Interleukin-2-inducible T-cell Kinase Research Articles

Conformational Selectivity of ITK Inhibitors: Insights from Molecular Dynamics Simulations.

Interleukin-2-inducible T-cell kinase (ITK) regulates the response to T-cell receptor signaling and is a drug target for inflammatory and immunological diseases. Molecules that bind preferentially to the active form of ITK have low selectivity between kinases, whereas those that bind preferentially to the inactive form have high selectivity for ITK. Therefore, computational methods to predict the conformational selectivity of compounds are required to design highly selective ITK inhibitors. In this study, we performed absolute binding free-energy perturbation (ABFEP) simulations for 11 compounds on both active and inactive forms of ITK, and the calculated binding free energies were compared with experimental data. The conformational selectivity of 10 of the 11 compounds was correctly predicted using ABFEP. To investigate the mechanism underlying the stabilization of the active and inactive structures by the compounds, we performed extensive, conventional molecular dynamics simulations, which revealed that the compound-induced stabilization of the P-loop and linkage of conformational changes in L489, V419, F501, and M410 upon compound binding were critical factors. A guideline for designing inactive-form binders is proposed based on these key structural factors. The ABFEP and the created guidelines are expected to facilitate the discovery of highly selective ITK inhibitors.

Targeting kinase ITK treats autoimmune arthritis via orchestrating T cell differentiation and function

IL-2 inducible T cell kinase (ITK) is critical in T helper subset differentiation and its inhibition has been suggested for the treatment of T cell-mediated inflammatory diseases. T follicular helper (Tfh), Th17 and regulatory T cells (Treg) also play important roles in the development of rheumatoid arthritis (RA), while the role of ITK in the development of RA and the intricate balance between effector T and regulatory T cells remains unclear. Here, we found that CD4+ T cells from RA patients presented with an elevated ITK activation. ITK inhibitor alleviated existing collagen-induced arthritis (CIA) and reduced antigen specific antibody production. Blocking ITK kinase activity interferes Tfh cell generation. Moreover, ITK inhibitor effectively rebalances Th17 and Treg cells by regulating Foxo1 translocation. Furthermore, we identified dihydroartemisinin (DHA) as a potential ITK inhibitor, which could inhibit PLC-γ1 phosphorylation and the progression of CIA by rebalancing Th17 and Treg cells. Out data imply that ITK activation is upregulated in RA patients, and therefore blocking ITK signal may provide an effective strategy to treat RA patients and highlight the role of ITK on the Tfh induction and RA progression.

Overcoming immune checkpoint blockade resistance in solid tumors with intermittent ITK inhibition

Cytotoxic CD8 + T cell (CTL) exhaustion is driven by chronic antigen stimulation. Reversing CTL exhaustion with immune checkpoint blockade (ICB) has provided clinical benefits in different types of cancer. We, therefore, investigated whether modulating chronic antigen stimulation and T-cell receptor (TCR) signaling with an IL2-inducible T-cell kinase (ITK) inhibitor, could confer ICB responsiveness to ICB resistant solid tumors. In vivo intermittent treatment of 3 ICB-resistant solid tumor (melanoma, mesothelioma or pancreatic cancer) with ITK inhibitor significantly improved ICB therapy. ITK inhibition directly reinvigorate exhausted CTL in vitro as it enhanced cytokine production, decreased inhibitory receptor expression, and downregulated the transcription factor TOX. Our study demonstrates that intermittent ITK inhibition can be used to directly ameliorate CTL exhaustion and enhance immunotherapies even in solid tumors that are ICB resistant.

