krn-7000 and Neoplasms

Natural killer T (NKT) cells are a specific T cell subset known to express the αβ-T cell receptor (TCR) for antigens identification and express typical NK cell specifications, such as surface expression of CD56 and CD16 markers as well as production of granzyme. Human NKT cells are divided into two subgroups based on their cytokine receptor and TCR repertoire. Both of them are CD1-restricted and recognize lipid antigens presented by CD1d molecules. Studies have demonstrated that these cells are essential in defense against malignancies. These cells secret proinflammatory and regulatory cytokines that stimulate or suppress immune system responses. In several murine tumor models, activation of type I NKT cells induces tumor rejection and inhibits metastasis's spread. However, type II NKT cells are associated with an inhibitory and regulatory function during tumor immune responses. Variant NKT cells may suppress tumor immunity via different mechanisms that require cross-talk with other immune-regulatory cells. NKT-like cells display high tumor-killing abilities against many tumor cells. In the recent decade, different studies have been performed based on the application of NKT-based immunotherapy for cancer therapy. Moreover, manipulation of NKT cells through administering autologous dendritic cell (DC) loaded with α-galactosylceramide (α-GalCer) and direct α-GalCer injection has also been tested. In this review, we described different subtypes of NKT cells, their function in the anti-tumor immune responses, and the application of NKT cells in cancer immunotherapy from bench to bed.

Topics: Animals; Galactosylceramides; Humans; Immunotherapy; Killer Cells, Natural; Mice; Natural Killer T-Cells; Neoplasms

NKT cells are CD1d-restricted innate-like T cells expressing both T cell receptor and NK cell markers. The major group of NKT cells in both human and mice is the invariant NKT (iNKT) cells and the best-known function of iNKT cells is their potent anti-tumor function in mice. Since its discovery 25 years ago, the prototype ligand of iNKT cells, α-galactosylceramide (α-GalCer) has been used in over 30 anti-tumor clinical trials with mostly suboptimal outcomes. To realize its therapeutic potential, numerous preclinical models have been developed to optimize the scheme and strategies for α-GalCer-based cancer immunotherapies. Nevertheless, since there is no standard protocol for α-GalCer delivery, we reviewed the preclinical studies with a focus on B16 melanoma model in the goal of identifying the best treatment schemes for α-GalCer treatment. We then reviewed the current progress in developing more clinically relevant mouse models for these preclinical studies, most notably the generation of new mouse models with a humanized CD1d/iNKT cell system. With ever-emerging novel iNKT cell ligands, invention of novel α-GalCer delivery strategies and significantly improved preclinical models for optimizing these new strategies, one can be hopeful that the full potential of anti-tumor potential for α-GalCer will be realized in the not too distant future.

Topics: Animals; Disease Models, Animal; Drug Evaluation, Preclinical; Galactosylceramides; Humans; Immunomodulation; Immunotherapy; Mice; Natural Killer T-Cells; Neoplasms; Treatment Outcome

α-Galactosylceramide (α-GalCer) represents a new class of immune stimulators and vaccine adjuvants that activate type I natural killer T (NKT) cells to swiftly release cytokines and to exert helper functions for acquired immune responses. This unique property prompted clinicians to exploit the antitumor potential of NKT cells. Here, we review the effects of α-GalCer in (pre)clinics and discuss current and future strategies that aim to optimize NKT cell-mediated antitumor therapy, with a particular focus on cell-based and nanovector vaccines.

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; Galactosylceramides; Humans; Immunity, Innate; Immunotherapy; Nanocapsules; Natural Killer T-Cells; Neoplasms

NKT cells are a subpopulation of T lymphocytes with phenotypic properties of both T and NK cells and a wide range of immune effector properties. In particular, one subset of these cells, known as invariant NKT cells (iNKT cells), has attracted substantial attention because of their ability to be specifically activated by glycolipid antigens presented by a cell surface protein called CD1d. The development of synthetic α-galactosylceramides as a family of powerful glycolipid agonists for iNKT cells has led to approaches for augmenting a wide variety of immune responses, including those involved in vaccination against infections and cancers. Here, we review basic, preclinical and clinical observations supporting approaches to improving immune responses through the use of iNKT cell-activating glycolipids. Results from preclinical animal studies and preliminary clinical studies in humans identify many promising applications for this approach in the development of vaccines and novel immunotherapies.

Topics: Adjuvants, Immunologic; Animals; Antigens; Antigens, Bacterial; Antigens, CD1d; Antigens, Neoplasm; Bacterial Infections; Bacterial Vaccines; Cancer Vaccines; Cytokines; Dendritic Cells; Galactosylceramides; Glycolipids; Humans; Immunologic Memory; Immunotherapy, Active; Ligands; Lymphocyte Activation; Mice; Natural Killer T-Cells; Neoplasms; Primates; Receptors, Antigen, T-Cell, alpha-beta; Vaccination

Despite advances in therapeutic strategies, the ability of cancer cells to evade destruction remains a significant obstacle to the development of effective anticancer treatment. In recent years a subset of immune cells, myeloid-derived suppressor cells (MDSCs), has been shown to play a key role in evasion of the patient's immune system by tumor cells. A number of different tumor types are associated with increased numbers of circulating MDSCs in cancer patients, suppressing the immune response and permitting continued tumor cell proliferation. Invariant NKT (iNKT) cells have recently been defined as a unique subset of immune cells that are able to act as a link between the innate and adaptive arms of the immune system. iNKT cells have the ability to carry out immune surveillance of tumor cells and control proliferation of malignant cells. Recently, we presented evidence that iNKT cells are able to interact with and decrease the numbers of circulating MDSCs in melanoma patients. This review discusses the evidence for MDSCs in tumor progression and the implication that iNKT cells could be developed as a potent therapeutic strategy.

Topics: Animals; Antigens, CD; Cell Proliferation; Disease Progression; Galactosylceramides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunotherapy; Lymphocyte Activation; Mice; Myeloid Cells; Natural Killer T-Cells; Neoplasms; Tumor Escape

KRN7000, an anticancer drug candidate developed by Kirin Brewery Co. in 1995, is an α-galactosyl ceramide. It is a ligand making a complex with CD1d protein, and it stimulates invariant natural killer T (NKT) cells, which are one of the lineages of immunocytes. NKT cells activated by recognition of the CD1d/KRN7000 complex with its invariant T-cell receptor (TCR) can induce both protective and regulatory immune responses. To determine the recognition and activation mechanisms of NKT cells and to develop drug candidates more effective than KRN7000, a large number of analogs of KRN7000 have been synthesized. Some of them show potent bioactivities and have the potential of being utilized as therapeutic agents. In this review, structure-activity relationship studies of novel glycolipids which stimulate NKT cells efficiently are summarized.

Topics: Animals; Antigens, CD1d; Antineoplastic Agents; Cytokines; Galactosylceramides; Glycosphingolipids; Humans; Lymphocyte Activation; Mice; Natural Killer T-Cells; Neoplasms; Receptors, Antigen, T-Cell; Structure-Activity Relationship; Th1-Th2 Balance

Natural killer T (NKT) cells constitute an important subset of T cells that can both directly and indirectly mediate antitumor immunity. However, we and others have reported that cancer patients have a reduction in both NKT cell number and function. NKT cells can be stimulated and expanded with α-GalCer and cytokines and these expanded NKT cells retain their phenotype, remain responsive to antigenic stimulation, and display cytotoxic function against tumor cell lines. These data strongly favor the use of ex vivo expanded NKT cells in adoptive immunotherapy. NKT cell based-immunotherapy has been limited by the use of autologous antigen-presenting cells, which can vary substantially in their quantity and quality. A standardized system that relies on artificial antigen-presenting cells (aAPCs) could produce the stimulating effects of dendritic cell (DC) without the pitfalls of allo- or xenogeneic cells. In this review, we discuss the progress that has been made using CD1d-based aAPC and how this acellular antigen presenting system can be used in the future to enhance our understanding of NKT cell biology and to develop NKT cell-specific adoptive immunotherapeutic strategies.

Topics: Antigen Presentation; Antigen-Presenting Cells; Antigens, CD1d; Cell Line, Tumor; Cytokines; Galactosylceramides; Humans; Immunotherapy, Adoptive; Lymphocyte Activation; Lymphocyte Count; Natural Killer T-Cells; Neoplasms

For over a century, research has sought ways to boost the immune system in order to eradicate tumors and viruses that exist after escaping immunosurveillance. For the treatment of cancer and hepatitis immunotherapeutic strategies have overall had limited clinical success. An urgent need exists therefore to introduce more effective therapeutic approaches. Invariant (i)NKT cells constitute a conserved T lymphocyte lineage with dominant immunoregulatory, antitumor and antiviral effector cell properties. iNKT specifically recognize the glycolipid α-galactosylceramide in the context of CD1d resulting in their activation. Activated iNKT can promote the development of a long-lasting Th1 biased proinflammatory immune response as demonstrated in multiple tumor-metastasis and viral infection models. Here, we will provide a brief overview of the preclinical data of α-galactosylceramide that formed the basis for subsequent clinical trials in patients with advanced cancer and chronic hepatitis B/C, and elaborate on our own clinical experience with α-galactosylceramide in these patient groups.

