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Summary Cell-based immunotherapy has become the new-generation cancer medicine, and “off-the-shelf” cell products that can be manufactured at large scale and distributed readily to treat patients are necessary. Invariant natural killer T (iNKT) cells are ideal cell carriers for developing allogeneic cell therapy because they are powerful immune cells targeting cancers without graft-versus-host disease (GvHD) risk. However, healthy donor blood contains extremely low numbers of endogenous iNKT cells. Here, by combining hematopoietic stem cell (HSC) gene engineering and in vitro differentiation, we generate human allogeneic HSC-engineered iNKT (AlloHSC-iNKT) cells at high yield and purity; these cells closely resemble endogenous iNKT cells, effectively target tumor cells using multiple mechanisms, and exhibit high safety and low immunogenicity. These cells can be further engineered with chimeric antigen receptor (CAR) to enhance tumor targeting or/and gene edited to ablate surface human leukocyte antigen (HLA) molecules and further reduce immunogenicity. Collectively, these preclinical studies demonstrate the feasibility and cancer therapy potential of AlloHSC-iNKT cell products and lay a foundation for their translational and clinical development., Graphical abstract, Highlights • Allogeneic HSC-iNKT cells are generated in vitro at high yield and purity • Allogeneic HSC-iNKT cells effectively target tumor cells using multiple mechanisms • Allogeneic HSC-iNKT cells exhibit high safety and low immunogenicity • A preclinical study demonstrated feasibility, safety, and cancer therapy potential, Li et al. report the preclinical development of allogeneic-hematopoietic-stem-cell-engineered invariant natural killer T (HSC-iNKT) cells for off-the-shelf cancer therapy. Allogeneic HSC-iNKT cells demonstrate cancer therapy potential and a high safety profile.

International audience; The glycosphingolipid, α-galactosylceramide (αGalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (iNKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of αGalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS2, we screened murine intestinal tracts to identify and quantify αGalCers, and we investigated the αGalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of αGalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor αGalCer. The identified αGalCer contained a β(R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis Unlike β-anomeric structures, but similar to αGalCers from B. fragilis, the synthetic form of the murine αGalCer induced iNKT cell activation in vitro. Last, we observed a decrease in αGalCer production in mice exposed to conditions that alter the composition of the gut microbiota, including Western type diet, colitis, and influenza A virus infection. Collectively, this study suggests that αGalCer is produced by commensals in the mouse intestine and reveals that stressful conditions causing dysbiosis alter its synthesis. The consequences of this altered production on iNKT cell-mediated local and systemic immune responses are worthy of future studies.

The nervous system serves numerous critical roles in the regulation of immune responses. Consequently, psychological stress can result in immunosuppressive states that are conducive to the development of infection and cancer. Yet, whether stress impacts the functions of innate-like T lymphocytes including invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, which participate in early host defense against pathogens and tumors, remains poorly understood. In this thesis, I leveraged multiple established methods with which to induce psychological stress in mice. I demonstrate that TH1- and TH2-type immune responses initiated by iNKT cells are abrogated during stress, effects which are lost upon habituation to homotypic stressors. Instead, iNKT cells in stressed mice trigger an abnormal systemic inflammatory response characterized by striking levels of interleukin (IL)-10, IL-23, and IL-27. These dysregulated responses are driven by iNKT cell-intrinsic glucocorticoid receptor (GR) signaling. Accordingly, iNKT cells upregulate the co-inhibitory molecule T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) in a GR-dependent manner and blockade of TIGIT partially restores their functional capacity in stressed mice. Ultimately, in a GR-dependent fashion, iNKT cells from stressed mice fail to prevent pulmonary metastases of B16 melanoma. MAIT cells also upregulate TIGIT and are incapable of mounting optimal TH1- and TH2-type responses during stress. Lastly, these inhibitory effects are not simply due to cell death since human and mouse iNKT and MAIT cells are unusually refractory to glucocorticoid-induced apoptosis. Collectively, my findings reveal a mechanism of stress-induced immunosuppression with implications for iNKT or MAIT cell-based immunotherapies.

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes characterized by the expression of an invariant T cell receptor (iTCR) that recognizes glycolipid antigens presented by the MHC I-like CD1d molecule. Following antigenic stimulation, iNKT cells rapidly produce large amounts of cytokines that can trans-activate dendritic cells (DC) and promote the anti-tumor functions of cytotoxic lymphocytes, such as natural killer (NK) and CD8 T cells. Additionally, iNKT cells can mediate robust and direct cytotoxicity against CD1d+ tumor targets. However, many tumors down-regulate CD1d and evade iNKT cell attack. To circumvent this critical barrier to iNKT cell anti-tumor activity, a novel monoclonal antibody (mAb), NKT14 has been recently developed. This agonistic antibody binds directly and specifically to the iTCR of murine iNKT cells. In the current study, we demonstrate that NKT14m mediates robust activation, cytokine production and degranulation of murine iNKT cells, in vitro. Consistently, NKT14m also promoted iNKT cell activation and immunomodulatory functions, in vivo. Finally, administration of NKT14m with low dose interleukin (IL)-12 further augmented iNKT cell IFN-&gamma, production in vivo, and this combination conferred superior suppression of tumor cell growth compared to NKT14m or IL-12 alone. Together, these data demonstrate that a combination treatment consisting of low dose IL-12 and iTCR-specific mAb may be an attractive alternative to activate iNKT cell anti-tumor functions.

Neuroblastoma (NB) is the most common extracranial solid tumor in children and, in the high-risk group, has a 5-year mortality rate of ~50%. The high mortality rate and significant treatment-related morbidities associated with current standard of care therapies belie the critical need for more tolerable and effective treatments for this disease. While the monoclonal antibody dinutuximab has demonstrated the potential for immunotherapy to improve overall NB outcomes, the 5-year overall survival of high-risk patients has not yet substantially changed. The frequency and type of invariant natural killer T cells (iNKTs) and natural killer cells (NKs) has been associated with improved outcomes in several solid and liquid malignancies, including NB. Indeed, iNKTs and NKs inhibit tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs), kill cancer stem cells (CSCs) and neuroblasts, and robustly secrete cytokines to recruit additional immune effectors. These capabilities, and promising pre-clinical and early clinical data suggest that iNKT- and NK-based therapies may hold promise as both stand-alone and combination treatments for NB. In this review we will summarize the biologic features of iNKTs and NKs that confer advantages for NB immunotherapy, discuss the barriers imposed by the NB tumor microenvironment, and examine the current state of such therapies in pre-clinical models and clinical trials.

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.

Recent studies suggest that murine invariant natural killer T (iNKT) cell development culminates in three terminally differentiated iNKT cell subsets denoted as NKT1, 2, and 17 cells. Although these studies corroborate the significance of the subset division model, less is known about the factors driving subset commitment in iNKT cell progenitors. In this review, we discuss the latest findings in iNKT cell development, focusing in particular on how T-cell receptor signal strength steers iNKT cell progenitors toward specific subsets and how early progenitor cells can be identified. In addition, we will discuss the essential factors for their sustenance and functionality. A picture is emerging wherein the majority of thymic iNKT cells are mature effector cells retained in the organ rather than developing precursors.

Malignant B cells in chronic lymphocytic leukemia serve an essential role in the whole immune response, so their interactions with other immune cells are more complex than observed in solid tumors. The latest study results indicate that the immune dysregulation in chronic lymphocytic leukemia (CLL) also affects a small population of invariant natural killer T cells (iNKT). Using peripheral blood iNKT cells obtained from patients with CLL, the objective of the present study was to assess the intracellular expression of typical cytokines involved in the Th1 (IFN-γ) and Th2 (IL-4) response pathways following stimulation with the iNKT-specific ligand α-galactosylceramide. iNKT cells from patients with CLL exhibited upregulated IL-4 and IFN-γ expression in comparison to those from HVs. No significant association between the ability of iNKT cells to produce IL-4 or IFN-γ and the expression of CD1d on leukemic B lymphocytes or monocytes was identified. However, the function of iNKT cells was compromised in patients with CLL by a strong Th2 bias (high IL-4 and low IFN-γ expression). The ratio of iNKT+IFN-γ+:iNKT+IL-4+ was significantly decreased in the CLL group when compared with HVs, and this decreased further as the disease progressed. This change may result in the promotion of leukemic B lymphocyte survival. Therefore, in the pathogenesis of CLL, Th2 bias may delay the antitumor response that relies on stimulation of the Th1 immune response.

