Injection Of α-GalCer Research Articles

Alpha-galactosylceramide as adjuvant induces protective cell-mediated immunity against Leishmania mexicana infection in vaccinated BALB/c mice.

Adjuvants represent a promising strategy to improve vaccine effectiveness against infectious diseases such as leishmaniasis. Vaccination with the invariant natural killer T cell ligand α-galactosylceramide (αGalCer) has been used successfully as adjuvant, generating a Th1-biased immunomodulation. This glycolipid enhances experimental vaccination platforms against intracellular parasites including Plasmodium yoelii and Mycobacterium tuberculosis. In the present study, we assessed the protective immunity induced by a single-dose intraperitoneal injection of αGalCer (2μg) co-administrated with a lysate antigen of amastigotes (100μg) against Leishmania mexicana infection in BALB/c mice. The prophylactic vaccination led to 5.0-fold reduction of parasite load at the infection site, compared to non-vaccinated mice. A predominant pro-inflammatory response was observed in challenged vaccinated mice, represented by a 1.9 and 2.8-fold-increase of IL-1β and IFN-γ producing cells, respectively, in the lesions, and by 23.7-fold-increase of IFN-γ production in supernatants of restimulated splenocytes, all compared to control groups. The co-administration of αGalCer also stimulated the maturation of splenic dendritic cells and modulated a Th1-skewed immune response, with high amounts of IFN-γ production in serum. Furthermore, peritoneal cells of αGalCer-immunized mice exhibited an elevated expression of Ly6G and MHCII. These findings indicate that αGalCer improves protection against cutaneous leishmaniasis, supporting evidence for its potential use as adjuvant in Leishmania-vaccines.

Role of NKT cells in cancer immunotherapy-from bench to bed.

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.

Expansion of iNKT Cells Promotes Liver Repair Following Hepatic Ischemia Reperfusion Injury.

Invariant natural killer T (iNKT) cells are involved in the initiation and resolution of inflammatory responses. We previously reported that activated iNKT cells facilitate liver repair after hepatic ischemia reperfusion (I/R) injury by accelerating macrophage polarization during the early phase of hepatic I/R injury. Upon activation with α-galactosylceramide (α-GalCer), iNKT cell numbers transiently decrease before increasing within 72 h of stimulation. In the present study, we examined the role of expanded hepatic iNKT cells in the late phase of hepatic I/R injury. iNKT cells were activated by intraperitoneal injection of α-GalCer in male C57/BL6 mice at the induction of hepatic ischemia followed by reperfusion. Numbers of activated hepatic iNKT cells immediately diminished after hepatic I/R and reached minimal levels at 24 h and 48 h post-reperfusion. Numbers of hepatic iNKT cells then increased at 72 h and 96 h post-reperfusion to levels approximately 2-fold higher than in mice that underwent a sham operation. Liver repair as demonstrated by decreased necrotic area and increased expression of proliferating cell nuclear antigen (PCNA) was enhanced in α-GalCer-treated mice at 96 h post-reperfusion. Interleukin (IL)-13 production by proliferating iNKT cells was observed at 96 h post-reperfusion, which was associated with enhanced liver repair and increased numbers of reparative macrophages. Repopulation of hepatic iNKT cells promotes liver repair by stimulating macrophage phenotype switching in the late phase of hepatic I/R injury.

Migration, Distribution, and Safety Evaluation of Specific Phenotypic and Functional Mouse Spleen-Derived Invariant Natural Killer T2 Cells after Adoptive Infusion

