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

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.

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Mitochondrial metabolism is essential for invariant natural killer T cell development and function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021385118 ◽

2021 ◽

Vol 118 (13) ◽

pp. e2021385118

Author(s):

Xiufang Weng ◽

Amrendra Kumar ◽

Liang Cao ◽

Ying He ◽

Eva Morgun ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Natural Killer ◽

Cell Development ◽

Mitochondrial Metabolism ◽

Inkt Cell ◽

Inkt Cells ◽

And Function ◽

Invariant Natural Killer

Conventional T cell fate and function are determined by coordination between cellular signaling and mitochondrial metabolism. Invariant natural killer T (iNKT) cells are an important subset of “innate-like” T cells that exist in a preactivated effector state, and their dependence on mitochondrial metabolism has not been previously defined genetically or in vivo. Here, we show that mature iNKT cells have reduced mitochondrial respiratory reserve and iNKT cell development was highly sensitive to perturbation of mitochondrial function. Mice with T cell-specific ablation of Rieske iron-sulfur protein (RISP; T-Uqcrfs1−/−), an essential subunit of mitochondrial complex III, had a dramatic reduction of iNKT cells in the thymus and periphery, but no significant perturbation on the development of conventional T cells. The impaired development observed in T-Uqcrfs1−/− mice stems from a cell-autonomous defect in iNKT cells, resulting in a differentiation block at the early stages of iNKT cell development. Residual iNKT cells in T-Uqcrfs1−/− mice displayed increased apoptosis but retained the ability to proliferate in vivo, suggesting that their bioenergetic and biosynthetic demands were not compromised. However, they exhibited reduced expression of activation markers, decreased T cell receptor (TCR) signaling and impaired responses to TCR and interleukin-15 stimulation. Furthermore, knocking down RISP in mature iNKT cells diminished their cytokine production, correlating with reduced NFATc2 activity. Collectively, our data provide evidence for a critical role of mitochondrial metabolism in iNKT cell development and activation outside of its traditional role in supporting cellular bioenergetic demands.

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

Frontiers in Immunology ◽

10.3389/fimmu.2021.700374 ◽

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.

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

10.1101/2021.04.29.442021 ◽

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.

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Expansion of invariant natural killer T cells from systemic lupus erythematosus patients by alpha-Galactosylceramide and IL-15

PLoS ONE ◽

10.1371/journal.pone.0261727 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0261727

Author(s):

Chien-Ya Hsu ◽

Yu-Shan Chueh ◽

Ming-Ling Kuo ◽

Pei-Tzu Lee ◽

Hsiu-Shan Hsiao ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

T Cells ◽

Natural Killer ◽

Lupus Erythematosus ◽

Natural Killer T Cells ◽

Inkt Cell ◽

Inkt Cells ◽

Systemic Lupus ◽

Killer T Cells ◽

Invariant Natural Killer

CD1d-restricted invariant natural killer T cells (iNKT cells) may play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Interleukin (IL)-15 is a pro-inflammatory cytokine which is over-expressed in SLE patients. In the present study, we investigated the iNKT cell expansion of mononuclear cells (MNCs) from SLE patients following 10 days’ culture with α-galactosylceramide (α-Galcer) and /or IL-15. We sought to determine the phenotypic and functional characteristics of the expanded iNKT cells compared to healthy controls and correlated with disease activity. We observed that 1. The percentages of Vα24+/Vβ11+ iNKT cells following 10-day incubation was lower in SLE groups compared to controls; 2. The percentages and absolute numbers of Vα24+/Vβ11+ iNKT cells were expanded by α-galactosylceramide (α-Galcer), and further enhanced with IL-15 in SLE patient, but the effect of IL-15 was much lower than controls; 3.IL-15 +α-Galcer expanded CD3+/CD56+ NKT-like cells from SLE patients, especially with active disease 4. The CD161+ Vα24+/Vβ11+ iNKT cells in SLE were more responsive to α-Galcer stimulation than the CD161- counterpart; 5. IL-15 decreased apoptosis of α-Galcer activated SLE iNKT cells; 6. IL-15 enhanced CD69, CD1d and CD11a expression on α-Galcer treated iNKT cells; 7. The IL-4 production of iNKT cells was decreased in SLE patients compared to controls; 8. IL-15 increased IFN-γ and IL-4 production of SLE iNKT cells; 8. IL-15 failed to augment the ability of iNKT cells to aid NK-mediated K562 cytolysis in SLE patients; 9. CD161 positivity, granzyme B and perforin expression of α-Galcer+IL-15 expanded iNKT cells correlated with C3 levels in SLE patients. Taken together, our results demonstrated numeric and functional deficiency of iNKT cells and their response to IL-15 in SLE patients. Our finding may provide insight for using adoptive iNKT cell therapy in autoimmune diseases.

