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1.  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.
PMCID: PMC4470169  PMID: 25681342
migration; IL-4; allergic asthma
2.  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).
PMCID: PMC4772695  PMID: 26458831
RNA virus; RNA-dependent RNA polymerase; Polymerase fidelity; Live-attenuated virus; Vaccine; Attenuation; Poliovirus; Sequence homology
3.  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.
PMCID: PMC4352551  PMID: 25063868
Colitis; Foxp3; ICOS; IL-2; Tec kinase
4.  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.
PMCID: PMC4287144  PMID: 25568116
autoimmune encephalomyelitis; migration; multiple sclerosis; Th17; Treg
5.  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.
PMCID: PMC4033297  PMID: 24610010
Dendritic cell; IL-2-inducible T cell kinase; Innate memory phenotype; Major histocompatibility complex class II; Regulatory CD4+ T cells
6.  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.
PMCID: PMC4214770  PMID: 25360046
7.  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.
PMCID: PMC4286740  PMID: 25567129
8.  IL-2–inducible T-cell kinase modulates TH2-mediated allergic airway inflammation by suppressing IFN-γ in naive CD4+ T cells 
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.
We sought to better understand the role of Itk signaling in TH differentiation programs and in the development and molecular pathology of allergic asthma.
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.
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.
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.
PMCID: PMC4033298  PMID: 23768572
Tec kinase; TH2; allergic asthma; T-bet; Eomesodermin; IFN-γ
9.  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
10.  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.
PMCID: PMC4033300  PMID: 23454662
Tec kinase; G-protein; Btk homology domain; Serum response factor
11.  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.
PMCID: PMC4033301  PMID: 23408840
Antigen specific response; Innate memory phenotype; Homeostatic expansion; ITK; Non-conventional T cells
12.  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.
PMCID: PMC3846793  PMID: 24312486
13.  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.
PMCID: PMC3532926  PMID: 22981631
14.  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.
PMCID: PMC3337705  PMID: 22403441
Non-conventional T cells; Tec kinases; Fas; T cell development
15.  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.
PMCID: PMC3143500  PMID: 21558014
16.  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.
PMCID: PMC2941197  PMID: 20483745
17.  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.
PMCID: PMC2846297  PMID: 19948240
18.  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.
PMCID: PMC2846296  PMID: 19926338
19.  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.
PMCID: PMC2767186  PMID: 19818650
20.  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.
PMCID: PMC2770951  PMID: 19688746
Tec kinase; TFII-I; T cell signaling
21.  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.
PMCID: PMC2820903  PMID: 20016000
22.  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
23.  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.
PMCID: PMC2893210  PMID: 20596543
24.  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
25.  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

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