Background Systemic sclerosis (SSc) is a potentially fatal autoimmune disease that leads to extensive fibrosis of the skin and internal organs. Invariant natural killer T (iNKT) cells are potent immunoregulatory T lymphocytes being able to orchestrate dysregulated immune responses. The purpose of this study was to evaluate numbers and function of iNKT cells in patients with SSc and to analyze their correlation with disease parameters. Methods Human iNKT cells from 88 patients with SSc and 33 healthy controls were analyzed by flow cytometry. Their proliferative capacity and cytokine production were investigated following activation with CD1d ligand α-galactosylceramide (α-GalCer). Results We observed an absolute and relative decrease of iNKT cells in patients with SSc compared with healthy controls. Interestingly, the subtype of SSc, disease severity, or treatment with immunosuppressive drugs did not affect iNKT cell numbers. However, T helper (Th) cell immune polarization was biased towards a Th17 immunophenotype in SSc patients. Moreover, iNKT cells from patients with SSc showed a significantly decreased expansion capacity upon stimulation with α-GalCer. Conclusion iNKT cells are deficient and functionally impaired in patients with SSc. Therefore, adoptive transfer strategies using culture-expanded iNKT cells could be a novel approach to treat SSc patients.

Invariant natural killer T cells (iNKTs) directly kill tumor cells and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK) cells, and T and B cells. As such, iNKTs serve as a powerful tool for use in cell-based cancer immunotherapy. iNKT cell activation commonly requires engagement of the invariant T cell receptor (iTCR) by CD1d presenting glycolipid antigens. However, transformed cells often down-regulate CD1d expression, which results in a reduction of iNKT cell anti-tumor functions. One approach to circumvent this critical barrier to iNKT cell activation is to develop an agonistic antibody that binds directly to the iTCR without the requirement for CD1d-mediated antigen presentation. To this end, we have characterized the iNKT cell stimulatory properties of NKTT320, a novel, recombinant, humanized, monoclonal antibody that binds selectively and with high affinity to human iTCRs. Strikingly, immobilized NKTT320 mediated robust iNKT cell activation (upregulation of CD25 and CD69) and proliferation (carboxyfluorescein succinimidyl ester (CFSE) dilution), as well as Th1 and Th2 cytokine production. Additionally, iNKTs stimulated by plate-bound NKTT320 exhibited increased intracellular levels of granzyme B and degranulation (exposure of CD107 on the cell surface). Furthermore, both soluble and immobilized NKTT320 induced iNKT cell-mediated activation of bystander immune cells, suggesting that this novel anti-iTCR antibody facilitates both direct and indirect iNKT cell cytotoxicity. These studies are significant, as they provide a framework by which iNKT cell anti-cancer functions could be enhanced for therapeutic purposes.

Submitted by Cristina Santos (cmaria@ua.pt) on 2019-01-24T11:53:22Z No. of bitstreams: 1 Documento.pdf: 2845453 bytes, checksum: aecc9acd3f838c221f2d515e5e4644cb (MD5) Made available in DSpace on 2019-01-24T11:53:22Z (GMT). No. of bitstreams: 1 Documento.pdf: 2845453 bytes, checksum: aecc9acd3f838c221f2d515e5e4644cb (MD5) Previous issue date: 2018-12-21 Mestrado em Biomedicina Molecular

Invariant Natural Killer (iNKT) cells are T lymphocytes that recognize lipid antigens presented by CD1d molecules and have a semi-invariant T cell receptor. iNKT cells produce high quantities of cytokines after antigen recognition or activation. The regulation of iNKT cell activation is crucial for its role in infection and tumor control. CD5 and CD6 are transmembrane surface glycoproteins expressed by conventional T cells that regulate the activation of these cells, but their expression on human iNKTs has not been investigated yet. Here, we studied the basal expression of both CD5 and CD6 on human iNKTs and also their modulation after activation. The involvement of CD6 in antigen induced iNKT cell activation was also addressed. Our results show that human peripheral blood iNKT cells express both CD5 and CD6, at similar and higher levels, respectively, than conventional T cells. Activation of a human iNKT cell line with the non-specific stimulus Phytohaemagglutinin (PHA) down-regulated the expression of both CD5 and CD6, whereas when the prototypic antigen α-Galactosylceramide (α-GalCer) was used only a decrease in CD6 expression was observed. The use of Raji B cells, expressing or not CD166, a ligand for CD6, as antigen presenting cells did not disclose a major role for CD6 in α-GalCer induced iNKT cell activation As células Invariant Natural killer T (iNKT) são um subtipo de linfócitos T que reconhecem antigénios lipídicos através da molecula de CD1d e têm um recetor de células T semi-invariante. Estas células produzem grandes quantidades de citocinas após serem ativadas. A regulação das iNKTs é crucial para controlar o seu efeito na infeção e no cancro. CD5 e CD6 são glicoproteínas transmembranares expressas na superfície das células T e que regulam a sua activação, sendo que a sua expressão em células iNKTs humanas ainda não tinha sido estudada. Neste trabalho estudou-se a expressão basal destas moléculas em iNKTs humanas e também a sua modelação após activação. O envolvimento do CD6 na indução de ativação das iNKTs pelo antigénio α-Galactosylceramide (α-GalCer) também foi estudada. Os resultados mostraram que as iNKTs presentes no sangue humano periférico expressam CD5 e CD6, sendo que em comparação com as células T convencionais, o CD5 é expresso a níveis semelhantes e o CD6 é expresso em níveis superiores. A activação de iNKTs humanas pela Phytohaemagglutinin (PHA) (estímulo não específico) diminuiu a expressão das moléculas CD5 e CD6 enquanto que com o antigénio prototípico α-GalCer só se verificou uma descida na expressão de CD6. O uso de células Raji expressando ou não o CD166, um ligando do CD6, como células apresentadoras de antigénio, não revelou ter um papel importante na indução de activação da iNKTs pelo antigénio α-GalCer Mestrado em Biomedicina Molecular

CD1d-restricted invariant natural killer T (iNKT) cells are innate-like T cells that express an invariant T cell receptor (TCR) α-chain and recognize self and foreign glycolipid antigens. They can rapidly respond to agonist activation and stimulate an extensive array of immune responses. Thymic development and function of iNKT cells are regulated by many different cellular processes, including autophagy, a self-degradation mechanism. In this mini review, we discuss the current understanding of how autophagy regulates iNKT cell development and effector lineage differentiation. Importantly, we propose that iNKT cell development is tightly controlled by metabolic reprogramming.

Graft-versus-host disease (GVHD) is a major cause of significant morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are potent regulators of immune responses, protect from lethal GVHD, and promote graft-versus-leukemia effects in murine studies. Since iNKT cells constitute less than 0.5% of human peripheral blood mononuclear cells (PBMCs), in vitro expansion with their glycolipid ligands is required before they can be used for cytotherapy and experimental purposes. Three weeks of cell culture and autologous restimulation with either KRN7000, PBS44, or PBS57 resulted in a robust proliferation of iNKT cells from human PBMCs. Next, iNKT cells were sorted to a purity higher than 90% being crucial for further experimental and clinical applications. These iNKT cells significantly decreased activation and proliferation of allogeneic CD3+ T lymphocytes. In addition, leukemia cell lines and primary leukemia cells were efficiently lysed by culture-expanded iNKT cells. Importantly, culture-expanded donor iNKT cells promoted robust antileukemia activity against HLA-matched allogeneic patient leukemia cells. Our data indicate that the adoptive transfer of culture-expanded iNKT cells could be a powerful cytotherapeutic approach to induce immune tolerance and prevent leukemia relapse after allogeneic HCT in humans.

