Rationally Designed Highly Potent NKT Cell Agonists with Different Cytokine Selectivity through Hydrogen-Bond Interaction.

Synthetic α-galactosylceramide (αGalCer) and its analogues as powerful agonists for natural killer T (NKT) cell manipulation have received significant attention in immunotherapy and adjuvant development. However, identifying new potent NKT cell agonists, especially those with Th1 selectivity that promote anticancer effects, remains a challenging task. In this work, we introduced a sulfonamide group into the acyl chain of αGalCer to form additional hydrogen bonds to intensify the glycolipid/CD1d interaction. Two compounds GCS-11 and GCS-12 demonstrated remarkable potency while exhibiting different cytokine induction patterns. Compared to αGalCer, the Th1-biased GCS-11 exhibited a 6-fold increase in IFN-γ but not IL-4, while the Th1/2-balanced GCS-12 elicited 7- and 5-fold increase in IFN-γ and IL-4, respectively, in vivo. These findings place them among the most potent NKT cell agonists, with superior antitumor effects. Therefore, hydrogen-bond-involved derivatization could be a powerful strategy to develop potent and polarized NKT cell agonists for various immunotherapies.

Effective suppression of tumor growth and hepatic metastasis of neuroblastoma by NKT-stimulatory phenyl glycolipid.

Invariant natural killer T cell (iNKT) cells produce large amounts of cytokines in response to α-Galactosylceramide (α-GalCer) stimulation. An analog containing two phenyl rings on the acyl chain, C34, was previously found to be more Th1-biased than α-GalCer and triggered greater anticancer activities against breast cancer, melanoma and lung cancer in mice. Since liver is enriched in iNKT cells, we investigated anticancer efficacy of C34 on neuroblastoma with hepatic metastasis. C34 induced Th1-biased cytokine secretions in the liver, significantly suppressed neuroblastoma growth/metastasis and prolonged mouse survival. The anti-tumor efficacy might be attributed to greater expansions of hepatic NKT, NK, CD4+ T, and CD8+ T cells as well as reduction of the number of SSCloGr1intCD11b+ subset of myeloid-derived suppressor cells (MDSCs) in the liver of tumor-bearing mice, as compared to DMSO control group. C34 also upregulated expression of CD1d and CD11c, especially in the SSCloGr1intCD11b+ subset of MDSCs, which might be killed by C34-activated NKT cells, attributing to their reduced number. In addition, C34 also induced expansion of CD4+ T, CD8+ T, and NK cells, which might eliminate neuroblastoma cells. These immune-modulating effects of C34 might act in concert in the local milieu of liver to suppress the tumor growth. Further analysis of database of neuroblastoma revealed that patients with high CD11c expression in the monocytic MDSCs in the tumor had longer survival, suggesting the potential clinical application of C34 for treatment of neuroblastoma.

Sustained release system from PLGA particles co-encapsulated with inactivated influenza virus with natural killer T cell agonist α-galactosylceramide.

Vaccines against influenza and many other infectious diseases require multiple boosters in addition to the primary dose to improve efficacy, but this approach is not ideal for compliance. The multiple doses could potentially be replaced by sustained or pulsatile release of antigens encapsulated in degradable microparticles (MPs). The efficacy of a vaccine is improved by adding an adjuvant, which can be co-delivered from the particles to enhance immunogenicity. Here, we developed degradable poly-lactic-co-glycolic acid (PLGA) (7-17 kDa) MPs capable of sustained release of ultraviolet killed influenza virus (A/PR/8/34) (kPR8) vaccine and the natural killer T (NKT) cell agonist alpha-galactosylceramide (α-GalCer) and tested their effectiveness at providing long-term protection against influenza virus infection in mice. Multiple formulations were developed for encapsulating the virus and adjuvant separately, and in combination. The MPs exhibited sustained release of both the virus and the adjuvant lasting more than a month. Co-encapsulation significantly increased the encapsulation efficiency (EE) of the vaccine but reduced the release duration. On the other hand, co-encapsulation led to a reduction in EE for the α-GalCer and a change in release profile to a higher initial burst followed by a linear release compared to a low initial burst and slower linear release. The α-GalCer also had considerably longer release duration compared to the vaccine. Mice injected with particle formulations co-encapsulating kPR8 and α-GalCer were protected from a lethal influenza virus infection 30 weeks after vaccination. This study demonstrates that PLGA MP based vaccines are promising for providing effective vaccination and possibly for replacing multiple doses with a single injection.

