Preformed Cytokines Research Articles

Regulation of IFN-γ production by ZFP36L2 in T cells is time-dependent.

CD8+ T cells kill target cells by releasing cytotoxic molecules and proinflammatory cytokines, such as TNF and IFN-γ. The magnitude and duration of cytokine production are defined by posttranscriptional regulation, and critical regulator herein are RNA-binding proteins (RBPs). Although the functional importance of RBPs in regulating cytokine production is established, the kinetics and mode of action through which RBPs control cytokine production are not well understood. Previously, we showed that the RBP ZFP36L2 blocks the translation of preformed cytokine encoding mRNA in quiescent memory T cells. Here, we uncover that ZFP36L2 regulates cytokine production in a time-dependent manner. T cell-specific deletion of ZFP36L2 (CD4-cre) had no effect on T-cell development or cytokine production during early time points (2-6 h) of T-cell activation. In contrast, ZFP36L2 specifically dampened the production of IFN-γ during prolonged T-cell activation (20-48 h). ZFP36L2 deficiency also resulted in increased production of IFN-γ production in tumor-infiltrating T cells that are chronically exposed to antigens. Mechanistically, ZFP36L2 regulates IFN-γ production at late time points of activation by destabilizing Ifng mRNA in an AU-rich element-dependent manner. Together, our results reveal that ZFP36L2 employs different regulatory nodules in effector and memory T cells to regulate cytokine production.

Human monocytes store and secrete preformed CCL5, independent of de novo protein synthesis.

Monocytes are a subset of circulating peripheral blood mononuclear cells with diverse roles in immunity, including sentinel roles in cytokine secretion. Conventionally, cytokines require an inductive stimulus for their expression and secretion, resulting in a time lag from the time of stimulation to when the proteins are packaged and secreted. Because cytokines are the main communicators in the immune system, their temporal expression is a key factor in coordinating responses to efficiently resolve infection. Herein, we identify that circulating human monocytes contain preformed cytokines that are stored intracellularly, in both resting and activated states. Having preformed cytokines bypasses the time lag associated with de novo synthesis, allowing monocytes to secrete immune mediators immediately upon activation or sensing of microbe-associated molecular patterns. We demonstrate here that, out of several cytokines evaluated, human monocytes contain a previously undescribed reservoir of the preformed chemokine CCL5. Furthermore, we showed that CCL5 could be secreted from monocytes treated with the protein synthesis inhibitor (cycloheximide) and Golgi blocker (brefeldin A). We examined the possibility for uptake of extracellular CCL5 from platelet aggregates and observed no significant levels of platelet binding to our enriched monocyte preparations, indicating that the source of preformed CCL5 was not from platelets. Preformed CCL5 was observed to be distributed throughout the cytoplasm and partially colocalized with CD63+ and Rab11A+ membranes, implicating endosomal compartments in the intracellular storage and trafficking of CCL5.

Stabilization of cytokine mRNAs in iNKT cells requires the serine-threonine\xa0kinase IRE1alpha

Activated invariant natural killer T (iNKT) cells rapidly produce large amounts of cytokines, but how cytokine mRNAs are induced, stabilized and mobilized following iNKT activation is still unclear. Here we show that an endoplasmic reticulum stress sensor, inositol-requiring enzyme 1α (IRE1α), links key cellular processes required for iNKT cell effector functions in specific iNKT subsets, in which TCR-dependent activation of IRE1α is associated with downstream activation of p38 MAPK and the stabilization of preformed cytokine mRNAs. Importantly, genetic deletion of IRE1α in iNKT cells reduces cytokine production and protects mice from oxazolone colitis. We therefore propose that an IRE1α-dependent signaling cascade couples constitutive cytokine mRNA expression to the rapid induction of cytokine secretion and effector functions in activated iNKT cells.

