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1.  Cutting Edge: Drebrin regulated actin dynamics regulates IgE-dependent mast cell activation and allergic responses* 
Mast cells play critical roles in allergic responses, and calcium signaling controls the function of these cells, and a role for actin in regulating calcium influx into cells has been suggested. We have previously identified the actin reorganizing protein Drebrin as a target of the immunosuppressant BTP, which inhibits calcium influx into cells. We show here that Drebrin−/− mice exhibit reduced IgE-mediated histamine release and passive systemic anaphylaxis and Drebrin−/− mast cells also exhibit defects in FcεRI-mediated degranulation. Drebrin−/− mast cells exhibit defects in actin cytoskeleton organization and calcium responses downstream of the FcεRI, and agents that relieve actin reorganization rescue mast cell FcεRI induced degranulation. Our results indicate that Drebrin regulates the actin cytoskeleton and calcium responses in mast cells, thus regulating mast cell function in vivo.
doi:10.4049/jimmunol.1401442
PMCID: PMC4490998  PMID: 26056254
actin; BTP; calcium
2.  Differential Regulation of Th17 and T Regulatory Cell Differentiation by Aryl Hydrocarbon Receptor Dependent Xenobiotic Response Element Dependent and Independent Pathways 
Toxicological Sciences  2015;145(2):233-243.
The aryl hydrocarbon receptor (AHR) is regarded as an environmental sensor and has been shown to link environmental stresses with chronic inflammatory and autoimmune diseases. The AHR can be activated to regulate both the X/DRE (xenobiotic or dioxin response elements) as well as a non-X/DRE mediated pathway. Selective AHR modulators (SAhRMs) are recently identified compounds that activate non-X/DRE mediated pathway without activating the X/DRE-driven responses. Here, we have used 3 classes of AHR ligands; agonist, antagonist, and a SAhRM, to delineate the role of these AHR-driven pathways in T helper 17 (Th17)/T regulatory (Treg) regulation. We show that Th17 differentiation is primarily dependent on X/DRE-driven responses, whereas Treg differentiation can be suppressed by inhibiting non-X/DRE pathway. Using a model of Citrobacter rodentium infection, we further show that AHR agonist enhances Th17 production and promoted resolution of infection, whereas a SAhRM inhibited Th17 mediated responses with reduced resolution of infection. These data indicate that Th17/Treg function may be differentially regulated by SAhRMs that differentially activate the X/DRE and non-X/DRE mediated pathways, and point to a therapeutic strategy to leverage AHR function in the treatment of chronic inflammatory and autoimmune disease.
doi:10.1093/toxsci/kfv046
PMCID: PMC4471441  PMID: 25716673
AHR; Th17; Treg; SAhRMs
3.  Cutting Edge: STAT6 signaling in eosinophils is necessary for development of Allergic Airway Inflammation¶ 
Eosinophils are critical cellular mediators in allergic asthma and inflammation, however signals that regulate their functions are unclear. The transcription factor STAT6 regulates Th2 cytokine responses, acting downstream of IL-4 and -13. We have previously shown that eosinophil derived IL-13 plays an important role in the recruitment of T cells to the lung, and subsequent development of allergic asthma. However whether eosinophils respond to Th2 signals to control allergic airway inflammation is unclear. Here, we show that STAT6−/− eosinophils are unable to induce the development of allergic lung inflammation, including recruitment of CD4+ T cells, mucous production and development of airways hyperresponsiveness. This is likely due to the reduced migration of STAT6−/− eosinophils to the lung and in response to eotaxin. These data indicate that like Th cells, eosinophils need to respond to Th2 cytokines via STAT6 during the development of allergic airway inflammation.
doi:10.4049/jimmunol.1402096
PMCID: PMC4470169  PMID: 25681342
migration; IL-4; allergic asthma
4.  Polymerase Mechanism-Based Method of Viral Attenuation 
Vaccines remain the most effective way of preventing infection and spread of infectious diseases. These prophylactics have been used for centuries but still to this day only three main design strategies exist: (1) live attenuated virus (LAV) vaccines, (2) killed or inactivated virus vaccines, (3) and subunit vaccines of the three, the most efficacious vaccines remain LAVs. LAVs replicate in relevant tissues, elicit strong cellular and humoral responses, and often confer lifelong immunity. While this vaccine strategy has produced the majority of successful vaccines in use today, there are also important safety concerns to consider with this approach. In the past, the development of LAVs has been empirical. Blind passage of viruses in various cell types results in the accumulation of multiple attenuating mutations leaving the molecular mechanisms of attenuation unknown. Also, due to the high error rate of RNA viruses and selective pressures of the host environment, these LAVs, derived from such viruses, can potentially revert back to wild-type virulence. This not only puts the vaccinee at risk, but if shed can put those that are unvaccinated at risk as well. While these vaccines have been successful there still remains a need for a rational design strategy by which to create additional LAVs.
