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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  Differential sensitivity to Itk kinase signals for T helper 2 cytokine production and chemokine mediated migration* 
Allergic asthma is dependent on chemokine mediated Th2 cell migration and Th2 cytokine secretion into the lungs. The tyrosine kinase Itk regulates the production of Th2 cytokines as well as migration in response to chemokine gradients. Mice lacking Itk are resistant to developing allergic asthma, however, the role of kinase activity of Itk in the development of this disease is unclear. In addition, whether distinct Itk derived signals lead to T cell migration and secretion of Th2 cytokines is also unknown. Using transgenic mice specifically lacking Itk kinase activity, we show that active kinase signaling is required for control of Th2 responses and development of allergic asthma. Moreover, dominant suppression of Itk kinase activity led to normal Th2 responses, but significantly reduced chemokine mediated migration, resulting in prevention of allergic asthma. These observations indicate that signals required for Th2 responses and migration are differentially sensitive to Itk kinase activity. Manipulation of Itk’s kinase activity can thus provide a new strategy to treat allergic asthma by differentially affecting migration of T cells into the lungs, leaving Th2 responses intact.
PMCID: PMC2913463  PMID: 18322190
18.  Modeling Susceptibility versus Resistance in Allergic Airway Disease Reveals Regulation by Tec Kinase Itk 
PLoS ONE  2010;5(6):e11348.
Murine models of allergic asthma have been used to understand the mechanisms of development and pathology in this disease. In addition, knockout mice have contributed significantly to our understanding of the roles of specific molecules and cytokines in these models. However, results can vary significantly depending on the mouse strain used in the model, and in particularly in understanding the effect of specific knockouts. For example, it can be equivocal as to whether specific gene knockouts affect the susceptibility of the mice to developing the disease, or lead to resistance. Here we used a house dust mite model of allergic airway inflammation to examine the response of two strains of mice (C57BL/6 and BALB/c) which differ in their responses in allergic airway inflammation. We demonstrate an algorithm that can facilitate the understanding of the behavior of these models with regards to susceptibility (to allergic airway inflammation) (Saai) or resistance (Raai) in this model. We verify that both C57BL/6 and BALB/c develop disease, but BALB/c mice have higher Saai for development. We then use this approach to show that the absence of the Tec family kinase Itk, which regulates the production of Th2 cytokines, leads to Raai in the C57BL/6 background, but decreases Saai on the BALB/c background. We suggest that the use of such approaches could clarify the behavior of various knockout mice in modeling allergic asthma.
doi:10.1371/journal.pone.0011348
PMCID: PMC2893210  PMID: 20596543
19.  Selective expression rather than specific function of Txk and Itk regulate TH1 and TH2 responses§ 
Itk and Txk/Rlk are Tec family kinases expressed in T cells. Itk is expressed in both TH1 and TH2 cells. By contrast, Txk is preferentially expressed in TH1 cells. Although Itk is required for TH2 responses in vivo and Txk is suggested to regulate IFNγ expression and TH1 responses, it remains unclear whether these kinases have distinct roles in TH cell differentiation/function. We demonstrate here that Txk null CD4+ T cells are capable of producing both TH1 and TH2 cytokines similar to those produced by WT CD4+ T cells. To further examine whether Itk and Txk play distinct roles in TH cell differentiation and function we examined Itk-null mice carrying a transgene that expresses Txk at levels similar to the expression of Itk in TH2 cells. Using two TH2 model systems: allergic asthma and Schistosome egg-induced lung granulomas, we found that the Txk transgene rescued TH2 cytokine production and all TH2 symptoms without notable enhancement of IFNγ expression. These results suggest that Txk is not a specific regulator of TH1 responses. Importantly, they suggest that Itk and Txk exert their effects on TH cell differentiation/function at the level of expression.
