release syndrome. Collectively, these preclinical studies underscore the feasibility and significant therapeutic potential of UCAR-NKT cell products and lay a foundation for their translational and clinical development.

Cholecystohepatic shunt pathway reduces secondary bile acid accumulation to enhance natural killer T cell-mediated anti-hepatocellular carcinoma immunity.

BACKGROUND AND AIM: The impact of cholecystectomy, which blocks the cholecystohepatic shunt pathway (CHSP), on the prognosis of patients with hepatocellular carcinoma (HCC) is unclear. Hepatic secondary bile acids (BAs) inhibit natural killer T (NKT) cell-mediated immunity against HCC, and the regulation of homeostasis of hepatic secondary BAs is controlled by the CHSP. However, the influence of CHSP on NKT cell-mediated immunity against HCC remains unclear. METHODS: The clinical data of hospitalized patients undergoing HCC resection were collected. Meanwhile, an in situ HCC mouse model was established, and the CHSP was augmented using oleanolic acid (OA). RESULTS: After 1:1 propensity score matching, Cox regression analysis revealed that cholecystectomy was an independent risk factor for HCC recurrence after hepatectomy (P = 0.027, hazard ratio: 1.599, 95% confidence interval: 1.055-2.422). Experimentally, when OA enhanced CHSP, a significant decrease was observed in the accumulation of secondary BAs in the livers of mice. Additionally, a significant increase was observed in the levels of C-X-C ligand 16 and interferon γ in the serum and tumor tissues. Further, the percentage of C-X-C receptor 6 (+) NKT cells in the tumor tissues increased significantly, and the growth of liver tumors was inhibited. CONCLUSIONS: This clinical study revealed that cholecystectomy promoted the recurrence after radical hepatectomy in patients with HCC. Preserving the normal-functioning gallbladder as much as possible during surgery may be beneficial to the patient's prognosis. Further investigation into the mechanism revealed that CHSP enhanced NKT cell-mediated immunity against HCC by reducing the hepatic accumulation of secondary BAs.

Control of the Development, Distribution, and Function of Innate-Like Lymphocytes and Innate Lymphoid Cells by the Tissue Microenvironment.

Recently, considerable attention has been directed toward innate-like T cells (ITCs) and innate lymphoid cells (ILCs) owing to their indispensable contributions to immune responses, tissue homeostasis, and inflammation. Innate-like T cells include NKT cells, MAIT cells, and γδ T cells, whereas ILCs include NK cells, type 1 ILCs (ILC1s), type 2 ILCs (ILC2s), and type 3 ILCs (ILC3s). Many of these ITCs and ILCs are distributed to specific tissues and remain tissue-resident, while others, such as NK cells and some γδ T cells, circulate through the bloodstream. Nevertheless, recent research has shed light on novel subsets of innate immune cells that exhibit characteristics intermediate between tissue-resident and circulating states under normal and pathological conditions. The local microenvironment frequently influences the development, distribution, and function of these innate immune cells. This review aims to consolidate the current knowledge on the functional heterogeneity of ITCs and ILCs, shaped by local environmental cues, with particular emphasis on IL-15, which governs the activities of the innate immune cells involved in type 1 immune responses.

A randomised controlled trial of long NY-ESO-1 peptide-pulsed autologous dendritic cells with or without alpha-galactosylceramide in high-risk melanoma.