We have exploited the properties of filamentous bacteriophage fd to deliver immunologically active lipids together with antigenic peptides. Filamentous bacteriophages resemble for size, capability to be permeable to blood vessels and high density antigen expression, a nature-made nanoparticle. In addition, their major coat protein pVIII, which is arranged to form a tubular shield surrounding the phage genome, has a high content of hydrophobic residues promoting lipid association. We conjugated bacteriophages to alpha-GalactosylCeramide (alpha-GalCer), a lipid antigen stimulating invariant Natural Killer T (iNKT) cells and capable of inducing their anti-tumoral activities. We found that bacteriophage fd/alpha-GalCer conjugates could repeatedly stimulate iNKT cells in vitro and in vivo, without inducing iNKT anergy. Moreover, co-delivery of alpha-GalactosylCeramide and a MHC class I restricted tumor associated antigenic determinant to APCs via bacteriophages strongly boosted adaptive CD8+ T cell response and efficiently delayed tumor progression. Co-delivery of a tumor antigen and iNKT-stimulatory lipid on the surface of filamentous bacteriophages is a novel approach to potentiate adaptive anti-cancer immune responses, overcoming the current limitations in the use of free alpha-GalactosylCeramide and may represent an attractive alternative to existing delivery methods, opening the path to a potential translational usage of this safe inexpensive and versatile tool.

Increasing drug resistance and the lack of an effective vaccine are the main factors contributing to Mycobacterium tuberculosis (Mtb) being a major cause of death globally. Despite intensive research efforts, it is not well understood why some individuals control Mtb infection and some others develop active disease. HIV-1 infection is associated with an increased incidence of active tuberculosis, even in virally suppressed individuals. Mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells are innate T cells that can recognize Mtb-infected cells. Contradicting results regarding the frequency of MAIT cells in latent Mtb infection have been reported. In this confirmatory study, we investigated the frequency, phenotype, and IFNγ production of MAIT and iNKT cells in subjects with latent or active Mtb infection. We found that the frequency of both cell types was increased in subjects with latent Mtb infection compared with uninfected individuals or subjects with active infection. We found no change in the expression of HLA-DR, PD-1, and CCR6, as well as the production of IFNγ by MAIT and iNKT cells, among subjects with latent Mtb infection or uninfected controls. The proportion of CD4− CD8+ MAIT cells in individuals with latent Mtb infection was, however, increased. HIV-1 infection was associated with a loss of MAIT and iNKT cells, and the residual cells had elevated expression of the exhaustion marker PD-1. Altogether, the results suggest a role for MAIT and iNKT cells in immunity against Mtb and show a deleterious impact of HIV-1 infection on those cells.

Adipose tissue is a metabolic organ that plays a central role in controlling systemic energy homeostasis. Compelling evidence indicates that immune system is closely linked to healthy physiologic functions and pathologic dysfunction of adipose tissue. In obesity, the accumulation of pro-inflammatory responses in adipose tissue subsequently leads to dysfunction of adipose tissue as well as whole body energy homeostasis. Simultaneously, adipose tissue also activates anti-inflammatory responses in an effort to reduce the unfavorable effects of pro-inflammation. Notably, the interplay between adipocytes and resident invariant natural killer T (iNKT) cells is a major component of defensive mechanisms of adipose tissue. iNKT cells are leukocytes that recognize lipids loaded on CD1d as antigens, whereas most other immune cells are activated by peptide antigens. In adipose tissue, adipocytes directly interact with iNKT cells by presenting lipid antigens and stimulate iNKT cell activation to alleviate pro-inflammation. In this review, we provide an overview of the molecular and cellular determinants of obesity-induced adipose tissue inflammation. Specifically, we focus on the roles of iNKT cell-adipocyte interaction in maintaining adipose tissue homeostasis as well as the consequent modulation in systemic energy metabolism. We also briefly discuss future research directions regarding the interplay between adipocytes and adipose iNKT cells in adipose tissue inflammation.

Invariant natural killer T (iNKT) cells are a CD1d-restricted T cell population that can respond to lipid antigenic stimulation within minutes by secreting a wide variety of cytokines. This broad functional scope has placed iNKT cells at the frontlines of many kinds of immune responses. Although the diverse functional capacities of iNKT cells have long been acknowledged, only recently have distinct iNKT cell subsets, each with a marked functional predisposition, been appreciated. Furthermore, the subsets can frequently occupy distinct niches in different tissues and sometimes establish long-term tissue residency where they can impact homeostasis and respond quickly when they sense perturbations. In this review, we discuss the developmental origins of the iNKT cell subsets, their localization patterns, and detail what is known about how different subsets specifically influence their surroundings in conditions of steady and diseased states.

Background CD1d restricted invariant natural killer T (iNKT) cells are important in the activation and regulation of immune responses. Limited information is available regarding the functional role of iNKT cells in the human immunodeficiency virus (HIV) disease progression. Methodology α-GalCer stimulated iNKT cells were characterized for their functionality in terms of cytokine production (IFN-γ, TNF-α, IL-2, IL-4, and IL-21) and CD107a expression in HIV-1 infected [23 long-term non progressors (LTNPs), 28 progressors, 18 patients before and after suppressive anti-retroviral treatment (ART)] along with 25 HIV-1 negative subjects using multicolor flow cytometry. Results The functional profile of α-GalCer stimulated iNKT cells was similar in LTNPs and healthy controls. The number of LTNPs showing functional response in terms of secretion of cytokines (IFN-γ/IL2/TNF-α) and CD107a expression was significantly higher than seen in the progressors. The cytokine secretion by the stimulated iNKT cells was predominantly Th1 type. The frequencies of iNKT cells showing secretion of IFN-γ or IL2 or TNF-α or expression of CD107a were higher in LTNPs (p

Sepsis is the third leading cause of death in the neonatal population, due to susceptibility to infection conferred by immaturity of both the innate and adaptive components of the immune system. Invariant natural killer T (iNKT) cells are specialized adaptive immune cells that possess important innate-like characteristics and have not yet been well-studied in septic neonates. We hypothesized that iNKT cells would play an important role in mediating the neonatal immune response to sepsis. To study this, we subjected 5- to 7-day-old neonatal C57BL/6 mice to sepsis by intraperitoneal (i.p.) cecal slurry (CS) injection. Thirty hours prior to or immediately following sepsis induction, pups received i.p. injection of the iNKT stimulator KRN7000 (KRN, 0.2 µg/g) or vehicle. Ten hours after CS injection, blood and tissues were collected for various analyses. Thirty-hour pretreatment with KRN resulted in better outcomes in inflammation, lung injury, and survival, while immediate treatment with KRN resulted in worse outcomes compared to vehicle treatment. We further analyzed the activation status of neonatal iNKT cells for 30 h after KRN administration, and showed a peak in frequency of CD69 expression on iNKT cells and serum IFN-γ levels at 5 and 10 h, respectively. We then used CD1d knockout neonatal mice to demonstrate that KRN acts through the major histocompatibility complex-like molecule CD1d to improve outcomes in neonatal sepsis. Finally, we identified that KRN pretreatment exerts its protective effect by increasing systemic levels of TGF-β1. These findings support the importance of iNKT cells for prophylactic immunomodulation in neonates susceptible to sepsis.

Invariant natural killer T (iNKT) cells are a unique T cell subset that exhibits characteristics of both innate immune cells and T cells. They express Vα14-Jα18 (Trav11-Traj18) as an invariant chain of the T cell receptor (TCR) and are restricted to the MHC class I-like monomorphic antigen presenting molecule CD1d. iNKT cells are known as immune regulators that bridge the innate and acquired immune systems by rapid and massive production of a wide range of cytokines, which could enable them to participate in immune responses during various disease states. Thus, Traj18-deficient mice, Cd1d-deficient mice, or iNKT cell-overexpressing mice such as iNKT TCRα transgenic mice and iNKT cell cloned mice which contain a Vα14-Jα18 rearrangement in the TCRα locus are useful experimental models for the analysis of iNKT cells in vivo and in vitro. In this review, we describe the pros and cons of the various available genetically manipulated mice and summarize the insights gained from their study, including the possible roles of iNKT cells in obesity and diabetes.