Herein, the migration distribution and safety of specific phenotypic and functionally identified spleen-derived invariant natural killer T2 (iNKT2) cells after adoptive infusion in mice were studied. The proliferation and differentiation of iNKT cells were induced by intraperitoneal injection of α-galactosylceramide (α-GalCer) in vivo. Mouse spleens were isolated in a sterile environment. iNKT cells were isolated by magnetic-activated cell sorting columns (MS columns). Cytometric bead array (CBA) assay was used to detect cytokine secretion in the supernatant stimulated by iNKT cells. The basic life status of the mice was observed, and systematic quantitative scoring was conducted after injecting spleen-derived iNKT cells through the tail vein. An in vivo imaging system was used to trace the migration and distribution of iNKT cells in DBA mice. The percentage of the iNKT2 subgroup was the highest in 3 days after intraperitoneal injection of α-GalCer, and iNKT2 subsets accounted for more than 92% after separation and purification by magnetic-activated cell sorting (MACS). Anti-inflammatory cytokine IL-4 was mainly found in the supernatant of cell cultures. The adoptive infusion of iNKT cells into healthy mice resulted in no significant change in the basic life status of mice compared with the noninjected group. iNKT cells were detected in the lung, spleen, and liver, but no fluorescence was detected in lymph nodes and thymus. After dissecting the mice, it was found that there were no significant abnormalities in the relevant immune organs, brain, heart, kidney, lung, and other organs. Intraperitoneal injection of α-GalCer results in a large number of iNKT2 cells, mainly secreting anti-inflammatory cytokine IL-4, from the spleen of mice. After adoptive infusion, the iNKT2 cells mainly settled in the liver and spleen of mice with a satisfactory safety profile.

Proliferation and Directional Differentiation of iNKT Cells Derived from DBA/1 Mice Thymus.

The rates of invariant natural killer T (iNKT) cells in vivo are very low, and the amounts of cells obtained directly from the body are hard enough to fulfill their potential in clinical application. To overcome this problem, we subcutaneously injected alpha-galactosylceramide (α-GalCer) into DBA/1 mice and thymic single cells were isolated and cultured in vitro. Fluorescence-activated cell sorting was used to detect the iNKT cells and their subsets in the thymus after the injection of α-GalCer by different methods. In addition, in vitro changes of single-cell suspensions and their cytokines in culture supernatants were assessed. Compared with the α-GalCer multiple subcutaneous injection group, the rates of iNKT cells in the α-GalCer single subcutaneous injection group were markedly higher at each time point, while the highest levels of iNKT1 and iNKT2 cells were observed on day 4 and 8, respectively. In α-GalCer single subcutaneous injection for 8 days and thymic mononuclear cell cultured for 14 days group, the expansion rate of iNKT cells was significantly faster than the other groups, while it reached a peak for iNKT1 cells. Interferon-gamma was consistent with the development of iNKT1 cells, however no difference was found between the cultured iNKT cells in vitro and the natural iNKT cells in vivo in terms of cytokine production. Herein, we introduced a method in which antigenic stimulation in vivo and directed induction in vitro yielded high levels of iNKT cells with specific functions.

Inflammation-Induced Lactate Leads to Rapid Loss of Hepatic Tissue-Resident NK Cells

The liver harbors two main innate lymphoid cell (ILC) populations: conventional NK (cNK) cells and tissue-resident NK (trNK) cells. Using the MCMV model of infection, we find that, in contrast to liver cNK cells, trNK cells initially undergo a contraction phase followed by a recovery phase to homeostatic levels. The contraction is MCMV independent because a similar phenotype is observed following poly(I:C)/CpG or α-GalCer injection. The rapid contraction phase is due to apoptosis, whereas the recovery phase occurs via proliferation in situ. Interestingly, trNK cell apoptosis is not mediated by fratricide and not induced by liver lymphocytes or inflammatory cytokines. Instead, we find that trNK cell apoptosis is the consequence of an increased sensitivity to lactic acid. Mechanistic analysis indicates that trNK cell sensitivity to lactate is linked to impaired mitochondrial function. These findings underscore the distinctive properties of the liver-resident NK cell compartment.

Activation of Invariant Natural Killer T Cell Subsets in C57BL/6J Mice by Different Injection Modes of α-galactosylceramide.