AIM: We have previously reported that polyfunctional T cell responses can be induced to the cancer testis antigen NY-ESO-1 in melanoma patients injected with mature autologous monocyte-derived dendritic cells (DCs) loaded with long NY-ESO-1-derived peptides together with α-galactosylceramide (α-GalCer), an agonist for type 1 Natural Killer T (NKT) cells. OBJECTIVE: To assess whether inclusion of α-GalCer in autologous NY-ESO-1 long peptide-pulsed DC vaccines (DCV + α-GalCer) improves T cell responses when compared to peptide-pulsed DC vaccines without α-GalCer (DCV). DESIGN, SETTING AND PARTICIPANTS: Single-centre blinded randomised controlled trial in patients ≥ 18 years old with histologically confirmed, fully resected stage II-IV malignant cutaneous melanoma, conducted between July 2015 and June 2018 at the Wellington Blood and Cancer Centre of the Capital and Coast District Health Board. INTERVENTIONS: Stage I. Patients were randomised to two cycles of DCV or DCV + α-GalCer (intravenous dose of 10 × 106 cells, interval of 28 days). Stage II. Patients assigned to DCV + α-GalCer were randomised to two further cycles of DCV + α-GalCer or observation, while patients initially assigned to DCV crossed over to two cycles of DCV + α-GalCer. OUTCOME MEASURES: Primary: Area under the curve (AUC) of mean NY-ESO-1-specific T cell count detected by ex vivo IFN-γ ELISpot in pre- and post-treatment blood samples, compared between treatment arms at Stage I. Secondary: Proportion of responders in each arm at Stage I; NKT cell count in each arm at Stage I; serum cytokine levels at Stage I; adverse events Stage I; T cell count for DCV + α-GalCer versus observation at Stage II, T cell count before versus after cross-over. RESULTS: Thirty-eight patients gave written informed consent; 5 were excluded before randomisation due to progressive disease or incomplete leukapheresis, 17 were assigned to DCV, and 16 to DCV + α-GalCer. The vaccines were well tolerated and associated with increases in mean total T cell count, predominantly CD4+ T cells, but the difference between the treatment arms was not statistically significant (difference - 6.85, 95% confidence interval, - 21.65 to 7.92; P = 0.36). No significant improvements in T cell response were associated with DCV + α-GalCer with increased dosing, or in the cross-over. However, the NKT cell response to α-GalCer-loaded vaccines was limited compared to previous studies, with mean circulating NKT cell levels not significantly increased in the DCV + α-GalCer arm and no significant differences in cytokine response between the treatment arms. CONCLUSIONS: A high population coverage of NY-ESO-1-specific T cell responses was achieved with a good safety profile, but we failed to demonstrate that loading with α-GalCer provided an additional advantage to the T cell response with this cellular vaccine design. CLINICAL TRIAL REGISTRATION: ACTRN12612001101875. Funded by the Health Research Council of New Zealand.

Adipose invariant NKT cells interact with CD1d-expressing macrophages to regulate obesity-related inflammation.

Obesity is accompanied by and accelerated with chronic inflammation in adipose tissue, especially visceral adipose tissue (VAT). This low-level inflammation predisposes the host to the development of metabolic disease, most notably type 2 diabetes. We have focused on the capacity of glycolipid-reactive, CD1d-restricted natural killer T (NKT) cells to modulate obesity and its associated metabolic sequelae. We previously reported that CD1d knockout (KO) mice are partially protected against the development of obesity-associated insulin resistance, and these findings were recapitulated in mice with an adipocyte-specific CD1d deficiency, suggesting that NKT cell-adipocyte interactions play a critical role in exacerbating disease. However, many other CD1d-expressing cells contribute to the in vivo responses of NKT cells to lipid antigens. In the present study, we examined the role of CD1d expression by macrophages (Mϕ) in the development of obesity-associated metabolic inflammation using LysMcre-cd1d1f/f mice where the CD1d1 gene is disrupted in a Mϕ-specific manner. Unexpectedly, these animals contained a higher frequency of T-bet+ CD4+ T cells in VAT with increased production of Th1 cytokines that aggravated VAT inflammation. Mϕ from mutant mice displayed increased production of IL-12p40, suggesting M1 polarization. These findings indicate that interactions of CD1d on Mϕ with NKT cells play a beneficial role in obesity-associated VAT inflammation and insulin resistance with a sharp contrast to an aggravating role of CD1d in another type of antigen-presenting cell, dendritic cells.

Cancer immunotherapy using artificial adjuvant vector cells to deliver NY-ESO-1 antigen to dendritic cells in situ.