Harnessing Metformin's Immunomodulatory Effects on Immune Cells to Combat Breast Cancer.

Metformin, a medication known for its anti-glycemic properties, also demonstrates potent immune system activation. In our study, using a 4T1 breast cancer model in BALB/C WT mice, we examined metformin's impact on the functional phenotype of multiple immune cells, with a specific emphasis on natural killer T (NKT) cells due to their understudied role in this context. Metformin administration delayed the appearance and growth of carcinoma. Furthermore, metformin increased the percentage of IFN-γ+ NKT cells, and enhanced CD107a expression, as measured by MFI, while decreasing PD-1+, FoxP3+, and IL-10+ NKT cells in spleens of metformin-treated mice. In primary tumors, metformin increased the percentage of NKp46+ NKT cells and increased FasL expression, while lowering the percentages of FoxP3+, PD-1+, and IL-10-producing NKT cells and KLRG1 expression. Activation markers increased, and immunosuppressive markers declined in T cells from both the spleen and tumors. Furthermore, metformin decreased IL-10+ and FoxP3+ Tregs, along with Gr-1+ myeloid-derived suppressor cells (MDSCs) in spleens, and in tumor tissue, it decreased IL-10+ and FoxP3+ Tregs, Gr-1+, NF-κB+, and iNOS+ MDSCs, and iNOS+ dendritic cells (DCs), while increasing the DCs quantity. Additionally, increased expression levels of MIP1a, STAT4, and NFAT in splenocytes were found. These comprehensive findings illustrate metformin's broad immunomodulatory impact across a variety of immune cells, including stimulating NKT cells and T cells, while inhibiting Tregs and MDSCs. This dynamic modulation may potentiate its use in cancer immunotherapy, highlighting its potential to modulate the tumor microenvironment across a spectrum of immune cell types.

A natural killer T cell nanoagonist-initiated immune cascade for hepatocellular carcinoma synergistic immunotherapy.

Natural killer T (NKT) cell-mediated immunotherapy shows great promise in hepatocellular carcinoma featuring an inherent immunosuppressive microenvironment. However, targeted delivery of NKT cell agonists remains challenging. Here, we developed a hyaluronic acid (HA) modified metal organic framework (zeolitic imidazolate framework-8, ZIF-8) to encapsulate α-galactosylceramide (α-Galcer), a classic NKT cell agonist, and doxorubicin (DOX) for eliminating liver cancer, denoted as α-Galcer/DOX@ZIF-8@HA. In the tumor microenvironment (TME), these pH-responsive nano-frameworks can gradually collapse to release α-Galcer for activating NKT cells and further boosting other immune cells in order to initiate an antitumor immune cascade. Along with DOX, the released α-Galcer enabled efficient NKT cell activation in TME for synergistic immunotherapy and tumor elimination, leading to evident tumor suppression and prolonged animal survival in both subcutaneous and orthotopic liver tumor models. Manipulating NKT cell agonists into functional nano-frameworks in TME may be matched with other advanced managements applied in a wider range of cancer therapies.

Long-Chain Acylcarnitines Induce Senescence of Invariant Natural Killer T Cells in Hepatocellular Carcinoma.

