scholarly journals Distinct bioenergetic features of human invariant natural killer T (iNKT) cells enable retained functions in nutrient-deprived states

2021 ◽  
Author(s):  
Priya Khurana ◽  
Chakkapong Burudpakdee ◽  
Stephan A. Grupp ◽  
Ulf H. Beier ◽  
David M. Barrett ◽  
...  
Keyword(s):  
Natural Killer ◽  
Metabolic Flux ◽  
Cytokine Production ◽  
Inkt Cell ◽  
Flux Analysis ◽  
Inkt Cells ◽  
Effector Functions ◽  
Functional Features ◽  
Invariant Natural Killer ◽  
Natural Killer T

ABSTRACTInvariant natural killer T (iNKT) cells comprise a unique subset of lymphocytes that are primed for activation and possess innate NK-like functional features. Currently, iNKT cell-based immunotherapies remain in early clinical stages, and little is known about the ability of these cells to survive and retain effector functions within the solid tumor microenvironment (TME) long-term. In conventional T cells (TCONV), cellular metabolism is linked to effector functions and their ability to adapt to the nutrient-poor TME. In contrast, the bioenergetic requirements of iNKT cells – particularly those of human iNKT cells – at baseline and upon stimulation are not well understood; neither is how these requirements affect cytokine production or anti-tumor effector functions. We find that unlike TCONV, human iNKT cells are not dependent upon glucose or glutamine for cytokine production and cytotoxicity upon stimulation with anti-CD3 and anti-CD28. Additionally, transcriptional profiling revealed that stimulated human iNKT cells are less glycolytic than TCONV and display higher expression of fatty acid oxidation (FAO) and adenosine monophosphate-activated protein kinase (AMPK) pathway genes. Furthermore, stimulated iNKT cells displayed higher mitochondrial mass and membrane potential relative to TCONV. Real-time Seahorse metabolic flux analysis revealed that stimulated human iNKT cells utilize fatty acids as substrates for oxidation more than stimulated TCONV. Together, our data suggest that human iNKT cells possess different bioenergetic requirements from TCONV and display a more memory-like metabolic program relative to effector TCONV. Importantly, iNKT cell-based immunotherapeutic strategies could co-opt such unique features of iNKT cells to improve their efficacy and longevity of anti-tumor responses.

scholarly journals Distinct Bioenergetic Features of Human Invariant Natural Killer T Cells Enable Retained Functions in Nutrient-Deprived States

2021 ◽  
Vol 12 ◽  
Author(s):  
Priya Khurana ◽  
Chakkapong Burudpakdee ◽  
Stephan A. Grupp ◽  
Ulf H. Beier ◽  
David M. Barrett ◽  
...  
Keyword(s):  
T Cells ◽  
Natural Killer ◽  
Metabolic Flux ◽  
Inkt Cell ◽  
Flux Analysis ◽  
Inkt Cells ◽  
Effector Functions ◽  
Functional Features ◽  
Invariant Natural Killer ◽  
Natural Killer T

Invariant natural killer T (iNKT) cells comprise a unique subset of lymphocytes that are primed for activation and possess innate NK-like functional features. Currently, iNKT cell-based immunotherapies remain in early clinical stages, and little is known about the ability of these cells to survive and retain effector functions within the solid tumor microenvironment (TME) long-term. In conventional T cells (TCONV), cellular metabolism is linked to effector functions and their ability to adapt to the nutrient-poor TME. In contrast, the bioenergetic requirements of iNKT cells – particularly those of human iNKT cells – at baseline and upon stimulation are not well understood; neither is how these requirements affect effector functions such as production of cytokines and cytolytic proteins. We find that unlike TCONV, human iNKT cells are not dependent upon glucose or glutamine for these effector functions upon stimulation with anti-CD3 and anti-CD28. Additionally, transcriptional profiling revealed that stimulated human iNKT cells are less glycolytic than TCONV and display higher expression of fatty acid oxidation (FAO) and adenosine monophosphate-activated protein kinase (AMPK) pathway genes. Furthermore, stimulated iNKT cells displayed higher mitochondrial mass and membrane potential relative to TCONV. Real-time Seahorse metabolic flux analysis revealed that stimulated human iNKT cells utilize fatty acids as substrates for oxidation more than stimulated TCONV. Together, our data suggest that human iNKT cells possess different bioenergetic requirements from TCONV and display a more oxidative metabolic program relative to effector TCONV. Importantly, iNKT cell-based immunotherapeutic strategies could co-opt such unique features of iNKT cells to improve their efficacy and longevity of anti-tumor responses.