One approach for rational vaccine design involves increasing the fidelity of the viral RdRp. Increased fidelity decreases the viral mutational frequency thereby reducing the genetic variation the virus needs in order to evade the host imposed bottlenecks to infection. While polymerase mutants exist which decrease viral mutation frequency the mutations are not in conserved regions of the polymerase, which doesn’t lend itself toward using a common mutant approach toward developing a universal vaccine strategy for all RNA viruses. We have identified a conserved lysine residue in the active site of the PV RdRp that acts as a general acid during nucleotide incorporation. Mutation from a lysine to an arginine results in a high fidelity polymerase that replicates slowly thus creating an attenuated virus that is genetically stable and less likely to revert to a wild-type phenotype. This chapter provides detailed methods in which to identify the conserved lysine residue and evaluating fidelity and attenuation in cell culture (in vitro) and in the PV transgenic murine model (in vivo).
doi:10.1007/978-1-4939-3008-1_6
PMCID: PMC4772695  PMID: 26458831
RNA virus; RNA-dependent RNA polymerase; Polymerase fidelity; Live-attenuated virus; Vaccine; Attenuation; Poliovirus; Sequence homology
5.  Role(s) of IL-2 inducible T cell kinase and Bruton's tyrosine kinase in mast cell response to lipopolysaccharide 
Genomics Data  2016;8:18-20.
Mast cells play critical roles during immune responses to the bacterial endotoxin lipopolysaccharide (LPS) that can lead to fatal septic hypothermia [1], [2], [3]. IL-2 inducible T cell kinase (ITK) and Bruton's tyrosine kinase (BTK) are non-receptor tyrosine kinases that act downstream of numerous receptors, and have been shown to modulate mast cell responses downstream of FcεRIα [4], however, their roles in regulating mast cell responses to endotoxic stimuli were unclear. We found that the absence of ITK and BTK alters the mast cell response to LPS, and leads to enhanced pro-inflammatory cytokine production by mast cells and more severe LPS-induced hypothermia in mice [5]. Here, we detail our investigation using microarray analysis to study the transcriptomic profiles of mast cell responses to LPS, and the roles of ITK and/or BTK expression in this process. Mouse whole genome array data of WT, Itk−/−, Btk−/−, and Itk−/− Btk−/− bone marrow-derived mast cells (BMMCs) stimulated by PBS (control) or LPS for 1 h were used in our latest research article [5] and is available in the Gene Expression Omnibus under accession number GSE64287.
doi:10.1016/j.gdata.2016.02.010
PMCID: PMC4818347  PMID: 27081634
Mast cells; Kinases; Endotoxin; Inflammation; Immune regulation
6.  Post-transcriptional Regulation of Immunological Responses through Riboclustering 
Immunological programing of immune cells varies in response to changing environmental signals. This process is facilitated by modifiers that regulate the translational fate of mRNAs encoding various immune mediators, including cytokines and chemokines, which in turn determine the rapid activation, tolerance, and plasticity of the immune system. RNA-binding proteins (RBPs) recruited by the specific sequence elements in mRNA transcripts are one such modifiers. These RBPs form RBP–RNA complexes known as “riboclusters.” These riboclusters serve as RNA sorting machinery, where depending upon the composition of the ribocluster, translation, degradation, or storage of mRNA is controlled. Recent findings suggest that this regulation of mRNA homeostasis is critical for controlling the immune response. Here, we present the current knowledge of the ribocluster-mediated post-transcriptional regulation of immune mediators and highlight recent findings regarding their implications for the pathogenesis of acute or chronic inflammatory diseases.