CD1d-restricted invariant natural killer T (iNKT) cells actively patrol the liver and possess valuable antitumor potential. However, clinical trials evaluating administration of iNKT cell-specific agonist α-galactosylceramide (α-GalCer) have failed to achieve obvious tumor regression. Improving the efficacy of iNKT cell-based immunotherapy requires a better understanding of the factors restraining the clinical benefits. In the context of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), we found circulating and hepatic iNKT cells were hyperactivated but demonstrated imbalances in ratio and defective α-GalCer responsiveness. Exogenous IL2 helped to expand residual α-GalCer-responsive clones with reduced T-cell receptor diversity. However, transcriptome-wide analysis revealed activation of the senescence-associated secretory phenotype and dampened cytotoxicity in iNKT cells, weakening their immune surveillance capacity. The senescent status of iNKT cells from the patients was further illustrated by cell-cycle arrest, impaired telomere maintenance, perturbed calcium transport-related biological processes, and altered metabolism. Lipidomic profiling revealed the accumulation of long-chain acylcarnitines (LCAC) and aberrant lipid metabolism in HCC tissue. Exogenous LCACs, especially palmitoyl-carnitine and stearoyl-carnitine, inhibited iNKT cell expansion and promoted senescence. Collectively, our results provide deeper insights into iNKT cell dysregulation and identify a cell senescence-associated challenge for iNKT cell-based immunotherapy in HBV-related HCC. The mechanistic links between iNKT cell senescence and accumulated LCACs suggest new targets for anti-HCC immunotherapies. SIGNIFICANCE: Patients with HBV-related HCC exhibit a cell senescence-associated dysregulation of invariant natural killer cells that is related to altered lipid metabolism and accumulated LCACs in tumor tissue.

Activation of iNKT Cells Facilitates Liver Repair After Hepatic Ischemia Reperfusion Injury Through Acceleration of Macrophage Polarization.

Macrophage polarization is critical for liver tissue repair following acute liver injury. However, the underlying mechanisms of macrophage phenotype switching are not well defined. Invariant natural killer T (iNKT) cells orchestrate tissue inflammation and tissue repair by regulating cytokine production. Herein, we examined whether iNKT cells played an important role in liver repair after hepatic ischemia-reperfusion (I/R) injury by affecting macrophage polarization. To this end, we subjected male C57BL/6 mice to hepatic I/R injury, and mice received an intraperitoneal (ip) injection of α-galactosylceramide (α-GalCer) or vehicle. Compared with that of the vehicle, α-GalCer administration resulted in the promotion of liver repair accompanied by acceleration of macrophage differentiation and by increases in the numbers of Ly6Chigh pro-inflammatory macrophages and Ly6Clow reparative macrophages. iNKT cells activated with α-GalCer produced interleukin (IL)-4 and interferon (IFN)-γ. Treatment with anti-IL-4 antibodies delayed liver repair, which was associated with an increased number of Ly6Chigh macrophages and a decreased number of Ly6Clow macrophages. Treatment with anti-IFN-γ antibodies promoted liver repair, associated with reduced the number of Ly6Chigh macrophages, but did not change the number of Ly6Clow macrophages. Bone marrow-derived macrophages up-regulated the expression of genes related to both a pro-inflammatory and a reparative phenotype when co-cultured with activated iNKT cells. Anti-IL-4 antibodies increased the levels of pro-inflammatory macrophage-related genes and decreased those of reparative macrophage-related genes in cultured macrophages, while anti-IFN-γ antibodies reversed the polarization of macrophages. Cd1d-deficient mice showed delayed liver repair and suppressed macrophage switching, compared with that in wild-type mice. These results suggest that the activation of iNKT cells by α-GalCer facilitated liver repair after hepatic I/R injury by both IL-4-and IFN-γ-mediated acceleration of macrophage polarization. Therefore, the activation of iNKT cells may represent a therapeutic tool for liver repair after hepatic I/R injury.

Potent Neutralizing Antibodies Elicited by RBD-Fc-Based COVID-19 Vaccine Candidate Adjuvanted by the Th2-Skewing iNKT Cell Agonist.