Whether different injection modes of α-galactosylceramide (α-GalCer) affect the activation of different subsets of invariant natural killer T (iNKT) cells in different tissues and organs of mice is unclear. This study included healthy control, subcutaneous injection, and intraperitoneal injection groups (n=10 in each group). The subcutaneous and intraperitoneal injection groups were injected with α-Galcer (0.1 mg/kg weight), and then the changes in thymus, spleen, and liver iNKT cell frequencies and subsets were observed. The intraperitoneal injection of α-GalCer could increase the frequency of splenic iNKT cells, but the subcutaneous injection did not affect the frequency. Neither injection had any effect on the frequency of iNKT cells in the thymus and liver. The subcutaneous injection of α-GalCer increased the rate of iNKT2 subsets in the thymus but did not affect the rate of iNKT1 subsets. However, the intraperitoneal injection of α-GalCer did not affect thymus iNKT1 and iNKT2 subsets. Interestingly, the subcutaneous injection of α-GalCer significantly increased the proportion of iNKT1 in the spleen and liver but did not significantly change the proportion of iNKT2. The intraperitoneal injection of α-GalCer significantly increased the rate of iNKT2 in spleen and liver but decreased the rate of iNKT1. Subsets of iNKT1 or iNKT2 cells in the spleen and liver were selectively activated by the subcutaneous or intraperitoneal injection of α-GalCer. It provides a valuable means for treating tumors and certain autoimmune diseases. Further exploration of the activation mechanism may provide new ideas about the development of related vaccines.

Natural killer T cell activation increases iNOS+CD206- M1 macrophage and controls the growth of solid tumor

BackgroundNKT cells play an important role in anti-tumor immunity. Alpha-galactosylceramide (α-GalCer), a synthetic glycolipid is presented to natural killer T (NKT) cells by most antigen-presenting cells through CD1d molecules leading to activation of NKT cells. However, the precise mechanisms of how α-GalCer-activated NKT regulate the polarization of the macrophages and effector T cells in the solid tumor are not studied adequately.MethodsWe induced solid tumor in C57BL/6 mice by subcutaneous injection of B16F10 cell line (1 X 106 cells) and monitored the tumor growth. Animals were given an intraperitoneal injection of α-GalCer (2 μg/injection) in 200 μl PBS on day + 1, + 5, + 10, + 15, and + 20 (with respect to tumor cell injection). Immune cells were characterized using flow cytometry and immunofluorescence staining. NK cells, Gr1+ cells, and F4/80+ macrophages in the mice were depleted by intravenous injection of cell-specific antibodies. Statistical analysis was performed using Student’s t-test or one-way ANOVA.ResultsOur results showed that intratumoral NKT cells have a lower frequency of CD69, CD25, CD122, and IFN-γR expression; produced less inflammatory cytokines such as IFN-γ, TNF-α, and GM-CSF; higher frequency CD62L+ NKT cells; and also showed reduced proliferation as compared to the splenic NKT cells. Mice treated with α-GalCer showed a significantly increased frequency of IFN-γ-producing NKT cells, CD8+ T cells, and effector Th1 cells. Depletion of NK cells in α-GalCer-treated mice showed a lower frequency of IFN-γ-producing CD4+ and CD8+ T cells in the tumor and prevented the α-GalCer-induced tumor growth. NKT cell activation with α-GalCer treatment significantly increased the iNOS+CD206− M1-macrophages and reduced the iNOS−CD206+ M2-macrophages in the spleen and tumor, and depletion of F4/80+ macrophages prevented the α-GalCer-induced reduction in the tumor growth.ConclusionsWe showed that activation of NKT cell with α-GalCer modulates the frequency of M1-macrophages and effector Th1 cells in the secondary lymphoid tissues and tumor microenvironment and inhibit tumor growth. The finding suggests that activation of NKT cells with α-GalCer may provide an effective anti-cancer outcome.

