A mutation within the SH2 domain of slp‐76 regulates the tissue distribution and cytokine production of iNKT cells in mice

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

Scientific Reports ◽

10.1038/s41598-021-02062-z ◽

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.

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Perifosine Affects Phenotype and Function of Human Myeloid Dendritic Cells

Blood ◽

10.1182/blood.v116.21.3911.3911 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3911-3911

Author(s):

Weihua Song ◽

Teru Hideshima ◽

Yu-Tzu Tai ◽

Rao H Prabhala ◽

Kenneth C. Anderson ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Immune Responses ◽

Cell Activation ◽

Production Control ◽

Cytokine Production ◽

Inkt Cells ◽

Uptake Capacity ◽

Antigen Uptake ◽

Ifn Gamma

Abstract Abstract 3911 Perifosine is a synthetic novel alkylphospholipid, a new class of antitumor agent which targets cell membranes and inhibits Akt activation. Perifosine inhibits multiple myeloma (MM) cell growth in vitro and in vivo. Currently perifosine is under phase III clinical evaluation in MM. Although perifosine has shown significant direct antitumor effects, its effect on immune system has not yet been clarified. Dendritic cells (DCs) play a crucial role in immune system via mediating antigen-specific immune responses as well as regulating the innate and adaptive immunity through secreted cytokines. In this study, effect of perifosine on phenotype, antigen uptake, processing and presentation, and cytokine production on human monocyte-derived dendritic cells (DCs) were evaluated at clinically relevant concentrations (2.5μM to 10μM). The effect of perifosine on survival of DCs was tested by annexin V and PI staining. We observed that up to 48 hours of perifosine treatment had no effect on viability of both immature DCs and DCs during maturation by LPS (100ng/ml) or poly(IC) (25μg/ml) (>90%). Alteration of DC phenotype by perifosine was further examined by flow cytometry. Our results demonstrated that perifosine treatment led to a dose-dependent downregulation of surface antigen expression, associated with costimulation (CD40), antigen presentation (HLA-ABC) and maturation (CD83) on both immature and mature DCs at 24 and 48 hours. We next evaluated whether perifosine affected the antigen uptake capacity by immature DCs, using various sizes and forms of antigens. Compared to control, 24 hours-perifosine (10μM) treatment significantly reduced uptake capacity of both protein antigens (Alexa Fluor 488-conjugated 45-KDa protein A and 20-KDa protein G) and polysaccharide antigen (Alexa Fluor 488-conjugated 40-KD dextran) by immature DCs. Importantly, we systematically investigated the impact of perifosine on DCs involved in both classic (MHC-mediated) and non-classic (CD1d-mediated) antigen specific immune responses and report a significant impairment upon perifosine treatment. Perifosine inhibited DC-mediated T cell activation. Following 24 hours treatment, control or perifosine (10μM)-treated immature DCs were pulsed with tetanus toxoid (0.5μg/ml) overnight in the presence of LPS, and then used to stimulate autologous T cells. T cell response was inhibited as evidenced by significantly reduced IFN-gamma production detected by ELISA (control vs treatment = 15106 pg/ml vs 8332 pg/ml). We also confirmed that perifosine pretreatment of freshly isolated blood myeloid DCs led to significant inhibition of allogenic T cell immune responses (control vs treatment = 982 pg/ml vs 605 pg/ml). In addtion to presenting peptide antigens, DCs also present glycolipids to activate invariant NKT (iNKT) cells. Our data demonstrated that perifosine impairs DCs-mediated iNKT cell activation. The production of both Th1-type cytokines and Th2-type cytokines by iNKT cells were significantly repressed upon perifosine (10μM)-treated alpha-GalCer-pulsing DCs, compared to control (IFN-gamma production (control vs treatment= 11631 pg/ml vs 6768 pg/ml) and IL-4 production (control vs treatment= 1285 pg/ml vs 783 pg/ml). Since DCs play a crucial regulatory role via cytokine production, we next determined IL-12p70 and IL-10 secretion by LPS-induced DCs with and without perifosine treatment. Compared to controls, perifosine treatment at 24 hours significantly inhibited LPS-induced-IL-12p70 production by DCs (control vs treatment = 192 pg/ml vs 166 pg/ml (2.5μM), 111 pg/ml (5μM) and 45 pg/ml (10μM)), as well as inhibited IL-10 production (control vs treatment = 472 pg/ml vs 371 pg/ml (2.5μM), 306 pg/ml (5μM) and 179 pg/ml (10μM)). Mechanism study revealed that RelB, a component of NF-kappaB signaling pathway, was downregulated in immature DCs by 18 hours of perifosine treatment (10μM). We have also evaluated the effect of perifosine on the expression of IDO, a repressor of DCs activation. However, we did not observe the significant alteration of perifosine treatment compared to the control. In summary, our preclinical data suggest that perifosine is able to affect both immature and mature DCs and could contribute to inhibition of DC-mediated immune responses, indicating a need to monitor immune functions in patients under the Akt inhibitor treatment. Disclosures: No relevant conflicts of interest to declare.