Identification of IL-2 inducible tyrosine kinase inhibitors by quantum mechanics and ligand based virtual screening approaches

Interleukin-2-inducible T-cell kinase (ITK) is a crucial intracellular signaling mediator in normal and malignant T-cells and natural killer cells. Selective inhibition of ITK might be useful for treating a variety of disorders including; autoimmune, inflammatory, and neoplastic disorders. Over the past two decades, the clinical management of ITK inhibitors has progressed dramatically. So far, specific inhibitor with no off-target effects against ITK is available. Herein, we aim to discover potential virtual hits to fasten the process of drug design and development against ITK. In this regard, the key chemical characteristics of ITK inhibitors were identified using ligand-based pharmacophore modeling. The validated pharmacophore comprises one hydrogen bond donor and three hydrogen bond acceptors and was utilized as a 3D query in virtual screening using ZINC, Covalent, and in-house databases. A total of 12 hit compounds were chosen on the basis of their critical interactions with the significant amino acids of ITK. The orbital energies such as HOMO and LUMO of the hit compounds were calculated to evaluate the inhibitor’s potencies. Further, molecular dynamics simulation demonstrated the stability of ITK upon binding of selected virtual hits. Binding energy using the MMGBSA method showed the potential binding affinity of all the hits with ITK. The research identifies key chemical characteristics with geometric restrictions that lead to ITK inhibition. Communicated by Ramaswamy H. Sarma

ITK inhibition improves the response to immune checkpoint blockade in solid tumors

Abstract Cytotoxic CD8+ T cell (CTL) exhaustion is driven by persistent T cell receptor (TCR) stimulation in chronic infections and tumors. The exhausted state of T cells is characterized by impaired effector function and proliferative capacity, upregulation of multiple inhibitory receptors and altered transcriptional profiles. The phenotypic and epigenetic changes of these cells hinder effective antitumor immunity and immunotherapy. Therefore, reinvigorating exhausted T cells may provide significant clinical benefits to patients with cancer. Our approach is to reduce chronic TCR stimulation by inhibiting IL2-inducible T-cell kinase (ITK), a pivotal component of TCR signaling. In our study, we found that intermittent ITK inhibitor treatment significantly improved ICB response in three immune checkpoint blockade (ICB) -resistant solid tumors (melanoma, mesothelioma or pancreatic cancer) in vivo. In vitro, we demonstrated that specific ITK inhibitor and ibrutinib, an inhibitor of Bruton’s tyrosine kinase (BTK) and ITK, directly reversed the exhaustion-related phenotypes in exhausted CTLs. The changes included enhanced cytokine production, decreased inhibitory receptor expression and changes in transcription factors. Subsequently, RNA-seq data revealed that exhaustion-related transcriptional profile of in vitro CTLs is reduced by ibrutinib treatment. Ibrutinib mediated reversal of CTL exhaustion was BTK independent. To conclude, our study demonstrates that ITK inhibition can be used to directly ameliorate CTLs exhaustion and overcomes immune checkpoint blockade resistance in solid tumors. This work was supported by the department of Immunology, KWF Grant 128371 (PDK), in part from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779295 (PDK), and by the China Scholarship Council for funding PhD fellowships (No. 201506160120 for MZ and 201906210055 for LL).

Regulation of Cx43 Gap Junction Intercellular Communication by Bruton's Tyrosine Kinase and Interleukin-2-Inducible T-Cell Kinase.

T and B cell receptor signaling involves the activation of Akt, MAPKs, and PKC as well as an increase in intracellular Ca2+ and calmodulin activation. While these coordinate the rapid turnover of gap junctions, also implicated in this process is Src, which is not activated as part of T and B cell receptor signaling. An in vitro kinase screen identified that Bruton's tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) phosphorylate Cx43. Mass spectroscopy revealed that BTK and ITK phosphorylate Cx43 residues Y247, Y265, and Y313, which are identical to the residues phosphorylated by Src. Overexpression of BTK or ITK in the HEK-293T cells led to increased Cx43 tyrosine phosphorylation as well as decreased gap junction intercellular communication (GJIC) and Cx43 membrane localization. In the lymphocytes, activation of the B cell receptor (Daudi cells) or T cell receptor (Jurkat cells) increased the BTK and ITK activity, respectively. While this led to increased tyrosine phosphorylation of Cx43 and decreased GJIC, the cellular localization of Cx43 changed little. We have previously identified that Pyk2 and Tyk2 also phosphorylate Cx43 at residues Y247, Y265, and Y313 with a similar cellular fate to that of Src. With phosphorylation critical to Cx43 assembly and turnover, and kinase expression varying between different cell types, there would be a need for different kinases to achieve the same regulation of Cx43. The work presented herein suggests that in the immune system, ITK and BTK have the capacity for the tyrosine phosphorylation of Cx43 to alter the gap junction function in a similar manner as Pyk2, Tyk2, and Src.