Increasing drug resistance and the lack of an effective vaccine are the main factors contributing to Mycobacterium tuberculosis (Mtb) being a major cause of death globally. Despite intensive research efforts, it is not well understood why some individuals control Mtb infection and some others develop active disease. HIV-1 infection is associated with an increased incidence of active tuberculosis, even in virally suppressed individuals. Mucosal-associated invariant T (MAIT) and invariant natural killer T (iNKT) cells are innate T cells that can recognize Mtb-infected cells. Contradicting results regarding the frequency of MAIT cells in latent Mtb infection have been reported. In this confirmatory study, we investigated the frequency, phenotype, and IFNγ production of MAIT and iNKT cells in subjects with latent or active Mtb infection. We found that the frequency of both cell types was increased in subjects with latent Mtb infection compared with uninfected individuals or subjects with active infection. We found no change in the expression of HLA-DR, PD-1, and CCR6, as well as the production of IFNγ by MAIT and iNKT cells, among subjects with latent Mtb infection or uninfected controls. The proportion of CD4− CD8+ MAIT cells in individuals with latent Mtb infection was, however, increased. HIV-1 infection was associated with a loss of MAIT and iNKT cells, and the residual cells had elevated expression of the exhaustion marker PD-1. Altogether, the results suggest a role for MAIT and iNKT cells in immunity against Mtb and show a deleterious impact of HIV-1 infection on those cells.

Tolerance against self-antigens is regulated by a variety of cell types with immunoregulatory properties, such as CD1d-restricted invariant natural killer T (iNKT) cells. In many experimental models of autoimmunity, iNKT cells promote self-tolerance and protect against autoimmunity. These findings are supported by studies with patients suffering from autoimmune diseases. Based on these studies, the therapeutic potential of iNKT cells in autoimmunity has been explored. Many of these studies have been performed with the potent iNKT cell agonist KRN7000 or its structural variants. These findings have generated promising results in several autoimmune diseases, although mechanisms by which iNKT cells modulate autoimmunity remain incompletely understood. Here, we will review these preclinical studies and discuss the prospects for translating their findings to patients suffering from autoimmune diseases.

Invariant natural killer T (iNKT) cells are a distinct subset of innate-like lymphocytes bearing an invariant T-cell receptor, through which they recognize lipid antigens presented by monomorphic CD1d molecules. Upon activation, iNKT cells are capable of not only having a direct effector function but also transactivating NK cells, maturing dendritic cells, and activating B cells, through secretion of several cytokines and cognate TCR-CD1d interaction. Endowed with the ability to orchestrate an all-encompassing immune response, iNKT cells are critical in shaping immune responses against pathogens and cancer cells. In this review, we examine the critical role of iNKT cells in antitumor responses from two perspectives: (i) how iNKT cells potentiate antitumor immunity and (ii) how CD1d+ tumor cells may modulate their own expression of CD1d molecules. We further explore hypotheses to explain iNKT cell activation in the context of cancer and how the antitumor effects of iNKT cells can be exploited in different forms of cancer immunotherapy, including their role in the development of cancer vaccines.

Gout is an inflammatory arthritis caused by deposition of intra-articular monosodium urate (MSU) crystal. Previous studies have focused on resident macrophage, infiltrating monocyte, and neutrophil responses to MSU crystal; yet the mechanisms of cellular changes and the potential involvement of other regulatory immune cells remain largely unknown. Invariant natural killer T (iNKT) cells, an innate type of T cell, are involved in the development of various inflammatory diseases. Here, we investigate the role of iNKT cells in MSU crystal-induced gouty inflammation. MSU crystal-induced inflammatory profiles in an air-pouch model were examined in iNKT-deficient CD1d knockout (KO) and wild-type (WT) control mice. To explore potential mechanisms of iNKT cell regulation of gouty inflammation, we cocultured CD4+ or CD4−iNKT cells with bone marrow-derived macrophages (BMDMs). We found that iNKT cells quickly migrated to the site of inflammation upon MSU crystal stimulation in WT mice. The total number of infiltrating cells in CD1d KO mice, especially neutrophils, was dramatically increased at 6 and 12 h (P

We have shown that invariant natural killer T (iNKT) cells mediate sepsis-induced end-organ changes and immune responses, including macrophage bacterial phagocytosis, a finding regulated by the check point protein program cell death receptor-1 (PD-1). Furthermore, PD-1 mediates mortality in both adult and neonatal murine sepsis as well as in surgical patients. Given our previous findings, we hypothesize that iNKT cells will also modulate neonatal sepsis survival, and that this effect is regulated in part through PD-1. We utilized a polymicrobial intra-peritoneal cecal slurry (CS) sepsis model in wild type (WT), iNKT−/− or PD-1−/− 5–7 day old neonatal pups. Typically, tissues were harvested at 24 h for various bioassays/histology and, in some cases, survival was assessed for up to 7 days. Interestingly, similar to what we recently reported for PD-1−/− mice following CS, iNKT−/−-deficient animals exhibit a markedly improved survival vs. WT. Histologically, minor alterations in liver architectural, which were noted in WT pups, were attenuated in both iNKT−/− and PD-1−/− pups. Following CS, PECAM-1 expression was unchanged in the WT pups but increased in both iNKT−/− and PD-1−/− pups. In WT, following CS the emergence of a Ly6Clow subpopulation was noted among the influxed peritoneal macrophage population. Conversely, within iNKT−/− pups, there were fewer peritoneal macrophages and a greater percentage of Ly6Chigh macrophages. We show not only a key role for iNKT cells in affecting end-organ damage as well as alterations in phagocytes phenotypes in neonatal sepsis but that this iNKT cell mediated effect is driven by the central checkpoint protein PD-1.

International audience; The role of sphingolipids (SLs) in the immune system has come under increasing scrutiny recently due to the emerging contributions that these important membrane components play in regulating a variety of immunological processes. The acyl chain length of SLs appears particularly critical in determining SL function. Here, we show a role for very-long acyl chain SLs (VLC-SLs) in invariant natural killer T (iNKT) cell maturation in the thymus and homeostasis in the liver. Ceramide synthase 2-null mice, which lack VLC-SLs, were susceptible to a hepatotropic strain of lymphocytic choriomeningitis virus, which is due to a reduction in the number of iNKT cells. Bone marrow chimera experiments indicated that hematopoietic-derived VLC-SLs are essential for maturation of iNKT cells in the thymus, whereas parenchymal-derived VLC-SLs are crucial for iNKT cell survival and maintenance in the liver. Our findings suggest a critical role for VLC-SL in iNKT cell physiology.

The ubiquitously expressed tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2 (SHP-2, encoded by Ptpn11) is required for constitutive cellular processes including proliferation, differentiation, and the regulation of immune responses. During development and maturation, subsets of T cells express a variety of inhibitory receptors known to associate with phosphatases, which in turn, dephosphorylate key players of activating receptor signaling pathways. We hypothesized that SHP-2 deletion would have major effects on T cell development by altering the thresholds for activation, as well as positive and negative selection. Surprisingly, using mice conditionally deficient for SHP-2 in the T cell lineage, we show that the development of these lymphocytes is globally intact. In addition, our data demonstrate that SHP-2 absence does not compromise T cell effector functions, suggesting that SHP-2 is dispensable in these cells. Unexpectedly, in aging mice, Ptpn11 gene deletion driven by CD4 Cre recombinase leads to cartilage tumors in wrist bones in a T cell-independent manner. These tumors resemble miniature cartilaginous growth plates and contain CD4-lineage positive chondrocyte-like cells. Altogether these results indicate that SHP-2 is a cartilage tumor suppressor during aging.

c-Maf belongs to the large Maf family of transcription factors and plays a key role in the regulation of cytokine production and differentiation of TH2, TH17, TFH, and Tr1 cells. Invariant natural killer T (iNKT) cells can rapidly produce large quantity of TH-related cytokines such as IFN-γ, IL-4, and IL-17A upon stimulation by glycolipid antigens, such as α-galactosylceramide (α-GalCer). However, the role of c-Maf in iNKT cells and iNKT cells-mediated diseases remains poorly understood. In this study, we demonstrate that α-GalCer-stimulated iNKT cells express c-Maf transcript and protein. By using c-Maf-deficient fetal liver cell-reconstituted mice, we further show that c-Maf-deficient iNKT cells produce less IL-17A than their wild-type counterparts after α-GalCer stimulation. While c-Maf deficiency does not affect the development and activation of iNKT cells, c-Maf is essential for the induction of IL-17-producing iNKT (iNKT17) cells by IL-6, TGF-β, and IL-1β, and the optimal expression of RORγt. Accordingly, c-Maf-deficient iNKT17 cells lose the ability to recruit neutrophils into the lungs. Taken together, c-Maf is a positive regulator for the expression of IL-17A and RORγt in iNKT17 cells. It is a potential therapeutic target in iNKT17 cell-mediated inflammatory disease.