doi:10.3389/fimmu.2016.00161
PMCID: PMC4850162  PMID: 27199986
inflammation; stress granules; polysomes; mRNA stability; T cell maturation; thymic and peripheral tolerance
7.  IL-2 inducible T cell kinase (ITK) tunes T regulatory cell development and is required for suppressive function1 
ITK is a key signaling mediator downstream of TcR, mediating T cell positive selection, innate T cell and CD4+ Th2/Th17 differentiation. Here we show that ITK also negatively tunes IL-2-induced expansion of Foxp3+ regulatory T cells (Treg). In vivo, Treg abundance is inversely correlated with ITK expression, and iTreg development is inversely dependent on ITK kinase activity. While Treg development normally requires both hematopoietic and thymic MHC class 2 (MHC2) expression, the absence of ITK allows Treg development with MHC2 expression in either compartment with preference for selection by thymic MHC2, suggesting a gatekeeper role of ITK in ensuring that only Treg cells selected by both thymic and hematopoietic MHC2 survive selection. Although ITK suppresses Treg development and is not required for maintenance of NRP1+ natural Treg cells in the periphery, it is indispensable for Treg functional suppression of naïve CD4+ T cell-induced colitis in Rag−/− recipients. ITK thus regulates the development and function of Treg cells.
doi:10.4049/jimmunol.1400968
PMCID: PMC4352551  PMID: 25063868
Colitis; Foxp3; ICOS; IL-2; Tec kinase
8.  Itk Signals Promote Neuroinflammation by Regulating CD4+ T-Cell Activation and Trafficking 
The Journal of Neuroscience  2015;35(1):221-233.
Here we demonstrate that interleukin-2-inducible T-cell kinase (Itk) signaling in cluster of differentiation 4-positive (CD4+) T cells promotes experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). We show that Itk−/− mice exhibit reduced disease severity, and transfer of Itk−/− CD4+ T cells into T cell-deficient recipients results in lower disease severity. We observed a significant reduction of CD4+ T cells in the CNS of Itk−/− mice or recipients of Itk−/− CD4+ T cells during EAE, which is consistent with attenuated disease. Itk−/− CD4+ T cells exhibit defective response to myelin antigen stimulation attributable to displacement of filamentous actin from the CD4+ coreceptor. This results in inadequate transmigration of Itk−/− CD4+ T cells into the CNS and across brain endothelial barriers in vitro. Finally, Itk−/− CD4+ T cells show significant reduction in production of T-helper 1 (Th1) and Th17 cytokines and exhibit skewed T effector/T regulatory cell ratios. These results indicate that signaling by Itk promotes autoimmunity and CNS inflammation, suggesting that it may be a viable target for treatment of MS.
doi:10.1523/JNEUROSCI.1957-14.2015
PMCID: PMC4287144  PMID: 25568116
autoimmune encephalomyelitis; migration; multiple sclerosis; Th17; Treg
9.  Dendritic cell-MHCII and Itk regulate functional development of regulatory innate memory CD4+ T cells in bone marrow transplantation1 
MHCII-influenced CD4+ T cell differentiation and function play critical roles in regulating the development of autoimmunity. The lack of hematopoietic MHCII causes autoimmune disease that leads to severe wasting in syngeneic recipients. Using murine models of bone marrow transplantation (BMT), we find that MHCII−/−→WT BMT developed disease, with defective development of innate memory phenotype (IMP, CD44hi/CD62Llo) CD4+ T cells. While conventional regulatory T cells (Treg) are unable to suppress pathogenesis, IMP CD4+ T cells, which include Tregs, can suppress pathogenesis in MHCII−/−→WT chimeras. The functional development of IMP CD4+ T cells requires hematopoietic but not thymic MHCII. B cells and hematopoietic CD80/86 regulate the population size, while MHCII expression by dendritic cells (DC) is sufficient for IMP CD4+ T cell functional development and prevention of pathogenesis. Furthermore, the absence of Tec kinase Itk in MHCII-/- donors leads to preferential development of IMP CD4+ T cells and partially prevents pathogenesis. We conclude that DC-MHCII and Itk regulate the functional development of IMP CD4+ T cells, which suppresses the development of autoimmune disorder in syngeneic BMTs.
doi:10.4049/jimmunol.1303176
PMCID: PMC4033297  PMID: 24610010
Dendritic cell; IL-2-inducible T cell kinase; Innate memory phenotype; Major histocompatibility complex class II; Regulatory CD4+ T cells
10.  People's instinctive travels and the paths to science 
Molecular Biology of the Cell  2014;25(21):3263-3266.