Topics: Antigens, CD1d; Antineoplastic Agents; Clinical Trials as Topic; Galactosylceramides; Hepatitis B, Chronic; Hepatitis C, Chronic; Humans; Killer Cells, Natural; Lymphocyte Activation; Neoplasms

Mouse and human livers contain innate immune leukocytes, NK cells, NKT cells, and macrophage-lineage Kupffer cells. Various bacterial components, including Toll-like receptor (TLR) ligands and an NKT cell ligand (α-galactocylceramide), activate liver Kupffer cells, which produce IL-1, IL-6, IL-12, and TNF. IL-12 activates hepatic NK cells and NKT cells to produce IFN-γ, which further activates hepatic T cells, in turn activating phagocytosis and cytokine production by Kupffer cells in a positive feedback loop. These immunological events are essentially evoked to protect the host from bacterial and viral infections; however, these events also contribute to antitumor and antimetastatic immunity in the liver by activated liver NK cells and NKT cells. Bystander CD8(+)CD122(+) T cells, and tumor-specific memory CD8(+)T cells, are also induced in the liver by α-galactocylceramide. Furthermore, adoptive transfer experiments have revealed that activated liver lymphocytes may migrate to other organs to inhibit tumor growth, such as the lungs and kidneys. The immunological mechanism underlying the development of hepatocellular carcinoma in cirrhotic livers in hepatitis C patients and liver innate immunity as a double-edged sword (hepatocyte injury/regeneration, septic shock, autoimmune disease, etc.) are also discussed.

Topics: Aging; Animals; Bacteria; CD8-Positive T-Lymphocytes; Galactosylceramides; Hepatocytes; Humans; Interleukin-12; Interleukin-2 Receptor beta Subunit; Killer Cells, Natural; Kupffer Cells; Liver Neoplasms; Lymphocyte Activation; Multiple Organ Failure; Natural Killer T-Cells; Neoplasms; Shock, Septic; T-Lymphocytes, Cytotoxic

To optimize vaccination strategies, it is important to use protocols that can 'jump-start' immune responses by harnessing cells of the innate immune system to assist the expansion of antigen-specific B and T cells. In this Review, we discuss the evidence indicating that invariant natural killer T (iNKT) cells can positively modulate dendritic cells and B cells, and that their pharmacological activation in the presence of antigenic proteins can enhance antigen-specific B- and T-cell responses. In addition, we describe structural and kinetic analyses that assist in the design of optimal iNKT-cell agonists that could be used in the clinical setting as vaccine adjuvants.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD1d; B-Lymphocytes; Cell Differentiation; Clinical Trials as Topic; Cytokines; Dendritic Cells; Forecasting; Galactosylceramides; Glycolipids; Humans; Immunity, Innate; Immunologic Surveillance; Infections; Lymphocyte Activation; Mice; Models, Molecular; Natural Killer T-Cells; Neoplasms; Receptors, Antigen, T-Cell, alpha-beta; Structure-Activity Relationship; Vaccination

Tumor immunosurveillance is a part of the dynamic process of interaction between abnormal cells and the host immune system. Tumor immunosurveillance is actively and continuously regulated in both positive and negative ways. Natural killer T (NKT) cells are cells that have been shown to play a role in both positive and negative regulation of tumor immunosurveillance. Recent studies suggest that NKT cells are a heterogeneous cell population with multiple subsets with distinct functions.. This review discusses the functions of those NKT cell subsets in regulating tumor immunity and potential interactions or counter-regulation among the NKT cell subsets.. Selected literature is reviewed.. Manipulation of the balance among those subsets may provide new modes of intervention for tumor immunotherapy.

Topics: Animals; Antigens, Neoplasm; CD28 Antigens; CD40 Antigens; Cytokines; Disease Susceptibility; Galactosylceramides; Humans; Immunocompetence; Immunologic Memory; Immunologic Surveillance; Killer Cells, Natural; Lipids; Lymphocyte Activation; Lymphocytes, Null; Lymphocytes, Tumor-Infiltrating; Mice; Mice, Inbred NOD; Mice, Transgenic; Neoplasms; T-Lymphocyte Subsets

CD1d-restricted T cells (NKT cells) are potent regulators of a broad range of immune responses. In particular, an abundance of research has focussed on the role of NKT cells in tumor immunity. This field of research has been greatly facilitated by the finding of agonist ligands capable of potently stimulating NKT cells and also animal models where NKT cells have been shown to play a natural role in the surveillance of tumors. Herein, we review the capability of NKT cells to promote the rejection of tumors and the mechanisms by which this occurs. We also highlight a growing field of research that has found that NKT cells are capable of suppressing anti-tumor immunity and discuss the progress to date for the immunotherapeutic use of NKT cells.

Topics: Animals; Antigens, CD1; Clinical Trials, Phase I as Topic; Disease Models, Animal; Galactosylceramides; Humans; Killer Cells, Natural; Mice; Monitoring, Immunologic; Neoplasms

The observation that the glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent stimulator of natural killer T (NKT) cells has provided an important means for investigating NKT cell biology. alpha-GalCer is presented on CD1d to the invariant NKT receptor, leading to interleukin-12 (IL-12) production by dendritic cells (DCs) and to NK cell activation. We review our research on the tumor-protective properties of alpha-GalCer, particularly the major role played by DCs. We compared administration of alpha-GalCer on mature DCs with soluble glycolipid and found that DCs induced more prolonged interferon-gamma (IFN-gamma) production by NKT cells and better protection against B16 melanoma. Human alpha-GalCer-loaded DCs also expanded NKT cell numbers in cancer patients. alpha-GalCer-activated NKT cells were then found to induce DC maturation in vivo. The maturing DCs produced IL-12, upregulated co-stimulatory molecules, and induced adaptive immunity to captured cellular antigens, including prolonged, combined CD4(+)/CD8(+) T-cell immunity to dying tumor cells. Surprisingly, co-stimulator-poor tumor cells, if directly loaded with alpha-GalCer ('tumor/Gal') and injected intravenously, also induced strong NKT- and NK-cell responses. The latter killed the tumor/Gal, which were subsequently cross presented by CD1d on DCs to elicit DC maturation and prolonged adaptive T-cell immunity, which lasted 6-12 months. These findings help explain tumor protection via alpha-GalCer and urge development of the DC-NKT axis to provide innate and adaptive immunity to human cancers.

Topics: Animals; Cross-Priming; Dendritic Cells; Galactosylceramides; Histocompatibility Antigens Class I; Humans; Immunity, Cellular; Interferon-gamma; Killer Cells, Natural; Mice; Neoplasms; T-Lymphocytes

NKT cells are a unique subset of T cells that recognize glycolipid antigens presented by CD1d molecules. NKT cells have the potential to produce key cytokines of both Th1 and Th2 T cells and are involved in the control of several types of immune response. Furthermore, NKT cells perform spontaneous tumor immunosurveillance. Upon specific activation with alpha-GalCer, NKT cells show strong antitumor immune responses through direct cytotoxicity and indirect activation of a cascade of antitumor effector cells such as natural killer (NK) cells and CD8+ cytotoxic T cells. In addition to alpha-GalCer, many other CD1d ligands, including self and bacterial glycolipids and modified synthetic glycolipid antigens, have also been discovered. Structurally different glycolipid antigens have the distinct ability to activate NKT cells. Thus, it seems that we are now close to a position in which we can control the activation status of NKT cells; this makes NKT cells an ideal target of anticancer immunotherapies. Clinical trials with soluble alpha-GalCer or alpha-GalCer-pulsed dendritic cells aimed at in vivo reconstitution and activation of human NKT cells have provided both promising and challenging results. In this review, we discuss NKT-cell-mediated antitumor immune responses, as well as the early outcomes and implications of recent clinical studies.