Abstract 541: Specific Deletion of LDL Receptor--Related Protein on Macrophages Skews Cytokine Production by Invariant Natural Killer T Cells

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Roman Covarrubias ◽  
Amy S Major
Keyword(s):  
Natural Killer ◽  
Cell Activation ◽  
Ldl Receptor ◽  
Inkt Cell ◽  
Related Protein ◽  
Inkt Cells ◽  
Glycolipid Antigen ◽  
Invariant Natural Killer ◽  
Natural Killer T

Invariant Natural Killer T (iNKT) cells are specialized lymphocytes that when activated can regulate chronic inflammatory conditions and atherosclerotic processes. The activation of iNKT cells occurs when glycolipid antigens bind the MHC class-I like molecule CD1d present on antigen presenting cells (APCs). The pathways by which glycolipid antigens target CD1d for presentation and activation of iNKT cells remain unclear, yet the expression of surface receptors associated with lipid homeostasis, such as the LDL receptor (LDLr), have been implicated in the modulation of iNKT cell activation. The LDLr has been shown to modulate this process by binding apoE-containing lipoproteins, which can carry antigenic glycolipids for iNKT cell activation. The LDL receptor-related protein (LRP), a transmembrane receptor from the LDL receptor family of proteins, shares structural homology with LDLr and can bind a number of ligands including apoE-containing lipoproteins. We hypothesized that LRP can play an active role in glycolipid antigen presentation and subsequent activation of iNKT cells. Here, we demonstrate that LRP is preferentially expressed at high levels on F4/80 + macrophages, when compared to other APCs. We also show that a specialized subset of macrophages expressing CD169, known for their ability to present glycolipid antigen to iNKT cells, have increased levels of LRP when compared to CD169 - macrophages. Using mice with a targeted deletion of LRP in macrophages, we observed decreased activation of iNKT cells in vitro (24, 48 hours) and normal IFN-gamma but blunted IL-4 response in vivo. Further flow cytometric analysis showed normal surface expression of CD1d in LRP-cKO macrophages as well as normal uptake of fluorescently labeled glycolipid in vitro . Additionally, analysis of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages of iNKT cells and no homeostatic disruption as evidenced by absence of programmed death-1 and LY-49. Collectively, these data suggest that macrophage LRP contributes to early iNKT cell activation by enhancing early IL-4 responses.

scholarly journals TNF receptor associated factor 3 plays a key role in development and function of invariant natural killer T cells

2013 ◽  
Vol 210 (6) ◽  
pp. 1079-1086 ◽  
Author(s):  
Zuoan Yi ◽  
Laura L. Stunz ◽  
Gail A. Bishop
Keyword(s):  
Natural Killer ◽  
Developmental Stages ◽  
Critical Role ◽  
Inkt Cell ◽  
Tnf Receptor ◽  
Inkt Cells ◽  
Tcr Signaling ◽  
Associated Factor ◽  
Invariant Natural Killer ◽  
Natural Killer T

TCR signaling is a prerequisite for early stage development of invariant natural killer T (iNKT) cells, whereas IL-15 signaling is required for expansion and maturation at later stages. In this study, we show that TNF receptor associated factor 3 (TRAF3) plays a critical role in the transition between these two distinct signaling pathways and developmental stages. TRAF3-deficient iNKT cells in CD4CreTRAF3flox/flox (T-TRAF3−/−) mice exhibit defective up-regulation of T-bet and CD122, two critical molecules for IL-15 signaling, and as a consequence, IL-15–mediated iNKT cell proliferation and survival are impaired. Consistently, development of iNKT cells in T-TRAF3−/− mice shows a major defect at developmental stages 2 and 3, but not stages 0 and 1. We further demonstrated that defective T-bet up-regulation occurring during the stage 1 to stage 2 transition results from reduced TCR signaling in TRAF3−/− iNKT cells. In addition, mature TRAF3−/− iNKT cells displayed defective cytokine responses upon TCR stimulation. Collectively, our results reveal that by modulating the relative strength of TCR signaling, TRAF3 is an important regulator of iNKT cell development and functions.

scholarly journals Invariant Natural Killer T-cell Dynamics in Human Immunodeficiency Virus–associated Tuberculosis