The development of a safe and effective COVID-19 vaccine is of paramount importance to terminate the current pandemic. An adjuvant is crucial for improving the efficacy of the subunit COVID19 vaccine. α-Galactosylceramide (αGC) is a classical iNKT cell agonist which causes the rapid production of Th1- and Th2-associated cytokines; we, therefore, expect that the Th1- or Th2-skewing analogues of αGC can better enhance the immunogenicity of the receptor-binding domain in the spike protein of SARS-CoV-2 fused with the Fc region of human IgG (RBD-Fc). Herein, we developed a universal synthetic route to the Th1-biasing (α-C-GC) and Th2-biasing (OCH and C20:2) analogues. Immunization of mice demonstrated that αGC-adjuvanted RBD-Fc elicited a more potent humoral response than that observed with Alum and enabled the sparing of antigens. Remarkably, at a low dose of the RBD-Fc protein (2 µg), the Th2-biasing agonist C20:2 induced a significantly higher titer of the neutralizing antibody than that of Alum.

Miltefosine enhances infectivity of a miltefosine-resistant Leishmania infantum strain by attenuating its innate immune recognition.

BACKGROUND: Miltefosine (MIL) is currently the only oral drug available to treat visceral leishmaniasis but its use as first-line monotherapy has been compromised by an increasing treatment failure. Despite the scarce number of resistant clinical isolates, MIL-resistance by mutations in a single aminophospholipid transporter gene can easily be selected in a laboratory environment. These mutations result in a reduced survival in the mammalian host, which can partially be restored by exposure to MIL, suggesting a kind of drug-dependency. METHODOLOGY/PRINCIPAL FINDINGS: To enable a combined study of the infection dynamics and underlying immunological events for differential in vivo survival, firefly luciferase (PpyRE9) / red fluorescent protein (DsRed) double-reporter strains were generated of MIL-resistant (MIL-R) and syngeneic MIL-sensitive (MIL-S) Leishmania infantum. Results in C57Bl/6 and BALB/c mice show that MIL-R parasites induce an increased innate immune response that is characterized by enhanced influx and infection of neutrophils, monocytes and dendritic cells in the liver and elevated serum IFN-γ levels, finally resulting in a less efficient establishment in liver macrophages. The elevated IFN-γ levels were shown to originate from an increased response of hepatic NK and NKT cells to the MIL-R parasites. In addition, we demonstrated that MIL could increase the in vivo fitness of MIL-R parasites by lowering NK and NKT cell activation, leading to a reduced IFN-γ production. CONCLUSIONS/SIGNIFICANCE: Differential induction of innate immune responses in the liver was found to underlie the attenuated phenotype of a MIL-R parasite and its peculiar feature of drug-dependency. The impact of MIL on hepatic NK and NKT activation and IFN-γ production following recognition of a MIL-R strain indicates that this mechanism may sustain infections with resistant parasites and contribute to treatment failure.

Chronic stress physically spares but functionally impairs innate-like invariant T cells.

The deleterious effects of psychological stress on mainstream T lymphocytes are well documented. However, how stress impacts innate-like T cells is unclear. We report that long-term stress surprisingly abrogates both T helper 1 (TH1)- and TH2-type responses orchestrated by invariant natural killer T (iNKT) cells. This is not due to iNKT cell death because these cells are unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice exhibit a "split" inflammatory signature and trigger sudden serum interleukin-10 (IL-10), IL-23, and IL-27 spikes. iNKT cell dysregulation is mediated by cell-autonomous glucocorticoid receptor signaling and corrected upon habituation to predictable stressors. Importantly, under stress, iNKT cells fail to potentiate cytotoxicity against lymphoma or to reduce the burden of metastatic melanoma. Finally, stress physically spares mouse mucosa-associated invariant T (MAIT) cells but hinders their TH1-/TH2-type responses. The above findings are corroborated in human peripheral blood and hepatic iNKT/MAIT cell cultures. Our work uncovers a mechanism of stress-induced immunosuppression.