INKT Cell Stimulation with Liposomal Alpha/Gal/Cer Amplifies Proliferation of Adoptively Transferred Regulatory T Cells In Vivo and Reduces GvHD

Abstract Cellular therapy with regulatory T cells (Tregs) has shown promising results for suppressing graft versus host diseases (GvHD) in animal models and phase I/II clinical trials. However, a paucity of Tregs in the peripheral blood makes it difficult to acquire sufficient numbers of cells and hampers further clinical applications. Invariant natural killer T (iNKT) cells are another compartment of regulatory cells that ameliorate GvHD by interacting with Tregs upon either transplantation with activation with α-galactosylceramide (αGalCer) or adoptive transfer of iNKT cells. The aim of this study was to explore the proliferation and anti-GvHD effect of Treg following stimulation of iNKT cells with αGalCer.We utilized a liposomal formulation of αGalCer that was found to have the potential of preventing GvHD in a recently published phase I/IIa clinical trial. Lethally (8.8Gy) irradiated BALB/c recipient mice were transferred with 5 x 106T cell depleted bone marrow cells and 1 x 106conventional CD4+and CD8+T cells (Tcons) from WT C57BL/6 donor mice, with or without 1 x 105CD4+CD8-CD25hiFoxp3-GFP+cells (Tregs) that were freshly isolated from Foxp3-GFP+C57BL/6 mice. Immediately after cell injection, αGalCer-loaded or unloaded (Ctrl) liposomes were injected IV at 10 μg/kg. We utilized a subtherapeutic dose of Treg (1 x 105) that did not provide survival benefit compared to those without Treg transfer. αGalCer injection alone did not result in improved survival, suggested that a single injection of αGalCer is not sufficiently potent to prevent GvHD. Mice transferred with a subtherapeutic dose of Treg and liposomal αGalCer injection showed significant improvement in survival.Tissue damage examined 10 days after BMT was less severe in the Treg plus αGalCer group compared to those of the other groups. We analyzed the Treg population among CD4+T cells in the spleen, superficial/mesenteric lymph nodes, and liver on day 10 following transplant. As αGalCer stimulation promoted both Tcon-derived (CD45.1+Foxp3+) and transferred (CD45.2+Foxp3-GFP+) Treg, the proportion of Treg in the mice treated with the combination of Treg plus αGalCer stimulation significantly increased in all organs compared to those of the other groups. We evaluated the transferred Tregs in vivo by bioluminescent imaging. Treg-signals in αGalCer treated mice did not show significant difference compared to those in Ctrl mice until day 7 and increased thereafter especially in the gastrointestinal tract and became significantly higher 2-3 weeks after BMT. Host iNKT cells rather than donor iNKT cells contributed more to GvHD suppression because survival benefit of αGalCer stimulation was not shown in the transplantation of WT C57BL/6 mice into Jα18-/- BALB/c mice, whereas it was in that from Jα18-/-C57BL/6 mice into WT BALB/c mice. In conclusion, liposomal αGalCer injection enhances Treg function through activation of host derived iNKT cells and could make Treg cell therapy more feasible and safer by reducing the requirement of Treg number. Disclosures No relevant conflicts of interest to declare.

Kinetics of the liver resident NK cell compartment

Abstract The presence of tissue-resident NK (trNK) cells in the liver has only recently been appreciated. While the role of conventional NK cells (cNK) has been well-characterized, the trNK cell function is only beginning to be elucidated. Using the MCMV model of infection, we found that in contrast to liver cNK cells, trNK cells initially undergo a contraction phase followed by a recovery phase to homeostatic levels. The contraction phase is independent of the virus itself as a similar phenotype is observed following Poly I:C/CpG or α-GalCer injection. Interestingly, this initial contraction phase is independent of other liver lymphocyte subsets as it occurs even in infected lymphopenic animals, suggesting it is provoked by the inflammatory milieu. We also found that the contraction phase is partially due to cell apoptosis while the recovery phase occurs via proliferation in situ. These findings underscore the unique homeostatic properties and kinetics of liver resident NK cell compartment.

Functional role of natural killer T cells in non-obese pre-diabetes model mice.