To be the recipient of the E. E. Just Award for 2014 is one of my greatest honors, as this is a truly rarefied group. In this essay, I try to trace my path to becoming a scientist to illustrate that multiple paths can lead to science. I also highlight that I did not build my career alone. Rather, I had help from many and have tried to pay it forward. Finally, as the country marches toward a minority majority, I echo the comments of previous E. E. Just Award recipients on the state of underrepresented minorities in science.
doi:10.1091/mbc.E14-06-1120
PMCID: PMC4214770  PMID: 25360046
11.  The tyrosine kinase Itk suppresses CD8+ memory T cell development in response to bacterial infection 
Scientific Reports  2015;5:7688.
Vaccine efficacy depends on strong long-term development of immune memory and the formation of memory CD8+ T cells is critical for recall responses to infection. Upon antigen recognition by naïve T cells, the strength of the TcR signal influences the subsequent effector and memory cells differentiation. Here, we have examined the role of Itk, a tyrosine kinase critical for TcR signaling, in CD8+ effector and memory T cell differentiation during Listeria monocytogenes infection. We found that the reduced TcR signal strength in Itk deficient naïve CD8+ T cells enhances the generation of memory T cells during infection. This is accompanied by increased early Eomesodermin, IL-7Rα expression and memory precursor effector cells. Furthermore, Itk is required for optimal cytokine production in responding primary effector cells, but not secondary memory responses. Our data suggests that Itk-mediated signals control the expression of Eomesodermin and IL-7Rα, thus regulating the development of memory CD8+ T cells, but not subsequent response of memory cells.
doi:10.1038/srep07688
PMCID: PMC4286740  PMID: 25567129
12.  IL-2–inducible T-cell kinase modulates TH2-mediated allergic airway inflammation by suppressing IFN-γ in naive CD4+ T cells 
Background
Asthma is a predominantly TH2 cell–dominated inflammatory disease characterized by airway inflammation and a major public health concern affecting millions of persons. The Tec family tyrosine kinase IL-2–inducible T-cell kinase (Itk) is primarily expressed in T cells and critical for the function and differentiation of TH cells. Itk−/− mice have a defective TH2 response and are not susceptible to allergic asthma.
Objective
We sought to better understand the role of Itk signaling in TH differentiation programs and in the development and molecular pathology of allergic asthma.
Methods
Using a murine model of allergic airway inflammation, we dissected the role of Itk in regulating TH cell differentiation through genetic ablation of critical genes, chromatin immunoprecipitation assays, and house dust mite–driven allergic airway inflammation.
Results
Peripheral naive Itk−/− CD4+ T cells have substantially increased transcripts and expression of the prototypic TH1 genes Eomesodermin, IFN-γ, T-box transcription factor (T-bet), and IL-12Rβ1. Removal of IFN-γ on the Itk−/− background rescues expression of TH2-related genes in TH cells and allergic airway inflammation in Itk−/− mice. Furthermore, small hairpin RNA–mediated knockdown of Itk in human peripheral blood T cells results in increased expression of mRNA for IFN-γ and T-bet and reduction in expression of IL-4.
Conclusion
Our results indicate that Itk signals suppress the expression of IFN-γ in naive CD4+ T cells, which in a positive feed-forward loop regulates the expression of TH1 factors, such as T-bet and Eomesodermin, and suppress development of TH2 cells and allergic airway inflammation.
doi:10.1016/j.jaci.2013.04.033
PMCID: PMC4033298  PMID: 23768572
Tec kinase; TH2; allergic asthma; T-bet; Eomesodermin; IFN-γ
13.  MicroRNA-570-3p regulates HuR and cytokine expression in airway epithelial cells 
Asthma is a chronic lung disease that affects people of all ages and is characterized by high morbidity. The mechanisms of asthma pathogenesis are unclear, and there is a need for development of diagnostic biomarkers and greater understanding of regulation of inflammatory responses in the lung. Post-transcriptional regulation of cytokines, chemokines, and growth factors by the action of microRNAs and RNA-binding proteins on stability or translation of mature transcripts is emerging as a central means of regulating the inflammatory response. In this study, we demonstrate that miR-570-3p expression is increased with TNFα stimuli in normal human bronchial epithelial cells (2.6 ± 0.6, p = 0.01) and the human airway epithelial cell line A549 (4.6 ± 1.4, p = 0.0068), and evaluate the functional effects of its overexpression on predicted mRNA target genes in transfected A549 cells. MiR-570-3p upregulated numerous cytokines and chemokines (CCL4, CCL5, TNFα, and IL-6) and also enhanced their induction by TNFα. For other cytokines (CCL2 and IL-8), the microRNA exhibited an inhibitory effect to repress their upregulation by TNFα. These effects were mediated by a complex pattern of both direct and indirect regulation of downstream targets by miR-570-3p. We also show that the RNA-binding protein HuR is a direct target of miR-570-3p, which has implications for expression of numerous other inflammatory mediators that HuR is known regulate post-transcriptionally. Finally, expression of endogenous miR-570-3p was examined in both serum and exhaled breath condensate (EBC) from asthmatic and healthy patients, and was found to be significantly lower in EBC of asthmatics and inversely correlated to their lung function. These studies implicate miR-570-3p as a potential regulator of asthmatic inflammation with potential as both a diagnostic and therapeutic target in asthma.