We and others have found that the functions of hepatic natural killer (NK) cells are inhibited but invariant NKT (iNKT) cells become activated after alcohol drinking, leaving a possibility that there exists interplay between NK cells and iNKT cells during alcoholic liver disease. Here, in a chronic plus single-binge ethanol consumption mouse model, we observed that NK cells and interferon-γ (IFN-γ) protected against ethanol-induced liver steatosis, as both wild-type (WT) mice treated with anti-asialo GM1 antibody and IFN-γ-deficient GKO mice developed more severe alcoholic fatty livers. As expected, IFN-γ could directly downregulate lipogenesis in primary hepatocytes in vitro. On the contrary, iNKT cell-deficient Jα18−/− or interleukin-10 (IL-10)−/− mice showed fewer alcoholic steatosis, along with the recovered number and IFN-γ release of hepatic NK cells, and exogenous IL-10 injection was sufficient to compensate for iNKT cell deficiency. Furthermore, NK cell depletion in Jα18−/− or IL-10−/− mice caused more severe hepatosteatosis, implying NK cells are the direct effector cells to inhibit liver steatosis. Importantly, adoptive transfer of iNKT cells purified from normal but not IL-10−/− mice resulted in suppression of the number and functions of NK cells and aggravated alcoholic liver injury in Jα18−/− mice, indicating that IL-10-producing iNKT (NKT10) cells are the regulators on NK cells. Conclusion: Ethanol exposure-triggered NKT10 cells antagonize the protective roles of NK cells in alcoholic hepatosteatosis.

Invariant natural killer T (iNKT) cells serve as a bridge between innate and adaptive immunity and have been shown to play an important role in immune regulation, defense against pathogens, and cancer immunity. Recent data also suggest that this compartment of the immune system plays a significant role in reducing graft-versus-host disease (GVHD) in the setting of allogeneic hematopoietic stem cell transplantation. Murine studies have shown that boosting iNKT numbers through certain conditioning regimens or adoptive transfer leads to suppression of acute or chronic GVHD. Preclinical work reveals that iNKT cells exert their suppressive function by expanding regulatory T cells in vivo, though the exact mechanism by which this occurs has yet to be fully elucidated. Human studies have demonstrated that a higher number of iNKT cells in the graft or in the peripheral blood of the recipient post-transplantation are associated with a reduction in GVHD risk, importantly without a loss of graft-versus-tumor effect. In two separate analyses of many immune cell subsets in allogeneic grafts, iNKT cell dose was the only parameter associated with a significant improvement in GVHD or in GVHD-free progression-free survival. Failure to reconstitute iNKT cells following allogeneic transplantation has also been associated with an increased risk of relapse. These data demonstrate that iNKT cells hold promise for future clinical application in the prevention of GVHD in allogeneic stem cell transplantation and warrant further study of the immunoregulatory functions of iNKT cells in this setting.

The CD1d-restricted invariant natural killer T (iNKT) cells are implicated in innate immune responses against human immunodeficiency virus (HIV). However, the determinants of cellular dysfunction across the iNKT cells subsets are seldom defined in HIV disease. Herein, we provide evidence for the involvement of the negative checkpoint regulator (NCR) 2B4 in iNKT cell alteration in a well-defined cohort of HIV-seropositive anti-retroviral therapy (ART) naive, ART-treated, and elite controllers (ECs). We report on exaggerated 2B4 expression on iNKT cells of HIV-infected treatment-naive individuals. In sharp contrast to CD4-iNKT cells, 2B4 expression was significantly higher on CD4+ iNKT cell subset. Notably, an increased level of 2B4 on iNKT cells was strongly correlated with parameters associated with HIV disease progression. Further, iNKT cells from ARTnaive individuals were defective in their ability to produce intracellular IFN-gamma Together, our results suggest that the levels of 2B4 expression and the downstream co-inhibitory signaling events may contribute to impaired iNKT cell responses. Funding Agencies|University of Malaya Research [RG50113HTM]; Health and Translational Medicine Cluster; European Molecular Biology Organization (EMBO) Short-Term Fellowship; Deutsches Zentrum fur Infektions forschung [04.810, 04.811]; High Impact Research Grant of the Ministry of Higher Education (MoHE) Malaysia [HIRGA E000001-20001]

Toxic shock syndrome (TSS) is an acute, potentially fatal condition characterized by high-grade fever, hypotensive shock and systemic inflammation. It is caused by exposure to staphylococcal and streptococcal superantigens (SAgs), which can activate up to 50% of T cells resulting in a hyperinflammatory ‘cytokine storm’ within hours. This inflammatory cascade progresses to a life-threatening illness with alarming rapidity, and SAg-exposed individuals can develop multi-organ failure within hours of onset of symptoms. However, there are currently no available treatments that efficiently mitigate the cytokine storm, which drives TSS immunopathology. Therefore, identifying and understanding the critical components underlying this process should hold the key to designing effective therapeutics to reduce TSS severity. In this thesis, I have utilized a clinically relevant humanized HLA-DR4 transgenic (DR4tg) mouse model of TSS to reveal the previously unrecognized roles of three rapid host responses in the initiation or control of the cytokine storm. First, genetic and antibody-mediated depletion of invariant natural killer T (iNKT) cells in DR4tg mice show that iNKT cells are pathogenic in TSS and contribute to the cytokine storm. Targeting iNKT cell responses with the T helper type-2 (Th2)-polarizing glycolipid agonist OCH also reduces TSS morbidity and mortality. Second, I found that granulocytic myeloid-derived suppressor cells (MDSCs) are rapidly recruited to the liver of DR4tg mice during TSS. These hepatic MDSCs potently suppress SAg-induced T cell responses and may therefore mitigate tissue injury in TSS. Lastly, I define the rapid production of interleukin-17A (IL-17A) by effector memory T cells as a novel mechanism promoting immunopathology in TSS. Blockade of IL-17A signaling in human blood mononuclear cells reduces the expression of multiple inflammatory mediators of TSS, suggesting that IL-17A contributes to the cytokine storm. Importantly, the treatment of DR4tg mice with an IL-17A-neutralizing antibody attenuates TSS-induced tissue damage, morbidity and mortality. Collectively, the results presented in this thesis delineate the novel contributions of iNKT cells, MDSCs and IL-17A to the early phase of TSS pathogenesis. Furthermore, my findings suggest that therapeutic approaches targeting iNKT cells or IL-17A responses may be effective in reducing TSS mortality.