Mast cells in atherosclerotic cardiovascular disease – Activators and actions

Mast cells are potent actors involved in inflammatory reactions in various tissues, including both in the intimal and the adventitial layers of atherosclerotic arteries. In the arterial intima, the site of atherogenesis, mast cells are activated to degranulate, and thereby triggered to release an abundance of preformed inflammatory mediators, notably histamine, heparin, neutral proteases and cytokines stored in their cytoplasmic secretory granules. Depending on the stimulus, mast cell activation may also launch prolonged synthesis and secretion of single bioactive molecules, such as cytokines and derivatives of arachidonic acid. The mast cell-derived mediators may impede the functions of different types of cells present in atherosclerotic lesions, and also compromise the structural and functional integrity of the intimal extracellular matrix. In the adventitial layer of atherosclerotic coronary arteries, mast cells locate next to peptidergic sensory nerve fibers, which, by releasing neuropeptides may activate mast cells to release vasoactive compounds capable of triggering local vasoconstriction. The concerted actions of arterial mast cells have the potential to contribute to the initiation and progression of atherosclerosis, and ultimately to destabilization and rupture of an advanced atherosclerotic plaque with ensuing atherothrombotic complications.

Homeostatic Eosinophils: Characteristics and Functions.

Eosinophils are typically considered to be specialized effector cells that are recruited to the tissues as a result of T helper type 2 (Th2) cell responses associated with helminth infections or allergic diseases such as asthma. Once at the site of injury, eosinophils release their cytotoxic granule proteins as well as preformed cytokines and lipid mediators, contributing to parasite destruction but also to exacerbation of inflammation and tissue damage. Accumulating evidence indicates that, besides their roles in Th2 responses, eosinophils also regulate homeostatic processes at steady state, thereby challenging the exclusive paradigm of the eosinophil as a destructive and inflammatory cell. Indeed, under baseline conditions, eosinophils rapidly leave the bloodstream to enter tissues, mainly the gastrointestinal tract, lungs, adipose tissue, thymus, uterus, and mammary glands, where they regulate a variety of important biological functions, such as immunoregulation, control of glucose homeostasis, protection against obesity, regulation of mammary gland development, and preparation of the uterus for pregnancy. This article provides an overview of the characteristics and functions of these homeostatic eosinophils.

Human mast cells capture, store, and release bioactive, exogenous IL-17A.

IL-17A, a major proinflammatory cytokine, can be produced by a variety of leukocytes, but its exact cellular source in human inflammatory diseases remains incompletely understood. IL-17A protein is abundantly found in mast cells in human tissues, such as inflamed synovium, but surprisingly, mechanistic murine studies failed to demonstrate IL-17A production by mast cells. Here, we demonstrate that primary human tissue mast cells do not produce IL-17A themselves but actively capture exogenous IL-17A through receptor-mediated endocytosis. The exogenous IL-17A is stored in intracellular granules and can subsequently be released in a bioactive form. This novel mechanism confers to mast cells the capacity to steer IL-17A-mediated tissue inflammation by the rapid release of preformed cytokine.

IL-16 and MIF: messengers beyond neutrophil cell death.