PMCID: PMC4138130  PMID: 25143867
MicroRNA; asthma; inflammation; airway; biomarker; cytokines; post-transcriptional regulation
14.  The zinc-binding region of IL-2 inducible T cell kinase (Itk) is required for interaction with Gα13 and activation of Serum Response Factor 
Tec family kinases play critical roles in the activation of immune cells. In particular, Itk is important for the activation of T cells via the T cell Receptor (TcR), however, molecules that cooperate with Itk to activate downstream targets remain little explored. Here we show that Itk interacts with the heterotrimeric G-protein ! subunit Gα13 during TcR triggering. This interaction requires membrane localization of both partners, and is partially dependent on GDP-and GTP-bound states of Gα13. Furthermore, we find that Itk interacts with Gα13 via the zinc binding regions within its Tec homology domain. The interaction between Itk and Gα13 also results in tyrosine phosphorylation of Gα13, however this is not required for the interaction. Itk enhances Gα13 mediated activation of Serum Response Factor (SRF) transcriptional activity dependent on its ability to interact with Gα13, but its kinase activity is not required to enhance SRF activity. These data reveal a new pathway regulated by Itk in cells, and suggest cross talk between Itk and G-protein signaling downstream of the TcR.
doi:10.1016/j.biocel.2013.02.011
PMCID: PMC4033300  PMID: 23454662
Tec kinase; G-protein; Btk homology domain; Serum response factor
15.  Cutting Edge: Innate memory CD8+ T cells are distinct from homeostatic expanded CD8+ T cells and rapidly respond to primary antigenic stimuli§ 
Innate memory phenotype (IMP) CD8+ T cells are non-conventional αβ T cells exhibiting features of innate immune cells, and are significantly increased in the absence of ITK. Their developmental path and function are not clear. Here we show hematopoietic MHCI dependent generation of antigen specific IMP CD8+ T cells using bone marrow chimeras. WT bone marrow gives rise to IMP CD8+ T cells in MHCI−/− recipients, resembling those in Itk−/− mice, but distinct from those derived via homeostatic proliferation (HP), and independent of recipient thymus. By contrast, MHCI−/− bone marrow does not lead to IMP CD8+ T cells in WT recipients. OTI IMP CD8+ T cells generated via this method exhibited enhanced early response to antigen without prior primary stimulation. Our findings suggest a method to generate antigen specific “naïve” CD8+ IMP T cells, demonstrate that they are not HP cells and can promptly respond in an antigen specific fashion.
doi:10.4049/jimmunol.1202988
PMCID: PMC4033301  PMID: 23408840
Antigen specific response; Innate memory phenotype; Homeostatic expansion; ITK; Non-conventional T cells
16.  Evaluation of the Stability, Bioavailability, and Hypersensitivity of the Omega-3 Derived Anti-Leukemic Prostaglandin: Δ12-Prostaglandin J3 
PLoS ONE  2013;8(12):e80622.