PMCID: PMC2849304  PMID: 18941202
20.  Naïve and Innate Memory phenotype CD4+ T-cells have different requirements for active Itk for their development* 
The Tec family kinase Itk regulates the development of conventional and innate CD8+ T cells, however little is known about the role of Itk in the development of CD4+ T cell lineages, although the role of Itk in the T cell activation and function is well defined. We show here that the Itk null mice have increased percentage of CD62LLoCD44Hi memory phenotype CD4+ T cells compared to WT mice. These cells arise directly in the thymus, express high levels of transcripts for the T-bet and IFNγ and are able to produce IFNγ directly ex vivo in response to stimulation. Itk deficiency greatly decreases the number of CD4+ T cells with CD62LHiCD44Lo naïve phenotype, but has no effect on the number of memory phenotype CD4+ T cells, indicating that the development of memory phenotype CD4+ T cells is Itk independent. We further show that the development of the naïve phenotype CD4+ T cells is dependent on active Itk kinase signals and can be rescued by expression of Itk specifically in T cells. Our data also show that the Itk is required for functional TCR signaling in these cells, but not for the innate function in response to IL-12/IL-18 or L. monocytogenes stimulation. These results indicate that CD62LHiCD44Lo “naïve” and CD62LLoCD44Hi “innate memory phenotype” CD4+ T cells may be independent populations that differ in their requirement for Itk’s signals for development. Our data also suggest that CD4+CD62LLoCD44Hi memory phenotype T cells have innate immune function.
PMCID: PMC2836934  PMID: 18453573
Listeria monocytogenes; IFNγ; IL-12; T cell lineage
21.  Elevated non-specific immunity and normal Listeria clearance in young and old vitamin D receptor knockout mice 
International Immunology  2008;21(2):113-122.
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and the vitamin D receptor (VDR) are important regulators of autoimmunity. The effect of the VDR on the ability of mice to fight a primary or secondary infection has not been determined. Young and old VDR knockout (KO) mice were able to clear both primary and secondary infections with Listeria monocytogenes. However, the kinetics of clearance was somewhat delayed in the absence of the VDR. Memory T cell development was not different in young VDR KO and wild-type (WT) mice; however, old VDR KO mice had significantly less memory T cells than their WT counterparts but still mounted an adequate immune response as determined by the complete clearance of L. monocytogenes. Although the primary and secondary immune responses were largely intact in the VDR KO mice, the old VDR KO mice had increased cytokines and antibody responses compared with the old WT mice. In particular, old VDR KO mice had elevated antigen non-specific antibodies; however, these magnified immune responses did not correspond to more effective Listeria clearance. The increased antibody and cytokine responses in the old VDR KO mice are consistent with the increased susceptibility of these mice to autoimmunity.
doi:10.1093/intimm/dxn129
PMCID: PMC2721678  PMID: 19088060
aging; antibodies; 1,25-dihydroxyvitamin D3; Listeria monocytogenes
22.  Itk Derived Signals Regulate the Expression of Th-POK and Controls the Development of CD4+ T Cells 
PLoS ONE  2010;5(1):e8891.
T cell development is critically dependent on both the environment and signals delivered by the T cell Receptor (TCR). The Tec family kinase Itk has been suggested to be an amplifier of signals emanating from the TCR and the loss of Itk partially affects most stages of thymopoiesis. Loss of Itk also differentially affects the development of conventional vs. non-conventional or innate memory phenotype T cells. Here, we examine whether these lineage choices are affected by a combination of TCR affinity and Itk by analyzing mice lacking Itk and carrying two TCR transgenes with differing affinities, OT-II and DO11.10. Our results show that developing thymocytes receive a gradient of signals, DO11.10>OT-II>DO11.10/Itk−/−>OT-II/Itk−/−. We also show that the development of CD4+ T cells is controlled by TCR signaling via Itk, which regulates the expression of the transcription factor, Th-POK, an enforcement factor for CD4 commitment. This results in a reduction in CD4+ T cell development, and an increase in the development of MHC class II restricted TCR transgenic CD8+ T cells that resemble non-conventional or innate memory phenotype CD8 T cells. This alteration accompanies increased expression of Runx3 and its target genes Eomesodermin, Granzyme B and Perforin in Itk null OT-II CD4+ thymocytes. All together, these data suggest that Itk plays an important role in CD4/CD8 commitment by regulating signal thresholds for the lineage commitment. Our data also suggest that the lower level of TCR signaling that occurs with a low affinity TCR in the absence of Itk can redirect some MHC class II restricted CD4+ T cell to class II-restricted CD8+ innate memory phenotype T cells.