Abstract 1770: CD28-Y218 phosphorylation in CAR-T cells is mediated by the IL-2-inducible T-cell kinase (ITK)

Abstract CD28-derived co-stimulation modules are frequently used as part of the signaling domain of chimeric antigen receptors (CAR). These modules contain well characterized tyrosine-containing moieties (YMNM and PYAP) that become phosphorylated upon CAR activation, and result in IL-2 secretion and T cell expansion. Previous work by our group has shown that an additional tyrosine residue, located in the C-terminus of CD28 (Y218), was also phosphorylated upon CAR activation. The kinase responsible for this phosphorylation remains unknown, but in silico studies point towards the IL-2-inducible T-cell kinase (ITK), the protein-tyrosine kinase TEC and the bone marrow tyrosine kinase (BMX). In this work we sought to elucidate the molecular mechanism and functional relevance of CD28-Y218 phosphorylation in CAR-T cells.To analyze the kinetics of Y218 phosphorylation, prostate stem cell antigen (PSCA)-specific second-generation CAR-T cells were stimulated through co-culture with HPAC pancreatic cancer cells for 0, 1, 10, 30 and 60 minutes. Phosphorylation of CD28 Y218, Zap70, PLCγ and Vav1 was analyzed by Western blotting. To determine the role of ITK in CD28-218 phosphorylation: 1) PSCA-specific CAR-T cells were treated with ITK inhibitors 10 μM (BMS-509744 or Ibrutinib) for 24h and 2) ITK-deficient Jurkat cells expressing a PSCA-specific CAR were cocultured with HPAC cells for 0 or 10 min. To determine the functional relevance of this phosphorylation event, we generated mutant CARs where the tyrosine residue was replaced with a non-phosphorylatable amino acid (Y218F) and evaluated its effects in vivo and in vitro. CAR-T cell cytotoxicity was monitored using the xCELLigence Real Time Cytotoxicity Assay (RTCA) and cytokine production was assessed following overnight co-culture with target cells using the ELLA system (Biotechne, CA, USA). To evaluate the in vivo antitumor efficacy of PSCA-specific CAR-T cells, NSG mice were injected (s.c.) with 1 HPAC cells and infused 14 days later with 5 CAR-T cells (i.v.). Tumor growth was monitored for 35 days.Our results show that phosphorylation of CD28-Y218 occurs after the phosphorylation of early TCR signaling mediators, reaching maximum levels around 10-30 min post-stimulation. Pharmacological inhibition and genomic ablation of ITK impairs CD28-Y218 phosphorylation, suggesting an essential role of ITK in this signaling event. We observed that mutation of the Y218 residue did not affect CAR expression, in vitro cytotoxicity, or INFγ production. However, this mutation was associated with reduced IL-2 and TNFα secretion. In vivo, this mutation completely abrogated the therapeutic effect of CAR-T cells in a pancreatic cancer model. In conclusion, these results indicate that ITK is required for CD28-Y218 phosphorylation, a signaling event that plays an important role in CAR-T cell function, with direct impact on IL-2 secretion and antitumor efficacy. Citation Format: Elena Martinez-Planes, Miguel G. Fontela, Cecilia Ramello, Daniel Abate-Daga. CD28-Y218 phosphorylation in CAR-T cells is mediated by the IL-2-inducible T-cell kinase (ITK) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1770.

