Invariant Natural Killer T-Cell Control of Type 1 Diabetes: A Dendritic Cell Genetic Decision of a Silver Bullet or Russian Roulette

OBJECTIVE: In part, activation of invariant natural killer T (iNKT)-cells with the superagonist alpha-galactosylceramide (alpha-GalCer) inhibits the development of T-cell-mediated autoimmune type 1 diabetes in NOD mice by inducing the downstream differentiation of antigen-presenting dendritic cells (DCs) to an immunotolerogenic state. However, in other systems iNKT-cell activation has an adjuvant-like effect that enhances rather than suppresses various immunological responses. Thus, we tested whether in some circumstances genetic variation would enable activated iNKT-cells to support rather than inhibit type 1 diabetes development. RESEARCH DESIGN AND METHODS: We tested whether iNKT-conditioned DCs in NOD mice and a major histocompatibility complex-matched C57BL/6 (B6) background congenic stock differed in capacity to inhibit type 1 diabetes induced by the adoptive transfer of pathogenic AI4 CD8 T-cells. RESULTS: Unlike those of NOD origin, iNKT-conditioned DCs in the B6 background stock matured to a state that actually supported rather than inhibited AI4 T-cell-induced type 1 diabetes. The induction of a differing activity pattern of T-cell costimulatory molecules varying in capacity to override programmed death-ligand-1 inhibitory effects contributes to the respective ability of iNKT-conditioned DCs in NOD and B6 background mice to inhibit or support type 1 diabetes development. Genetic differences inherent to both iNKT-cells and DCs contribute to their varying interactions in NOD and B6.H2(g7) mice. CONCLUSIONS: This great variability in the interactions between iNKT-cells and DCs in two inbred mouse strains should raise a cautionary note about considering manipulation of this axis as a potential type 1 diabetes prevention therapy in genetically heterogeneous humans

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Extracellular NAD and ATP: Partners in immune cell modulation

Extracellular NAD and ATP exert multiple, partially overlapping effects on immune cells. Catabolism of both nucleotides by extracellular enzymes keeps extracellular concentrations low under steady-state conditions and generates metabolites that are themselves signal transducers. ATP and its metabolites signal through purinergic P2 and P1 receptors, whereas extracellular NAD exerts its effects by serving as a substrate for ADP-ribosyltransferases (ARTs) and NAD glycohydrolases/ADPR cyclases like CD38 and CD157. Both nucleotides activate the P2X7 purinoceptor, although by different mechanisms and with different characteristics. While ATP activates P2X7 directly as a soluble ligand, activation via NAD occurs by ART-dependent ADP-ribosylation of cell surface proteins, providing an immobilised ligand. P2X7 activation by either route leads to phosphatidylserine exposure, shedding of CD62L, and ultimately to cell death. Activation by ATP requires high micromolar concentrations of nucleotide and is readily reversible, whereas NAD-dependent stimulation begins at low micromolar concentrations and is more stable. Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and non-lytic mechanisms, and may serve as ‘danger signals–to alert the immune response to tissue damage. Since ART expression is limited to naïve/resting T cells, P2X7-mediated NAD-induced cell death (NICD) specifically targets this cell population. In inflamed tissue, NICD may inhibit bystander activation of unprimed T cells, reducing the risk of autoimmunity. In draining lymph nodes, NICD may eliminate regulatory T cells or provide space for the preferential expansion of primed cells, and thus help to augment an immune response

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes

Molecular mechanisms and function of ADP-ribosyltransferase ART2 mediated apoptosis in murine T lymphocytes