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Transcription factor Bcl11b sustains iNKT1 and iNKT2 cell programs, restricts iNKT17 cell program, and governs iNKT cell survival

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1521846113 ◽

2016 ◽

Vol 113 (27) ◽

pp. 7608-7613 ◽

Cited By ~ 12

Author(s):

Mohammad Nizam Uddin ◽

Dil Afroz Sultana ◽

Kyle J. Lorentsen ◽

Jonathan J. Cho ◽

Mariana E. Kirst ◽

...

Keyword(s):

T Cells ◽

Transcription Factor ◽

Immune Responses ◽

Cytokine Production ◽

Inkt Cells ◽

Double Positive ◽

Cell Numbers ◽

Function Identity ◽

Invariant Natural Killer

Invariant natural killer T (iNKT) cells are innate-like T cells that recognize glycolipid antigens and play critical roles in regulation of immune responses. Based on expression of the transcription factors (TFs) Tbet, Plzf, and Rorγt, iNKT cells have been classified in effector subsets that emerge in the thymus, namely, iNKT1, iNKT2, and iNKT17. Deficiency in the TF Bcl11b in double-positive (DP) thymocytes has been shown to cause absence of iNKT cells in the thymus and periphery due to defective self glycolipid processing and presentation by DP thymocytes and undefined intrinsic alterations in iNKT precursors. We used a model of cre-mediated postselection deletion of Bcl11b in iNKT cells to determine its intrinsic role in these cells. We found that Bcl11b is expressed equivalently in all three effector iNKT subsets, and its removal caused a reduction in the numbers of iNKT1 and iNKT2 cells, but not in the numbers of iNKT17 cells. Additionally, we show that Bcl11b sustains subset-specific cytokine production by iNKT1 and iNKT2 cells and restricts expression of iNKT17 genes in iNKT1 and iNKT2 subsets, overall restraining the iNKT17 program in iNKT cells. The total numbers of iNKT cells were reduced in the absence of Bcl11b both in the thymus and periphery, associated with the decrease in iNKT1 and iNKT2 cell numbers and decrease in survival, related to changes in survival/apoptosis genes. Thus, these results extend our understanding of the role of Bcl11b in iNKT cells beyond their selection and demonstrate that Bcl11b is a key regulator of iNKT effector subsets, their function, identity, and survival.

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The A2aR adenosine receptor controls cytokine production in iNKT cells

European Journal of Immunology ◽

10.1002/eji.200939897 ◽

2010 ◽

Vol 40 (3) ◽

pp. 682-687 ◽

Cited By ~ 53

Author(s):

Michael Nowak ◽

Lydia Lynch ◽

Simon Yue ◽

Akio Ohta ◽

Michail Sitkovsky ◽

...

Keyword(s):

Adenosine Receptor ◽

Cytokine Production ◽

Inkt Cells

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HDAC Inhibition by LBH589 Affects Phenotype and Function of Human Dendritic Cells.

Blood ◽

10.1182/blood.v114.22.1646.1646 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1646-1646

Author(s):

Weihua Song ◽

Yu-Tzu Tai ◽

Ze Tian ◽

Teru Hideshima ◽

Dharminder Chauhan ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Hdac Inhibitor ◽