ITK degradation to block T cell receptor signaling and overcome therapeutic resistance in T cell lymphomas

Interleukin (IL)-2-inducible Tcell kinase (ITK) is essential for Tcell receptor (TCR) signaling and plays an integral role in Tcell proliferation and differentiation. Unlike the ITK homolog BTK, no inhibitors of ITK are currently US Food and Drug Administration (FDA) approved. In addition, recent studies have identified mutations within BTK that confer resistance to both covalent and non-covalent inhibitors. Here, as an alternative strategy, we report the development of BSJ-05-037, a potent and selective heterobifunctional degrader of ITK. BSJ-05-037 displayed enhanced anti-proliferative effects relative to its parent inhibitor BMS-509744, blocked the activation of NF-kB/GATA-3 signaling, and increased the sensitivity of Tcell lymphoma cells to cytotoxic chemotherapy both invitro and invivo. In summary, targeted degradation of ITK is a novel approach to modulate TCR signal strength that could have broad application for the investigation and treatment of Tcell-mediated diseases.

Discovery of Potent and Highly Selective Interleukin-2-Inducible T-Cell Kinase Degraders with In Vivo Activity.

Interleukin-2-inducible T-cell kinase (ITK) is a promising therapeutic target for human autoimmune diseases and T-cell malignant lymphomas. This paper reports the development of a series of cereblon-recruiting ITK proteolysis targeting chimeras based on a structure-based design strategy. The representative compounds 23 and 28 exhibited potent ITK degradation and IL-2 inhibition activities in Jurkat cells. Global proteomic profiling assays indicated that compounds 23 and 28 are highly selective ITK degraders. Moreover, compound 28 showed good plasma exposure levels and elicited efficient, rapid, and prolonged ITK degradation in Balb/c mice. Furthermore, it significantly suppressed IL-2 secretion stimulated by anti-CD3 antibody. Compound 28 represents the first effective and highly selective ITK degrader. Thus, 28 is a valuable tool compound for further in vitro and in vivo studies exploring the underlying biological effects and potential therapeutic utility of ITK degradation in human diseases.

IL2-inducible T-cell Kinase is Required for HBV-induced Type T Interferon Expression and Antiviral Response.

Hepatitis B is a vaccine-preventable liver infection caused by the hepatitis B virus (HBV), and is seen as a serious global health problem. HBV infection induces the expression of type I interferon (IFN), including IFN-α and IFN-β, which have anti-HBV activities, and have been used for HBV treatment. IL2-inducible T-cell kinase (ITK) is a tyrosine kinase, which regulates T-cell differentiation and activation, while its precise effects on type I IFN production during HBV infection remain unknown. We monitored the ITK expression in peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with acute and chronic HBV infection. We used ITK inhibitor ibrutinib to treat hepatocytes and evaluated the type I IFN expression after HBV infection. We also administrated ibrutinib to mice and evaluated its effect on HBV infection in vivo. We generated ITK, suppressor of cytokine signaling 1 (SOCS1) knockout and ITK/SOCS1 double knockout cells using CRISPR, and monitored the HBV-induced type I IFN production. ITK and type I IFN were upregulated in patients with acute HBV infection. Inhibition of ITK by ibrutinib suppressed HBV-induced expression of type I IFN mRNA in mice. ITK knockout cells had decreased IRF3 activation but promoted the expression of SOCS1. ITK negatively regulated SOSC1 expression. The down regulation of type I IFN in ITK knockout cells after HBV stimulation was abolished in the absence of SOCS1. ITK regulated HBV-induced expression of type I IFN mRNA by modulating SOCS1.

ASK120067 potently suppresses B-cell or T-cell malignancies in vitro and in vivo by inhibiting BTK and ITK.