Topics: Antigen Presentation; Antigens, CD1; Antigens, CD1d; Cytokines; Cytotoxicity, Immunologic; Dendritic Cells; Galactosylceramides; Glycolipids; Humans; Immunotherapy; Killer Cells, Natural; Ligands; Lymphocyte Activation; Neoplasms; T-Lymphocyte Subsets; T-Lymphocytes, Cytotoxic

A unique lymphocyte population, CD1d-restricted NKT cells, has been revealed to be a key player in both the innate and acquired immune responses, including antitumor effects. Recent studies revealed that at least two subsets of CD1d-restricted NKT cells exist: type I, having invariant Valpha14 receptor; and type II, having heterogeneous non-Valpha14 receptor. The specific glycolipid ligand, alpha-GalCer, effectively stimulates mouse and human type I NKT cells. The activation of type I NKT cells substantially influences function of other various cell types, particularly DC, NK cells, CD4 Th1 cells, and CD8 cytotoxic T cells, all contributing to the antitumor immune responses. Recent studies also indicated that, unlike type I NKT cells, type II NKT cells have a potential to repress antitumor immune responses. In this review, we summarize the characteristics of the antitumor immune responses mediated by both mouse and human CD1d-restricted NKT cells and discuss their potential in clinical applications against cancer.

Topics: Animals; Antigens, CD1; Antigens, CD1d; Cell Communication; Clinical Trials as Topic; Dendritic Cells; Galactosylceramides; Humans; Immunotherapy; Killer Cells, Natural; Lymphocyte Activation; Lymphocyte Subsets; Mice; Neoplasms

A unique lymphocyte population, Valpha14 NKT cells, has recently been revealed to be a key player in the immune responses against tumors. Activation of Valpha14 NKT cells affects various cell types, particularly dendritic cells (DCs), NK cells, CD4 Th1 cells, and CD8 cytotoxic T cells in the innate and acquired immune systems, eventually resulting in the enhanced activation of NKT cell-mediated cellular cascade in the anti-tumor responses. The specific ligand, alpha-galactosylceramide (alpha-GalCer), effectively stimulates mouse and human NKT cells, making NKT cells an ideal target for the development of cancer immunotherapy. Clinical trials using alpha-GalCer have actually started in several centers in the world. In this review, we summarize the Valpha14 NKT cell-mediated cellular cascade in the anti-tumor response in mice and discuss potential clinical applications of alpha-GalCer-pulsed DC therapy.

Topics: Animals; Clinical Trials as Topic; Cytotoxicity, Immunologic; Galactosylceramides; Humans; Killer Cells, Natural; Neoplasms; Receptors, Immunologic; Receptors, Natural Killer Cell

Invariant natural killer T (iNKT) cells are an unusual group of T lymphocytes that recognize glycolipid antigens presented by the major histocompatibility complex class I-related protein CD1d. Because iNKT cells play a regulatory role in the immune system, they are attractive targets for immunotherapy. The marine-sponge-derived glycolipid alpha-galactosylceramide (alpha-GalCer) potently activates iNKT cells. In vivo administration of alpha-GalCer to mice or humans results in rapid and robust cytokine secretion by iNKT cells, followed by the activation of a variety of cell types of the innate and adaptive immune systems. These potent immunomodulatory activities of alpha-GalCer are being exploited for therapeutic purposes. Preclinical studies in mice have demonstrated that alpha-GalCer and related glycolipids can protect mice against a variety of diseases, including cancer, infections, and several autoimmune and inflammatory conditions. Although alpha-GalCer treatment of mice is associated with unwanted side-effects, it has been proven safe in clinical trials with cancer patients. These studies have raised significant enthusiasm for the development of effective and safe iNKT-cell-based immunotherapies for a variety of human diseases.

Topics: Animals; Antigens, CD1; Autoimmune Diseases; Carbohydrate Sequence; Galactosylceramides; Glycolipids; Humans; Infections; Inflammation; Killer Cells, Natural; Mice; Molecular Sequence Data; Molecular Structure; Neoplasms; T-Lymphocyte Subsets

Natural killer T (NKT) cells constitute a T cell subpopulation that shares several characteristics with NK cells. NKT cells are characterized by a narrow T cell antigen receptor (TCR) repertoire, recognize glycolipid antigen in the context of the monomorphic CD1d antigen-presenting molecule, and have the unique capacity to rapidly produce large amounts of both T helper (Th) 1 and Th2 cytokines. Important roles of NKT cells have now been demonstrated in the regulation of autoimmune, allergic, antimicrobial, and antitumor immune responses. Here, we review the immunoregulatory role of NKT cells in disease and discuss NKT cell based immunotherapeutic strategies.

Topics: Adjuvants, Immunologic; Animals; Antigens, CD1; Antigens, CD1d; Autoimmune Diseases; Communicable Diseases; Galactosylceramides; Graft Survival; Humans; Hypersensitivity; Immunotherapy; Killer Cells, Natural; Mice; Neoplasms; T-Lymphocytes

CD1d-restricted Natural Killer T cells (NKT cells), a novel lymphocyte lineage, are considered to play an intermediary role bridging innate and acquired immunity. This review discusses the characteristics of NKT cells and their biological significance in the immune system, and summarizes their in vivo functions observed in a number of pathological settings, including infectious diseases, cancer, autoimmunity, and transplantation. Further, we discuss recent data that have generated considerable interest in utilizing NKT cells as targets of new therapeutic interventions in various human diseases.

Topics: Animals; Antigens, CD1; Antigens, CD1d; Apoptosis; Autoimmunity; Cell Transplantation; Cytokines; Diabetes Mellitus, Type 1; Encephalomyelitis, Autoimmune, Experimental; Galactosylceramides; Glycolipids; Humans; Immune System; Immunity, Innate; Killer Cells, Natural; Multiple Sclerosis; Neoplasms

Natural killer T (NKT) cells are distinct glycolipid reactive innate lymphocytes that are implicated in the resistance to pathogens and tumors. Earlier attempts to mobilize NKT cells, specifically, in vivo in humans met with limited success. Here, we evaluated intravenous injection of monocyte-derived mature DCs that were loaded with a synthetic NKT cell ligand, alpha-galactosyl-ceramide (alpha-GalCer; KRN-7000) in five patients who had advanced cancer. Injection of alpha-GalCer-pulsed, but not unpulsed, dendritic cells (DCs) led to >100-fold expansion of several subsets of NKT cells in all patients; these could be detected for up to 6 mo after vaccination. NKT activation was associated with an increase in serum levels of interleukin-12 p40 and IFN-gamma inducible protein-10. In addition, there was an increase in memory CD8+ T cells specific for cytomegalovirus in vivo in response to alpha-GalCer-loaded DCs, but not unpulsed DCs. These data demonstrate the feasibility of sustained expansion of NKT cells in vivo in humans, including patients who have advanced cancer, and suggest that NKT activation might help to boost adaptive T cell immunity in vivo.

Topics: Adult; Blood Chemical Analysis; CD8-Positive T-Lymphocytes; Cell Proliferation; Chemokine CXCL10; Chemokines; Chemokines, CXC; Cytokines; Cytomegalovirus; Dendritic Cells; Flow Cytometry; Galactosylceramides; Humans; Immunotherapy; Interleukin-12; Killer Cells, Natural; Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Vaccination

alpha-galactosylceramide (KRN7000) is a glycosphingolipid that has been shown to inhibit tumor growth and to prolong survival in inoculated mice through activation of natural killer (NK) T cells. We performed a dose escalation study of KRN7000 in advanced cancer patients.. Patients with solid tumors received i.v. KRN7000 (50-4,800 micro g/m(2)) on days 1, 8, and 15 of a 4-weekly cycle. Patients were given 1 cycle and, in the absence of dose-limiting toxicity or progression, treatment was continued. Pharmacokinetics (PK) and immunomonitoring were performed in all patients.. Twenty-four patients were entered into this study. No dose-limiting toxicity was observed over a wide range of doses (50-4,800 micro g/m(2)). PK was linear in the dose range tested. Immunomonitoring demonstrated that NKT cells (CD3+Valpha24+Vbeta11+) typically disappeared from the blood within 24 h of KRN7000 injection. Additional biological effects included increased serum cytokine levels (tumor necrosis factor alpha and granulocyte macrophage colony-stimulating factor) in 5 of 24 patients and a transient decrease in peripheral blood NK cell numbers and cytotoxicity in 7 of 24 patients. Importantly, the observed biological effects depended on pretreatment NKT-cell numbers rather than on the dose of KRN7000. Pretreatment NKT-cell numbers were significantly lower in patients compared with healthy controls (P = 0.0001). No clinical responses were recorded and seven patients experienced stable disease for a median duration of 123 days.. i.v. KRN7000 is well tolerated in cancer patients over a wide range of doses. Biological effects were observed in several patients with relatively high pretreatment NKT-cell numbers. Other therapeutic strategies aiming at reconstitution of the deficient NKT-cell population in cancer patients may be warranted.