NY-ESO-1 is a cancer/testis antigen expressed in various cancer types. However, the induction of NY-ESO-1-specific CTLs through vaccines is somewhat difficult. Thus, we developed a new type of artificial adjuvant vector cell (aAVC-NY-ESO-1) expressing a CD1d-NKT cell ligand complex and a tumor-associated antigen, NY-ESO-1. First, we determined the activation of invariant natural killer T (iNKT) and natural killer (NK) cell responses by aAVC-NY-ESO-1. We then showed that the NY-ESO-1-specific CTL response was successfully elicited through aAVC-NY-ESO-1 therapy. After injection of aAVC-NY-ESO-1, we found that dendritic cells (DCs) in situ expressed high levels of costimulatory molecules and produced interleukn-12 (IL-12), indicating that DCs undergo maturation in vivo. Furthermore, the NY-ESO-1 antigen from aAVC-NY-ESO-1 was delivered to the DCs in vivo, and it was presented on MHC class I molecules. The cross-presentation of the NY-ESO-1 antigen was absent in conventional DC-deficient mice, suggesting a host DC-mediated CTL response. Thus, this strategy helps generate sufficient CD8+ NY-ESO-1-specific CTLs along with iNKT and NK cell activation, resulting in a strong antitumor effect. Furthermore, we established a human DC-transferred NOD/Shi-scid/IL-2γcnull immunodeficient mouse model and showed that the NY-ESO-1 antigen from aAVC-NY-ESO-1 was cross-presented to antigen-specific CTLs through human DCs. Taken together, these data suggest that aAVC-NY-ESO-1 has potential for harnessing innate and adaptive immunity against NY-ESO-1-expressing malignancies.

CD1d-mediated lipid presentation by CD11c+ cells regulates intestinal homeostasis.

Intestinal homeostasis relies on a continuous dialogue between the commensal bacteria and the immune system. Natural killer T (NKT) cells, which recognize CD1d-restricted microbial lipids and self-lipids, contribute to the regulation of mucosal immunity, yet the mechanisms underlying their functions remain poorly understood. Here, we demonstrate that NKT cells respond to intestinal lipids and CD11c+ cells (including dendritic cells (DCs) and macrophages) are essential to mediate lipid presentation within the gut ultimately controlling intestinal NKT cell homeostasis and activation. Conversely, CD1d and NKT cells participate in the control of the intestinal bacteria composition and compartmentalization, in the regulation of the IgA repertoire and in the induction of regulatory T cells within the gut. These changes in intestinal homeostasis require CD1d expression on DC/macrophage populations as mice with conditional deletion of CD1d on CD11c+ cells exhibit dysbiosis and altered immune homeostasis. These results unveil the importance of CD11c+ cells in controlling lipid-dependent immunity in the intestinal compartment and reveal an NKT cell-DC crosstalk as a key mechanism for the regulation of gut homeostasis.

Invariant NKT cell-augmented GM-CSF-secreting tumor vaccine is effective in advanced prostate cancer model.

Invariant natural killer T cells (iNKT cells) express a semi-invariant T cell receptor that recognizes certain glycolipids (including α-galactosylceramide, αGC) bound to CD1d, and can induce potent antitumor responses. Here, we assessed whether αGC could enhance the efficacy of a GM-CSF-producing tumor cell vaccine in the transgenic SV40 T antigen-driven TRAMP prostate cancer model. In healthy mice, we initially found that optimal T cell responses were obtained with αGC-pulsed TRAMP-C2 cells secreting GM-CSF and milk fat globule epidermal growth factor protein-8 (MFG-E8) with an RGD to RGE mutation (GM-CSF/RGE TRAMP-C2), combined with systemic low dose IL-12. In a therapeutic model, transgenic TRAMP mice were then castrated at ~ 20 weeks, followed by treatment with the combination vaccine. Untreated mice succumbed to tumor by ~ 40 weeks, but survival was markedly prolonged by vaccine treatment, with most mice surviving past 80 weeks. Prostates in the treated mice were heavily infiltrated with T cells and iNKT cells, which both secreted IFNγ in response to tumor cells. The vaccine was not effective if the αGC, IL-12, or GM-CSF secretion was eliminated. Finally, immunized mice were fully resistant to challenge with TRAMP-C2 cells. Together these findings support further development of therapeutic vaccines that exploit iNKT cell activation.

α-Galactosylceramide-Reactive NKT Cells Increase IgG1 Class Switch against a Clostridioides difficile Polysaccharide Antigen and Enhance Immunity against a Live Pathogen Challenge.