Die NAD-abhängige ADP-Ribosylierung ist - ähnlich der Phosphorylierung - eine posttranslationale Protein-Modifikation. Über diesen Mechanismus entfalten Cholera-, Pertussis- und andere bakterielle Toxine ihre pathogene Wirkung. Unsere Arbeitsgruppe hat toxinverwandte Mono-ADP-Ribosyltransferasen (ART1-ART5) bei Säugetieren kloniert, die als sezernierte oder GPI-verankerte Ektoenzyme exprimiert werden. Diese Arbeit konzentriert sich auf die murine ADP-Ribosyltransferase ART2, die von ruhenden T-Zellen auf der Zelloberfläche exprimiert wird und nach T-Zell Aktivierung von der Zelloberfläche abgestoßen werden kann. Die Inkubation von ART2-exprimierenden T-Zellen mit NAD, dem ART-Substrat, führt zur ADP-Ribosylierung wichtiger Membranproteine und kann die Apoptose der Zelle auslösen. Im Rahmen dieser Arbeit wurden die molekularen Mechanismen des NAD induzierten Zelltodes (NICD) untersucht. Durch vergleichende Untersuchungen von Wildtyp- und ART2 defizienten Mäusen und mit Hilfe von Blockade-Experimenten mittels monoklonaler Antikörper oder spezifischer Inhibitoren konnte gezeigt werden, dass ART2 und der Purinozeptor P2X7 essentiell für die NAD-vermittelte Signaltransduktion sind. P2X7 ist ein ATP-gesteuerter nicht-selektiver Kationen-Kanal, der als Membranprotein die Cytoplasmamembran zweimal durchspannt. Es konnte gezeigt werden, dass durch ART2 katalysierte ADP-ribosylierung von P2X7 ein kovalent gebundener Ligand präpariert, und P2X7 hierdurch dauerhaft aktiviert wird. Mit Dosis-Wirkungs-Analysen konnte gezeigt werden, dass NAD (über die ART2-katalyiserte ADP-Ribosylierung) P2X7 bereits in 10-fach geringerer Konzentration aktivieren kann als der lösliche Ligand ATP. Pulse-Chase-Experimente zeigten, dass das Entfernen von ATP nach kurzen Inkubationen zur Deaktivierung von P2X7 führt. Kurze Inkubationen mit NAD hingegen führen durch ADP-Ribosylierung zur Bildung eines kovalent gebundenen Liganden, der auch nach Entfernen des NAD P2X7 dauerhaft aktiviert. ATP- und NAD-vermittelte Aktivierung von P2X7 führen zu klassischen Zeichen der T-Zell Apoptose: das nach außen Kehren des Membranlipids Phosphatidylserin von der Innenseite der Zytoplasmamembran auf die Außenseite, Schrumpfen der Zellen, Caspaseaktivierung, Zusammenbruch der mitochondrialen Membranintegrität und Fragmentierung der DNA. Mit Hilfe von Durchfluss-zytometrischen Echt-Zeit-Untersuchungen konnte aber gezeigt werden, dass anders als bei klassischen Apoptosewegen das nach außen Kehren des Phosphatidylserins bereits nach wenigen Sekunden induziert wird. Darüber hinaus wurden zwei Lymphomzelllinien identifiziert, die sich als Zellkulturmodelle für den NAD induzierten Zelltod eignen und nützliche Werkzeuge für weiterführende Untersuchungen darstellen. Es ist denkbar dass NAD und ATP aus lysierten oder beschädigten Zellen freigesetzt werden. In dieser Arbeit konnte gezeigt werden, dass Erythrozytenlysate ausreichende Mengen an NAD und auch ATP enthalten um P2X7 zu aktivierten. Durch vergleichende Untersuchungen von Wildtyp und ART2-defizienten T-Zellen konnte darüber hinaus gezeigt werden, dass NAD auch in höheren Verdünnungen der Lysate wirksam war, während nur stark konzentrierte Lysate ausreichende ATP Konzentrationen enthielten. Beide Nukleotide unterlagen in den Lysaten einer raschen Degradation. Es konnte ferner gezeigt werden, dass bereits bei der Präparation von T-Zellen aus Lymphknoten NAD, nicht aber ATP, in ausreichenden Mengen zur Aktivierung von P2X7 freigesetzt werden. Vergleichende Untersuchungen eingezüchteter Mausstämme zeigten, dass der Anteil „spontan“ apoptotischer Zellen in frischen T-Zell-Präparationen mit dem Expressionsniveau bzw. Genotyp von ART2 und P2X7 eng korreliert. Das übliche Schicksal apoptotischer Zellen in vivo ist die Entfernung durch Phagozyten – noch bevor das Todesprogramm vollständig abgelaufen ist. Dabei wird das nach außen Kehren von Phosphatidylserin als ein „friß mich“-Signal für Makrophagen gewertet. In dieser Arbeit konnte gezeigt werden, dass T-Zellen nach Aktivierung von P2X7 durch NAD oder ATP durch Peritonealmakrophagen tatsächlich innerhalb von einer Stunde phagozytiert werden. Die Ergebnisse dieser Arbeit unterstützen die Vorstellung, dass der NAD induzierte Zelltod ein Mechanismus für die Eliminierung von naiven T-Zellen in Situationen von Gewebeverletzungen oder in Entzündungsherden sein könnte. Bereits aktivierte T-Zellen, die ART2 von der Oberfläche abgestoßen haben, sind resistent gegenüber extrazellulärem NAD, während potentiell autoreaktive Bystanderzellen ART2 auf der Zelloberfläche tragen und somit eliminiert werden können