2019 ◽  
Vol 70 (9) ◽  
pp. 1865-1874 ◽  
Author(s):  
Naomi F Walker ◽  
Charles Opondo ◽  
Graeme Meintjes ◽  
Nishtha Jhilmeet ◽  
Jon S Friedland ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiretroviral Therapy ◽  
Natural Killer ◽  
Inkt Cell ◽  
Inkt Cells ◽  
Cross Sectional ◽  
Immunodeficiency Virus ◽  
Invariant Natural Killer ◽  
Hiv 1 ◽  
Natural Killer T

Abstract Background Tuberculosis (TB) is the leading cause of mortality and morbidity in people living with human immunodeficiency virus (HIV) infection (PLWH). PLWH with TB disease are at risk of the paradoxical TB-associated immune reconstitution inflammatory syndrome (TB-IRIS) when they commence antiretroviral therapy. However, the pathophysiology is incompletely understood and specific therapy is lacking. We investigated the hypothesis that invariant natural killer T (iNKT) cells contribute to innate immune dysfunction associated with TB-IRIS. Methods In a cross-sectional study of 101 PLWH and HIV-uninfected South African patients with active TB and controls, iNKT cells were enumerated using α-galactosylceramide-loaded CD1d tetramers and subsequently functionally characterized by flow cytometry. In a second study of 49 people with HIV type 1 (HIV-1) and active TB commencing antiretroviral therapy, iNKT cells in TB-IRIS patients and non-IRIS controls were compared longitudinally. Results Circulating iNKT cells were reduced in HIV-1 infection, most significantly the CD4+ subset, which was inversely associated with HIV-1 viral load. iNKT cells in HIV-associated TB had increased surface CD107a expression, indicating cytotoxic degranulation. Relatively increased iNKT cell frequency in patients with HIV-1 infection and active TB was associated with development of TB-IRIS following antiretroviral therapy initiation. iNKT cells in TB-IRIS were CD4+CD8– subset depleted and degranulated around the time of TB-IRIS onset. Conclusions Reduced iNKT cell CD4+ subsets as a result of HIV-1 infection may skew iNKT cell functionality toward cytotoxicity. Increased CD4– cytotoxic iNKT cells may contribute to immunopathology in TB-IRIS.

scholarly journals Graft invariant natural killer T-cell dose predicts risk of acute graft-versus-host disease in allogeneic hematopoietic stem cell transplantation

Blood ◽  
2012 ◽  
Vol 119 (21) ◽  
pp. 5030-5036 ◽  
Author(s):  
Aristeidis Chaidos ◽  
Scott Patterson ◽  
Richard Szydlo ◽  
Mohammed Suhail Chaudhry ◽  
Francesco Dazzi ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Natural Killer ◽  
Inkt Cell ◽  
Inkt Cells ◽  
Cell Dose ◽  
Grade Ii ◽  
Clinically Significant ◽  
Invariant Natural Killer ◽  
Natural Killer T

Abstract Invariant natural killer T (iNKT) cells are powerful immunomodulatory cells that in mice regulate a variety of immune responses, including acute GVHD (aGVHD). However, their clinical relevance and in particular their role in clinical aGVHD are not known. We studied whether peripheral blood stem cell (PBSC) graft iNKT-cell dose affects on the occurrence of clinically significant grade II-IV aGVHD in patients (n = 57) undergoing sibling, HLA-identical allogeneic HSCT. In multivariate analysis, CD4− iNKT-cell dose was the only graft parameter to predict clinically significant aGVHD. The cumulative incidence of grade II-IV aGVHD in patients receiving CD4− iNKT-cell doses above and below the median were 24.2% and 71.4%, respectively (P = .0008); low CD4− iNKT-cell dose was associated with a relative risk of grade II-IV aGVHD of 4.27 (P = .0023; 95% CI, 1.68-10.85). Consistent with a role of iNKT cells in regulating aGVHD, in mixed lymphocyte reaction assays, CD4− iNKT cells effectively suppressed T-cell proliferation and IFN-γ secretion in a contact-dependent manner. In conclusion, higher doses of CD4− iNKT cells in PBSC grafts are associated with protection from aGVHD. This effect could be harnessed for prevention of aGVHD.

scholarly journals Liver X receptors regulate natural killer T cell population and antitumor activity in the liver of mice