New Paradigm in NKT Cell Antigens: MCS-0208 (2-(Hydroxymethyl)phenylthio-phytoceramide) - an Aryl-Phytoceramide Compound with a Single Hydroxyl Group Stimulates NKT Cells.

Natural Killer T (NKT) cells play an important role in the immune response and can be activated by glycolipids presented by CD1d protein. We present MCS-0208, an unprecedented arylthioether-phytoceramide able to induce potent invariant NKT (iNKT) cell activation, notably when tested in human iNKT cells. This arylsphingolipid analog has a simple phenyl group containing a single hydroxyl substituent as a surrogate of the sugar ring. The phenylthioether structure contrasts with α-galactosylceramide (1), the prototypical glycolipid used to induce iNKT cell stimulation, where the galactose 2'-OH and 3'-OH substituents are accepted as the minimal footprint and considered critical for NKT T cell receptor (TCR) recognition. A computational study supports the recognition of aromatic compound by the CD1d and TCR proteins as radically new structures for NKT cell stimulation.

Lactic acid inhibits iNKT cell functions via a phosphodiesterase-5 dependent pathway.

Lactic acid in tumor microenvironment inhibits iNKT cell functions and thus dampens their anti-tumor efficacy. The underlying mechanisms remain unclear. Here, we show that phosphodiesterase-5 inhibitors, sildenafil and tadalafil, promote IFN-γ and IL-4 production in iNKT cells in a cGMP-PKG pathway dependent manner. To favor their cytokine production, iNKT cells reduce Pde5a mRNA lever after activation. In line with the reduction of cytokines caused by lactic acid, lactic acid elevates Pde5a mRNA lever in activated iNKT cells. As a result, phosphodiesterase-5 inhibitor partially restores the cytokine production in lactic acid-treated cells. Our results demonstrate that phosphodiesterase-5 inhibits cytokine production in iNKT cells, and that contributes to the lactic acid-caused dysfunction of iNKT cells.

Antibiotic-associated dysbiosis affects the ability of the gut microbiota to control intestinal inflammation upon fecal microbiota transplantation in experimental colitis models.

BACKGROUND: The gut microbiota plays a central role in host physiology and in several pathological mechanisms in humans. Antibiotics compromise the composition and functions of the gut microbiota inducing long-lasting detrimental effects on the host. Recent studies suggest that the efficacy of different clinical therapies depends on the action of the gut microbiota. Here, we investigated how different antibiotic treatments affect the ability of the gut microbiota to control intestinal inflammation upon fecal microbiota transplantation in an experimental colitis model and in ex vivo experiments with human intestinal biopsies. RESULTS: Murine fecal donors were pre-treated with different antibiotics, i.e., vancomycin, streptomycin, and metronidazole before FMT administration to colitic animals. The analysis of the gut microbiome, fecal metabolome, and the immunophenotyping of colonic lamina propria immune cells revealed that antibiotic pre-treatment significantly influences the capability of the microbiota to control intestinal inflammation. Streptomycin and vancomycin-treated microbiota failed to control intestinal inflammation and were characterized by the blooming of pathobionts previously associated with IBD as well as with metabolites related to the presence of oxidative stress and metabolism of simple sugars. On the contrary, the metronidazole-treated microbiota retained its ability to control inflammation co-occurring with the enrichment of Lactobacillus and of innate immune responses involving iNKT cells. Furthermore, ex vivo cultures of human intestinal lamina propria mononuclear cells and iNKT cell clones from IBD patients with vancomycin pre-treated sterile fecal water showed a Th1/Th17 skewing in CD4+ T-cell populations; metronidazole, on the other hand, induced the polarization of iNKT cells toward the production of IL10. CONCLUSIONS: Diverse antibiotic regimens affect the ability of the gut microbiota to control intestinal inflammation in experimental colitis by altering the microbial community structure and microbiota-derived metabolites. Video Abstract.