Topics: Adult; Aged; Antigens, CD; Antineoplastic Agents; Area Under Curve; Cell Division; Cell Survival; Cytotoxicity, Immunologic; Female; Galactosylceramides; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-gamma; Interleukin-12; Killer Cells, Natural; Ligands; Lymphocyte Activation; Male; Maximum Tolerated Dose; Middle Aged; Neoplasms; Receptors, Antigen, T-Cell, alpha-beta; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocytes

Tumor-associated antigens (TAAs) represent attractive targets in the development of anti-cancer vaccines. The filamentous bacteriophage is a safe and versatile delivery nanosystem, and recombinant bacteriophages expressing TAA-derived peptides at a high density on the viral coat proteins improve TAA immunogenicity, triggering effective in vivo anti-tumor responses. To enhance the efficacy of the bacteriophage as an anti-tumor vaccine, we designed and generated phage particles expressing a CD8+ peptide derived from the human cancer germline antigen NY-ESO-1 decorated with the immunologically active lipid alpha-GalactosylCeramide (α-GalCer), a potent activator of invariant natural killer T (iNKT) cells. The immune response to phage expressing the human TAA NY-ESO-1 and delivering α-GalCer, namely fdNY-ESO-1/α-GalCer, was analyzed either in vitro or in vivo, using an HLA-A2 transgenic mouse model (HHK). By using NY-ESO-1-specific TCR-engineered T cells and iNKT hybridoma cells, we observed the efficacy of the fdNY-ESO-1/α-GalCer co-delivery strategy at inducing activation of both the cell subsets. Moreover, in vivo administration of fdNY-ESO-1 decorated with α-GalCer lipid in the absence of adjuvants strongly enhances the expansion of NY-ESO-1-specific CD8+ T cells in HHK mice. In conclusion, the filamentous bacteriophage delivering TAA-derived peptides and the α-GalCer lipid may represent a novel and promising anti-tumor vaccination strategy.

Topics: Animals; Antibodies; Antigens, Neoplasm; CD8-Positive T-Lymphocytes; Galactosylceramides; Humans; Membrane Proteins; Mice; Mice, Transgenic; Neoplasms; Peptides

We report a promising cancer vaccine candidate comprising antigen/adjuvant-displaying enveloped viral replica as a novel vaccine platform. The artificial viral capsid, which consists of a self-assembled β-annulus peptide conjugated with an HER2-derived antigenic CH401 peptide, was enveloped within a lipid bilayer containing the lipidic adjuvant α-GalCer. The use of an artificial viral capsid as a scaffold enabled precise control of its size to ∼100 nm, which is generally considered to be optimal for delivery to lymph nodes. The encapsulation of the anionically charged capsid by a cationic lipid bilayer dramatically improved its stability and converted its surface charge to cationic, enhancing its uptake by dendritic cells. The developed CH401/α-GalCer-displaying enveloped viral replica exhibited remarkable antibody-production activity. This study represents a pioneering example of precise vaccine design through bottom-up construction and opens new avenues for the development of effective vaccines.

Topics: Adjuvants, Immunologic; Antigens; Cancer Vaccines; Lipid Bilayers; Neoplasms; Peptides

Invariant natural killer T (iNKT) cells have the capacity to mount potent anti-tumor reactivity and have therefore become a focus in the development of cell-based immunotherapy. iNKT cells attack tumor cells using multiple mechanisms with a high efficacy; however, their clinical application has been limited because of their low numbers in cancer patients and difficulties in infiltrating solid tumors. In this study, we aimed to overcome these critical limitations by using α-GalCer, a synthetic glycolipid ligand specifically activating iNKT cells, to recruit iNKT to solid tumors. By adoptively transferring human iNKT cells into tumor-bearing humanized NSG mice and administering a single dose of tumor-localized α-GalCer, we demonstrated the rapid recruitment of human iNKT cells into solid tumors in as little as one day and a significantly enhanced tumor killing ability. Using firefly luciferase-labeled iNKT cells, we monitored the tissue biodistribution and pharmacokinetics/pharmacodynamics (PK/PD) of human iNKT cells in tumor-bearing NSG mice. Collectively, these preclinical studies demonstrate the promise of an αGC-driven iNKT cell-based immunotherapy to target solid tumors with higher efficacy and precision.

Topics: Animals; Antigens, CD1d; Galactosylceramides; Humans; Mice; Natural Killer T-Cells; Neoplasms; Tissue Distribution

Adoptive immunotherapy with T cells engineered with tumor-specific T cell receptors (TCRs) holds promise for cancer treatment. However, suppressive cues generated in the tumor microenvironment (TME) can hinder the efficacy of these therapies, prompting the search for strategies to overcome these detrimental conditions and improve cellular therapeutic approaches. CD1d-restricted invariant natural killer T (iNKT) cells actively participate in tumor immunosurveillance by restricting suppressive myeloid populations in the TME. Here, we showed that harnessing iNKT cells with a second TCR specific for a tumor-associated peptide generated bispecific effectors for CD1d- and major histocompatibility complex (MHC)-restricted antigens in vitro. Upon in vivo transfer, TCR-engineered iNKT (TCR-iNKT) cells showed the highest efficacy in restraining the progression of multiple tumors that expressed the cognate antigen compared with nontransduced iNKT cells or CD8

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Engineering; Humans; Mice; Myeloid Cells; Natural Killer T-Cells; Neoplasms; Receptors, Antigen, T-Cell; Tumor Microenvironment

Invariant natural killer T (iNKT) cells display important properties that could bridge the innate and adaptive immunity, and they have been shown to play key roles in cancer immunotherapy. However, administration of iNKT cell agonist αGalCer fails to induce sustained antitumor immunity due to the rapid anergy induction after an initial strong activation. To this end, we have designed a recombinant CD1d protein that is fused to an anti-TAA scFv, which is able to recruit iNKT cells to the tumor site and induce tumor regression. Importantly, recombinant CD1d fusion proteins loaded with α-GalCer demonstrated sustained activation of iNKT cells upon repeated injections and superior tumor control, as compared to α-GalCer treatment.

Topics: Antigens, CD1d; Galactosylceramides; Humans; Lymphocyte Activation; Natural Killer T-Cells; Neoplasms; Recombinant Fusion Proteins; Single-Chain Antibodies

The capacity of α-galactosylceramide (α-GalCer) to act as an anti-cancer agent in mice through the specific stimulation of type I NKT (iNKT) cells has prompted extensive investigation to translate this finding to the clinic. However, low frequencies of iNKT cells in cancer patients and their hypo-responsiveness to repeated stimulation have been seen as barriers to its efficacy. Currently the most promising clinical application of α-GalCer, or its derivatives, is as stimuli for ex vivo expansion of iNKT cells for adoptive therapy, although some encouraging clinical results have recently been reported using α-GalCer pulsed onto large numbers of antigen presenting cells (APCs). In on-going preclinical studies, attempts to improve efficacy of injected iNKT cell agonists have focussed on optimising presentation in vivo, through encapsulation in particulate vectors, making structural changes that help binding to the presenting molecule CD1d, or injecting agonists covalently attached to recombinant CD1d. Variations on these same approaches are being used to enhance the APC-licencing function of iNKT cells in vivo to induce adaptive immune responses to associated tumour antigens. Looking ahead, a unique capacity of in vivo-activated iNKT cells to facilitate formation of resident memory CD8

Topics: Adjuvants, Immunologic; Animals; Antigen-Presenting Cells; Cancer Vaccines; CD8-Positive T-Lymphocytes; Galactosylceramides; Humans; Immunotherapy; Lymphocyte Activation; Natural Killer T-Cells; Neoplasms

The last few decades have witnessed groundbreaking research geared towards immune surveillance mechanisms and have yielded significant improvements in the field of cancer immunotherapy. This approach narrows down on the development of therapeutic agents that either activate or enhance the recognitive function of the immune system to facilitate the destruction of malignant cells. The α -galactosylceramide derivative, KRN7000, is an immunotherapeutic agent that has gained attention due to its pharmacological ability to activate CD1d-restricted invariant natural killer T(iNKT) cells with notable potency against cancer cells in mouse models; a therapeutic success was not well replicated in human models. Dual structural modification of KRN7000 entailing the incorporation of hydrocinnamoyl ester on C6" and C4-OH truncation of the sphingoid base led to the development of AH10-7 which, interestingly, exhibited high potency in human cells.. Therefore, to gain molecular insights into the structural dynamics and selective mechanisms of AH10-7 for human variants, we employed integrative molecular dynamics simulations and thermodynamic calculations to investigate the inhibitory activities of KRN7000 andAH10-7 on hTCR-CD1d towards activating iNKT.. Interestingly, our findings revealed that AH10-7 exhibited higher affinity binding and structural effects on hTCR-CD1d, as mediated by the incorporated hydrocinnamoyl ester moiety which accounted for stronger intermolecular interactions with 'non-common' binding site residues.. Findings extracted from this study further reveal important molecular and structural perspectives that could aid in the design of novel α-GalCer derivatives for cancer immunotherapeutics.