Cell Activation ◽

Production Control ◽

Cytokine Production ◽

Hdac Inhibition ◽

Inkt Cells ◽

Antigen Uptake ◽

And Function

Abstract Abstract 1646 Poster Board I-672 LBH589 is a novel pan-HDAC inhibitor which has potent antitumor activity in both solid tumors and hematologic malignancies. LBH589 induces hyperacetylation of core histone proteins, resulting in modulation of protein expression, leading to cell cycle arrest in the G2/M phase and apoptosis. Currently, LBH589 is undergoing phase I/II clinical evaluation in multiple myeloma and other hematologic tumors. Although the role of HDAC inhibition by LBH589 has been widely studied, its effects on immune function have not yet been defined. Dendritic cells (DCs) play a crucial role in immune system via mediating antigen-specific immune responses as well as regulating the innate and adaptive immunity through secreted cytokines. In this study, effect of LBH589 on phenotype, antigen uptake, processing and presentation, and the cytokine production on human monocyte-derived dendritic cells (DCs) were evaluated at clinically relevant concentrations (2.5nM to 20nM). The effect of LBH589 on survival of DCs was tested by annexin V and PI staining. Following 24 hours treatment, LBH589 up to 10nM did not affect the viability of immature DCs (> 90%); Mature DCs induced by LPS (100ng/mL) showed stronger anti-apoptotic activity, no significant apoptosis appeared up to 20 nM of LBH589 treatment. HDAC inhibition by LBH589 led to both up- and down-regulation of important surface molecules on both immature and mature DCs, associated with DC maturation (CD83↓), antigen presentation (HLA-ABC↓ and HLA-DR↓), and T cell co-stimulation (CD40↓ and CD86↓), analyzed by flow cytometry. We next evaluated whether HDAC inhibition affected the antigen uptake capacity by immature DCs, using various sizes and forms of antigens. Compared to control, 24 hours-LBH589 (10nM) treatment significantly reduced uptake capacity of both protein antigens (Alexa Fluor 488-conjugated 45-KDa protein A and 20-KDa protein G) and polysaccharide antigen (Alexa Fluor 488-conjugated 40-KD dextran) by immature DCs. Importantly, we systematically investigated the impact of HDAC inhibition on DCs involved in both classic (MHC-mediated) and non-classic (CD1d-mediated) antigen specific immune responses and report a significant impairment upon LBH589 treatment. HDAC inhibition by LBH589 inhibited DC-mediated T cell activation. Following 24 hours treatment, control or LBH589 (10nM)-treated immature DCs were pulsed with tetanus toxoid (0.5μg/mL) overnight in the presence of LPS, and then used to stimulate autologous T cells. T cell response was inhibited as evidenced by significantly reduced IFN-gamma production detected by ELISA (control vs treatment = 14289 pg/mL vs 5154 pg/mL). Similar effect was observed by LBH589 (10nM) treatment on DCs during maturation (control vs treatment = 19629 pg/mL vs 9486 pg/mL). In addtion to presenting peptide antigens, DCs also present glycolipids to activate invariant NKT (iNKT) cells. Our data demonstrated that HDAC inhibition by LBH589 impairs DCs-mediated iNKT cell activation. The production of both Th1-type cytokines and Th2-type cytokines by iNKT cells were significantly repressed upon LBH589 (10nM)-treated alpha-GalCer-pulsing DCs, compared to control (IFN-gamma production (control vs treatment=12333 pg/mL vs 7892 pg/ mL), IL-2 production (control vs treatment=253 pg/ mL vs 153 pg/ mL), and IL-4 production (control vs treatment= 1279 pg/ mL vs 845 pg/mL). Moreover, we demonstrated that 24 hours LBH589 treatment dramatically repressed LPS-induced cytokine production by DCs, which play crucial roles in the Th1 and Th17 cell polarization, NK cell activation, and inflammation, including IL-6 (control vs LBH589 2.5nM/10nM/20nM) (4127pg/mL vs 2379pg/mL /1977pg/mL /1048pg/mL), IL-10 (458pg/mL vs 106pg/mL /11pg/mL /9pg/mL), IL-12 (68pg/mL vs 9pg/mL /0pg/mL /0pg/mL), IL-23 (575pg/mL vs 12pg/mL/ 0.1 pg/mL /0pg/mL) and TNF-alpha (45% positive cells (control) vs 24% positive cells (LBH589 2.5nM) and 5% positive cells(LBH589 10nM)). In summary, our study demonstrates that HDAC inhibition by LBH589 significantly impairs the phenotype and function of both immature and mature DCs. Our pre-clinical data indicates the need to monitor immune functions in patients receiving the HDAC inhibitor therapy, and to possibly avoid the combination of HDAC inhibitor with DC vaccination strategies. In contrast, the significantly reduced pro-inflammatory cytokine production may suggest need to investigate use of this agent in treatment of inflammatory disease as well as autoimmune disorders. Disclosures No relevant conflicts of interest to declare.

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400 Persistence and tissue distribution of agent-797 – a native allogeneic iNKT cell-therapy drug product

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.400 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A432-A432

Author(s):

Marco Purbhoo ◽

Burcu Yigit ◽

Darrian Moskowitz ◽

Min Lim ◽

Irina Shapiro ◽

...

Keyword(s):