Pre-diabetic patients have a high risk of developing diabetes as well as other associated diseases. From the viewpoint of risk assessment and to assist the development of protective therapies, we focused on the functional role of natural killer T (NKT) cells in pre-diabetes. We found that the expression of an NKT cell marker gene, Va14-Ja18, was significantly lower in specific tissues/organs such as adipose tissue and pancreas in non-obese pre-diabetes model mice than in their normal littermates. Subsequently, in the pre-diabetes model mice, Va14-Ja18 was activated with α-galactosylceramide (α-GalCer) and its effect on glucose tolerance was estimated. The simultaneous injection of α-GalCer and lymphocytes improved glucose tolerance with its maximum effect on the 3rd day. An analysis of circulating cytokine levels revealed that interferon-γ, which is a pro-inflammatory cytokine, was secreted only on the 1st day after treatment with α-GalCer and that interleukin (IL)-4, which is an anti-inflammatory cytokine, was secreted from the 1st to the4th day. The prolonged secretion of IL-4 was thought to substantially contribute to the improvement of glucose tolerance. Based on these results, the functional role of NKT cells in pre-diabetes is to improve metabolic dysfunctions.

Interferon-γ constrains cytokine production of group 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2s) produce a significant amount of interleukin-5 (IL-5), which supports eosinophil responses in various tissues; they also produce IL-13, which induces mucus production and contributes to tissue repair or fibrosis. The ILC2s are activated by alarmins, such as IL-33 released from epithelia, macrophages and natural killer T (NKT) cells in response to infection and allergen exposure, leading to epithelial injury. We examined gene expression in lung ILC2s and found that ILC2s expressed Ifngr1, the receptor for interferon-γ (IFN-γ). Interferon-γ severely inhibited IL-5 and IL-13 production by lung and kidney ILC2s. To evaluate the effects in vivo, we used α-galactosylceramide (α-GalCer) to induce NKT cells to produce IL-33 and IFN-γ. Intraperitoneal injection of α-GalCer in mice induced NKT cell activation resulting in IL-5 and IL-13 production by ILC2s. Administration of anti-IFN-γ together with α-GalCer significantly enhanced the production of IL-5 and IL-13 by ILC2s in lung and kidney. Conversely, cytokine production from ILC2s was markedly suppressed after injection of exogenous IL-33 in Il33(-/-) mice pre-treated with α-GalCer. Hence, IFN-γ induced or already present in tissues can impact downstream pleiotropic functions mediated by ILC2s, such as inflammation and tissue repair.

Administration of α-Galactosylceramide Improves Adenine-Induced Renal Injury.

Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration.

In contrast to other species, α-Galactosylceramide (α-GalCer) is not an immunostimulatory NKT cell agonist in horses

α-GalCer is a potent immunomodulatory molecule that is presented to NKT cells via the CD1 antigen-presenting system. We hypothesized that when used as an adjuvant α-GalCer would induce protective immune responses against Rhodococcus equi, an important pathogen of young horses. Here we demonstrate that the equine CD1d molecule shares most features found in CD1d from other species and has a suitable lipid-binding groove for presenting glycolipids to NKT cells. However, equine CTL stimulated with α-GalCer failed to kill cells infected with R. equi, and α-GalCer did not increase killing by CTL co-stimulated with R. equi antigen. Likewise, α-GalCer did not induce the lymphoproliferation of equine PBMC or increase the proliferation of R. equi-stimulated cells. Intradermal injection of α-GalCer in horses did not increase the recruitment of lymphocytes or cytokine production. Furthermore, α-GalCer-loaded CD1d tetramers, which have been shown to be broadly cross-reactive, did not bind equine lymphocytes. Altogether, our results demonstrate that in contrast to previously described species, horses are unable to respond to α-GalCer. This raises questions about the capabilities and function of NKT cells and other lipid-specific T lymphocytes in horses.