Topics: Adjuvants, Immunologic; Antigens, CD1d; Galactosylceramides; Humans; Immunotherapy; Molecular Dynamics Simulation; Natural Killer T-Cells; Neoplasms; Protein Binding; Receptors, Antigen, T-Cell; Thermodynamics

Activation of invariant natural killer T (iNKT) cells by α-galactosylceramides (α-GalCers) stimulates strong immune responses and potent anti-tumor immunity. Numerous modifications of the glycolipid structure have been assessed to derive activating ligands for these T cells with altered and potentially advantageous properties in the induction of immune responses. Here, we synthesized variants of the prototypical α-GalCer, KRN7000, with amide-linked phenyl alkane substitutions on the C4″-position of the galactose ring. We show that these variants have weak iNKT cell stimulating activity in mouse models but substantially greater activity for human iNKT cells. The most active of the C4″-amides in our study showed strong anti-tumor effects in a partially humanized mouse model for iNKT cell responses.

Topics: Amides; Animals; Galactosylceramides; Glycolipids; Humans; Killer Cells, Natural; Mice; Models, Animal; Neoplasms; Sugars

The spatiotemporal distribution of cytokines orchestrates immune responses in vivo, yet the underlying mechanisms remain to be explored. We showed here that the spatial distribution of interleukin-4 (IL4) in invariant natural killer T (iNKT) cells regulated crosstalk between iNKT cells and dendritic cells (DCs) and controlled iNKT cell-mediated T-helper type 1 (Th1) responses. The persistent polarization of IL4 induced by strong lipid antigens, that is, α-galactosylceramide (αGC), caused IL4 accumulation at the immunological synapse (IS), which promoted the activation of the IL4R-STAT6 (signal transducer and activator of transcription 6) pathway and production of IL12 in DCs, which enhanced interferon-γ (IFNγ) production in iNKT cells. Conversely, the nonpolarized secretion of IL4 induced by Th2 lipid antigens with a short or unsaturated chain was incapable of enhancing this iNKT cell-DC crosstalk and thus shifted the immune response to a Th2-type response. The nonpolarized secretion of IL4 in response to Th2 lipid antigens was caused by the degradation of Cdc42 in iNKT cells. Moreover, reduced Cdc42 expression was observed in tumor-infiltrating iNKT cells, which impaired IL4 polarization and disturbed iNKT cell-DC crosstalk in tumors.

Topics: Animals; cdc42 GTP-Binding Protein; Dendritic Cells; Galactosylceramides; Humans; Immunological Synapses; Interleukin-4; Mice, Inbred C57BL; Microtubule-Organizing Center; Natural Killer T-Cells; Neoplasms; Paclitaxel; Receptors, Interleukin-4; Signal Transduction; STAT6 Transcription Factor; Th1 Cells; Th2 Cells

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that play a crucial role in the tumor surveillance. The activation of iNKT cells by their specific ligand α-galactosylceramide (α-GalCer) induces the activation of dendritic cells (DCs) via reciprocal interaction, which results in the generation of cellular immunity against cancer. Here we describe a method to detect DC-mediated cellular adjuvant properties of human iNKT cells in vitro.

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Communication; Cell Differentiation; Cell Line, Tumor; Culture Media, Conditioned; Cytokines; Dendritic Cells; Flow Cytometry; Fluorescent Antibody Technique, Direct; Galactosylceramides; Healthy Volunteers; Humans; Immunologic Surveillance; Immunomagnetic Separation; Induced Pluripotent Stem Cells; Mesenchymal Stem Cells; Mice; Natural Killer T-Cells; Neoplasms; Primary Cell Culture; Recombinant Proteins

The glycosphingolipid α-galactosylceramide (α-GalCer) is a well-described immune activator with strong anti-tumor properties in animal models. It is presented on CD1d and acts by stimulating the invariant, type I, natural killer T (iNKT) lymphocytes to rapidly secrete TH1 and TH2 associated cytokines. This in turn promotes activation of a diversity of immune cells including natural killer (NK) cells with anti-tumor functions. Prior to tumor development, iNKT cells can also perform tumor surveillance and naturally protect from emergence of cancer. In contrast, we have recently demonstrated that iNKT cells naturally promote polyps in the spontaneous murine adenomatous polyposis coli (Apc)

Topics: Animals; Antigens, CD1d; CD8-Positive T-Lymphocytes; Cytokines; Female; Galactosylceramides; Immunosuppression Therapy; Immunotherapy; Inflammation; Intestinal Polyps; Ligands; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Natural Killer T-Cells; Neoplasms; Th1 Cells; Th2 Cells; Tumor Microenvironment

Glycosylceramides that activate CD1d-restricted invariant natural killer T (iNKT) cells have potential therapeutic applications for augmenting immune responses against cancer and infections. Previous studies using mouse models identified sphinganine variants of α-galactosylceramide as promising iNKT cell activators that stimulate cytokine responses with a strongly proinflammatory bias. However, the activities of sphinganine variants in mice have generally not translated well to studies of human iNKT cell responses. Here, we show that strongly proinflammatory and anti-tumor iNKT cell responses were achieved in mice by a variant of α-galactosylceramide that combines a sphinganine base with a hydrocinnamoyl ester on C6″ of the sugar. Importantly, the activities observed with this variant were largely preserved for human iNKT cell responses. Structural and in silico modeling studies provided a mechanistic basis for these findings and suggested basic principles for capturing useful properties of sphinganine analogs of synthetic iNKT cell activators in the design of immunotherapeutic agents.

Topics: Adolescent; Adult; Aged; Animals; Antigens, CD1d; Antineoplastic Agents, Immunological; Cell Line, Tumor; Cells, Cultured; Female; Galactosylceramides; Humans; Immunotherapy; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Molecular Docking Simulation; Natural Killer T-Cells; Neoplasms

A major obstacle to the success rate of chimeric antigen receptor (CAR-) T-cell therapy against solid tumors is the microenvironment antagonistic to T cells that solid tumors create. Conventional checkpoint blockade can silence lymphocyte antisurvival pathways activated by tumors, but because they are systemic, these treatments disrupt immune homeostasis and induce autoimmune side effects. Thus, new technologies are required to remodel the tumor milieu without causing systemic toxicities. Here, we demonstrate that targeted nanocarriers that deliver a combination of immune-modulatory agents can remove protumor cell populations and simultaneously stimulate antitumor effector cells. We administered repeated infusions of lipid nanoparticles coated with the tumor-targeting peptide iRGD and loaded with a combination of a PI3K inhibitor to inhibit immune-suppressive tumor cells and an α-GalCer agonist of therapeutic T cells to synergistically sway the tumor microenvironment of solid tumors from suppressive to stimulatory. This treatment created a therapeutic window of 2 weeks, enabling tumor-specific CAR-T cells to home to the lesion, undergo robust expansion, and trigger tumor regression. CAR-T cells administered outside this therapeutic window had no curative effect. The lipid nanoparticles we used are easy to manufacture in substantial amounts, and we demonstrate that repeated infusions of them are safe. Our technology may therefore provide a practical and low-cost strategy to potentiate many cancer immunotherapies used to treat solid tumors, including T-cell therapy, vaccines, and BITE platforms.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Combined Modality Therapy; Disease Models, Animal; Drug Compounding; Female; Galactosylceramides; Humans; Immunotherapy, Adoptive; Liposomes; Mice; Mice, Inbred BALB C; Nanoparticles; Neoplasms; Oligopeptides; Phosphoinositide-3 Kinase Inhibitors; Receptors, Chimeric Antigen; Treatment Outcome; Tumor Microenvironment

Invariant natural killer T (iNKT) cells recognize CD1d/glycolipid complexes and upon activation with synthetic agonists display immunostimulatory properties. We have previously described that the non-glycosidic CD1d-binding lipid, threitolceramide (ThrCer) activates murine and human iNKT cells. Here, we show that incorporating the headgroup of ThrCer into a conformationally more restricted 6- or 7-membered ring results in significantly more potent non-glycosidic analogs. In particular, ThrCer 6 was found to promote strong anti-tumor responses and to induce a more prolonged stimulation of iNKT cells than does the canonical α-galactosylceramide (α-GalCer), achieving an enhanced T-cell response at lower concentrations compared with α-GalCer both in vitro, using human iNKT-cell lines and in vivo, using C57BL/6 mice. Collectively, these studies describe novel non-glycosidic ThrCer-based analogs that have improved potency in iNKT-cell activation compared with that of α-GalCer, and are clinically relevant iNKT-cell agonists.