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Longitudinal analysis of invariant natural killer T cell activation reveals a cMAF-associated transcriptional state of NKT10 cells

10.1101/2021.12.29.474454 ◽

2021 ◽

Author(s):

Lydia Lynch ◽

Harry Kane ◽

Nelson M LaMarche ◽

Áine Ní Scannail ◽

Michael P. Brenner

Keyword(s):

T Cells ◽

Natural Killer ◽

T Cell Activation ◽

Cell Activation ◽

Inkt Cell ◽

Inkt Cells ◽

Innate T Cells ◽

Nonpeptide Antigens ◽

Invariant Natural Killer ◽

Natural Killer T

Innate T cells, including CD1d-restricted invariant natural killer T (iNKT) cells, are characterized by their rapid activation in response to nonpeptide antigens, such as lipids. While the transcriptional profiles of naive, effector and memory adaptive T cells have been well studied, less is known about transcriptional regulation of different iNKT cell activation states. Here, using single cell RNA-sequencing, we performed longitudinal profiling of activated iNKT cells, generating a transcriptomic atlas of iNKT cell activation states. We found that transcriptional signatures of activation are highly conserved among heterogeneous iNKT cell populations, including NKT1, NKT2 and NKT17 subsets, and human iNKT cells. Strikingly, we found that regulatory iNKT cells, such as adipose iNKT cells, undergo blunted activation, and display constitutive enrichment of memory-like cMAF+ and KLRG1+ populations. Moreover, we identify a conserved cMAF-associated transcriptional network among NKT10 cells, providing novel insights into the biology of regulatory and antigen experienced iNKT cells.

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Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection

Journal of Experimental Medicine ◽

10.1084/jem.20102555 ◽

2011 ◽

Vol 208 (6) ◽

pp. 1163-1177 ◽

Cited By ~ 186

Author(s):

Manfred Brigl ◽

Raju V.V. Tatituri ◽

Gerald F.M. Watts ◽

Veemal Bhowruth ◽

Elizabeth A. Leadbetter ◽

...

Keyword(s):

T Cells ◽

Natural Killer ◽

T Cell Activation ◽

Cell Activation ◽

Constitutive Expression ◽

Microbial Pathogens ◽

Inkt Cell ◽

Microbial Infection ◽

Inkt Cells ◽

Natural Killer T

Invariant natural killer T cells (iNKT cells) are critical for host defense against a variety of microbial pathogens. However, the central question of how iNKT cells are activated by microbes has not been fully explained. The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection. However, when we compared the role of microbial antigens versus innate cytokine-driven mechanisms, we found that iNKT cell interferon-γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor–driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12–induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections.

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Third Party Invariant Natural Killer T Cells Protect from Graft-Versus-Host Disease Lethality through Expansion of Donor CD4+FoxP3+ Regulatory T Cells

Blood ◽

10.1182/blood.v124.21.3825.3825 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3825-3825

Author(s):