Activation of invariant natural killer T cells impedes liver regeneration by way of both IFN-γ- and IL-4-dependent mechanisms

Invariant natural killer T (iNKT) cells are a major subset of lymphocytes found in the liver. These cells mediate various functions, including hepatic injury, fibrogenesis, and carcinogenesis. However, the function of iNKT cells in liver regeneration remains unclear. In the present study, partial hepatectomy (PHx) was used to study liver regeneration. α-Galactosylceramide (α-GalCer), a specific ligand for iNKT cells, was used to induce iNKT cell activation. After PHx, two strains of iNKT cell-deficient mice, CD1d(-/-) and Jα281(-/-) mice, showed normal liver regeneration. Injection of α-GalCer before or after PHx, which rapidly stimulated interferon-gamma (IFN-γ) and interleukin (IL)-4 production by iNKT cells, markedly inhibited liver regeneration. In vitro treatment with IFN-γ inhibited hepatocyte proliferation. In agreement with this in vitro finding, genetic disruption of IFN-γ or its downstream signaling molecule signal transducer and activator of transcription (STAT)1 significantly abolished the α-GalCer-mediated inhibition of liver regeneration. In vitro exposure to IL-4 did not affect hepatocyte proliferation, but surprisingly, genetic ablation of IL-4 or its downstream signaling molecule STAT6 partially eliminated the inhibitory effect of α-GalCer on liver regeneration. Further studies revealed that IL-4 contributed to α-GalCer-induced iNKT cell expansion and IFN-γ production, thereby inhibiting liver regeneration. iNKT cells play a minor role in controlling liver regeneration after PHx under healthy conditions. Activation of iNKT cells by α-GalCer induces the production of IFN-γ, which directly inhibits liver regeneration, and IL-4, which indirectly attenuates liver regeneration by stimulating iNKT cell expansion and IFN-γ production.

Invariant NKT cell activation induces neutrophil accumulation and hepatitis: Opposite regulation by IL-4 and IFN-γ

Alpha-Galactosylceramide (α-Galcer), a specific agonist for invariant natural killer T (iNKT) cells, is being evaluated in clinical trials for the treatment of viral hepatitis and liver cancer. However, the results from α-Galcer treatment are mixed, partially because of the variety of cytokines produced by activated iNKT cells that have an unknown synergistic effect on the progression of liver disease. It is well documented that injection of α-Galcer induces mild hepatitis with a rapid elevation in the levels of interleukin (IL)-4 and a delayed elevation in the levels of interferon-gamma (IFN-γ), and both of these cytokines are thought to mediate many functions of iNKT cells. Surprisingly, genetic deletion of both IL-4 and IFN-γ aggravated, rather than abolished, α-Galcer-induced iNKT hepatitis. Moreover, genetic ablation of IL-4, the IL-4 receptor, or its downstream signaling molecule signal transducer and activator of transcription (STAT)6 ameliorated α-Galcer-induced neutrophil infiltration, liver injury, and hepatitis. In contrast, genetic deletion of IFN-γ, the IFN-γ receptor, or its downstream signaling molecule STAT1 enhanced liver neutrophil accumulation, thereby exacerbating liver injury and hepatitis. Moreover, depletion of neutrophils eradicated α-Galcer-induced liver injury in wild-type, STAT1 knockout, and IFN-γ knockout mice. Our results propose a model in which activated iNKT cells rapidly release IL-4, which promotes neutrophil survival and hepatitis but also sequentially produce IFN-γ, which acts in a negative feedback loop to ameliorate iNKT hepatitis by inducing neutrophil apoptosis. Thus, modification of iNKT production of IL-4 and IFN-γ may have the potential to improve the efficacy of α-Galcer in the treatment of liver disease.