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaori Endo-Umeda ◽  
Hiroyuki Nakashima ◽  
Shigeyuki Uno ◽  
Shota Toyoshima ◽  
Naoki Umeda ◽  
...  
Keyword(s):  
T Cell ◽  
Antitumor Activity ◽  
Natural Killer ◽  
Cytokine Production ◽  
Nkt Cells ◽  
Inkt Cell ◽  
Cell Mediated Immunity ◽  
Inkt Cells ◽  
Nkt Cell ◽  
Natural Killer T

AbstractThe nuclear receptors liver X receptor α (LXRα) and LXRβ are lipid sensors that regulate lipid metabolism and immunity. Natural killer T (NKT) cells, a T cell subset expressing surface markers of both natural killer cells and T lymphocytes and involved in antitumor immunity, are another abundant immune cell type in the liver. The potential function of the metabolic regulators LXRα/β in hepatic NKT cells remains unknown. In this study, we examined the role of LXRα and LXRβ in NKT cells using mice deficient for LXRα and/or LXRβ, and found that hepatic invariant NKT (iNKT) cells are drastically decreased in LXRα/β-KO mice. Cytokine production stimulated by the iNKT cell activator α-galactosylceramide was impaired in LXRα/β-KO hepatic mononuclear cells and in LXRα/β-KO mice. iNKT cell-mediated antitumor effect was also disturbed in LXRα/β-KO mice. LXRα/β-KO mice transplanted with wild-type bone marrow showed decreased iNKT cells in the liver and spleen. The thymus of LXRα/β-KO mice showed a decreased population of iNKT cells. In conclusion, LXRα and LXRβ are essential for NKT cell-mediated immunity, such as cytokine production and hepatic antitumor activity, and are involved in NKT cell development in immune tissues, such as the thymus.

scholarly journals Combination of NKT14m and Low Dose IL-12 Promotes Invariant Natural Killer T Cell IFN-γ Production and Tumor Control

2020 ◽  
Vol 21 (14) ◽  
pp. 5085
Author(s):  
Peng Guan ◽  
Robert Schaub ◽  
Kim E. Nichols ◽  
Rupali Das
Keyword(s):  
T Cell ◽  
Natural Killer ◽  
Low Dose ◽  
Inkt Cell ◽  
Tumor Control ◽  
Inkt Cells ◽  
Ifn Γ ◽  
Invariant Natural Killer ◽  
Natural Killer T

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-γ 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.

scholarly journals Nur77 controls tolerance induction, terminal differentiation, and effector functions in semi-invariant natural killer T cells

2020 ◽  
Vol 117 (29) ◽  
pp. 17156-17165 ◽  
Author(s):  
Amrendra Kumar ◽  
Timothy M. Hill ◽  
Laura E. Gordy ◽  
Naveenchandra Suryadevara ◽  
Lan Wu ◽  
...  
Keyword(s):  
T Cells ◽  
Natural Killer ◽  
Cell Subset ◽  
Terminal Differentiation ◽  
Tolerance Induction ◽  
Cell Development ◽  
Inkt Cell ◽  
Inkt Cells ◽  
Effector Functions ◽  
Invariant Natural Killer

Semi-invariant natural killer T (iNKT) cells are self-reactive lymphocytes, yet how this lineage attains self-tolerance remains unknown. iNKT cells constitutively express high levels ofNr4a1-encoded Nur77, a transcription factor that integrates signal strength downstream of the T cell receptor (TCR) within activated thymocytes and peripheral T cells. The function of Nur77 in iNKT cells is unknown. Here we report that sustained Nur77 overexpression (Nur77tg) in mouse thymocytes abrogates iNKT cell development. Introgression of a rearrangedVα14-Jα18TCR-α chain gene into the Nur77tg(Nur77tg;Vα14tg) mouse rescued iNKT cell development up to the early precursor stage, stage 0. iNKT cells in bone marrow chimeras that reconstituted thymic cellularity developed beyond stage 0 precursors and yielded IL-4–producing NKT2 cell subset but not IFN-γ–producing NKT1 cell subset. Nonetheless, the developing thymic iNKT cells that emerged in these chimeras expressed the exhaustion marker PD1 and responded poorly to a strong glycolipid agonist. Thus, Nur77 integrates signals emanating from the TCR to control thymic iNKT cell tolerance induction, terminal differentiation, and effector functions.