Serial Stimulation of Invariant Natural Killer T Cells with Covalently Stabilized Bispecific T-cell Engagers Generates Antitumor Immunity While Avoiding Anergy.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. SIGNIFICANCE: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.

Immune-Related Adverse Events Associated With Immune Checkpoint Inhibitor Therapy.

As part of immune surveillance, killer T lymphocytes search for cancer cells and destroy them. Some cancer cells, however, develop escape mechanisms to evade detection and destruction. One of these mechanisms is the expression of cell surface proteins which allow the cancer cell to bind to proteins on T cells called checkpoints to switch off and effectively evade T-cell-mediated destruction. Immune checkpoint inhibitors (ICIs) are antibodies that block the binding of cancer cell proteins to T-cell checkpoints, preventing the T-cell response from being turned off by cancer cells and enabling killer T cells to attack. In other words, ICIs restore innate antitumor immunity, as opposed to traditional chemotherapies that directly kill cancer cells. Given their relatively excellent risk-benefit ratio when compared to other forms of cancer treatment modalities, ICIs are now becoming ubiquitous and have revolutionized the treatment of many types of cancer. Indeed, the prognosis of some patients is so much improved that the threshold for admission for intensive care should be adjusted accordingly. Nevertheless, by modulating immune checkpoint activity, ICIs can disrupt the intricate homeostasis between inhibition and stimulation of immune response, leading to decreased immune self-tolerance and, ultimately, autoimmune complications. These immune-related adverse events (IRAEs) may virtually affect all body systems. Multiple IRAEs are common and may range from mild to life-threatening. Management requires a multidisciplinary approach and consists mainly of immunosuppression, cessation or postponement of ICI treatment, and supportive therapy, which may require surgical intervention and/or intensive care. We herein review the current literature surrounding IRAEs of interest to anesthesiologists and intensivists. With proper care, fatality (0.3%-1.3%) is rare.

Using agonists for iNKT cells in cancer therapy.

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+ T cells is a new observation that could find a role in cancer therapy.

Recombinant Human Thyroid-Stimulating Hormone Increases the Percentages of Natural Killer T Cells and B Lymphocytes in Human Peripheral Blood In Vivo.

Multiple cellular and humoral components of the immune system play a significant role in the physiology and pathophysiology of various organs including the thyroid. On the other hand, both thyroid hormones and thyroid-stimulating hormone (TSH) have been shown to exert immunoregulatory activities, which are difficult to assess independently in vivo. In our study we employed a unique clinical model for the assessment of TSH biological function in humans. The structure of peripheral blood mononuclear cell populations was investigated, using flow cytometry, in athyroid patients (n = 109) after treatment because of the differentiated thyroid carcinoma (DTC) at two time-points: directly before and five days after recombinant human TSH (rhTSH) administration. The analysis revealed significant increase in the percentage of natural killer T cells and B lymphocytes in the peripheral blood of rhTSH treated patients, whereas, we did not observe any effects on investigated subpopulations of dendritic cells and monocytes, T cells and natural killer cells. The findings of the study indicate the immune regulatory role of TSH, directed specifically on selected cell subtypes.

Impact of Aging on the Phenotype of Invariant Natural Killer T Cells in Mouse Thymus.