Pharmacokinetics and Pharmacodynamics after Subcutaneous Dosing of the iNKT Cell Depleting Antibody NKTT120

Abstract The iNKT cell (invariant Natural Killer T cell) is a unique immune cell that has been shown to be associated with chronic inflammation in sickle cell disease. One approach to reducing iNKT mediated inflammation would be to reduce the number of iNKT cells in the tissue of SCD patients. NKTT120 is a humanized monoclonal antibody directed to the unique T cell receptor of invariant NKT cells that can deplete iNKT cells by antibody dependent cellular cytotoxicity. It recently completed a first in man Phase 1 study that demonstrated that intravenous single doses in adults with SCD specifically reduced iNKT cells without dose limiting toxicity in the therapeutic range of 0.3 and 1.0 mg/kg. Although intravenous dosing was found to be effective and safe, the ability to dose patients by subcutaneous administration would be an asset to a chronic treatment strategy and patient compliance. The purpose of this study was to assess the bioavailability and the efficacy of NKTT120 administered subcutaneously (SC) to cynomolgus monkeys. Female adult cynomolgus monkeys were administered NKTT120 as a single subcutaneous dose of 0.03, 0.1, 0.3 mg/kg (n=3 each) or as a single intravenous (IV) dose of 0.3 mg/kg (n=3). Animals were monitored for iNKT cell depletion of peripheral blood by FACS analysis and NKTT120 concentration was monitored by ELISA. All doses resulted in complete depletion of blood iNKT cells without any change in other blood cells. The return of measureable peripheral blood iNKT cells occurred in each group when the plasma concentration was ≤ 100 ng/mL. The time to recovery was dose dependent with measureable iNKT cells found at 4-6 weeks with the 0.03 mg/kg dose and 8-14 weeks with the 0.3 mg/kg IV and SC doses. NKTT120 was dose proportional with time to maximum plasma concentration of 48 hours following SC dosing. Bioavailability of NKTT120 after SC dosing was ~60% and T1/2 was 15 days for the IV dose group and ranged from 11-19 days for the SC dose groups. The duration of iNKT cell depletion and time to recovery for the 0.3 mg/kg SC dose was similar to that observed for the 0.3 mg/kg IV dose group. The results support a SC dosing strategy for NKTT120 and suggest that similar pharmacodynamic responses could be expected when a therapeutic intravenous dose (0.3 mg/kg) is administered by the subcutaneous route in SCD patients every 3-4 months. Plasma Concentration vs. Time for NKTT120 Following IV or SC doses to Cynomolgus Monkeys Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Schaub: NKT Therapeutics: Employment, Equity Ownership. Thariath:NKT Therapeutics: Employment, Equity Ownership. Scheuplein:NKT Therapeutics: Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership. </jats:sec

Mouse Invariant TCR Specific Monoclonal Antibody NKT14: A Novel Tool To Manipulate Invariant NKT Cell Function In Vivo