Influence of Genetic Modifiers on Levels of Invariant Natural Killer T-Cells in Patients with Sickle Cell Anemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4843-4843
Author(s):  
Ahmed Al-Yarabi ◽  
Tahar Al-Shabibi ◽  
Nasser Ambu-Ali ◽  
Shoaib Al-Zadjali ◽  
Abeer Al-Zubaidi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Natural Killer ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Inkt Cell ◽  
Genetic Modifiers ◽  
Inkt Cells ◽  
Invariant Natural Killer ◽  
Hydroxyurea Therapy ◽  
Natural Killer T

Abstract Although patients homozygous for the sickle cell disease (SCD) mutation have an identical genotype, the severity of the disease can be extremely variable. The hemoglobin (Hb) S mutation has been associated with five different beta S-globin gene cluster haplotypes (βS-haplotypes) that show different clinical expression. Because genetic modifiers can modulate treatment response, we hypothesized that βS-haplotypes can affect levels of invariant natural killer T (iNKT) cells, which are known to play a key role in the pathogenesis of SCD, and are being considered as a potential target to treat acute crises in SCD patients. Herein we evaluated the impact of βS-haplotypes and HbF concentration on iNKT cell and dendritic cell (DC) subsets in a well-defined group of patients with SCD in a steady state. Sickle cell anemia patients were selected based on the history of patients, clinical examination and hematological findings. The βS-haplotypes were identified by polymerase chain reaction-restriction fragment length polymorphism analysis for seven restriction sites. The iNKT cell subsets were characterized by the positive-staining of Vα24Jα18 T cell receptor alpha chain, along with CD3, CD4 and CD8 surface markers and the intra-cellular cytokine production of interferon-gamma (Th1-like), interleukin-4 (Th2-like) and interleukin-17 (Th17-like) cells using flow cytometry and cell culture. The myeloid DC (mDC) and plasmacytoid DC (pDC) cells were identified by the expression of HLA-DR, CD123, CD11c, Lin and CD1d, a non-classical molecule that induces the activation of iNKT cells by flow cytometry. Comparisons among βS-haplotypes were performed using ANOVA and unpaired t test, while the Spearman's correlation was used to assess associations. Among the 125 sickle cell anemia patients studied, Benin haplotype (40%) was the most common followed by Bantu (21%), Arab/Indian (18%), Atypical (12%) and Benin/Bantu or Benin/Arab-Indian or Bantu/Arab-Indian (9%). The majority of subjects with Benin/Benin βS-haplotype had a severe to moderate clinical profile similar to Bantu/Bantu or Arab/Indian βS-haplotype groups (P=0.23). A trend toward increased levels of CD3iNKT, CD4iNKT and CD8iNKT cell subsets was observed in the subjects with the Benin/Benin βS-haplotype, when compared to other βS-haplotype groups (P=0.06). Interestingly, subjects with the Benin/Benin βS-haplotype exhibited slightly higher levels of Th1-like cells, but not Th2-like and Th17-like cells, when compared to subjects with the Bantu/Bantu or Arab/Indian βS-haplotype groups (P=0.047). Comparisons between the levels of mDC and pDC cell subsets, as well as the expression of CD1d on these DC cells, showed no statistically significant differences among the βS-haplotype groups (P=0.42). Similarly, levels of iNKT cell subsets, Th1-like, Th2-like, and Th17-like cells as well as DC subsets were similar among patients who received or not hydroxyurea therapy, independently of βS-haplotype groups (P= 0.09). Likewise, coexistence of high HbF and βS-haplotypes did not show any significant association with iNKT cell subsets. Collectively, our findings suggest that neither the βS-haplotypes or HbF levels nor the hydroxyurea therapy or clinical severity appeared to be associated with iNKT cell or DC cell subsets in patients with SCD. The effects of other genetic modifiers such as alpha/beta-thalassemia and G6PD deficiency on iNKT cell subsets need to be evaluated in future studies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Genetic engineering of hematopoietic stem cells to generate invariant natural killer T cells

2015 ◽  
Vol 112 (5) ◽  
pp. 1523-1528 ◽  
Author(s):  
Drake J. Smith ◽  
Siyuan Liu ◽  
Sunjong Ji ◽  
Bo Li ◽  
Jami McLaughlin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Natural Killer ◽  
Inkt Cell ◽  
Cell Phenotype ◽  
Inkt Cells ◽  
Hematopoietic Stem ◽  
Invariant Natural Killer ◽  
Natural Killer T

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.

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