Topics: Animals; Antigens, CD1d; Ceramides; Cytokines; Galactosylceramides; Humans; Immunotherapy; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Natural Killer T-Cells; Neoplasms; Sugar Alcohols

Cancer progression involves carcinogenesis, an increase in tumour size, and metastasis. Here, we investigated the effect of overexpressed CXC chemokine ligand 14 (CXCL14) on these processes by using CXCL14/BRAK (CXCL14) transgenic (Tg) mice. The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that for isogenic wild type C57BL/6 (Wt) mice. When tumour cells were injected into these mice, the size of the tumours that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumour cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity played an important role during CXCL14-mediated suppression of tumour growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed in B16 melanoma cells. Further, the survival rates after tumour cell injection were significantly increased for the Tg mice. As these Tg mice showed no obvious abnormality, we propose that CXCL14 to be a promising molecular target for cancer suppression/prevention.

Topics: Animals; Antigens, Ly; Autoantibodies; Cell Transformation, Neoplastic; Chemokines, CXC; Chronic Disease; Colitis; Disease Models, Animal; Female; G(M1) Ganglioside; Galactosylceramides; Killer Cells, Natural; Lung Neoplasms; Lymphocyte Depletion; Melanoma, Experimental; Mice; Mice, Transgenic; Neoplasms; NK Cell Lectin-Like Receptor Subfamily B; Tumor Burden

Most studies characterizing antitumor properties of invariant natural killer T (iNKT) cells have used the agonist, α-galactosylceramide (α-GalCer). However, α-GalCer induces strong, long-lasting anergy of iNKT cells, which could be a major detriment for clinical therapy. A novel iNKT cell agonist, β-mannosylceramide (β-ManCer), induces strong antitumor immunity through a mechanism distinct from that of α-GalCer. The objective of this study was to determine whether β-ManCer induces anergy of iNKT cells.. Induction of anergy was determined by ex vivo analysis of splenocytes from mice pretreated with iNKT cell agonists as well as in the CT26 lung metastasis in vivo tumor model.. β-ManCer activated iNKT cells without inducing long-term anergy. The transience of anergy induction correlated with a shortened duration of PD-1 upregulation on iNKT cells activated with β-ManCer, compared with α-GalCer. Moreover, whereas mice pretreated with α-GalCer were unable to respond to a second glycolipid stimulation to induce tumor protection for up to 2 months, mice pretreated with β-ManCer were protected from tumors by a second stimulation equivalently to vehicle-treated mice.. The lack of long-term functional anergy induced by β-ManCer, which allows for a second dose to still give therapeutic benefit, suggests the strong potential for this iNKT cell agonist to succeed in settings where α-GalCer has failed.

Topics: Animals; Cell Line, Tumor; Ceramides; Clonal Anergy; Disease Models, Animal; Female; Galactosylceramides; Humans; Mice; Natural Killer T-Cells; Neoplasms; Programmed Cell Death 1 Receptor

Vγ9Vδ2-T cells constitute a proinflammatory lymphocyte subpopulation with established antitumor activity. Phosphoantigens activate Vγ9Vδ2-T cells in vivo and in vitro. We studied whether the antitumor activity of Vγ9Vδ2-T cells can be potentiated by invariant NKT cells (iNKT), an important immunoregulatory T cell subset. When activated by the glycolipid α-galactosylceramide (α-GalCer), iNKT produce large amounts of cytokines involved in antitumor immune responses. Monocyte-derived dendritic cells were loaded with both phosphoantigens (using aminobisphosphonates) and α-GalCer during maturation and subsequently co-cultured with Vγ9Vδ2-T and iNKT cells. Aminobisphosphonates dose-dependently enhanced Vγ9Vδ2-T cell activation, and this was potentiated by α-GalCer-induced iNKT co-activation. iNKT co-activation also enhanced the IFN-γ production and cytolytic potential of Vγ9Vδ2-T cells against tumor cells. Using transwell experiments and neutralizing antibodies cross-talk between iNKT and Vγ9Vδ2-T cells was found to be mediated by TNF-α. Our data provide a rationale for combining both activating ligands to improve Vγ9Vδ2-T cell based approaches in cancer-immunotherapy.

Topics: Antigens, Neoplasm; Cells, Cultured; Dendritic Cells; Diphosphonates; Galactosylceramides; Hemiterpenes; Humans; Immunologic Factors; Immunotherapy; Interferon-gamma; Lymphocyte Activation; Natural Killer T-Cells; Neoplasms; Organophosphorus Compounds; T-Lymphocytes, Cytotoxic; Tumor Necrosis Factor-alpha

Type 1 or invariant NKT (iNKT) cell agonists, epitomized by α-galactosylceramide, protect against cancer largely by IFN-γ-dependent mechanisms. Here we describe what we believe to be a novel IFN-γ-independent mechanism induced by β-mannosylceramide, which also defines a potentially new class of iNKT cell agonist, with an unusual β-linked sugar. Like α-galactosylceramide, β-mannosylceramide directly activates iNKT cells from both mice and humans. In contrast to α-galactosylceramide, protection by β-mannosylceramide was completely dependent on NOS and TNF-α, neither of which was required to achieve protection with α-galactosylceramide. Moreover, at doses too low for either alone to protect, β-mannosylceramide synergized with α-galactosylceramide to protect mice against tumors. These results suggest that treatment with β-mannosylceramide provides a distinct mechanism of tumor protection that may allow efficacy where other agonists have failed. Furthermore, the ability of β-mannosylceramide to synergize with α-galactosylceramide suggests treatment with this class of iNKT agonist may provide protection against tumors in humans.

Topics: Animals; Cell Line; Ceramides; Female; Galactosylceramides; Humans; Immune Tolerance; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Molecular Structure; Natural Killer T-Cells; Neoplasm Transplantation; Neoplasms

Invariant natural killer T (iNKT) cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. Here, we report the structure and function of a novel class of aromatic α-galactosylceramide structurally related glycolipids with marked Th1 bias in both mice and men, leading to superior tumour protection in vivo. The strength of the Th1 response correlates well with enhanced lipid binding to CD1d as a result of an induced fit mechanism that binds the aromatic substitution as a third anchor, in addition to the two lipid chains. This induced fit is in contrast to another Th1-biasing glycolipid, α-C-GalCer, whose CD1d binding follows a conventional key-lock principle. These findings highlight the previously unexploited flexibility of CD1d in accommodating galactose-modified glycolipids and broaden the range of glycolipids that can stimulate iNKT cells. We speculate that glycolipids can be designed that induce a similar fit, thereby leading to superior and more sustained iNKT cell responses in vivo.

Topics: Animals; Antigens, CD1d; Galactosylceramides; Mice; Natural Killer T-Cells; Neoplasm Metastasis; Neoplasms; Protein Binding

We had previously shown that activated NKT cells licensed B cells to be immunogenic antigen-presenting cells and helped to elicit a wide spectrum of cancer targeted immune responses. In the current study, we sought to verify the safety of αGalCer-loaded, and adenovirus-transduced B cell-based vaccines, together with mechanism of action. Intravenously injected αGalCer-loaded, antigen-expressing B cells rapidly localized in the spleen and directly primed CD8(+) T cells in an antigen-specific manner. The transferred antigen was sustained for at least 30 days. While some injected B cells produced nonspecific IgG, the antigen-specific IgG response was completely dependent on endogenous B cells. The liver was one of the main tissues where injected B cells were retained; however, we could not find the signs of liver toxicity. Our results demonstrate that αGalCer-loaded, antigen-expressing B cells behave as "antigen-presenting" cells that stimulate endogenous antigen-specific T cells and B cells in vivo without significant toxicity.

Topics: Adenoviridae; Animals; Antibody Specificity; Antigen-Presenting Cells; B-Lymphocytes; Cancer Vaccines; Galactosylceramides; Immunoglobulin G; Immunotherapy, Active; Ligands; Liver; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Natural Killer T-Cells; Neoplasms; Spleen; Transfection

Dendritic cells (DC) present α-galactosylceramide (αGalCer) to invariant T-cell receptor-expressing natural killer T cells (iNKT) activating these cells to secrete a variety of cytokines, which in turn results in DC maturation and activation of other cell types, including NK cells, B cells, and conventional T cells. In this study, we showed that αGalCer-pulsing of antigen-activated CD8 T cells before adoptive transfer to tumor-bearing mice caused a marked increase in donor T-cell proliferation, precursor frequency, and cytotoxic lymphocyte activity. This effect was interleukin (IL)-2 dependent and involved both natural killer T cells (NKT) and DCs, as mice lacking IL-2, NKTs, and DCs lacked any enhanced response to adoptively transferred αGalCer-loaded CD8 T cells. iNKT activation was mediated by transfer of αGalCer from the cell membrane of the donor CD8 T cells onto the αGalCer receptor CD1d which is present on host DCs. αGalCer transfer was increased by prior activation of the donor CD8 T cells and required AP-2-mediated endocytosis by host DCs. In addition, host iNKT cell activation led to strong IL-2 synthesis, thereby increasing expansion and differentiation of donor CD8 T cells. Transfer of these cells led to improved therapeutic efficacy against established solid tumors in mice. Thus, our findings illustrate how αGalCer loading of CD8 T cells after antigen activation in vitro may leverage the therapeutic potential of adoptive T-cell therapies.