Most cytokines are synthesized de novo upon appropriate stimulation, packed into vesicles and secreted from the producing cell. However, cells can also contain preformed cytokines, which means that these cytokines are synthesized prior to activation and stored inside the cell. Harboring of preformed effector molecules is essential for cells of low transcription and translation activity, as seen in neutrophils. Being part of the first line of defense against invading pathogens, neutrophils are rapidly recruited from the circulation into the infected tissue, where they immediately execute a multitude of antimicrobial effector functions.1 Several antimicrobial effector substances are stored inside neutrophils as preformed molecules. This allows a fast action, as these preformed substances can act on demand, without the need of time-consuming de novo synthesis. The presence of preformed cytokines in neutrophils emphasizes that these cells are not solely fast-acting effector cells but they can also regulate very rapidly the inflammatory and immune responses.2 Recently, we reported the storage of IL-16 and MIF in primary human neutrophils in the new journal Cell Death Discovery.3 Analysis of neutrophil granule fractions and confocal immune fluorescent microscopy revealed that IL-16 and MIF are stored in the cytosol rather than in neutrophil granules.3 Notably, IL-16 is synthesized as a biologically inactive precursor, pre-IL-16 (formerly also termed pro-IL-16), which is processed in a caspase-3-dependent manner. The C-terminal fragment IL-16C is the biologically active, secreted pro-inflammatory cytokine.4 Intracellular proteolytic activation by cytosolic proteases has also been described for other pro-inflammatory cytokines such as IL-18, IL-1β and IL-33.5, 6 Neutrophils are short-lived cells. After undergoing apoptosis they are cleared from the circulation in liver, spleen and bone marrow. Although their life span is extended at sites of infection/inflammation, large numbers of neutrophils undergo apoptosis after executing their effector functions. Apoptotic cells and blebs maintain their membrane integrity and are cleared by phagocytes in a process called efferocytosis. Since apoptotic cells/blebs are surrounded by an intact cell membrane, this process prevents the leakage/release of potentially harmful cellular content of neutrophils into the surrounding tissue. In case of insufficient or impaired efferocytosis, however, apoptotic neutrophils undergo secondary necrosis, resulting in the leakage of cell content through the disintegrated plasma membrane. Therefore, harboring preformed biologically active molecules not only favors rapid effector functions, but also bears the risk of uncontrolled release during primary necrosis or secondary necrosis. This passive release may lead to tissue damage, but is also believed to contribute to the development of autoimmunity to/against neutrophil constituents.7 The secretion mechanism of IL-16C and MIF from neutrophils, but also other cells, remains largely elusive. As both cytokines lack the signal peptide to be transferred to the ER, they are thought to be released by unconventional secretion mechanisms, as known for IL-1 and FGF2.8, 9 Previous studies hypothesized that IL-16 and MIF could be released actively from the producing cells.10, 11 However, our data indicate that in neutrophils both cytokines are released passively after the cells undergo secondary necrosis3 (Figure 1). After the screening of a large panel of substances, among others, TLR ligands, pro-inflammatory cytokines, lipid mediators, glucocorticoids, and the bacteria Escherichia coli and Staphylococcus aureus, no release of IL-16C and MIF was observed from viable or apoptotic neutrophils.3 Instead, release of IL-16C and MIF correlated strongly with the secondary necrosis of neutrophils.3 Figure 1 Storage, processing and release of IL-16 and MIF in human neutrophils. MIF and IL-16 are preformed cytokines stored in the cytosol of human neutrophils. During apoptosis, the precursor of IL-16 (pre-IL-16) is processed into the biologically active IL-16C ... As neutrophils release IL-16C and MIF only after undergoing secondary necrosis, it is tempting to speculate that IL-16C and MIF function as danger signals in case of insufficient clearance of apoptotic neutrophils. At sites of acute infection or inflammation, efferocytosis may not be sufficient to clear the large numbers of apoptotic neutrophils. Consequently, apoptotic neutrophils undergo secondary necrosis. Although the release of IL-16C and MIF from secondary necrotic neutrophils may be required to maintain and modulate the local defense and inflammatory functions, we have to keep in mind that they may also be detrimental in conditions of autoimmunity. Neutrophils, although known for a long time and studied extensively, are more complex and sophisticated in their functions than was imaginable one or two decades ago. By the release of neutrophil extracellular traps, they can fulfill antimicrobial effector functions even beyond their death.12 The study published in Cell Death Discovery3 extends this view by demonstrating that neutrophils can fulfill even their regulatory functions beyond their life span.

Systemic Inflammation Recruits Ccr7+ Dendritric-Biased Granulocyte-Macrophage Progenitors to Lymphatic Circulation in a Non-Canonical Traf6-Dependent Manner