Preclinical Evaluation of Invariant Natural Killer T-Cells in the 5T33 Multiple Myeloma Model

Abstract Abstract 938 Natural killer T (NKT) cells are T-lymphocytes that co-express conventional T-cell (CD3) and NK cell (NK1.1) surface receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain encoded by Vα14.Jα18 in mice and Vα24.Jα18 in human. These iNKTs can recognize glycolipid antigens such as α-Galactosylceramide (α-GalCer) presented by the class I-like major histocompatibility complex (MHC) molecule CD1d. Activation of iNKTs can lead to an anti-tumor Th1 (IFN-γ) response or an immunosuppressive Th2 (IL-4) response. Clinical studies in MM patients (1, 2) showed a low frequency and dysfunction of iNKTs which resulted in a low IFN-γ secretion. This defect could be overcome in vitro by stimulating the iNKTs with α-GalCer loaded DCs. Furthermore, when MM patients were injected with loaded DCs their iNKT pool expanded 100 fold. This make MM cells an interesting target for iNKT therapy. However, the data on NKT activity in MM patients is limited and the use of α-GalCer as a drug has not been preclinically evaluated yet. Therefore, in this study, we investigated the characteristics of iNKTs in the syngeneic 5T33MM murine model, which is an immunocompetent model of myeloma which mimics the human disease closely. We first investigated the frequency of iNKTs in the blood, BM, spleen and liver of both healthy and terminally diseased 5T33MM mice. The highest percentage of iNKTs was found in the liver (naive 7%) with a significant decrease in 5T33MM mice (2.6%). The percentage was also slightly decreased in spleen (from 1.5% in naive to 0.7% in 5T33MM mice) while no significant differences were observed in the other tissues. Next, we followed the frequency of iNKTs in the liver and spleen of MM mice during the development of the disease. We analyzed the number of iNKTs in the first, second, third and terminal week. We found that the percentage of iNKTs declined at the end stage of MM disease. To analyze the activity of iNKTs in vitro, liver iNKTs were cocultured with naive matured BM derived DCs in the presence or absence of 100 ng/ml α-GalCer. Naive iNKTs could secrete up to 2.3 ng/ml IFN-γ when stimulated with α-GalCer, and this level increased with the progression of MM to reach 3.3 ng/ml at week 2. However, the activity of the iNKTs dropped to undetectable levels upon further progression of the disease (week 4). In contrast, very slight IL-4 production was observed indicating that liver iNKTs are skewed to a Th1 profile and can therefore be used as an immunotherapeutic tool in MM. The activity of the NKTs was also followed in vivo. The serum level of IFN-γ peaked at 18h after α-GalCer injection in naive and non-terminal diseased mice and returned to baseline by 48h, however, the response of IFN-γ in diseased mice was twice (6 ng/ml) that measured in naive mice, confirming the possibility of inducing Th1 responses with α-GalCer in vivo in healthy and diseased mice. No response could be detected from terminally diseased mice. It has been described previously that CD1d is significantly downregulated in patients with advanced stages of MM (3). To investigate if this is similar in the 5T33MM model, we followed the expression of CD1d on spleen and BM cells during the course of the disease. Results showed a significant downregulation of CD1d expression on spleen cells from 93% CD1d (naive) to 68% at end stage. On BM cells, CD1d was less expressed compared to spleen cells, 52% in naive mice and expression declined significantly to 35%. CD1d expression on the MM cells themselves was high (79%) and did not alter during the course of the disease. We finally evaluated the effect of α-GalCer on the survival of MM mice. Survival was significantly increased when mice were injected with α-GalCer loaded DCs on the same day of 5T33MM inoculation (29 days survival) compared to mice injected with unloaded DCs (22 days survival). Taken together, our data demonstrate for the first time the possibility of using a murine model as a preclinical MM model to study the effects of α-GalCer on iNKTs and shows promising results of treating MM patients with a low tumorload. Disclosures: No relevant conflicts of interest to declare.