All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and, when conjugated to a protein carrier, induces a protective antibody response in animal models. Given that CD1d-restricted natural killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG titers were evident in sera from immunized mice. The inclusion of α-GC had a modest influence on isotype switch but increased the IgG1/IgG2c ratio. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18-/- mice were not protected by the PSII/α-GC immunization modality. Absence of NKT cells similarly had a modest effect on isotype switch, but ratios of IgG1/IgG2c decreased. These results indicate that α-GC-driven NKT cells move the humoral immune response against C. difficile PSII antigen toward Th2-driven IgG1 and may contribute to augmented protection. This study suggests that NKT activation represents a pathway for additional B-cell help that could be used to supplement existing efforts to develop vaccines against polysaccharides derived from C. difficile and other pathogens.

Negative control of diacylglycerol kinase ζ-mediated inhibition of T cell receptor signaling by nuclear sequestration in mice.

Diacylglycerol kinases (DGKs) play important roles in restraining diacylglycerol (DAG)-mediated signaling. Within the DGK family, the ζ isoform appears to be the most important isoform in T cells for controlling their development and function. DGKζ has been demonstrated to regulate T cell maturation, activation, anergy, effector/memory differentiation, defense against microbial infection, and antitumor immunity. Given its critical functions, DGKζ function should be tightly regulated to ensure proper signal transduction; however, mechanisms that control DGKζ function are still poorly understood. We report here that DGKζ dynamically translocates from the cytosol into the nuclei in T cells after TCR stimulation. In mice, DGKζ mutant defective in nuclear localization displayed enhanced ability to inhibit TCR-induced DAG-mediated signaling in primary T cells, maturation of conventional αßT and iNKT cells, and activation of peripheral T cells compared with WT DGKζ. Our study reveals for the first time nuclear sequestration of DGKζ as a negative control mechanism to spatially restrain it from terminating DAG mediated signaling in T cells. Our data suggest that manipulation of DGKζ nucleus-cytosol shuttling as a novel strategy to modulate DGKζ activity and immune responses for treatment of autoimmune diseases and cancer.

The protective function of invariant natural killer T cells in the relapse of experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) in mice provides a useful platform to study the pathogenesis and experimental therapeutics of human uveitis. One often used EAU model employs C57BL/6 (B6) mice sensitized with a peptide residue having 1 to 20 amino acids of human interphotoreceptor retinoid binding protein (hIRBP1-20). The model using the B6 background has permitted a liberal use of genetically engineered strains and has provided insights for understanding uveoretinitis. However, this is usually acute/monophasic and does not represent human uveoretinitis that is characterized as a chronic/recurrent disease. Several chronic/recurrent EAU models have been developed; of these, we employed administration of staphylococcal enterotoxin B (SEB) for relapse in the present study, and found that recurrence was induced at day 24 after primary immunization, which is thought to be the convalescent phase. We reported the activation of invariant natural killer T (iNKT)-cells upon primary immunization of the EAU model mice with the ligand RCAI-56, which was found to mitigate the disease in our previous study. Here, we first attempted to ameliorate EAU in the relapse model using a preventive regimen by activating iNKT cells at the same time relapse induction (day 24) or in a regimen after 3 days of relapse induction (day 27). The preventive as well as post-inductive regimens were successful in reducing histopathological scores by inhibiting the Ag-specific Th17-biased response. Collectively, activation of iNKT cells may be useful to mitigate the relapse response of EAU induced with SEB.

Structure, metabolism and biological functions of steryl glycosides in mammals.

Steryl glycosides (SGs) are sterols glycosylated at their 3ß-hydroxy group. They are widely distributed in plants, algae, and fungi, but are relatively rare in bacteria and animals. Glycosylation of sterols, resulting in important components of the cell membrane SGs, alters their biophysical properties and confers resistance against stress by freezing or heat shock to cells. Besides, many biological functions in animals have been suggested from the observations of SG administration. Recently, cholesteryl glucosides synthesized via the transglycosidation by glucocerebrosidases (GBAs) were found in the central nervous system of animals. Identification of patients with congenital mutations in GBA genes or availability of respective animal models will enable investigation of the function of such endogenously synthesized cholesteryl glycosides by genetic approaches. In addition, mechanisms of the host immune responses against pathogenic bacterial SGs have partially been resolved. This review is focused on the biological functions of SGs in mammals taking into consideration their therapeutic applications in the future.

IL-22 Signaling in the Tumor Microenvironment.