Hyperactivation of Bruton's tyrosine kinase (BTK) or interleukin-2-inducible T cell kinase (ITK) has been attributed to the pathogenesis of B-cell lymphoma or T-cell leukemia, respectively, which suggests that Bruton's tyrosine kinase and interleukin-2-inducible T cell kinase are critical targets for the treatment of hematological malignancies. We identified a novel third-generation epidermal growth factor receptor (EGFR) inhibitor, ASK120067 (limertinib) in our previous research, which has been applied as a new drug application against non-small cell lung cancer in China. In this work, we found that ASK120067 displayed potent in vitro inhibitory efficacy against Bruton's tyrosine kinase protein and interleukin-2-inducible T cell kinase protein via covalent binding. In cell-based assays, ASK120067 dose-dependently suppressed Bruton's tyrosine kinase phosphorylation and exhibited anti-proliferation potency by inducing apoptosis in numerous B-lymphoma cells. Meanwhile, it caused growth arrest and induced the apoptosis of T-cell leukemia cells by attenuating interleukin-2-inducible T cell kinase activation. Oral administration of ASK120067 led to significant tumor regression in B-cell lymphoma and T-cell leukemia xenograft models by weakening Bruton's tyrosine kinase and interleukin-2-inducible T cell kinase signaling, respectively. Taken together, our studies demonstrated that ASK120067 exerted preclinical anti-tumor activities against B-/T-cell malignancy by targeting BTK/ITK.

Panaxadiol targeting IL2 inducible T cell kinase promotes T cell immunity in radiotherapy.

Ginseng, as a traditional Chinese medicine, has a good protective effect against radiotherapy, but its mechanism in radiotherapy still needs to be further explored. The active ingredients of Ginseng were analyzed according to pharmacodynamics in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database, and the target genes of active ingredients were screened by UniProt, PubChem and Swiss target prediction database. The differentially expressed genes of GSE6871 and GSE20162 were analyzed from the GEO database. Further, cluster analysis and enrichment analysis were carried out through protein-protein interaction network to determine hub gene. Next, build the drug-disease target network, conduct molecular docking simulation, and determine the key ingredients and targets of Ginseng on radiotherapy. We screened 16 active ingredients of Ginseng and 747 target genes from the TCMSP database. Eighty-two common differentially expressed genes were obtained by the GEO database. After topological analysis, we finally determined CD28, FYN, IL2 inducible T cell kinase (ITK), MYC and CD247 as hub genes. After integrating the drug-disease target network and molecular docking, we found that Panaxadiol, as an active ingredient of Ginseng, can target ITK to participate in T cell signal receptor pathway and act on radiotherapy. Panaxadiol can act on the key target ITK of radiotherapy, participate in T cell signal receptor pathway, and then affect the proliferation, differentiation and immune response of radiotherapy T cells, so as to reduce the side effects of radiotherapy.

Targeting Ibrutinib to Tumor-Infiltrating T Cells with a Sialic Acid Conjugate-Modified Phospholipid Complex for Improved Tumor Immunotherapy.

Immune checkpoint blockade (ICB) treatment for the clinical therapy of numerous malignancies has attracted widespread attention in recent years. Despite being a promising treatment option, developing complementary strategies to enhance the proportion of patients benefiting from ICB therapy remains a formidable challenge because of the complexity of the tumor microenvironment. Ibrutinib (IBR), a covalent inhibitor of Bruton's tyrosine kinase (BTK), has been approved as a clinical therapy for numerous B-cell malignancies. IBR also irreversibly inhibits interleukin-2 inducible T cell kinase (ITK), an essential enzyme in Th2-polarized T cells that participates in tumor immunosuppression. Ablation of ITK by IBR can elicit Th1-dominant antitumor immune responses and potentially enhance the efficacy of ICB therapy in solid tumors. However, its poor solubility and rapid clearance in vivo restrict T cell targetability and tumor accumulation by IBR. A sialic acid derivative-modified nanocomplex (SA-GA-OCT@PC) has been reported to improve the efficacy of IBR-mediated combination immunotherapy in solid tumors. In vitro and in vivo experiments showed that SA-GA-OCT@PC effectively accumulated in tumor-infiltrating T cells mediated by Siglec-E and induced Th1-dominant antitumor immune responses. SA-GA-OCT@PC-mediated combination therapy with PD-L1 blockade agents dramatically suppressed tumor growth and inhibited tumor relapse in B16F10 melanoma mouse models. Overall, the combination of the SA-modified nanocomplex platform and PD-L1 blockade offers a treatment opportunity for IBR in solid tumors, providing novel insights for tumor immunotherapy.