Tissue Distribution ◽

Drug Product ◽

Xenograft Model ◽

Digital Pcr ◽

Inkt Cell ◽

Ethical Standards ◽

Inkt Cells ◽

Link Type ◽

Murine Xenograft Model ◽

Nog Mice

BackgroundInvariant Natural Killer T (iNKT) cells are key effectors and regulators of immune responses, making them an ideal immunotherapy. There is a paucity of evidence describing the persistence and trafficking of these cells in humans to inform the optimal clinical application. Here, we describe the development of a murine Xenograft model for the study of an unmodified human iNKT cell therapy (Agent-797) and present data on the persistence and tissue distribution of human iNKT cells in this model. We further describe the development and validation of a digital PCR-based methodology to track unmodified allogeneic human iNKT cells in blood and tissue and present exploratory clinical data on iNKT cell persistence in patients with cancer and viral ARDS treated with Agent-797.MethodsPersistence and tissue distribution of ex-vivo expanded human iNKT cells was investigated in immune compromised mice (NOG), as well as in NOG mice expressing human IL15 (NOG-hIL15), a key cytokine promoting iNKT cell survival. Persistence of iNKT cells was determined over a 35-day period, with takedowns on day 1, 7, 14, 21 and 35. iNKT cells were phenotyped for activation markers by flow cytometry. An assay based on Imegen Quimera digital PCR technology was developed and validated to quantify human iNKT in an allogeneic setting. We employed this assay to measure persistence of Agent-797 drug product in patients participating in clinical trials using iNKT cell-based immunotherapy in viral ARDS (NCT04582201) or multiple myeloma (NCT04754100).ResultsHuman IL15 was essential for the engraftment and persistence of human iNKT cells in NOG mice. Following injection, iNKT cells located to the blood, lung, liver, spleen, and bone marrow. iNKT cells persisted most prominently in bone marrow, where they demonstrated an activated phenotype. In mice challenged with hematological tumor cells (ALL cell line NALM6 expressing CD1d) persistence of iNKT cells in blood was prolonged. Initial data from human trials confirmed rapid translocation from peripheral blood of this tissue resident immune cell population following infusion of Agent-797.ConclusionsWe established a murine xenograft model and digital PCR-based methodology to characterize the persistence, trafficking, and efficacy of native allogeneic human iNKT cell-based products. Our models recapitulated the human iNKT distribution and demonstrated iNKTs induced preclinical efficacy in a tumor model. We further successfully developed a validated methodology to track unmodified allogeneic iNKT cells in humans.Trial RegistrationNCT04582201 and NCT04754100Ethics ApprovalAll procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards

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IL-7 During Antigenic Stimulation Using Allogeneic Dendritic Cells Promotes Expansion of CD45RA-CD62L+CD4+ Invariant NKT Cells With Th-2 Biased Cytokine Production Profile

Frontiers in Immunology ◽

10.3389/fimmu.2020.567406 ◽

2020 ◽

Vol 11 ◽

Author(s):

Abel Trujillo-Ocampo ◽

Hyun-Woo Cho ◽

Michael Clowers ◽

Sumedha Pareek ◽

Wilfredo Ruiz-Vazquez ◽

...

Keyword(s):

Dendritic Cells ◽

Mononuclear Cells ◽

Cytokine Production ◽

Ex Vivo ◽

Antigenic Stimulation ◽

Inkt Cells ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells ◽

Th 1 ◽

Stimulation Of

Invariant natural killer T (iNKT) cells are innate-like T lymphocytes cells that recognize glycolipid antigens associated with CD1d, non-classical antigen presenting proteins. They can drive either pro-inflammatory (Th-1) or anti-inflammatory (Th-2) immune microenvironment through the production of both Th-1 and Th-2 type cytokines upon activation, thus play a vital role in cancer, infection, and autoimmune diseases. Adoptive cell therapy using ex vivo expanded iNKT cells is a promising approach to enhance anti-tumor immunity or immunosuppression. However, overcoming phenotypic and functional heterogeneity and promoting in vivo persistency of iNKT cells remains to be a challenge. Here, we compared various methods for ex vivo expansion of human iNKT cells and assessed the quality of expansion, phenotype, and cytokine production profile of expanded iNKT cells. While a direct stimulation of iNKT cells in peripheral blood mononuclear cells with agonist glycolipid led to the expansion of iNKT cells in varying degrees, stimulation of enriched iNKT cells by irradiated autologous peripheral blood mononuclear cells or allogeneic dendritic cells resulted in consistent expansion of highly pure iNKT cells. Interestingly, the mode of antigenic stimulation influenced the dominant subtype of expanded iNKT cells. Further, we evaluated whether additional IL-7 or IL-15 during antigenic stimulation with allogeneic dendritic cells can improve the phenotypic heterogeneity and modify cytokine production profile of iNKT cells expanded from 18 consecutive donors. The presence of IL-7 or IL-15 during antigenic stimulation did not affect the fold of expansion or purity of expanded iNKT cells. However, IL-7, but not IL-15, led to a better expansion of CD4+ iNKT cells, enhanced Th-2 type cytokine production of CD4+ iNKT cells, and maintained the expansion of central memory (CD45RA-CD62L+) CD4+ iNKT cells. Our results suggest the addition of IL-7 during antigenic stimulation with allogeneic dendritic cells can promote the expansion of CD62L+Th-2+CD4+ human iNKT cells that can be used as novel immunotherapeutic to control excessive inflammation to treat various autoimmune diseases.

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