Abstract The iNKT cell represents a novel therapeutic target for important hematologic diseases such as sickle cell disease (SCD) and myeloma. While an antibody specifically targeting human iNKT cells is now in a clinical trial, no surrogate reagent that specifically recognizes murine iNKT cells has been previously reported. This abstract defines work on a unique, recently developed antibody specifically directed to the T cell receptor of the mouse iNKT cell. These cells are a small subset of T lymphocytes that share characteristics with adaptive as well as innate immune cells. In contrast to conventional T cells they recognize glycolipid antigens presented on the MHC-I like molecule CD1d. Upon activation they can rapidly release either pro-inflammatory or anti-inflammatory cytokines, depending on stimulus and microenvironment. This enables them to direct downstream immune functions into inflammatory or tolerizing modes. iNKT cell activation has been implicated as a mediator of the chronic inflammation that is found in patients with SCD (Field et al. Blood 121:3321, 2013) suggesting that reduction of activity or iNKT cell depletion may be an effective therapy. The activation of iNKT cells has been shown to have therapeutics effects in multiple hematologic tumors including myeloma, lymphoma, and leukemia (Dhodapkar and Richter Clin.Immunol.140:160, 2011). Until now, the role of iNKT cells in immune regulation has been studied using iNKT cell deficient inbred mouse strains like CD1d and Ja18 knockout mice or with the iNKT cell activating agent alpha-Galactosyl-Ceramide (aGalCer). These tools have weaknesses and limitations. CD1d deficient mice are not only deficient in invariant NKT cells but also other CD1d restricted cells, such as Type 2 NKT cells. Ja18 knockout mice have recently been shown to have a substantial decrease in TCR diversity in addition to their iNKT cell deficiency (Bedel et al.,Nat Immunol. 2012 Jul 19;13(8):705-6.). Furthermore, these mouse strains lack iNKT cells from birth and little is known about pharmacologic suppression in iNKT cell competent mouse strains. Although aGalCer can be used to activate iNKT cells in vivo, it induces a persistent iNKT cell anergy after activation. NKT Therapeutics has developed human iNKT cell specific humanized monoclonal antibodies, one of which is currently being evaluated in a Phase I study in patients with sickle cell disease. The human iNKT cell specific antibodies are not cross-reactive to murine iNKT cells. In order to better understand the potential of pharmacologic modulation of iNKT cell function in pre-clinical disease models, we developed a mouse iNKT specific monoclonal antibody. We have a generated both a depleting version (NKT-14) and by manipulating the FC-function through mutations we have also generated a non-depleting, activating version (NKT-14m). Both are highly specific for mouse iNKT cells and recognize all aGalCer -loaded CD1d tetramer binding cells (Fig. 1A) in multiple inbred mouse strains tested (C57BL/6, BALB/c, NOD, DBA, C3H,NZW, NZW/NZB F1, AKR, SJL and A/J). NKT-14 rapidly and very specifically depletes iNKT cells in vivo (Fig. 1B). NKT-14m can activate iNKT cells in vivo and induces release if IFn-Gamma (Fig. 1C). These novel mouse invariant TCR specific monoclonal antibodies will allow us to better understand the role of iNKT cells in health and disease in order to inform clinical trials of therapeutics which manipulate these unique immune regulatory cells for the treatment of disease. Disclosures: Scheuplein: NKT Therapeutics: Employment. Thariath:NKT Therapeutics: Employment. Mashal:NKT Therapeutics: Employment, Equity Ownership. Schaub:NKT Therapeutics: Employment. </jats:sec

Antibody Mediated Depletion of iNKT Cells Protects Against Hypoxia-Induced Pulmonary Injury in a Murine Model of Sickle Cell Disease