Dominik Schneidawind ◽

Jeanette Baker ◽

Corina Buechele ◽

Everett H. Meyer ◽

Robert S. Negrin

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Natural Killer ◽

Graft Versus Host Disease ◽

Adoptive Transfer ◽

Third Party ◽

Inkt Cells ◽

Graft Versus Host ◽

Donor T Cells ◽

Invariant Natural Killer

Abstract Graft-versus-host disease (GVHD) is driven by extensive activation and proliferation of alloreactive donor T cells causing significant morbidity and mortality following allogeneic hematopoietic cell transplantation (HCT). Invariant natural killer T (iNKT) cells are potent regulators of immune responses in both humans (TCRα Vα24-Jα18) and mice (TCRα Vα14-Jα18). As the iNKT cell receptor and the glycolipid-presenting molecule CD1d interaction is highly conserved, we explored the role of adoptively transferred third party CD4+ iNKT cells in a murine model of allogeneic HCT. BALB/c (H-2Kd) recipient mice were irradiated with 8 Gy and transplanted with T cell-depleted bone marrow together with 1x106 CD4+/CD8+ T cells (Tcon) from C57BL/6 (H-2Kb) donor mice. Adoptive transfer of purified (>95%) 5x104 CD4+ iNKT cells from FVB/N (H-2Kq) third party mice resulted in a significant survival benefit (p<0.001) while retaining Tcon mediated graft-versus-tumor (GVT) effects against A20 lymphoma cells (p=0.002). Consistently, weight and GVHD scores improved in mice that received a single injection of third party CD4+ iNKT cells as compared to animals that received Tcon alone. Notably, CD4+ iNKT cells from third party mice were as protective as CD4+ iNKT cells from donor mice (p=0.50). Signal intensity deriving from expanding luciferase expressing alloreactive Tcon was significantly lower in animals treated with third party CD4+ iNKT cells (p=0.003). Interestingly, inhibition of Tcon proliferation was similar to animals that received CD4+ iNKT cells from donor mice (p=0.90). In addition, adoptive transfer of third party CD4+ iNKT cells promoted a Th2-biased cytokine response of alloreactive donor T cells. Although we found that third party CD4+ iNKT cells were rejected by day +10 after allogeneic HCT, adoptive transfer of these cells resulted in a robust expansion of luciferase expressing donor CD4+FoxP3+ regulatory T cells (Treg) as measured by bioluminescence imaging (p=0.006). Using FoxP3DTR C57BL/6 donor mice, depletion of Treg from the graft abrogated both donor Treg expansion and protection from GVHD lethality through third party CD4+ iNKT cells. We conclude that low numbers of highly purified and adoptively transferred third party CD4+ iNKT cells protect from lethal GVHD through activation and expansion of donor Treg with retained GVT effects. Despite the fact that iNKT cells are a rare cell population, the in vivo activity of small numbers of cells and feasibility of in vitro expansion provide the basis for clinical translation. Disclosures No relevant conflicts of interest to declare.

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Relevance of sexual dimorphism to regulatory T cells: estradiol promotes IFN-γ production by invariant natural killer T cells

Blood ◽

10.1182/blood-2004-07-2819 ◽

2005 ◽

Vol 105 (6) ◽

pp. 2415-2420 ◽

Cited By ~ 98

Author(s):

Pierre Gourdy ◽

Luiza M. Araujo ◽

Ren Zhu ◽

Barbara Garmy-Susini ◽

Séverine Diem ◽

...

Keyword(s):

T Cells ◽

Natural Killer ◽

Interleukin 4 ◽

Response To Treatment ◽

Inkt Cells ◽

Gender Dimorphism ◽

Ifn Γ ◽

Invariant Natural Killer ◽

Natural Killer T

Abstract Mechanisms accounting for gender dimorphism during immune responses are still poorly understood. Since invariant natural killer T (iNKT) cells exert important regulatory functions through their capacity to produce both T helper 1 (Th1) and Th2 cytokines, we addressed the question of whether these activities could be modulated by sexual hormones. We found that in vivo challenge with the specific ligand of iNKT cells, α-galactosylceramide (α-GalCer), induced significantly higher concentrations of interferon γ (IFN-γ) in the serum of female than in that of male mice, while interleukin 4 (IL-4) production was not modified. In support of a crucial role of ovarian hormones in this phenomenon, a significant decrease of serum IFN-γ concentrations occurred in ovariectomized females, in response to treatment with α-GalCer, while orchidectomy affected neither IFN-γ nor IL-4 serum concentrations in males. The implication of estrogens in this selective enhancement of IFN-γ production by iNKT cells was demonstrated by (1) the increased α-GalCer–induced IFN-γ synthesis by iNKT cells upon both in vitro and in vivo exposure to estradiol and (2) the abolition of the sex-linked difference in α-GalCer–induced IFN-γ release in estrogen receptor α-deficient mice. These results provide the first evidence that estrogens influence iNKT cells leading to this gender dimorphism in their cytokine production profile.