Previous studies have demonstrated the ability of an eicosapentaenoic acid (EPA)-derived endogenous cyclopentenone prostaglandin (CyPG) metabolite, Δ12-PGJ3, to selectively target leukemic stem cells, but not the normal hematopoietic stems cells, in in vitro and in vivo models of chronic myelogenous leukemia (CML). Here we evaluated the stability, bioavailability, and hypersensitivity of Δ12-PGJ3. The stability of Δ12-PGJ3 was evaluated under simulated conditions using artificial gastric and intestinal juice. The bioavailability of Δ12-PGJ3 in systemic circulation was demonstrated upon intraperitoneal injection into mice by LC-MS/MS. Δ12-PGJ3 being a downstream metabolite of PGD3 was tested in vitro using primary mouse bone marrow-derived mast cells (BMMCs) and in vivo mouse models for airway hypersensitivity. ZK118182, a synthetic PG analog with potent PGD2 receptor (DP)-agonist activity and a drug candidate in current clinical trials, was used for toxicological comparison. Δ12-PGJ3 was relatively more stable in simulated gastric juice than in simulated intestinal juice that followed first-order kinetics of degradation. Intraperitoneal injection into mice revealed that Δ12-PGJ3 was bioavailable and well absorbed into systemic circulation with a Cmax of 263 µg/L at 12 h. Treatment of BMMCs with ZK118182 for 12 h resulted in increased production of histamine, while Δ12-PGJ3 did not induce degranulation in BMMCs nor increase histamine. In addition, in vivo testing for hypersensitivity in mice showed that ZK118182 induces higher airways hyperresponsiveness when compared Δ12-PGJ3 and/or PBS control. Based on the stability studies, our data indicates that intraperitoneal route of administration of Δ12-PGJ3 was favorable than oral administration to achieve effective pharmacological levels in the plasma against leukemia. Δ12-PGJ3 failed to increase histamine and IL-4 in BMMCs, which is in agreement with reduced airway hyperresponsiveness in mice. In summary, our studies suggest Δ12-PGJ3 to be a promising bioactive metabolite for further evaluation as a potential drug candidate for treating CML.
doi:10.1371/journal.pone.0080622
PMCID: PMC3846793  PMID: 24312486
17.  Signal Transduction via the T cell Antigen Receptor in naïve and effector/memory T cells 
T cells play an indispensable role in immune defense against infectious agents, but can also be pathogenic. These T cells develop in the thymus, are exported into the periphery as naïve cells and participate in immune responses. Upon recognition of antigen, they are activated and differentiate into effector and memory T cells. While effector T cells carry out the function of the immune response, memory T cells can last up to the life time of the individual, and are activated by subsequent antigenic exposure. Throughout this life cycle, the T cell uses the same receptor for antigen, the T cell Receptor, a complex multi-subunit receptor. Recognition of antigen presented by peptide/MHC complexes on antigen presenting cells unleashes signaling pathways that control T cell activation at each stage. In this review, we discuss the signals regulated by the T cell receptor in naïve and effector/memory T cells.
doi:10.1016/j.biocel.2012.08.023
PMCID: PMC3532926  PMID: 22981631
18.  A unique role for ITK in survival of iNKT cells associated with the p53 dependent pathway in mice§ 
Invariant natural killer T (iNKT) cells play important roles in the immune response. ITK and TXK/RLK are Tec family kinases that are expressed in iNKT cells, with the expression level of ITK about 7-fold higher than that of TXK. Itk−/− mice have reduced iNKT cell frequency and numbers, with defects in development and cytokine secretion, which are exacerbated in the Itk/Txk DKO mice. By contrast, there is no iNKT cell defect in Txk−/− mice. To determine whether ITK and TXK play distinct roles in iNKT cell development and function, we examined mice that over express TXK in T cells to similar levels as Itk. Over expression of TXK rescues the maturation and cytokine secretion of Itk−/− iNKT cells, as well as altered expression of transcription factors T-bet, eomesodermin and PLZF. By contrast, the increased apoptosis observed in Itk−/− splenic iNKT cells is not affected by TXK over expression, likely due to lack of effect on the elevated expression of p53 regulated pro-apoptotic pathways Fas, Bax and Bad in those cells. Supporting this idea, p53−/− and Bax−/− mice have increased splenic iNKT cells. Our results suggest that TXK plays an overlapping role with ITK in iNKT cell development and function, but that ITK also has a unique function in the survival of iNKT cells, likely via a p53 dependent pathway.