Multiple sclerosis (MS) is a chronic progressive inflammatory demyelinating disorder of the central nervous system, and in several countries is a leading cause of permanent neurological disability in young adults, particularly women. MS is considered an autoimmune disease, caused by an aberrant immune response to environmental triggers in genetically susceptible subjects. However, the contribution of the innate or of the adaptive immune system to the development and progression of the disease has not yet been fully elucidated. Innate-like T lymphocytes are unconventional T cells that bridge the innate and adaptive arms of the immune system, because they use a T cell receptor to sense external ligands, but behave like innate cells when they rapidly respond to stimuli. These cells could play an important role in the pathogenesis of MS. Here, we focus on invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells, and we review the current knowledge on their biology and possible involvement in MS. Although several studies have evaluated the frequency and functions of iNKT and MAIT cells both in MS patients and in experimental mouse models, contradictory observations have been reported, and it is not clear whether they exert a protective or a pro-inflammatory and harmful role. A better understanding of how immune cells are involved in MS, and of their interactions could be of great interest for the development of new therapeutic strategies.

Objective: In this study, we aimed to investigate the frequency and activation of invariant natural killer T (iNKT) cells and natural killer (NK) cells among HIV-1, HIV-2, or dually HIV-1/HIV-2 (HIV-D)-infected individuals, in relation to markers of disease progression. Design: Whole blood samples were collected from treatment-naive HIV-1 (n = 23), HIV-2 (n = 34), and HIV-D (n = 11) infected individuals, as well as HIV-seronegative controls (n = 25), belonging to an occupational cohort in Guinea-Bissau. Methods: Frequencies and activation levels of iNKT and NK cell subsets were analysed using multicolour flow cytometry, and results were related to HIV-status, CD4+ T-cell levels, viral load, and T-cell activation. Results: HIV-1, HIV-D, and viremic HIV-2 individuals had lower numbers of CD4+ iNKT cells in circulation compared with seronegative controls. Numbers of CD56bright NK cells were also reduced in HIV-infected individuals as compared with control study participants. Notably, iNKT cell and NK cell activation levels, assessed by CD38 expression, were increased in HIV-1 and HIV-2 single, as well as dual, infections. HIV-2 viremia was associated with elevated activation levels in CD4+ iNKT cells, CD56bright, and CD56dim NK cells, as compared with aviremic HIV-2 infection. Additionally, disease markers such as CD4+ T-cell percentages, viral load, and CD4+ T-cell activation were associated with CD38 expression levels of both iNKT and NK cells, which activation levels also correlated with each other. Conclusion: Our data indicate that elevated levels of iNKT-cell and NK-cell activation are associated with viremia and disease progression markers in both HIV-1 and HIV-2 infections.

XDurant l’infection par le virus Influenza A (IAV), les changements physiques et immunologiques du poumon prédisposent l’hôte aux surinfections bactériennes. Les cellules T Natural Killer invariantes (iNKT) sont des lymphocytes T innés pouvant avoir des rôles bénéfiques ou délétères durant l’infection. Nos objectifs ont visé à (i) étudier le rôle naturel des cellules iNKT et (ii) à rechercher l’effet d’une activation exogène des cellules iNKT dans la surinfection bactérienne post-influenza.Lors de mon arrivée, le laboratoire venait de décrire, pour la première fois en contexte infectieux, que les cellules iNKT étaient capables de produire de l’IL-22 au cours de l’infection grippale. Cette cytokine joue un rôle majeur dans les processus de maintien et de réparation des épithéliums. L’une desDuring the infection by the virus Influenza A ( IAV), the physical and immunological changes of the lung predispose the host to the bacterial secondary infections. The invariant cells(units) T Natural Killer iNKT ) are lymphocytes T innate being able to have beneficial or noxious roles during the infection. Our objectives aimed at i) to study the natural role of cells(units) iNKT and ii) to look for the effect of an exogenous activation of cells(units) iNKT in the bacterial secondary infection post-influenza. During my arrival, the laboratory had just described, for the first time in infectious context, that cells(units) iNKT were capable of producing of IL-22 during the flu-like infection. This cytokine plays a major role in the processes of preservation and repair of epitheliums [...], Durant l’infection par le virus Influenza A (IAV), les changements physiques et immunologiques du poumon prédisposent l’hôte aux surinfections bactériennes. Les cellules T Natural Killer invariantes (iNKT) sont des lymphocytes T innés pouvant avoir des rôles bénéfiques ou délétères durant l’infection. Nos objectifs ont visé à (i) étudier le rôle naturel des cellules iNKT et (ii) à rechercher l’effet d’une activation exogène des cellules iNKT dans la surinfection bactérienne post-influenza.Lors de mon arrivée, le laboratoire venait de décrire, pour la première fois en contexte infectieux, que les cellules iNKT étaient capables de produire de l’IL-22 au cours de l’infection grippale. Cette cytokine joue un rôle majeur dans les processus de maintien et de réparation des épithéliums. L’une des causes des surinfections bactériennes post-grippales étant l’altération et/ou la perte de l’intégrité de l’épithélium pulmonaire, nous nous sommes proposés d’étudier le rôle potentiel de cette cytokine dans un modèle expérimental de surinfection bactérienne à S. pneumoniae. Nous avons ainsi pu montrer que si cette cytokine ne joue pas un rôle majeur dans la réponse anti-virale de l’hôte, l’IL-22 participe au contrôle de l’inflammation au cours de l’infection grippale et joue un rôle protecteur dans la surinfection bactérienne.Par ailleurs, l’utilisation de souris dépourvues en cellules iNKT (Jα18-/-) a permis de montrer que les cellules iNKT limitent la susceptibilité aux surinfections et réduisent le synergisme létal de la coinfection virus/bactérie. Au moment de l’infection bactérienne, les cellules iNKT des souris grippées sont incapables de produire de l’IFN-γ, cytokine dont nous avons montré le rôle essentiel dans les mécanismes de défense antibactérienne. Le défaut d’activation des cellules iNKT chez les souris surinfectées est lié à l’interleukine-10 (IL-10), cytokine immunosuppressive induite par l’infection virale, plutôt qu’à un défaut intrinsèque des cellules iNKT. L’IL-10 inhibe l’activation des cellules iNKT en réponse au pneumocoque en inhibant la production d’IL-12 par les cellules dendritiques dérivées de monocytes (MoDCs). La neutralisation de l’IL-10 restaure l’activation des cellules iNKT et augmente la résistance à la surinfection. Ainsi, les cellules iNKT ont un rôle bénéfique (en amont de la colonisation bactérienne) dans le contrôle de la surinfection bactérienne de la grippe et représentent une cible de l’immunosuppression.Nous avons par la suite étudié la possibilité que le superagoniste des cellules iNKT, l’ α-galactosylceramide (α-GalCer) puisse limiter la surinfection bactérienne. Pour cela, les souris ont été traitées par voie intranasale avec de l’α-GalCer à différents temps post-influenza, juste avant l’infection par le pneumocoque. Le traitement à jour 3, au pic de la réplication virale, limite fortement la surinfection. Cependant, l’inoculation d’α-GalCer pendant la phase aiguë du virus (jour 7) ne permet pas d’activer les cellules iNKT pulmonaires et n’a pas d’effet sur la surinfection. L’absence d’activation des cellules iNKT n’est pas intrinsèque et est associée à une disparition complète des cellules dendritiques CD103+ respiratoires (cDCs), lesquelles sont cruciales dans l’activation des cellules iNKTs. À des temps plus tardifs (jour 14), les cDCs repeuplent le poumon et l’α-GalCer promeut l’activité antibactérienne des cellules iNKT.Pris dans son ensemble, cette étude souligne le rôle des cellules iNKT dans la surinfection bactérienne de la grippe et ouvre de nouvelles voies thérapeutiques afin de limiter les surinfections bactériennes post-influenza.