Activated iNKT Cells Promote Memory CD8+ T Cell Differentiation during Viral Infection

α-galactosylceramide (α-GalCer) is the prototypical lipid ligand for invariant NKT cells. Recent studies have proposed that α-GalCer is an effective adjuvant in vaccination against a range of immune challenges, however its mechanism of action has not been completely elucidated. A variety of delivery methods have been examined including pulsing dendritic cells with α-GalCer to optimize the potential of α-GalCer. These methods are currently being used in a variety of clinical trials in patients with advanced cancer but cannot be used in the context of vaccine development against pathogens due to their complexity. Using a simple delivery method, we evaluated α-GalCer adjuvant properties, using the mouse model for cytomegalovirus (MCMV). We measured several key parameters of the immune response to MCMV, including inflammation, effector, and central memory CD8+ T cell responses. We found that α-GalCer injection at the time of the infection decreases viral titers, alters the kinetics of the inflammatory response, and promotes both increased frequencies and numbers of virus-specific memory CD8+ T cells. Overall, our data suggest that iNKT cell activation by α-GalCer promotes the development of long-term protective immunity through increased fitness of central memory CD8+ T cells, as a consequence of reduced inflammation.

Role of γδ T Cells in α-Galactosylceramide–Mediated Immunity

Attempts to harness mouse type I NKT cells in different therapeutic settings including cancer, infection, and autoimmunity have proven fruitful using the CD1d-binding glycolipid α-galactosylceramide (α-GalCer). In these different models, the effects of α-GalCer mainly relied on the establishment of a type I NKT cell-dependent immune cascade involving dendritic cell, NK cell, B cell, or conventional CD4(+) and CD8(+) T cell activation/regulation as well as immunomodulatory cytokine production. In this study, we showed that γδ T cells, another population of innate-like T lymphocytes, displayed a phenotype of activated cells (cytokine production and cytotoxic properties) and were required to achieve an optimal α-GalCer-induced immune response. Using gene-targeted mice and recombinant cytokines, a critical need for IL-12 and IL-18 has been shown in the α-GalCer-induced IFN-γ production by γδ T cells. Moreover, this cytokine production occurred downstream of type I NKT cell response, suggesting their bystander effect on γδ T cells. In line with this, γδ T cells failed to directly recognize the CD1d/α-GalCer complex. We also provided evidence that γδ T cells increase their cytotoxic properties after α-GalCer injection, resulting in an increase in killing of tumor cell targets. Moreover, using cancer models, we demonstrated that γδ T cells were required for an optimal α-GalCer-mediated anti-tumor activity. Finally, we reported that immunization of wild-type mice with α-GalCer enhanced the adaptive immune response elicited by OVA, and this effect was strongly mediated by γδ T cells. We conclude that γδ T cells amplify the innate and acquired response to α-GalCer, with possibly important outcomes for the therapeutic effects of this compound.

α-Galactosylceramide–Induced Airway Eosinophilia Is Mediated through the Activation of NKT Cells

Invariant NKT (iNKT) cells bridge innate and adaptive immune responses, resulting in the expansion of Ag-specific B and T cell responses. α-Galactosylceramide (α-GalCer), the most studied glycolipid that activates iNKT cells, has been proposed to be an effective adjuvant against infections and tumors. We found that the activation of iNKT cells by intranasal injection of α-GalCer induced airway eosinophilia in naive mice. Eosinophils, which mediate tissue damage and dysfunction by secreting mediators, play important roles in the pathogenesis of allergic diseases. In this study, we investigated the mechanism of how eosinophils are recruited to the lung by α-GalCer. Our results demonstrated that α-GalCer-induced eosinophil inflammation was mediated through iNKT cells. These cells secreted IL-5 to recruit eosinophils directly to the lung and/or secreted IL-4 and IL-13 to recruit eosinophils indirectly by inducing lung epithelial cells, endothelial cells, and fibroblast to secrete the eosinophil chemoattractant eotaxin. In addition, in the OVA-alum murine model of allergic asthma, α-GalCer administration in OVA-immunized mice also increased airway eosinophilia after challenge. Given our findings, intranasal administration of α-GalCer induced airway eosinophilic inflammation in both naive and allergic mice. Hence, it remains to be determined whether the activation of iNKT cells would be applicable in therapeutics for human diseases.