doi:10.4049/jimmunol.1102475
PMCID: PMC3337705  PMID: 22403441
Non-conventional T cells; Tec kinases; Fas; T cell development
19.  Structural Requirements for the Inhibition of Calcium Mobilization and Mast Cell Activation by the Pyrazole Derivative BTP2 
Mast cells play a critical role in the development of the allergic response. Upon activation by allergens and IgE via the high affinity receptor for IgE (FcεRI), these cells release histamine and other functional mediators that initiate and propagate immediate hypersensitivity reactions. Mast cells also secrete cytokines that can regulate immune activity. These processes are controlled, in whole or part, by increases in intracellular Ca2+ induced by the FcεRI. We show here that N-(4-(3,5-bis(Trifluoromethyl)-1H-pyrazol-1-yl)phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP2), a pyrazole derivative, inhibits activation-induced Ca2+ influx in the rat basophil cell line RBL-2H3 and in bone marrow-derived mast cells (BMMCs), without affecting global tyrosine phosphorylation of cellular proteins or phosphorylation of the mitogen-activated protein kinases Erk1/2 and p38. BTP2 also inhibits activation-induced degranulation and secretion of Interleukin (IL)-2, IL-3, IL-4, IL-6, IL-13, Tumor Necrosis Factor (TNF)-α, and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) by BMMCs, which correlates with the inhibition of Nuclear Factor of Activated T cells (NFAT) translocation. In vivo, BTP2 inhibits antigen-induced histamine release. Structure-activity relationship analysis indicates that substitution at the C3 or C5 position of the pyrazole moiety on BTP2 (5-triflouromethyl-3-methyl-pyrazole or 3-triflouromethyl-5-methyl-pyrazole, respectively) affected its activity, with the trifluoromethyl group at the C3 position being critical to its activity. We conclude that BTP2 and related compounds may be potent modulators of mast cell responses and potentially useful for the treatment of symptoms of allergic inflammation.
doi:10.1016/j.biocel.2011.04.016
PMCID: PMC3143500  PMID: 21558014
20.  Differential roles of ITK-mediated TCR signals in tissue-specific localization and maintenance of skin intraepithelial T cells 
Tissue-specific innate-like γδ T cells are important components of the immune system critical for the first line of defense. But mechanisms underlying their tissue-specific development are poorly understood. Our study with prototypical skin-specific intraepithelial γδT lymphocytes (sIEL) found that among different thymic γδ T cell subsets, fetal thymic precursors of sIELs specifically acquire a unique skin-homing property after the positive selection, suggesting an important role of the TCR selection signaling in “programming” them for the tissue-specific development. Here we identified IL2-inducible T-cell kinase (ITK) as a critical signal molecule regulating the acquirement of the skin-homing property by the fetal thymic sIEL precursors. In ITK-knockout mice, the sIEL precursors could not undergo the positive selection-associated upregulation of thymus-exiting and skin-homing molecules S1PR1 and CCR10 and accumulated in the thymus. On the other hand, the survival and expansion of sIELs in the skin did not require the ITK-transduced TCR-signaling while its persistent activation impaired the sIEL development by inducing apoptosis. These findings provide insights into molecular mechanisms underlying differential requirements of the TCR signaling in peripheral localization and maintenance of the tissue specific T cells.
doi:10.4049/jimmunol.1000453
PMCID: PMC2941197  PMID: 20483745
21.  Chemico-Genetic Identification of Drebrin as a Regulator of Calcium Responses 
Store-operated calcium channels are plasma membrane Ca2+ channels that are activated by depletion of intracellular Ca2+ stores, resulting in an increase in intracellular Ca2+ concentration, which is maintained for prolonged periods in some cell types. Increases in intracellular Ca2+ concentration serve as signals that activate a number of cellular processes, however, little is known about the regulation of these channels. We have characterized the immuno-suppressant compound BTP, which blocks store-operated channel mediated calcium influx into cells. Using an affinity purification scheme to identify potential targets of BTP, we identified the actin reorganizing protein, drebrin, and demonstrated that loss of drebrin protein expression prevents store-operated channel mediated Ca2+ entry, similar to BTP treatment. BTP also blocks actin rearrangements induced by drebrin. While actin cytoskeletal reorganization has been implicated in store-operated calcium channel regulation, little is known about actin binding proteins that are involved in this process, or how actin regulates channel function. The identification of drebrin as a mediator of this process should provide new insight into the interaction between actin rearrangement and tore-operated channel mediated calcium influx.
doi:10.1016/j.biocel.2009.11.019
PMCID: PMC2846297  PMID: 19948240
22.  Eosinophils and allergic airway disease: there is more to the story 
Trends in immunology  2009;31(1):39-44.