Interleukin (IL)-22 belongs to the IL-10 cytokine family which performs biological functions by binding to heterodimer receptors comprising a type 1 receptor chain (R1) and a type 2 receptor chain (R2). IL-22 is mainly derived from CD4+ helper T cells, CD8+ cytotoxic T cells, innate lymphocytes, and natural killer T cells. It can activate downstream signaling pathways such as signal transducer and activator of transcription (STAT)1/3/5, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), mitogen-activated protein kinase (MAPK), and phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) through these heterodimer receptors. Although IL-22 is produced by immune cells, its specific receptor IL-22R1 is selectively expressed in nonimmune cells, such as hepatocytes, colonic epithelial cells, and pancreatic epithelial cells (Jiang et al. Hepatology 54(3):900-9, 2011; Jiang et al. BMC Cancer 13:59, 2013; Curd et al. Clin Exp Immunol 168(2):192-9, 2012). Immune cells do not respond to IL-22 stimulation directly within tumors, reports from different groups have revealed that IL-22 can indirectly regulate the tumor microenvironment (TME). In the present chapter, we discuss the roles of IL-22 in malignant cells and immunocytes within the TME, meanwhile, the potential roles of IL-22 as a target for drug discovery will be discussed.

Invariant natural killer T cells stimulated with cholesteryl glycosides modulate immune responses in allergy and delayed-type hypersensitivity.

Invariant NKT cells were stimulated with cholesteryl O-acyl α-glycosides in the context of CD1d. The activated NKT cells have potential to sustain the homeostasis in the body exposed to excess in either Th1- or Th2-immunity.

A focus on natural killer T-cell subset characterization and developmental stages.

Almost 20 years ago, CD1d tetramers were developed to track invariant natural killer T (NKT) cells based on their specificity, and to define developmental steps during which differentiation markers and functional features are progressively acquired from early NKT cell precursor to fully mature NKT cell subsets. Based on these findings, a linear developmental model was proposed and subsequently used by all studies investigating the specific role of factors that control NKT cell development. More recently, based on intracellular staining patterns of lineage-specific transcription factors such as T-bet, GATA-3, promyelocytic leukemia zinc finger and RORγt, a lineage differentiation model was proposed for NKT cell development. Currently, studies on NKT cells development present lineage differentiation model data in addition to the linear maturation model. In the perspective presented here, we discuss current knowledge relating to NKT cell developmental models and particularly focus on the approaches and strategies, some of which appear nebulous, used to define NKT cell developmental stages and subsets.

Fully Synthetic Invariant NKT Cell-Dependent Self-Adjuvanting Antitumor Vaccines Eliciting Potent Immune Response in Mice.

Constructing an effective therapeutic cancer vaccine is very attractive and promising for cancer immunotherapy. However, the poor immunogenicity of tumor antigens and suppression of the immune system in the tumor microenvironment are two major obstacles for developing effective cancer vaccines. Invariant NKT cells (iNKT cells), which are essential bridges between the innate and adaptive immune systems, can be rapidly activated by their agonists and, consequently, evoke whole immune systems. Herein, we conjugated a potent agonist of the iNKT cell, α-galactosylceramide (α-GalCer), with the tumor-associated MUC1 glycopeptide antigens as novel self-adjuvanting cancer vaccines through click chemistry. Immunological studies revealed that the mouse immune system was potently evoked and that high levels of tumor-specific IgG antibodies were elicited by vaccine conjugates without an external adjuvant. The produced antibodies could specifically recognize and bind to antigen-expressing cancer cells and, subsequently, induce cytotoxicity through complement-dependent cytotoxicity. Thus, the insertion of α-GalCer significantly improved the immunogenicity of the MUC1 glycopeptide and induced strong antigen-specific antitumor responses, indicating that α-GalCer is an effective built-in adjuvant for constructing potent chemical synthetic antitumor vaccines.

Role of Innate Immune Cells in Psoriasis.

Psoriasis is a chronic inflammatory skin condition caused by a combination of hereditary and environmental factors. Its development is closely related to the adaptive immune response. T helper 17 cells are major IL-17-producing cells, a function that plays an important role in the pathogenesis of psoriasis. However, recent findings have demonstrated that innate immune cells also contribute to the development of psoriasis. Innate lymphoid cells, γδ T cells, natural killer T cells, and natural killer cells are activated in psoriasis, contributing to disease pathology through IL-17-dependent and -independent mechanisms. The present review provides an overview of recent findings, demonstrating a role for innate immunity in psoriasis.