Exploring the Role of Chemical Reactions in the Selectivity of Tyrosine Kinase Inhibitors.

A variety of diseases are associated with tyrosine kinase enzymes that activate many proteins via signal transduction cascades. The similar ATP-binding pockets of these tyrosine kinases make it extremely difficult to design selective covalent inhibitors. The present study explores the contribution of the chemical reaction steps to the selectivity of the commercialized inhibitor acalabrutinib over the Bruton's tyrosine kinase (BTK) and the interleukin-2-inducible T-cell kinase (ITK). Ab initio and empirical valence bond (EVB) simulations of the two kinases indicate that the most favorable reaction path involves a water-assisted mechanism of the 2-butynamide reactive group of acalabrutinib. BTK reacts with acalabrutinib with a substantially lower barrier than ITK, according to our calculated free-energy profile and kinetic simulations. Such a difference is due to the microenvironment of the active site, as further supported by a sequence-based analysis of specificity determinants for several commercialized inhibitors. Our study involves a new approach of simulating directly the IC50 and inactivation efficiency keff, instead of using the standard formulas. This new strategy is particularly important in studies of covalent inhibitors with a very exothermic bonding step. Overall, our results demonstrate the importance of understanding the chemical reaction steps in designing selective covalent inhibitors for tyrosine kinases.

Effects of ibrutinib on T-cell immunity in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), a highly heterogeneous B-cell malignancy, is characterized by tumor microenvironment disorder and T-cell immune dysfunction, which play a major role in the proliferation and survival of CLL cells. Ibrutinib is the first irreversible inhibitor of Bruton’s tyrosine kinase (BTK). In addition to targeting B-cell receptor (BCR) signaling to kill tumor cells, increasing evidence has suggested that ibrutinib regulates the tumor microenvironment and T-cell immunity in a direct and indirect manner. For example, ibrutinib not only reverses the tumor microenvironment by blocking cytokine networks and toll-like receptor signaling but also regulates T cells in number, subset distribution, T-cell receptor (TCR) repertoire and immune function by inhibiting interleukin-2 inducible T-cell kinase (ITK) and reducing the expression of inhibitory receptors, and so on. In this review, we summarize the current evidence for the effects of ibrutinib on the tumor microenvironment and cellular immunity of patients with CLL, particularly for the behavior and function of T cells, explore its potential mechanisms, and provide a basis for the clinical benefits of long-term ibrutinib treatment and combined therapy based on T-cell-based immunotherapies.

MicroRNA-155 acts as an anti-inflammatory factor in orbital fibroblasts from Graves' orbitopathy by repressing interleukin-2-inducible T-cell kinase.

To investigate the role of microRNA (miR)-155 in inflammation in an in-vitro model of Graves’ orbitopathy (GO). The expression levels of miR-155 were compared between GO and non-GO orbital tissues. The effects of inflammatory stimulation of interleukin (IL)-1β and tumour necrosis factor alpha (TNF-α) on miR-155 expression on GO and non-GO orbital fibroblasts (OFs) were investigated. The effects of miR-155 mimics and inhibitors of inflammatory proteins and IL-2-inducible T-cell kinase (ITK) expression were examined, along with those related to the knockdown of ITK with siITK transfection on inflammatory proteins. We also examined how ITK inhibitors affect miR-155 expression in GO and non-GO OFs. The expression levels of miR-155 were higher in GO orbital tissues than in non-GO tissue. The overexpression of miR-155 was induced by IL-1β and TNF-α in OFs from GO and non-GO patients. IL-1β-induced IL-6 (ICAM1) protein production was significantly reduced (increased) by miR-155 mimics and inhibitors. The mRNA and protein levels of ITK were downregulated by overexpressed miR-155 via miR-155 mimics. Knockdown of ITK via siITK transfection induced a decrease in the expression levels of ITK, IL-17, IL-6, IL-1β, and TNF-α protein. The expression of miR-155 was significantly downregulated by treatment with ITK inhibitors and Bruton’s tyrosine kinase (BTK)/ITK dual inhibitors in a time-dependent manner. Our results indicated a potential relationship between miR-155 and ITK in the context of GO OFs. The overexpression of miR-155 repressed ITK expression and relieved inflammation. Thus, miR-155 appears to have anti-inflammatory effects in GO OFs. This discovery provides a new concept for developing GO treatment therapeutics.