Topics: Animals; Antigens, CD1d; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Dendritic Cells; Endocytosis; Flow Cytometry; Galactosylceramides; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-17; Interleukin-2; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Natural Killer T-Cells; Neoplasms; Time Factors

NKT cells demonstrate antitumor activity when activated to produce Th1 cytokines by DCs loaded with alpha-galactosylceramide, the prototypic NKT cell-activating glycolipid antigen. However, most patients do not have sufficient numbers of NKT cells to induce an effective immune response in this context, indicating a need for a source of NKT cells that could be used to supplement the endogenous cell population. Induced pluripotent stem cells (iPSCs) hold tremendous potential for cell-replacement therapy, but whether it is possible to generate functionally competent NKT cells from iPSCs has not been rigorously assessed. In this study, we successfully derived iPSCs both from embryonic fibroblasts from mice harboring functional NKT cell-specific rearranged T cell receptor loci in the germline and from splenic NKT cells from WT adult mice. These iPSCs could be differentiated into NKT cells in vitro and secreted large amounts of the Th1 cytokine IFN-gamma. Importantly, iPSC-derived NKT cells recapitulated the known adjuvant effects of natural NKT cells and suppressed tumor growth in vivo. These studies demonstrate the feasibility of expanding functionally competent NKT cells via an iPSC phase, an approach that may be adapted for NKT cell-targeted therapy in humans.

Topics: Animals; Cell Differentiation; Cytokines; Galactosylceramides; Glycolipids; Induced Pluripotent Stem Cells; Mice; Natural Killer T-Cells; Neoplasms; Receptors, Antigen, T-Cell; Spleen

APCs, such as dendritic cells (DC), can present glycolipid Ags on CD1d molecules to NKT cells. This interaction activates DC and NKT cells, leading to release of cytokines and enhanced T cell responses. Thus, glycolipid Ags are currently being tested as adjuvants for immunotherapy. We were interested in the interaction of murine skin DC with NKT cells in skin-draining lymph nodes. We observed that all skin DC subsets expressed CD1d upon migration to the lymph nodes. Moreover, skin DC were able to present the synthetic glycolipid Ag alpha-galactosylceramide (alpha-GalCer) to the NKT cell hybridoma DN32.D3. Intradermally injected alpha-GalCer was presented by migratory skin DC and lymph node DC to NKT hybridoma cells in vitro. When we injected alpha-GalCer intradermally into the skin, the numbers of various leukocyte subsets in the draining lymph nodes did not change significantly. However, T and B cells as well as NKT cells up-regulated the activation marker CD69. Coapplication of alpha-GalCer with the tumor model Ag OVA induced strong cytolytic CD8(+) T cell function that could inhibit the growth of B16 melanoma cells expressing OVA. However, mice that were devoid of migratory skin DC developed similar cytotoxic immune responses after intradermal immunization, indicating that skin DC are not required for the adjuvant properties of NKT cell activation and Ag presentation by this immunization route. In conclusion, migratory skin DC are able to interact with NKT cells; however, intradermally applied glycolipids are presented predominantly by lymph node DC to NKT cells.

Topics: Animals; Antigens, CD1d; Cell Line; Cell Movement; Disease Models, Animal; Disease Progression; Galactosylceramides; Glycolipids; Langerhans Cells; Lymph Nodes; Mice; Mice, Inbred C57BL; Natural Killer T-Cells; Neoplasm Transplantation; Neoplasms; Skin; T-Lymphocytes, Cytotoxic; Tissue Culture Techniques

The glycolipid alpha-galactosylceramide (alphaGalCer) has immunomodulatory properties, which have been exploited to combat cancer, chronic inflammatory diseases, and infections. However, its poor solubility makes alphaGalCer a suboptimal compound for in vivo applications. In this study, a pegylated derivative of alphaGalCer is characterized, which exhibits improved physical and biological properties. The new compound, alphaGalCerMPEG, is water-soluble and retains the specificity for the CD1d receptor of alphaGalCer. The in vitro stimulatory properties on immune cells (e.g., dendritic cells and splenocytes) are maintained intact, even when tested at a 33-fold lower concentration of the active moiety than alphaGalCer. NK cells isolated from mice treated with alphaGalCerMPEG also had stronger cytotoxic activity on YAC-1 cells than those obtained from animals receiving either alphaGalCer or CpG. Intranasal immunization studies performed in mice showed that alphaGalCerMPEG exerts stronger adjuvant activities than the parental compound alphaGalCer when tested at 0.35 vs 11.7 nM/dose. Coadministration of beta-galactosidase with alphaGalCerMPEG resulted not only in high titers of Ag-specific Abs in serum (i.e., 1:512,000), but also in the stimulation of stronger Th2 and secretory IgA responses, both at local and remote mucosal effector sites (i.e., nose, lung, and vagina). The new synthetic derivative alphaGalCerMPEG represents a promising tool for the development of immune interventions against infectious and noninfectious diseases.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Antibody Specificity; Antigens; beta-Galactosidase; Cell Line; Chronic Disease; Dendritic Cells; Galactosylceramides; Humans; Immunity, Mucosal; Immunization; Immunoglobulin A; Immunologic Factors; Infections; Inflammation; Mice; Mucous Membrane; Neoplasms; Oligonucleotides; Polyethylene Glycols; Solubility; Spleen

NKT cells play a role in immunological regulation of certain diseases, and their frequency and/or function may be related to disease prognosis. However, it is often difficult to evaluate NKT cell function in patients with malignancies due to reduced numbers of NKT cells as well as the dysfunction of the APCs used as stimulators. We found that NKT cell function could not be evaluated by conventional ELISPOT assays, confirming the impaired function of APCs in chronic myelogenous leukemia (CML)-chronic phase patients. To overcome this problem, we have established a sensitive assay using murine dendritic cells to evaluate the function of small numbers of human NKT cells independent of autologous APCs. We found that imatinib-treated CML-chronic phase patients showing a complete cytogenetic response had NKT cells capable of producing IFN-gamma, whereas NKT cells from patients who were only partially responsive to imatinib treatment did not produce IFN-gamma. Functional NKT cells found in imatinib-treated, CML-complete cytogenetic response patients may offer the promise of effective immunotherapy with ex vivo-generated alpha-galactosylceramide-pulsed dendritic cells. This new approach should be available for evaluating the functions of NKT cells and APCs in cancer patients.

Topics: Animals; Benzamides; Cell Differentiation; Cells, Cultured; Coculture Techniques; Dendritic Cells; Galactosylceramides; Humans; Imatinib Mesylate; Interferon-gamma; Interleukin-12; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphocyte Activation; Mice; Monocytes; Neoplasms; Piperazines; Pyrimidines; T-Lymphocytes, Regulatory

We examined the role of CD4+CD25+ regulatory T cells in the development of 3-methylcholanthrene (MCA)-induced tumors. Immunization of wild-type BALB/c mice with a series of SEREX (serological identification of antigens by recombinant expression cloning)-defined broadly expressed self-antigens results in the development of highly active CD4+CD25+ regulatory T cells. Accelerated tumor development was observed in mice immunized with self-antigens and was abolished by antibody-mediated depletion of CD4+ T cells or CD25+ T cells. A similar acceleration of tumorigenesis was also observed in mice adoptively transferred 2 or 4 weeks after MCA injection with CD4+CD25+ T cells derived from mice immunized with DnaJ-like 2, one of these self-antigens. Experiments with Jalpha281-/- mice lacking invariant natural killer (iNK) T cells indicated that iNK T cells, known for their protective role in the development of MCA-induced tumors, were suppressed in immunized hosts. NK cells, also known to play a protective role in MCA induced-tumorigenesis, were also suppressed in mice immunized with serologically defined self-antigens in a CD4+CD25+ T cell-dependent manner. We propose that CD4+CD25+ regulatory T cells generated by immunization with these self-antigens enhance susceptibility to MCA induced-tumorigenesis by down-regulating iNK T and NK reactivity, and suggest that these observations provide direct evidence for the existence of cancer immunosurveillance in this system of chemical carcinogenesis.