A small fraction of hematopoietic stem and progenitor cells (HSPCs) is cyclically released into the bloodstream from bone marrow (BM). The existence of HSCP-lymphatic-blood circulation with the potential to give rise to extramedullary tissue-resident myeloid cells has been described. However, the functional role and underlying mechanisms of this circulation are not known. We hypothesized that inflammation may be a mediator of HSPC mobilization to lymph. In order to determine if inflammation was related to increased circulation of HSPCs through lymph, we analyzed the HSPC content in lymph nodes (LN) of patients with lymphadenitis. Analysis of CFU and side population (SP+) cell content confirmed that LNs from patients with reactive lymphadenitis and patients with inflammatory Hodgkin's disease contained ~2-fold higher HSPCs than in patients diagnosed of non-Hodgkin's lymphoma. To identify the pattern and mechanisms that regulate HSPCs during inflammation. We analyzed the content of HSPCs in blood (PB), BM, LN, thoracic duct (TD) and other organs after systemic administration of lipopolysaccharide (LPS) and compared with the normal circadian migratory rhythms in control animals (PBS). Mobilization of BM myeloid progenitors (mostly granulo-macrophage progenitors-GMPs, but not stem cells), to LN and TD peaked as early as three hours after LPS administration, and followed a very different pattern than the mobilization kinetics in PB. By using 3D reconstruction of confocal microscopy imaging of complete LN from lymphatic endothelium reporter mice (Lyve1Cre-eGFP), we localized all myeloid progenitors in the mantle zone of secondary follicles. We note that they did not enter the follicle germ centers. To determine the molecular mechanisms at play to induce HSPC mobilization to the LN upon LPS administration, we analyzed whether the recruitment of HSPCs in LN required Traf6, a mediator downstream of the LPS/TLR4 signaling pathway. Inducible deficiency of Traf6 driven by Mx1-Cre recombinase completely abolished the migration of myeloid progenitors to LN, but not to PB, induced by LPS as compared with their Mx1-Cre;Wt littermates indicating that the mechanism controlling HSPC recruitment to the LN was distinct from the one controlling HSPC migration to PB. Using a combination of in vivo and in vitro assays, we found that Traf6 activity regulates HSPC migration in a non-cell autonomous manner that depends on its expression in a small (~1%) fraction of non-progenitor LN myeloid cells expressing high levels of Ccl19. In vivo neutralization of Ccr7, the receptor for Ccl19, results in abrogation of myeloid progenitor mobilization to LN. GMPs migrating to LN were biased to differentiate into dendritic cells within 7 days post-migration. Using Cx3cr1-GFP transgenic mice as macrophage-dendritic cell progenitor (MDP) reporters, we found that the vast majority of GMPs recruited to the LN were MDPs, and thus already poised to DC differentiation. Finally, a combination of genetic and pharmacological approaches revealed that Traf6 signaling effects are NFkB independent but dependent upon Irak1/4, Ucb13 and IKKβ resulting in SNAP23 phosphorylation and exocytosis of pre-formed cytokines from LN myeloid cells. This study identifies the cellular and molecular basis of inflammation dependent migration of DC progenitors and suggests that the mobilization of HSPCs from BM to LN results in homeostatic replenishment of highly specialized antigen presenting cells. DisclosuresNo relevant conflicts of interest to declare.

Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol.

Neutrophils harbor a number of preformed effector proteins that allow for immediate antimicrobial functions without the need for time-consuming de novo synthesis. Evidence indicates that neutrophils also contain preformed cytokines, including interleukin (IL)-1ra, CXCL8 and CXCL2. In the search for additional preformed cytokines, a cytokine array analysis identified IL-16 and macrophage migration inhibitory factor (MIF) as preformed cytokines in lysates from human primary neutrophils. Both IL-16 and MIF are unconventional cytokines because they lack a signal sequence. Using confocal immunofluorescence microscopy as well as western blot analysis of subcellular fractions, IL-16 and MIF were found to be stored in the cytosol rather than in the granules of human neutrophils, which implies an unconventional secretion mechanism for both cytokines. IL-16 is synthesized and stored as a precursor (pre-IL-16). We present evidence that the processing of pre-IL-16 to the biologically active IL-16C is mediated by caspase-3 and occurs during both spontaneous and UV-induced apoptosis of human neutrophils. Although IL-16 processing occurs during apoptosis, IL-16C and MIF release was observed only during secondary necrosis of neutrophils. Screening a panel of microbial substances and proinflammatory cytokines did not identify a stimulus that induced the release of IL-16C and MIF independent of secondary necrosis. The data presented here suggest that IL-16 and MIF are neutrophil-derived inflammatory mediators released under conditions of insufficient clearance of apoptotic neutrophils, as typically occurs at sites of infection and autoimmunity.

Editorial: secretion of cytokines and chemokines by innate immune cells.

Editorial: secretion of cytokines and chemokines by innate immune cells.

Cytokine Secretion in Macrophages: SNAREs, Rabs, and Membrane Trafficking.

Macrophages have the capacity to rapidly secrete a wide range of inflammatory mediators that influence the development and extent of an inflammatory response. Newly synthesized and/or preformed stored cytokines and other inflammatory mediators are released upon stimulation, the timing, and volume of which is highly regulated. To finely tune this process, secretion is regulated at many levels; at the level of transcription and translation and post-translationally at the endoplasmic reticulum (ER), Golgi, and at or near the cell surface. Here, we discuss recent advances in deciphering these cytokine pathways in macrophages, focusing on recent discoveries regarding the cellular machinery and mechanisms implicated in the synthesis, trafficking, and secretion of cytokines. The specific roles of trafficking machinery including chaperones, GTPases, cytoskeletal proteins, and SNARE membrane fusion proteins will be discussed.