Interleukin-2 inducible T cell kinase (ITK) may participate in the anti-bacterial immune response of Nile tilapia via regulating T-cell activation

Interleukin-2 inducible T cell kinase (ITK) plays a predominant role in the T-cell receptor (TCR) signaling cascade to ensure valid T-cell activation and function. Nevertheless, whether it regulates T-cell response of early vertebrates remains unknown. Herein, we investigated the involvement of ITK in the lymphocyte-mediated adaptive immune response, and its regulation to T-cell activation in the Nile tilapia Oreochromis niloticus. Both sequence and structure of O. niloticus ITK (OnITK) were remarkably conserved with its homologues from other vertebrates, implying its potential conserved function. OnITK mRNA was extensively expressed in lymphoid-related tissues, and with the relative highest level in peripheral blood. Once Nile tilapia was infected by Edwardsiella piscicida, OnITK in splenic lymphocytes was significantly up-regulated on 7-day post infection at both transcription and translation levels, suggesting that OnITK might involve in the primary adaptive immune response of teleost. Furthermore, upon splenic lymphocytes were stimulated by T-cell specific mitogen PHA, OnITK mRNA and protein levels were dramatically elevated. More importantly, treatment of splenic lymphocytes with specific inhibitor significantly crippled OnITK expression, which in turn impaired the inducible expression of T-cell activation markers IFN-γ, IL-2 and CD122, indicating the critical roles of ITK in regulating T-cell activation of Nile tilapia. Taken together, our results suggest that ITK takes part in the lymphocyte-mediated adaptive immunity of tilapia, and is indispensable for T-cell activation of teleost. Our findings thus provide novel evidences for understanding the mechanism regulating T-cell immunity of early vertebrates, as well as the evolution of adaptive immune system.

ITK tunes the Th17/Treg switch response by controlling calcium dependent signaling.

Abstract Naïve CD4+ T cells can differentiate into pro inflammatory T helper type 17 (Th17) and anti inflammatory T regulatory (Treg) lineages following T cell receptor (TCR) activation. The strength of the TCR signal is tuned by Interleukin-2 inducible T cell Kinase (ITK) and ITK can tune the development of Th17 and Treg cells. ITK signals can also tune a Th17/Treg switch under Th17 conditions. How ITK mediated tuning of TCR signal control naïve CD4+ T cell commitment into Th17/Treg lineages is unclear. Here we used ITK inhibitors, conventional ITK and allele specific ITK (ITKas) IL17 GFP/Foxp3 RFP reporter mice, to track naïve CD4+ T cell commitment into Th17/Treg lineages. Under Th17 conditions, reduced TCR signal strength in absence of ITK activity/expression, tuned naïve CD4+ T cell fate by reducing Th17 differentiation and inducing switch to cells resembling Tregs expressing Foxp3. The ITKas model showed this switch is due to specific ITK inhibition. The switched Treg like cells resemble Tregs by expression of Treg markers (CD25, CTLA4, PD1) and suppression of effector T cell proliferation. Transcriptomic analysis of switched Treg like cells by RNA Seq indicate they resemble Tregs, with reduced Th17 (Rorc, Il17) and increased Treg (Foxp3, Nrp1, Ikzf2) gene expression. Analysis of chromatin accessibility by ATAC Seq indicated closed loci for Th17 (Rorc, Il17) genes. Activating intracellular calcium dependent pathways led to a loss of the Th17/Treg switch response in naïve CD4+ T cell commitment, effectively bypassing ITK mediated tuning of the TCR signal critical for the switch response. In conclusion, we show here ITK tunes a Th17/Treg switch response in naïve CD4+ T cell commitment under Th17 conditions by controlling calcium dependent signaling. Supported by grants from NIH (NIH AI129422).