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Vaccine Based on Dendritic Cells Electroporated with an Exogenous Ovalbumin Protein and Pulsed with Invariant Natural Killer T Cell Ligands Effectively Induces Antigen-Specific Antitumor Immunity

Cancers ◽

10.3390/cancers14010171 ◽

2021 ◽

Vol 14 (1) ◽

pp. 171

Author(s):

Akihiro Watanabe ◽

Kimihiro Yamashita ◽

Mitsugu Fujita ◽

Akira Arimoto ◽

Masayasu Nishi ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Natural Killer ◽

Cd8 T Cells ◽

Antitumor Immunity ◽

Tumor Antigens ◽

Vaccine Strategy ◽

Inkt Cells ◽

Invariant Natural Killer ◽

Natural Killer T

(1) Background: Cancer vaccines are administered to induce cytotoxic CD8+ T cells (CTLs) specific for tumor antigens. Invariant natural killer T (iNKT) cells, the specific T cells activated by α-galactosylceramide (α-GalCer), play important roles in this process as they are involved in both innate and adaptive immunity. We developed a new cancer vaccine strategy in which dendritic cells (DCs) were loaded with an exogenous ovalbumin (OVA) protein by electroporation (EP) and pulsed with α-GalCer. (2) Methods: We generated bone marrow-derived DCs from C57BL/6 mice, loaded full-length ovalbumin proteins to the DCs by EP, and pulsed them with α-GalCer (OVA-EP-galDCs). The OVA-EP-galDCs were intravenously administered to C57BL/6 mice as a vaccine. We then investigated subsequent immune responses, such as the induction of iNKT cells, NK cells, intrinsic DCs, and OVA-specific CD8+ T cells, including tissue-resident memory T (TRM) cells. (3) Results: The OVA-EP-galDC vaccine efficiently rejected subcutaneous tumors in a manner primarily dependent on CD8+ T cells. In addition to the OVA-specific CD8+ T cells both in early and late phases, we observed the induction of antigen-specific TRM cells in the skin. (4) Conclusions: The OVA-EP-galDC vaccine efficiently induced antigen-specific antitumor immunity, which was sustained over time, as shown by the TRM cells.

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Enhancing the antitumor functions of invariant natural killer T cells using a soluble CD1d-CD19 fusion protein

Blood Advances ◽

10.1182/bloodadvances.2018028886 ◽

2019 ◽

Vol 3 (5) ◽

pp. 813-824 ◽

Cited By ~ 1

Author(s):

Rupali Das ◽

Peng Guan ◽

Susan J. Wiener ◽

Nishant P. Patel ◽

Trevor G. Gohl ◽

...

Keyword(s):

Fusion Protein ◽

B Cell ◽

Natural Killer ◽

Tumor Cells ◽

Inkt Cell ◽

Inkt Cells ◽

Cell Responses ◽

Invariant Natural Killer

Abstract Invariant natural killer T (iNKT) cells comprise a unique lineage of CD1d-restricted lipid-reactive T lymphocytes that potently kill tumor cells and exhibit robust immunostimulatory functions. Optimal tumor-directed iNKT cell responses often require expression of the antigen-presenting molecule CD1d on tumors; however, many tumor cells downregulate CD1d and thus evade iNKT cell recognition. We generated a soluble bispecific fusion protein designed to direct iNKT cells to the site of B-cell cancers in a tumor antigen-specific but CD1d-independent manner. This fusion protein is composed of a human CD1d molecule joined to a single chain antibody FV fragment specific for CD19, an antigen widely expressed on B-cell cancers. The CD1d-CD19 fusion protein binds specifically to CD19-expressing, but not CD19-negative cells. Once loaded with the iNKT cell lipid agonist α-galactosyl ceramide (αGC), the CD1d-CD19 fusion induces robust in vitro activation of and cytokine production by human iNKT cells. iNKT cells stimulated by the αGC-loaded CD1d-CD19 fusion also strongly transactivate T-, B-, and NK-cell responses and promote dendritic cell maturation. Importantly, the αGC-loaded fusion induces robust lysis of CD19+CD1d− Epstein-Barr virus immortalized human B-lymphoblastoid cell lines that are otherwise resistant to iNKT cell killing. Consistent with these findings; administration of the αGC-loaded fusion protein controlled the growth of CD19+CD1d− tumors in vivo, suggesting that it can “link” iNKT cells and CD19+CD1d− targets in a therapeutically beneficial manner. Taken together, these preclinical studies demonstrate that this B cell–directed fusion protein can be used to effectively induce iNKT cell antitumor responses in vitro and in vivo.

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