CARs: Beyond T Cells and T Cell-Derived Signaling Domains.

When optimizing chimeric antigen receptor (CAR) therapy in terms of efficacy, safety, and broadening its application to new malignancies, there are two main clusters of topics to be addressed: the CAR design and the choice of transfected cells. The former focuses on the CAR construct itself. The utilized transmembrane and intracellular domains determine the signaling pathways induced by antigen binding and thereby the cell-specific effector functions triggered. The main part of this review summarizes our understanding of common signaling domains employed in CARs, their interactions among another, and their effects on different cell types. It will, moreover, highlight several less common extracellular and intracellular domains that might permit unique new opportunities. Different antibody-based extracellular antigen-binding domains have been pursued and optimized to strike a balance between specificity, affinity, and toxicity, but these have been reviewed elsewhere. The second cluster of topics is about the cellular vessels expressing the CAR. It is essential to understand the specific attributes of each cell type influencing anti-tumor efficacy, persistence, and safety, and how CAR cells crosstalk with each other and bystander cells. The first part of this review focuses on the progress achieved in adopting different leukocytes for CAR therapy.

Miscarriage induced by adoptive transfer of dendritic cells and invariant natural killer T cells into mice.

Unexpected fetal loss is one of the common complications of pregnancy; however, the pathogenesis of many miscarriages, particularly those not associated with infections, is unknown. We previously found that activated DEC-205+ dendritic cells (DCs) and NK1.1+ invariant natural killer T (iNKT) cells are recruited into the myometrium of mice when miscarriage is induced by the intraperitoneal administration of α-galactosylceramide (α-GalCer). Here we demonstrate that the adoptive transfer of DEC-205+ bone marrow-derived DCs cocultured with α-GalCer (DEC-205+ BMDCs-c/w-α-GalCer) directly induced marked fetal loss by syngeneic pregnant C57BL/6 (B6) mice and allogeneic mice (B6 (♀) × BALB/c (♂)), which was accompanied by the accumulation of activated iNKT cells in the myometrium. Further, the adoptive transfer of NK1.1+ iNKT cells obtained from B6 mice injected with α-GalCer facilitated miscarriages in syngeneic Jα18(-/-) (iNKT cell-deficient) mice. These results suggest that DEC-205+ DCs and NK1.1+ iNKT cells play crucial roles required for the initiation of fetal loss associated with stimulation by glycolipid antigens and sterile inflammation.

Regulatory T cells induce CD4- NKT cell anergy and suppress NKT cell cytotoxic function.

BACKGROUND: Due to the strong tumoricidal activities of activated natural killer T (NKT) cells, invariant NKT cell-based immunotherapy has shown promising clinical efficacy. However, suppressive factors, such as regulatory T cells (Tregs), may be obstacles in the use of NKT cell-based cancer immunotherapy for advanced cancer patients. Here, we investigated the suppressive effects of Tregs on NKT cells and the underlying mechanisms with the aim to improve the antitumor activities of NKT cells. METHODS: Peripheral blood samples were obtained from healthy donors, patients with benign tumors, and patients with head and neck squamous cell carcinoma (HNSCC). NKT cells, induced with α-galactosylceramide (α-GalCer), and monocyte-derived dendritic cells (DCs) were co-cultured with naïve CD4+ T cell-derived Tregs to investigate the mechanism of the Treg suppressive effect on NKT cell cytotoxic function. The functions and phenotypes of NKT cells were evaluated with flow cytometry and cytometric bead array. RESULTS: Treg suppression on NKT cell function required cell-to-cell contact and was mediated via impaired DC maturation. NKT cells cultured under Treg-enriched conditions showed a decrease in CD4- NKT cell frequency, which exert strong tumoricidal responsiveness upon α-GalCer stimulation. The same results were observed in HNSCC patients with significantly increased effector Tregs. CONCLUSION: Tregs exert suppressive effects on NKT cell tumoricidal function by inducing more CD4- NKT cell anergy and less CD4+ NKT cell anergy. Both Treg depletion and NKT cell recovery from the anergy state may be important for improving the clinical efficacy of NKT cell-based immunotherapy in patients with advanced cancers.