ITK signaling regulates IL-10 production by CD8+ T cells and lung immunopathology during influenza infection

Abstract Influenza (flu) infections cause 250,000 deaths and 3–5 million cases of severe illness during the average flu season. Severe influenza infections are associated with a combination of strong pro-inflammatory and weak anti-inflammatory immune responses. Production of the immunomodulatory cytokine IL-10 by T cells restricts immunopathology during flu infections, however knowledge of the signaling pathways regulating IL-10 induction during flu is limited. Using IL-10GFP reporter mouse models, we found Interleukin-2 inducible T cell kinase (ITK), a critical component in T cell receptor (TCR) signaling, regulates the development of IL-10-producing CD8+ T cells during influenza A infection. Compared to wild type (WT) mice, Itk−/− mice displayed increased morbidity and mortality after influenza infection, accompanied by a significant reduction of IL-10-producing CD8+ T cells in the airways. The absence of ITK and chemical inhibition of the PI3K/AKT/mTOR signaling pathway impaired both BLIMP1 expression and IL-10 production in CD8+ T cells in vitro. Introduction of Blimp-1 or a constitutively active AKT mutant via retroviral transduction rescued IL-10 production and Blimp-1 expression in Itk−/− CD8+ T cells. Utilizing a transgenic allele sensitive Itk (ITKas) mouse model to specifically inhibit ITK kinase activity in coculture, we found ITK kinase activity is not required for the suppressive function of IL-10 producing CD8+ T cells. Together, our data suggests that ITK is a critical regulator of the development and suppressive function of IL-10 producing CD8+ T cells and flu-induced pulmonary immunopathology. Modulating ITK signaling may be a strategy for regulating immunopathology due to respiratory viral infections. This work was supported in part by grants from the National Institutes of Health (R01 AI151139, R56 AI146226 and P20 GM130555). W. Huang also received research support from MegaRobo Technologies Corporation.

Kinase Inhibition as Treatment for Acute and Chronic Graft-Versus-Host Disease.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a potentially curative therapy for patients suffering from hematological malignancies via the donor immune system driven graft-versus-leukemia effect. However, the therapy is mainly limited by severe acute and chronic graft-versus-host disease (GvHD), both being life-threatening complications after allo-HCT. GvHD develops when donor T cells do not only recognize remaining tumor cells as foreign, but also the recipient’s tissue, leading to a severe inflammatory disease. Typical GvHD target organs include the skin, liver and intestinal tract. Currently all approved strategies for GvHD treatment are immunosuppressive therapies, with the first-line therapy being glucocorticoids. However, therapeutic options for glucocorticoid-refractory patients are still limited. Novel therapeutic approaches, which reduce GvHD severity while preserving GvL activity, are urgently needed. Targeting kinase activity with small molecule inhibitors has shown promising results in preclinical animal models and clinical trials. Well-studied kinase targets in GvHD include Rho-associated coiled-coil-containing kinase 2 (ROCK2), spleen tyrosine kinase (SYK), Bruton’s tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) to control B- and T-cell activation in acute and chronic GvHD. Janus Kinase 1 (JAK1) and 2 (JAK2) are among the most intensively studied kinases in GvHD due to their importance in cytokine production and inflammatory cell activation and migration. Here, we discuss the role of kinase inhibition as novel treatment strategies for acute and chronic GvHD after allo-HCT.