Invariant natural killer T (iNKT) cells represent a subclass of T cells possessing a restricted repertoire of T cell receptors enabling them to recognize lipid derived ligands. iNKT cells are continuously generated in thymus and differentiate into three main subpopulations: iNKT1, iNKT2, and iNKT17 cells. We investigated the transcriptomes of these subsets comparing cells isolated from young adult (6-10 weeks old) and aged BALB/c mice (25-30 weeks of age) in order to identify genes subject to an age-related regulation of expression. These time points were selected to take into consideration the consequences of thymic involution that radically alter the existing micro-milieu. Significant differences were detected in the expression of histone genes affecting all iNKT subsets. Also the proliferative capacity of iNKT cells decreased substantially upon aging. Several genes were identified as possible candidates causing significant age-dependent changes in iNKT cell generation and/or function such as genes coding for granzyme A, ZO-1, EZH2, SOX4, IGF1 receptor, FLT4, and CD25. Moreover, we provide evidence that IL2 differentially affects homeostasis of iNKT subsets with iNKT17 cells engaging a unique mechanism to respond to IL2 by initiating a slow rate of proliferation.

Anti-GD2 CAR-NKT cells in patients with relapsed or refractory neuroblastoma: an interim analysis.

Vα24-invariant natural killer T (NKT) cells have shown potent anti-tumor properties in murine tumor models and have been linked to favorable outcomes in patients with cancer. However, low numbers of these cells in humans have hindered their clinical applications. Here we report interim results from all three patients enrolled on dose level 1 in a phase 1 dose-escalation trial of autologous NKT cells engineered to co-express a GD2-specific chimeric antigen receptor (CAR) with interleukin-15 in children with relapsed or resistant neuroblastoma (NCT03294954). Primary and secondary objectives were to assess safety and anti-tumor responses, respectively, with immune response evaluation as an additional objective. We ex vivo expanded highly pure NKT cells (mean ± s.d., 94.7 ± 3.8%) and treated patients with 3 × 106 CAR-NKT cells per square meter of body surface area after lymphodepleting conditioning with cyclophosphamide/fludarabine (Cy/Flu). Cy/Flu conditioning was the probable cause for grade 3-4 hematologic adverse events, as they occurred before CAR-NKT cell infusion, and no dose-limiting toxicities were observed. CAR-NKT cells expanded in vivo, localized to tumors and, in one patient, induced an objective response with regression of bone metastatic lesions. These initial results suggest that CAR-NKT cells can be expanded to clinical scale and safely applied to treat patients with cancer.

α-Galactosylceramide and its analog OCH differentially affect the pathogenesis of ISO-induced cardiac injury in mice.

Immunotherapies for cancers may cause severe and life-threatening cardiotoxicities. The underlying mechanisms are complex and largely elusive. Currently, there are several ongoing clinical trials based on the use of activated invariant natural killer T (iNKT) cells. The potential cardiotoxicity commonly associated with this particular immunotherapy has yet been carefully evaluated. The present study aims to determine the effect of activated iNKT cells on normal and ß-adrenergic agonist (isoproterenol, ISO)-stimulated hearts. Mice were treated with iNKT stimulants, α-galactosylceramide (αGC) or its analog OCH, respectively, to determine their effect on ISO-induced cardiac injury. We showed that administration of αGC (activating both T helper type 1 (Th1)- and T helper type 2 (Th2)-liked iNKT cells) significantly accelerated the progressive cardiac injury, leading to enhanced cardiac hypertrophy and cardiac fibrosis with prominent increases in collagen deposition and TGF-ß1, IL-6, and alpha smooth muscle actin expression. In contrast to αGC, OCH (mainly activating Th2-liked iNKT cells) significantly attenuated the progression of cardiac injury and cardiac inflammation induced by repeated infusion of ISO. Flow cytometry analysis revealed that αGC promoted inflammatory macrophage infiltration in the heart, while OCH was able to restrain the infiltration. In vitro coculture of αGC- or OCH-pretreated primary peritoneal macrophages with primary cardiac fibroblasts confirmed the profibrotic effect of αGC and the antifibrotic effect of OCH. Our results demonstrate that activating both Th1- and Th2-liked iNKT cells is cardiotoxic, while activating Th2-liked iNKT cells is likely cardiac protective, which has implied key differences among subpopulations of iNKT cells in their response to cardiac pathological stimulation.