Natural killer cells induce eosinophil activation and apoptosis.

Eosinophils are potent inflammatory cells with numerous immune functions, including antigen presentation and exacerbation of inflammatory responses through their capacity to release a range of largely preformed cytokines and lipid mediators. Thus, timely regulation of eosinophil activation and apoptosis is crucial to develop beneficial immune response and to avoid tissue damage and induce resolution of inflammation. Natural Killer (NK) cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and eosinophils. Co-culture experiments revealed that human NK cells could trigger autologous eosinophil activation, as shown by up-regulation of CD69 and down-regulation of CD62L, as well as degranulation, evidenced by increased CD63 surface expression, secretion of eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN). Moreover, NK cells significantly and dose dependently increased eosinophil apoptosis as shown by annexin V and propidium iodide (PI) staining. Direct contact was necessary for eosinophil degranulation and apoptosis. Increased expression of phosphorylated extracellular signal-regulated kinase (ERK) in cocultured eosinophils and inhibition of eosinophil CD63 expression by pharmacologic inhibitors suggest that MAPK and PI3K pathways are involved in NK cell-induced eosinophil degranulation. Finally, we showed that NK cells increased reactive oxygen species (ROS) expression by eosinophils in co-culture and that mitochondrial inhibitors (rotenone and antimycin) partially diminished NK cell-induced eosinophil apoptosis, suggesting the implication of mitochondrial ROS in NK cell-induced eosinophil apoptosis. Pan-caspase inhibitor (ZVAD-FMK) only slightly decreased eosinophil apoptosis in coculture. Altogether, our results suggest that NK cells regulate eosinophil functions by inducing their activation and their apoptosis.

Barrier-repair prescription moisturizers: Do we really need them? Facts and controversies

There is now scientific evidence of genetically driven skin-barrier anomalies in atopic patients. These barrier anomalies facilitate sustained antigen ingress through the defective barrier, which can bring about a Th2-dominant response. It enhances the transepidermal water loss, resulting in dry skin and leading to the release of preformed proinflammatory cytokines and to a cascade of events ending up in inflammation.

Eosinophil‐derived cytokines in health and disease: unraveling novel mechanisms of selective secretion

Over the past two decades, our understanding of eosinophils has evolved from that of categorically destructive effector cells to include active participation in immune modulation, tissue repair processes, and normal organ development, in both health and disease. At the core of their newly appreciated functions is the capacity of eosinophils to synthesize, store within intracellular granules, and very rapidly secrete a highly diverse repertoire of cytokines. Mechanisms governing the selective secretion of preformed cytokines from eosinophils are attractive therapeutic targets and may well be more broadly applicable to other immune cells. Here, we discuss recent advances in deciphering pathways of cytokine secretion, both from intact eosinophils and from tissue-deposited cell-free eosinophil granules, extruded from eosinophils undergoing a lytic cell death.

Mast Cells

Mast cells have long been known as effector cells in allergic disorders. By binding IgE, they rapidly release a plethora of preformed cytokines, chemokines, bradykinins, and other highly active substances that mediate bronchospasm and urticaria.[1][1] Over recent years, it has become clear that mast

Mast Cells Contribute to Scar Formation during Fetal Wound Healing

Scar formation is a potentially detrimental process of tissue restoration in adults, affecting organ form and function. During fetal development, cutaneous wounds heal without inflammation or scarring at early stages of development, but begin to heal with significant inflammation and scarring as the skin becomes more mature. One possible cell type that could regulate the change from scarless to fibrotic healing is the mast cell. We show here that dermal mast cells in scarless wounds generated at embryonic day 15 (E15) are fewer in number, less mature and do not degranulate in response to wounding as effectively as mast cells of fibrotic wounds made at embryonic day 18 (E18). Differences were also observed between cultured mast cells from E15 and E18 skin with regard to degranulation and preformed cytokine levels. Injection of mast cell lysates into E15 wounds disrupted scarless healing, suggesting that mast cells interfere with scarless repair. Finally, wounds produced at E18, which normally heal with a scar, healed with significantly smaller scars in mast cell-deficient KitW/W-v mice compared to Kit+/+ littermates. Together, these data suggest that mast cells enhance scar formation, and that these cells may mediate the transition from scarless to fibrotic healing during fetal development.