Topics: Animals; Antigens, CD1; Antigens, CD1d; Carcinogens; CD4 Antigens; CD4-Positive T-Lymphocytes; Cell Proliferation; Chromium Radioisotopes; Cloning, Molecular; Down-Regulation; Female; Galactosylceramides; Genetic Predisposition to Disease; Killer Cells, Natural; Male; Methylcholanthrene; Mice; Mice, Inbred BALB C; Mice, Transgenic; Neoplasms; Plasmids; Protein Binding; Receptors, Interleukin-2; Recombinant Proteins; T-Lymphocytes; Time Factors

The CD1d reactive glycolipid, alpha-galactosylceramide (alpha-GalCer), potently activates T cell receptor-alpha type I invariant NKT cells that secondarily stimulate the proliferation and activation of other leukocytes, including NK cells. Here we report a rational approach to improving the antitumor activity of alpha-GalCer by using delayed interleukin (IL)-21 treatment to mature the alpha-GalCer-expanded pool of NK cells into highly cytotoxic effector cells. In a series of experimental and spontaneous metastases models in mice, we demonstrate far superior antitumor activity of the alpha-GalCer/IL-21 combination above either agent alone. Superior antitumor activity was critically dependent upon the increased perforin-mediated cytolytic activity of NK cells. Transfer of alpha-GalCer-pulsed dendritic cells (DCs) followed by systemic IL-21 caused an even more significant reduction in established (day 8) metastatic burden and prolonged survival. In addition, this combination prevented chemical carcinogenesis more effectively. Combinations of IL-21 with other NK cell-activating cytokines, such as IL-2 and IL-12, were much less effective in the same experimental metastases models, and these cytokines did not substitute effectively for IL-21 in combination with alpha-GalCer. Overall, the data suggest that NK cell antitumor function can be enhanced greatly by strategies that are designed to expand and differentiate NK cells via DC activation of NKT cells.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cytotoxicity Tests, Immunologic; Dendritic Cells; Galactosylceramides; Immunotherapy; Interferon-gamma; Interleukins; Killer Cells, Natural; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Knockout; Neoplasm Metastasis; Neoplasms; Perforin; Pore Forming Cytotoxic Proteins

NKT cells can produce large amounts of both Th1- and Th2-type cytokines and are an important regulatory cell type. To elucidate their role in acquired immunity, we examined the effect of human Valpha24+Vbeta11+ NKT cells or CD1d-specific ligand alpha-galactosylceramide (alphaGalCer) on the in vitro generation of antigen-specific CTLs from PBMCs using autologous MART-1(26-35) peptide-pulsed dendritic cells as stimulators. Flow cytometry using tetramer for MART-1(26-35) peptide revealed that NKT cells have inhibitory effects on CTL generation. Cytokine analysis using cytometric bead array assay and ELISA showed higher IL-4 and IL-10 secretion in the alphaGalCer(+) and/or NKT cell(+) culture setting, whereas IL-13 secretion in the culture was not affected by the presence of alphaGalCer. The CD4+ NKT cell subset seemed to play a major role in this inhibitory effect by secreting large amounts of Th2-type cytokines. Interestingly however, unlike recent reports utilizing mouse models, IL-13 was not a main effector molecule in our human system. Culture with alphaGalCer in the presence of cytokine-neutralizing antibodies for the Th2 cytokines, IL-4, IL-5 and IL-10, resulted in enhanced CTL generation, suggesting the dominant role of Th2 cytokines over Th1 cytokines. Thus, CD4+ NKT cells can work as immunoregulatory T cells that suppress anti-tumor immune response and, therefore, NKT cells or alphaGalCer could be used as therapeutic modalities to modulate systemic immune responses, such as autoimmune diseases. Conversely, the use of NKT cells along with anti-Th2 cytokine-neutralizing antibodies or CD4-negative NKT cell subset could enhance the generation of antigen-specific CTLs for adoptive immunotherapy.

Topics: Antigens, Neoplasm; Cell Proliferation; Cells, Cultured; Cytokines; Galactosylceramides; Humans; Immunotherapy, Adoptive; Killer Cells, Natural; Neoplasms; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory

The importance of immunoregulatory T cells has become increasingly apparent. Both CD4+CD25+ T cells and CD1d-restricted NKT cells have been reported to down-regulate tumor immunity in mouse tumor models. However, the relative roles of both T cell populations have rarely been clearly distinguished in the same tumor models. In addition, CD1d-restricted NKT cells have been reported to play a critical role not only in the down-regulation of tumor immunity but also in the promotion of the immunity. However, the explanation for these apparently opposite roles in different tumor models remains unclear. We show that in four mouse tumor models in which CD1d-restricted NKT cells play a role in suppression of tumor immunity, depletion of CD4+CD25+ T cells did not induce enhancement of immunosurveillance. Surprisingly, among the two subpopulations of CD1d-restricted NKT cells, Valpha14Jalpha18+ (type I) and Valpha14Jalpha18- (type II) NKT cells, type I NKT cells were not necessary for the immune suppression. These unexpected results may now resolve the paradox in the role of CD1d-restricted NKT cells in the regulation of tumor immunity, in that type II NKT cells may be sufficient for negative regulation, whereas protection has been found to be mediated by alpha-galactosylceramide-responsive type I NKT cells.

Topics: Animals; Antigens, CD1; Antigens, CD1d; Cell Line, Tumor; Galactosylceramides; Immunologic Surveillance; Killer Cells, Natural; Mice; Mice, Inbred BALB C; Neoplasms; T-Lymphocytes, Regulatory

The close cooperation of both innate and acquired immunity is essential for the induction of truly effective antitumor immunity. We tested a strategy to enhance the cross-talk between NKT cells and conventional antigen-specific T cells with the use of alpha GalCer-loaded dendritic cells genetically engineered to express antigen plus chemokine, attracting both conventional T cells and NKT cells. DC genetically engineered to express a model antigen, OVA, along with SLC/CCL21 or monokine induced by IFN-gamma/CXCL9, had been generated using a method based on in vitro differentiation of DC from mouse ES cells. The ES-DC were loaded with alpha-GalCer and transferred to mice bearing MO4, an OVA-expressing melanoma, and their capacity to evoke antitumor immunity was evaluated. In vivo transfer of either OVA-expressing ES-DC, stimulating OVA-reactive T cells, or alpha-GalCer-loaded non-transfectant ES-DC, stimulating NKT cells, elicited a significant but limited degree of protection against the i.p. disseminated MO4. A more potent antitumor effect was observed when alpha-GalCer was loaded to ES-DC expressing OVA before in vivo transfer, and the effect was abrogated by the administration of anti-CD8, anti-NK1.1 or anti-asialo GM1 antibody. alpha-GalCer-loaded double transfectant ES-DC expressing SLC along with OVA induced the most potent antitumor immunity. Thus, alpha-GalCer-loaded ES-DC expressing tumor-associated antigen along with SLC can stimulate multiple subsets of effector cells to induce a potent therapeutic effect against peritoneally disseminated tumor cells. The present study suggests a novel way to use alpha-GalCer in immunotherapy for peritoneally

Topics: Animals; Antigens, Neoplasm; Antineoplastic Agents; Cell Communication; Cell Line, Tumor; Cell Survival; Chemokine CCL21; Chemokines, CC; Dendritic Cells; Galactosylceramides; Genetic Engineering; Granulocyte-Macrophage Colony-Stimulating Factor; Killer Cells, Natural; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Neoplasms; Ovalbumin; Recombinant Proteins; T-Lymphocytes

Recent studies have revealed significant efficacy of the marine sponge glycolipid, alpha-galactosylceramide (alpha-GalCer), in treatment of experimental metastatic cancers, infections, and autoimmune diseases. However, the capacity of alpha-GalCer to prevent tumor development had never, to our knowledge, been evaluated in mouse models of chemical- and oncogene-dependent carcinogenesis. In this study, we demonstrate that long-term administration of soluble alpha-GalCer, spanning the time of tumor initiation, inhibits primary tumor formation in three different models: methylcholanthrene-induced sarcomas, mammary carcinomas in Her-2/neu transgenic mice, and spontaneous sarcomas in p53-/- mice. Weekly treatment of mice with alpha-GalCer maintained lymphoid tissue natural killer cell and T cell activation and elevated serum IFN-gamma and IL-4 concentrations. Consistent with the antimetastatic activity of alpha-GalCer, prevention of methylcholanthrene-induced sarcoma was IFN-gammaand tumor necrosis factor-related apoptosis-inducing ligand dependent, but not perforin-dependent. Taken together, our results demonstrate that NK1.1+alphabetaTCR+ cell-based immune therapy can inhibit primary tumorigenesis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fibrosarcoma; Flow Cytometry; Galactosylceramides; Genes, p53; Humans; Interferon-gamma; Interleukin-12; Interleukin-4; Ligands; Methylcholanthrene; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neoplasms; Receptor, ErbB-2; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Protein p53