XDurant l’infection par le virus Influenza A (IAV), les changements physiques et immunologiques du poumon prédisposent l’hôte aux surinfections bactériennes. Les cellules T Natural Killer invariantes (iNKT) sont des lymphocytes T innés pouvant avoir des rôles bénéfiques ou délétères durant l’infection. Nos objectifs ont visé à (i) étudier le rôle naturel des cellules iNKT et (ii) à rechercher l’effet d’une activation exogène des cellules iNKT dans la surinfection bactérienne post-influenza.Lors de mon arrivée, le laboratoire venait de décrire, pour la première fois en contexte infectieux, que les cellules iNKT étaient capables de produire de l’IL-22 au cours de l’infection grippale. Cette cytokine joue un rôle majeur dans les processus de maintien et de réparation des épithéliums. L’une desDuring the infection by the virus Influenza A ( IAV), the physical and immunological changes of the lung predispose the host to the bacterial secondary infections. The invariant cells(units) T Natural Killer iNKT ) are lymphocytes T innate being able to have beneficial or noxious roles during the infection. Our objectives aimed at i) to study the natural role of cells(units) iNKT and ii) to look for the effect of an exogenous activation of cells(units) iNKT in the bacterial secondary infection post-influenza. During my arrival, the laboratory had just described, for the first time in infectious context, that cells(units) iNKT were capable of producing of IL-22 during the flu-like infection. This cytokine plays a major role in the processes of preservation and repair of epitheliums [...], Durant l’infection par le virus Influenza A (IAV), les changements physiques et immunologiques du poumon prédisposent l’hôte aux surinfections bactériennes. Les cellules T Natural Killer invariantes (iNKT) sont des lymphocytes T innés pouvant avoir des rôles bénéfiques ou délétères durant l’infection. Nos objectifs ont visé à (i) étudier le rôle naturel des cellules iNKT et (ii) à rechercher l’effet d’une activation exogène des cellules iNKT dans la surinfection bactérienne post-influenza.Lors de mon arrivée, le laboratoire venait de décrire, pour la première fois en contexte infectieux, que les cellules iNKT étaient capables de produire de l’IL-22 au cours de l’infection grippale. Cette cytokine joue un rôle majeur dans les processus de maintien et de réparation des épithéliums. L’une des causes des surinfections bactériennes post-grippales étant l’altération et/ou la perte de l’intégrité de l’épithélium pulmonaire, nous nous sommes proposés d’étudier le rôle potentiel de cette cytokine dans un modèle expérimental de surinfection bactérienne à S. pneumoniae. Nous avons ainsi pu montrer que si cette cytokine ne joue pas un rôle majeur dans la réponse anti-virale de l’hôte, l’IL-22 participe au contrôle de l’inflammation au cours de l’infection grippale et joue un rôle protecteur dans la surinfection bactérienne.Par ailleurs, l’utilisation de souris dépourvues en cellules iNKT (Jα18-/-) a permis de montrer que les cellules iNKT limitent la susceptibilité aux surinfections et réduisent le synergisme létal de la coinfection virus/bactérie. Au moment de l’infection bactérienne, les cellules iNKT des souris grippées sont incapables de produire de l’IFN-γ, cytokine dont nous avons montré le rôle essentiel dans les mécanismes de défense antibactérienne. Le défaut d’activation des cellules iNKT chez les souris surinfectées est lié à l’interleukine-10 (IL-10), cytokine immunosuppressive induite par l’infection virale, plutôt qu’à un défaut intrinsèque des cellules iNKT. L’IL-10 inhibe l’activation des cellules iNKT en réponse au pneumocoque en inhibant la production d’IL-12 par les cellules dendritiques dérivées de monocytes (MoDCs). La neutralisation de l’IL-10 restaure l’activation des cellules iNKT et augmente la résistance à la surinfection. Ainsi, les cellules iNKT ont un rôle bénéfique (en amont de la colonisation bactérienne) dans le contrôle de la surinfection bactérienne de la grippe et représentent une cible de l’immunosuppression.Nous avons par la suite étudié la possibilité que le superagoniste des cellules iNKT, l’ α-galactosylceramide (α-GalCer) puisse limiter la surinfection bactérienne. Pour cela, les souris ont été traitées par voie intranasale avec de l’α-GalCer à différents temps post-influenza, juste avant l’infection par le pneumocoque. Le traitement à jour 3, au pic de la réplication virale, limite fortement la surinfection. Cependant, l’inoculation d’α-GalCer pendant la phase aiguë du virus (jour 7) ne permet pas d’activer les cellules iNKT pulmonaires et n’a pas d’effet sur la surinfection. L’absence d’activation des cellules iNKT n’est pas intrinsèque et est associée à une disparition complète des cellules dendritiques CD103+ respiratoires (cDCs), lesquelles sont cruciales dans l’activation des cellules iNKTs. À des temps plus tardifs (jour 14), les cDCs repeuplent le poumon et l’α-GalCer promeut l’activité antibactérienne des cellules iNKT.Pris dans son ensemble, cette étude souligne le rôle des cellules iNKT dans la surinfection bactérienne de la grippe et ouvre de nouvelles voies thérapeutiques afin de limiter les surinfections bactériennes post-influenza.

Invariant natural killer T (NKT) cells might contribute to the amplified and prolonged T-cell immune response that characterizes sarcoidosis. Therefore, we want to investigate the frequency and distribution of pulmonary invariant NKT cells in corticosteroid-naïve patients with sarcoidosis. We used multi-parameter flow cytometry with antibodies against CD3, CD4, CD8, CD14, CD19, CD45, CD16/56, TCR Valpha24, and TCR Vbeta11, on bronchoalveolar lavage fluid (BALF), to examine the frequency and distribution of pulmonary invariant NKT cells in 47 newly diagnosed sarcoidosis patients and in 8 control subjects. The frequencies of BALF Valpha24 Vbeta11 invariant NKT cells were significantly lower in patients with sarcoidosis in comparison to control subjects. Moreover, lower invariant NKT cell frequencies in patients with sarcoidosis significantly correlated with exaggerated BALF lymphocytosis and CD4 T cell responses. This study demonstrated a pulmonary deficiency in the frequency of a subset of T cells with immunoregulatory function in patients with sarcoidosis.

The global obesity epidemic and its associated co-morbidities, including type 2 diabetes, cardiovascular disease and certain types of cancers, have drawn attention to the pivotal role of adipocytes in health and disease. Besides their 'classical' function in energy storage and release, adipocytes interact with adipose-tissue resident immune cells, among which lipid-responsive invariant Natural Killer T (iNKT) cells. iNKT cells are activated by lipid antigens presented by antigen-presenting cells (APC) as CD1d/lipid complexes. Upon activation, iNKT cells can rapidly secrete soluble mediators that either promote or oppose inflammation. In lean adipose tissue, iNKT cells elicit a predominantly anti-inflammatory immune response, while obesity is associated with declining iNKT cell numbers. Recent work showed that adipocytes act as non-professional APC for lipid antigens. Here, we discuss endogenous lipid antigen processing and presentation by adipocytes, and speculate on how these lipid antigens together with 'environmental factors' such as tissue/organ environment and co-stimulatory signals are able to influence the fate of AT-resident iNKT cells, and thereby the role of these cells in obesity and its associated pathologies. This article is protected by copyright. All rights reserved.

To clarify the effect of γδ T cells and invariant Natural Killer T (iNKT) cells in pathophysiology of dyspeptic disorders, number of these two cells in patients with non-ulcer dyspepsia (NUD), peptic ulcer disease (PUD), and gastric cancer (GC) were compared.Patients with dyspepsia were divided into three groups of NUD, PUD, and GC according to their endoscopic and histopathological examinations. Helicobacter pylori infection was diagnosed by rapid urease test and histopathology. The number of peripheral blood CD3+TCRgd+ T cells and CD3+Va24Ja18+ iNKT cells were determined by flow cytometry. Immunohistochemistry (IHC) was also used for identifying the TCRgd+ cells.Forty two patients with NUD (31.6%), 44 with PUD (33.1%), and 47 with GC (35.3%) were included in the study. The frequency of CD3+TCRgd+ T cells in peripheral blood of patients with GC (2.71±0.25) was significantly lower than that in NUD (3.97±0.32, p

Invariant natural killer T (iNKT) cells become activated during a wide variety of infections. This includes organisms lacking cognate CD1d-binding glycolipid antigens recognized by the semi-invariant T cell receptor of iNKT cells. Additional studies have shown that iNKT cells also become activated in vivo in response to microbial products such as bacterial lipopolysaccharide, a potent inducer of cytokine production in antigen-presenting cells (APCs). Other studies have shown that iNKT cells are highly responsive to stimulation by cytokines such as interleukin-12. These findings have led to the concept that microbial pathogens can activate iNKT cells either directly via glycolipids, or indirectly by inducing cytokine production in APCs. iNKT cells activated in this manner produce multiple cytokines that can influence the outcome of infection, usually in favor of the host, although potent iNKT cell activation may contribute to an uncontrolled cytokine storm and sepsis. One aspect of the response of iNKT cells to microbial pathogens is that it is short-lived and followed by an extended time period of unresponsiveness to reactivation. This refractory period may represent a means to avoid chronic activation and cytokine production by iNKT cells, thus protecting the host against some of the negative effects of iNKT cell activation, but potentially putting the host at risk for secondary infections. These effects of microbial pathogens and their products on iNKT cells are not only important for understanding the role of these cells in immune responses against infections but also for the development of iNKT cell-based therapies.