Abstract Background: Painful vaso-occlusive crises (pVOC) are caused by the polymerization of sickle deoxyhemoglobin, red cell sickling and vaso-occlusion. Vaso-occlusion is exacerbated by an inflammatory cascade that is initiated by iNKT cell activation. Townes sickle cell mice have mouse globins replaced by human globins, including the mutant human sickle b-globin gene (SS). NKT-14 is a monoclonal antibody directed specifically to the mouse invariant T cell receptor (iTCR). The antibody is a chimeric mouse IgG2a that, when bound to the mouse iNKT cell, promotes a rapid, specific and long lasting (&gt; 14 days) depletion. Aims: Our primary goal was to determine if selective iNKT cell depletion with NKT-14 reduces lung inflammation and injury in response to hypoxia-induced RBC sickling in Townes SS mice. Methods: Hypoxic pulmonary injury was induced by placing mice in 8% O2 (Coy Lab products)followed by reoxygenation. Some mice were subject to a second period of hypoxia and continuously monitored to determine carotid Hb-saturation (Starr MouseOx). Total cell numbers and activation state of iNKT cells and neutrophils in lung were determined by FACS. Pulmonary vascular leak was quantified with Evans Blue dye. Results: Twelve hours of 8% hypoxia followed 4 hours of reoxygenation (H/R) was found to produce non-lethal lung inflammation and injury in SS mice. Compared to isotype Ab controls, injection of four mice with 100µg/mouse of NKT-14 Ab three days prior to HR was found to decrease the number of pulmonary iNKT cells by 91% (P &lt; 0.01). Following H/R, NKT-14 Ab treatment resulted in a significant 65% decrease in lung neutrophil accumulation (Fig. A, P &lt; 0.01), and a 54% decrease in pulmonary vascular leak (Fig. B, P &lt; 0.05). In addition, arterial Hb-Sat during a second exposure of SS mice to hypoxia was improved (Fig. C). Figure 1 Figure 1. Conclusions: Selective depletion of mouse iNKT cells by injection of NKT-14 Ab three days prior to H/R results in substantial reductions in pulmonary inflammation, vascular leak and injury as measured by Hb-Sat in SS mice. The results suggest that prophylactic Ab-mediated depletion of iNKT cells in humans with SCD may be useful for reducing the incidence and severity of pVOC and acute chest syndrome. Disclosures Schaub: NKT Therapeutics Inc: Employment, Equity Ownership. Scheuplein:NKT Therapeutics Inc: Employment, Equity Ownership. Mashal:NKT Therapeutics Inc: Employment, Equity Ownership. </jats:sec

Abstract 4294: Cancer immunotherapeutic potential of NKTT320, a novel human invariant natural killer T-cell activating monoclonal antibody

Abstract Invariant natural killer T cells (iNKTs) directly kill tumor cells and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK), 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 antigen. 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. Towards this end, we characterized the iNKT cell stimulatory properties of NKTT320, a novel recombinant humanized monoclonal antibody that binds selectively and with high affinity to the human iTCR. Strikingly, both soluble and immobilized NKTT320 induced a dose-dependent iNKT cell activation (upregulation of activation markers [CD25, CD69]), proliferation (by measuring CFSE dilution), degranulation (measured by exposure of CD107 on the cell surface) and activation of bystander immune cells. Additionally, iNKT cells stimulated by plate-bound NKTT320 also robustly secreted Th1 (IL-1β, IFN-γ, TNF-α, IL-2, IL-6) and Th2-type (IL-4, IL-5, IL-10) cytokines as well as chemokines. Our in vitro studies are consistent with in vivo data in Vα24 transgenic mice, which express the human iTCR alpha chain. Dosing of NKTT320 in these animals led to iNKT cell activation and IFN-γ production, as well as incorporation of BRDU indicating in vivo iNKT cell proliferation. Similarly, NKT14m (a murine iNKT-agonistic antibody) induced murine iNKT cell activation, cytokine production, and degranulation in vitro. Consistently, treatment of C57BL/6 (B6) mice with NKT14m led to rapid and robust iNKT cell activation and IFN-γ production. Furthermore, in vivo injection of NKT 14m in B6 mice induced robust T, B, and NK cell activation as well as DC maturation (as assessed by increased expression of MHC class II and co-stimulatory molecules [CD80 and CD86]). Importantly, treatment of T-lymphoma-bearing B6 mice with a single dose of NKT14m substantially repressed the growth of subcutaneous tumors. Taken together, these data suggest that iNKT-activating antibodies hold great potential for use in cell-based cancer immunotherapy. Studies are underway to examine whether treatment with NKT14m in combination with interleukin-12 (a potent iNKT and NK cell activating cytokine) can further augment iNKT cell activation in vivo. These studies are significant, as they provide a framework by which iNKT cell anti-cancer functions could be enhanced for therapeutic purposes. Citation Format: Rupali Das, Felix Scheuplein, Peng Guan, Robert Schaub, Kim E. Nichols. Cancer immunotherapeutic potential of NKTT320, a novel human invariant natural killer T-cell activating monoclonal antibody. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4294. doi:10.1158/1538-7445.AM2015-4294</jats:p