doi:10.1371/journal.pone.0008891
PMCID: PMC2811181  PMID: 20126642
23.  ITK Inhibitors in Inflammation and Immune-mediated Disorders 
Interleukin-2-inducible T cell kinase (ITK) is a non-receptor tyrosine kinase expressed in T cells, NKT cells and mast cells which plays a crucial role in regulating the T cell receptor (TCR), CD28, CD2, chemokine receptor CXCR4 and FcεR mediated signaling pathways. In T cells, ITK is an important mediator for actin reorganization, activation of PLCγ, mobilization of calcium, and activation of the NFAT transcription factor. ITK plays an important role in the secretion of IL-2, but more critically, also has a pivotal role in the secretion of Th2 cytokines, IL-4, IL-5 and IL-13. As such, ITK has been shown to regulate the development of effective Th2 response during allergic asthma as well as infections against parasitic worms. This ability of ITK to regulate Th2 responses, along with it's pattern of expression, has led to the proposal that it would represent an excellent target for Th2 mediated inflammation. We discuss here the possibilities and pitfalls of targeting ITK for inflammatory disorders.
PMCID: PMC2770952  PMID: 19689375
24.  Memory phenotype CD8+ T-cells with innate function selectively develop in the absence of active Itk 
European journal of immunology  2007;37(10):2892-2899.
Summary
T cells with a memory like phenotype and possessing innate immune function have been previously identified as CD8+CD44hi cells. These cells rapidly secrete IFNγ upon stimulation with IL-12/IL-18 and are involved in innate responses to infection with Listeria monocytogenes. The signals regulating these cells are unclear. The Tec kinase Itk regulates T cell activation and we report here that a majority of the CD8+ T cells in Itk null mice have a phenotype of CD44hi similar to memory like innate T cells. These cells are observed in mice carrying an Itk mutant lacking the kinase domain, indicating that active Tec kinase signaling suppresses their presence. These cells carry preformed message for and are able to rapidly produce IFNγ upon stimulation in vitro with IL-12/IL-18, and endow Itk null mice the ability to effectively respond to infection with L. monocytogenes or exposure to LPS by secretion of IFNγ. Transfer of these cells rescues the ability of IFNγ null mice to reduce bacterial burden following L. monocytogenes infection indicating that these cells are functional CD8+CD44hi T cells previously detected in vivo. These results indicate that active signals from Tec kinases regulate the development of memory like CD8+ T cells with innate function.
doi:10.1002/eji.200737311
PMCID: PMC2770953  PMID: 17724684
Listeria monocytogenes; Itk; IFNγ; IL-12
25.  Keeping the (Kinase) Party Going: SLP-76 and ITK Dance to the Beat 
The Tec-family protein tyrosine kinase IL-2–inducible T cell kinase (ITK) mediates T cell activation, as does the adaptor protein SLP-76 (SH2-domain–containing leukocyte protein of 76 kD), which forms a complex with ITK and other intracellular signaling enzymes. One of these enzymes is phospholipase C–γ1 (PLC-γ1), which mediates T cell receptor (TCR)–stimulated intracellular calcium mobilization leading to the activation of transcription factors such as nuclear factor of activated T cells. The Src-family tyrosine kinase Lck and the Syk-family tyrosine kinase ζ chain–associated protein kinase of 70 kD (ZAP-70), together with ITK, are necessary for the phosphorylation of PLC-γ1 in response to TCR stimulation. ITK is thought to phosphorylate a specific tyrosine residue of PLC-γ1 that is required for its activation. The mechanism of activation of ITK appears to involve the interaction between SLP-76 and ITK, which not only initiates ITK activity but is also important to maintain the kinase activity of ITK. This suggests that SLP-76 acts as more than a neutral adaptor in mediating T cell activation; SLP-76 also directly influences the kinase activity of ITK, allowing ITK to phosphorylate PLC-γ1.
doi:10.1126/stke.3962007pe39
PMCID: PMC2756487  PMID: 17652306

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