The eosinophil has been perceived as a terminal effector cell in allergic airway diseases. However, recent work has shown that this multifunctional cell could be more involved in the initial stages of allergic disease development than was previously thought, particularly with regard to the ability of the eosinophil to modulate T-cell responses. In this review, we discuss recent advances that suggest that eosinophils can present antigen to naïve as well as to antigen experienced T cells, induce T helper 2 cell development, cytokine production or both, and affect T-cell migration to sites of inflammation. These findings are changing the way that eosinophil function in disease is perceived, and represent a shift in the dogma of allergic disease development.
doi:10.1016/j.it.2009.10.001
PMCID: PMC2846296  PMID: 19926338
23.  Differential expression of IL-17A and IL-17F is coupled to TCR signaling via Itk-mediated regulation of NFATc1 
Immunity  2009;31(4):587-597.
Th17 cells play major roles in autoimmunity and bacterial infections, yet how T cell receptor (TCR) signaling affects Th17 differentiation is relatively unknown. We demonstrate that CD4+ T cells deficient in Itk, a tyrosine kinase required for full TCR-induced activation of PLC-γ, exhibit decreased IL-17A expression, yet relatively normal expression of RORγT, RORα and IL-17F. IL-17A expression was rescued by pharmacologically-induced Ca2+ influx or expression of activated NFATc1. Conversely, decreased TCR stimulation or FK506 treatment preferentially reduced expression of IL-17A. The promoter of IL-17A but not IL-17F has conserved NFAT binding sites that bind NFATc1 in WT, but not Itk-deficient cells, even though both promoters exhibit epigenetic modifications consistent with open chromatin. Finally, defective IL-17A expression and differential regulation of IL-17A and IL-17F were observed in vivo in Itk−/− mice in an allergic asthma model. Our results suggest that Itk specifically couples TCR signaling strength to IL-17A expression through NFATc1.
doi:10.1016/j.immuni.2009.07.009
PMCID: PMC2767186  PMID: 19818650
24.  ITK finds another (dance) partner…TFII-I 
European journal of immunology  2009;39(9):2354-2357.
The signals that regulate T cell activation have been studied for some time. We know that upon interaction with antigen/MHC complex, the T cell Receptor (TCR) triggers the activation of a number of kinases, including tyrosine and serine/threonine kinases. The Tec family kinase ITK plays a role in this response, but the signaling pathways it regulates are less well known. Even less well known are its binding partners and substrates. Sacristan and colleagues have now extended the known partners and substrates of ITK by reporting in this issue of the European Journal of Immunology, that ITK interacts with the transcriptional regulator TFII-I. The implications of this finding are discussed.
doi:10.1002/eji.200939813
PMCID: PMC2770951  PMID: 19688746
Tec kinase; TFII-I; T cell signaling
25.  Activation of the N-Terminally Truncated Form of the Stk Receptor Tyrosine Kinase Sf-Stk by Friend Virus-Encoded gp55 Is Mediated by Cysteine Residues in the Ecotropic Domain of gp55 and the Extracellular Domain of Sf-Stk ▿  
Journal of Virology  2009;84(5):2223-2235.
Friend virus induces an erythroleukemia in susceptible mice that is initiated by the interaction of the Friend virus-encoded glycoprotein gp55 with the erythropoietin (Epo) receptor and the product of the host Fv2 gene, a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). We have previously demonstrated that the activation of Sf-Stk, recruitment of a Grb2/Gab2/Stat3 signaling complex, and induction of Pu.1 expression by Stat3 are required for the development of the early stage of Friend disease both in vitro and in vivo. Here we demonstrate that the interaction of gp55 with Sf-Stk is dependent on cysteine residues in the ecotropic domain of gp55 and the extracellular domain of Sf-Stk. Point mutation of these cysteine residues or deletion of these domains inhibits the ability of gp55 to interact with Sf-Stk, resulting in the inability of these proteins to promote the Epo-independent growth of erythroid progenitor cells. We also demonstrate that the interaction of gp55 with Sf-Stk does not promote dimerization of Sf-Stk but results in enhanced phosphorylation of Sf-Stk and the relocalization of Sf-Stk from the cytosol to the plasma membrane. Finally, we demonstrate that a constitutively active form of Sf-Stk (Sf-StkM330T), as well as its human counterpart, Sf-Ron, promotes Epo-independent colony formation in the absence of gp55 and that this response is also dependent on the cysteines in the extracellular domains of Sf-StkM330T and Sf-Ron. These data suggest that the cysteines in the extracellular domains of Sf-Stk and Sf-Ron may also mediate the interaction of these truncated receptors with other cellular factors that regulate their ability to promote cytokine-independent growth.
doi:10.1128/JVI.02090-09
PMCID: PMC2820903  PMID: 20016000

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