Type 1 diabetes (T1D) is an autoimmune disease that is characterized by the specific destruction of insulin-producing pancreatic β cells. Invariant natural killer T (iNKT) cells have been associated with development of T1D. Class I MHC-restricted T cell-associated molecule (CRTAM) is expressed on activated iNKT, CD8(+), and CD4(+) T cells, and it is associated with the pro-inflammatory profiles of these cells. Crtam gene expression in CD3(+) lymphocytes from non-obese diabetic (NOD) mice is associated with T1D onset. However, expression of CRTAM on T cells from patients with T1D has not yet been evaluated. We compared iNKT cell (CD3(+)Vα24(+)Vβ11(+)) numbers and CRTAM expression in a Mexican population with recent-onset T1D and their first-degree relatives with control families. Remarkably, we found lower iNKT cell numbers in T1D families, and we identified two iNKT cell populations in some of the families. One iNKT cell population expressed high iTCR levels (iNKT(hi)), whereas another expressed low levels (iNKT(lo)) and also expressed CRTAM. These findings support a probable genetic determinant of iNKT cell numbers and a possible role for these cells in T1D development. This study also suggests that CRTAM identifies recently activated iNKT lymphocytes.

© 2015, National Academy of Sciences. All rights reserved. Invariant natural killer T (iNKT) cells comprise a small population of αβ T lymphocytes. They bridge the innate and adaptive immune systems and mediate strong and rapid responses to many diseases, including cancer, infections, allergies, and autoimmunity. However, the study of iNKT cell biology and the therapeutic applications of these cells are greatly limited by their small numbers in vivo (∼0.01-1% in mouse and human blood). Here, we report a new method to generate large numbers of iNKT cells in mice through T-cell receptor (TCR) gene engineering of hematopoietic stem cells (HSCs). We showed that iNKT TCR-engineered HSCs could generate a clonal population of iNKT cells. These HSC-engineered iNKT cells displayed the typical iNKT cell phenotype and functionality. They followed a two-stage developmental path, first in thymus and then in the periphery, resembling that of endogenous iNKT cells. When tested in a mouse melanoma lung metastasis model, the HSC-engineered iNKT cells effectively protected mice from tumor metastasis. This method provides a powerful and high-throughput tool to investigate the in vivo development and functionality of clonal iNKT cells in mice. More importantly, this method takes advantage of the self-renewal and longevity of HSCs to generate a long-term supply of engineered iNKT cells, thus opening up a new avenue for iNKT cell-based immunotherapy.

The expression of N-glycolyl-monosialodihexosyl-ganglioside (NGcGM3) in humans is restricted to cancer cells; therefore, it is a tumor antigen. There are measurable quantities of circulating anti-NGcGM3 antibodies (aNGcGM3 Abs) in human serum. Interestingly, some people have circulating Ag-specific immunoglobulins G (IgGs) that are capable of complement mediated cytotoxicity against NGcGM3 positive cells, which is relevant for tumor surveillance. In light of the chemical nature of Ag, we postulated it as a candidate ligand for CD1d. Furthermore, we hypothesize that the immune mechanism involved in the generation of these Abs entails cross talk between B lymphocytes (Bc) and invariant natural killer T cells (iNKT). Combining cellular techniques, such as flow cytometry and biochemical assays, we demonstrated that CD1d binds to NGcGM3 and that human Bc present NGcGM3 in a CD1d context according to two alternative strategies. We also showed that paraformaldehyde treatment of cells expressing CD1d affects the presentation. Finally, by co-culturing primary human Bc with iNKT and measuring Ki-67 expression, we detected a reproducible increment in the proliferation of the iNKT population when Ag was on the medium. Our findings identify a novel, endogenous, human CD1d ligand, which is sufficiently competent to stimulate iNKT. We postulate that CD1d-restricted Bc presentation of NGcGM3 drives effective iNKT activation, an immunological mechanism that has not been previously described for humans, which may contribute to understanding aNGcGM3 occurrence. Fil: Gentilini, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Pérez, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Fernandez, Pablo Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Fainboim, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina Fil: Arana, Eloisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina

Broad clinical interest rapidly followed the recent discovery of different subpopulations of T cells that have immune regulatory properties and a number of studies have been conducted aiming to dissect the translational potential of these promising cells. In this review we will focus on forkhead box P3 (FoxP3) positive regulatory T cells, T regulatory type 1 cells and invariant natural killer T cells (iNKT). We will analyze their ability to correct immune dysregulation in animal models of immune mediated diseases and we will examine the first clinical approaches where these cells have been directly or indirectly employed. We will discuss successes, challenges and limitations that rose in the road to the clinical use of regulatory T cells.

Summary Sepsis is characterized by a severe systemic inflammatory response to infection that is associated with high morbidity and mortality despite optimal care. Invariant natural killer T (iNK T) cells are potent regulatory lymphocytes that can produce pro- and/or anti-inflammatory cytokines, thus shaping the course and nature of immune responses; however, little is known about their role in sepsis. We demonstrate here that patients with sepsis/severe sepsis have significantly elevated proportions of iNK T cells in their peripheral blood (as a percentage of their circulating T cells) compared to non-septic patients. We therefore investigated the role of iNK T cells in a mouse model of intra-abdominal sepsis (IAS). Our data show that iNK T cells are pathogenic in IAS, and that T helper type 2 (Th2) polarization of iNK T cells using the synthetic glycolipid OCH significantly reduces mortality from IAS. This reduction in mortality is associated with the systemic elevation of the anti-inflammatory cytokine interleukin (IL)-13 and reduction of several proinflammatory cytokines within the spleen, notably interleukin (IL)-17. Finally, we show that treatment of sepsis with OCH in mice is accompanied by significantly reduced apoptosis of splenic T and B lymphocytes and macrophages, but not natural killer cells. We propose that modulation of iNK T cell responses towards a Th2 phenotype may be an effective therapeutic strategy in early sepsis.

Sepsis is characterized by a severe systemic inflammatory response to infection that is associated with high morbidity and mortality despite optimal care. Invariant natural killer T (iNKT) cells are potent regulatory lymphocytes that can produce pro- and/or anti-inflammatory cytokines, thus shaping the course and nature of immune responses; however, little is known about their role in sepsis. We demonstrate here that patients with sepsis/severe sepsis have significantly elevated proportions of circulating iNKT cells in their peripheral blood, as compared to non-septic patients. We therefore investigated iNKT cells in mice with intra-abdominal sepsis (IAS). Our data show that iNKT cells are pathogenic in IAS, and that T helper (Th)2-type polarization of iNKT cells using the synthetic glycolipid OCH significantly reduced mortality from IAS. This reduction in mortality is associated with the systemic elevation of the anti-inflammatory cytokine interleukin (IL)-13, and reduction of several pro-inflammatory cytokines within the spleen, notably IL-17. Finally, we show that administration of OCH in septic mice is associated with significantly reduced apoptosis of splenic T and B lymphocytes, as well as macrophages, but not natural killer cells. We propose that modulation of iNKT cell responses towards a Th2 phenotype may be an effective therapeutic strategy in sepsis.