Modulation of Mast Cell and Basophil Functions by Benzene Metabolites

Benzene is a carcinogenic compound used in industrial manufacturing and a common environmental pollutant mostly derived from vehicle emissions and cigarette smoke. Benzene exposure is associated with a variety of clinical conditions ranging from hematologic diseases to chronic lung disorders. Beside its direct toxicity, benzene exerts multiple effects after being converted to reactive metabolites such as hydroquinone and benzoquinone. Mast cells and basophils are primary effector cells involved in the development of respiratory allergies such as rhinitis and bronchial asthma and they play an important role in innate immunity. Benzene and its metabolites can influence mast cell and basophil responses either directly or by interfering with other cells, such as T cells, macrophages and monocytes, which are functionally connected to mast cells and basophils. Hydroquinone and benzoquinone inhibit the release of preformed mediators, leukotriene synthesis and cytokine production in human basophils stimulated by IgE- and non IgE-mediated agonists. Furthermore, these metabolites reduce IgE-mediated degranulation of mast cells and the development of allergic lung inflammation in rats. Both in vitro and in vivo studies indicate that benzene metabolites alter biochemical and functional activities of other immunocompetent cells and may impair immune responses in the lung. These inhibitory effects of benzene metabolites are primarily mediated by interference with early transduction signals such as PI3 kinase. Together, currently available studies indicate that benzene metabolites interfere by multiple mechanisms with the role of basophils and mast cells in innate immunity and in chronic inflammation in the lung.

The Role of Eosinophils and Mast Cells in Intestinal Functional Disease

Functional gastrointestinal disorders (FGIDs) are common and currently defined by a symptom-based classification with no discernable pathology. In functional dyspepsia (FD), the duodenum is now implicated as a key area where symptoms originate.This is attributed to immune activation with increasing evidence indicating a role for duodenal eosinophilia. In irritable bowel syndrome (IBS), mastocytosis has been documented throughout the small and large intestine. Eosinophils and mast cells are an important link between innate and adaptive immunity, and are important in allergic type TH2 inflammation. Eosinophils may give rise to symptoms due to release of preformed cytokine proteins, which trigger neural excitation, muscle spasm, and pain. The close relationship of mast cells to nerves in IBS may similarly give rise to symptoms. Genetic studies also support of the role of innate immunity in FGIDs. The data supporting a prime role for eosinophils and mast cells in subsets of FD and IBS has become credible, and these data should be used to implement advances in diagnosis and therapeutic trials.

Differential Expression of Pre-formed Cytokines in Human Basophils and Eosinophils

RATIONALE: As basophils and eosinophils have both been implicated as mediators of Th2 immunity through rapid secretion of cytokines, we examined their differential pre-formed cytokine content to further delineate their respective roles in the induction of Th2 responses.

Interleukin-2-inducible T Cell Kinase (Itk) Network Edge Dependence for the Maturation of iNKT Cell

Invariant natural killer T (iNKT) cells are a unique subset of innate T lymphocytes that are selected by CD1d. They have diverse immune regulatory functions via the rapid production of interferon-γ (IFN-γ) and interleukin-4 (IL-4). In the absence of signaling nodes Itk and Txk, Tec family non-receptor tyrosine kinases, mice exhibit a significant block in iNKT cell development. We now show here that although the Itk node is required for iNKT cell maturation, the kinase domain edge of Itk is not required for continued maturation iNKT cells in the thymus compared with Itk-null mice. This rescue is dependent on the expression of the Txk node. Furthermore, this kinase domain independent edge rescue correlates with the increased expression of the transcription factors T-bet, the IL-2/IL-15 receptor β chain CD122, and suppression of eomesodermin expression. By contrast, α-galactosyl ceramide induced cytokine secretion is dependent on the kinase domain edge of Itk. These findings indicate that the Itk node uses a kinase domain independent edge, a scaffolding function, in the signaling pathway leading to the maturation of iNKT cells. Furthermore, the findings indicate that phosphorylation of substrates by the Itk node is only partially required for maturation of iNKT cells, while functional activation of iNKT cells is dependent on the kinase domain/activity edge of Itk.