Absence Of Itk Research Articles

Type I Diabetes induced by OT-I T-Cells requires ITK-mediated TCR signaling.

Abstract Activation of CD8+ T-cells induces changes in T cell trafficking, promoting effector T cell migration into tissues. ITK, a tyrosine kinase activated by TCR signaling, has previously been shown to be required for CD8+ T cell migration into the gastrointestinal tract during viral infection. This finding prompted us to investigate whether ITK signaling was important for effector T cell trafficking into non-infected tissue, as might occur in autoimmune disease. Using the RIP-mOVA mouse model, in which the OVA protein is expressed in the β-islet cells of the pancreas, we examined the ability of WT versus ITK-deficient OT-I T cells to infiltrate the pancreas inducing Type I diabetes. To test this, WT or ITK-deficient OT-I cells were adoptively transferred into RIP-mOVA recipients. Mice were immunized with OVA peptide plus LPS, and monitored for urine glucose. We also examined OT-I cell numbers in the pancreatic draining lymph node and in the pancreas 72hr post-immunization. We found that in the absence of ITK, OT-I cells were impaired in their ability to induce diabetes, with only ~1/3 of the recipients progressing to disease; in contrast 100% of recipients receiving WT OT-I cells developed Type I diabetes. In addition, the numbers of infiltrating ITK-deficient OT-I cells in the pancreas was significantly reduced compared to the numbers of WT OT-I cells detected. These findings indicate that ITK has a key role in generating pathogenic tissue-infiltrating CD8+ T cells capable of migration into β-islets of the mouse pancreas. While exploration of the mechanism underlying this impaired migration needs further investigation, these data provide support for therapeutic potential of an ITK inhibitor in patients with tissue-specific autoimmune diseases. Supported by NAID TRC SIGNLG

ITK independent development of Th17 responses during hypersensitivity pneumonitis driven lung inflammation

T helper 17 (Th17) cells develop in response to T cell receptor signals (TCR) in the presence of specific environments, and produce the inflammatory cytokine IL17A. These cells have been implicated in a number of inflammatory diseases and represent a potential target for ameliorating such diseases. The kinase ITK, a critical regulator of TCR signals, has been shown to be required for the development of Th17 cells. However, we show here that lung inflammation induced by Saccharopolyspora rectivirgula (SR) induced Hypersensitivity pneumonitis (SR-HP) results in a neutrophil independent, and ITK independent Th17 responses, although ITK signals are required for γδ T cell production of IL17A. Transcriptomic analysis of resultant ITK independent Th17 cells suggest that the SR-HP-induced extrinsic inflammatory signals may override intrinsic T cell signals downstream of ITK to rescue Th17 responses in the absence of ITK. These findings suggest that the ability to pharmaceutically target ITK to suppress Th17 responses may be dependent on the type of inflammation.

The Tec Kinase Itk Integrates Naïve T Cell Migration and In Vivo Homeostasis.

Naïve T cells (TN) constitutively recirculate through secondary lymphatic organs (SLOs), where they scan dendritic cells (DCs) for cognate peptide-loaded major histocompatibility complexes (pMHC). Continuous trafficking between SLOs not only enables rapid clonal selection but also ensures TN homeostasis by providing access to prosurvival signals from TCR, IL-7R, and the chemokine receptor CCR7. Inside the lymphoid tissue, CCR7-mediated TN motility is mainly driven by the Rac activator DOCK2, with a separate contribution by a phosphoinositide-3-kinase γ (PI3Kγ)-dependent pathway. Tec tyrosine kinases and the Rac activator Tiam1 constitute prominent downstream effectors of PI3K signaling. Yet, the precise role of Tec kinase versus Tiam1 signaling during CCR7-mediated TN migration and homeostasis remains incompletely understood. Here, we examined the function of the Tec family member interleukin-2-inducible T-cell kinase (Itk) and Tiam1 during TN migration in vitro and in vivo using intravital microscopy. Itk deficiency caused a mild decrease in CCR7-triggered TN migration, mirroring observations made with PI3Kγ;-/- T cells, while lack of Tiam1 did not affect TN motility. In silico modeling suggested that reduced migration in the absence of Itk does not result in a substantial decrease in the frequency of TN encounters with DCs within the lymphoid tissue. In contrast, Itk was important to maintain in vivo homeostasis of CD4+ TN, also in MHCII-deficient hosts. Taken together, our data suggest that Itk contributes to TN migration and survival by integrating chemokine receptor and TCR signaling pathways.

The Role of Tyrosine Kinase Itk in T helper 17 and T regulatory cells in Hypersensitivity Pneumonitis

Abstract The balance of inflammatory and suppressive cytokines is critical in controlling inflammatory responses, and the pro- and anti-inflammatory cytokines IL17A and IL10 has been implicated in numerous pulmonary inflammatory diseases. The tyrosine kinase, Itk, plays a critical role in T cell activation. Itk is required for the development of Th17 cells and their production of IL17A in allergic lung inflammation. Furthermore, Type I regulatory and Foxp3+ T regulatory (Tregs) cells, producers of IL10, are positively and negatively regulated by Itk respectively. Farmer’s lung, a subset of hypersensitivity pneumonitis, develops due to repeated exposure to the bacteria Saccharopolyspora rectivirgula (SR) and is dependent on IL17A and regulated by IL10. Surprisingly, exposure to SR drives robust CD4+ T cell IL17A response even in the absence of Itk, with pulmonary inflammation. Transcriptomic analysis of sort purified WT and Itk−/− IL17A producing CD4+ T cells from SR-exposed mice revealed an enrichment of Notch signaling pathway in the absence of Itk. SR also induced the Itk independent development of a population of IL17A producing Foxp3+ Tregs cells, and a significant decrease in IL10 producing Tr1 cells. These data suggest that Itk regulates the expression of IL10, and pathogenic Th17 cells via Notch signaling. These studies suggest that TCR signaling through Itk differentially regulates the development of inflammatory Th17 cells and suppressive Tregs and Tr1 cells in response to SR exposure. Understanding how Itk modulates the development of Th17/Treg cytokine responses will allow us to better understand the precise role of Itk in the regulating the balance of pro- and anti-inflammatory cytokine production during airway inflammation.

The role of tyrosine kinase Itk in T helper 17 and T regulatory cells in IL17 mediated hypersensitivity pneumonitis

Abstract The balance of inflammatory and suppressive cytokines is critical in controlling inflammatory responses, and the pro- and anti-inflammatory cytokines IL17A and IL10 has been implicated in numerous pulmonary inflammatory diseases. The tyrosine kinase, Itk, plays a critical role in T cell activation. We have previously shown that Itk−/− mice do not develop allergic airway disease coupled with reduced production of IL17A in the lungs. In vitro, Itk is required for the development of Th17 cells and their production of the pro-inflammatory cytokine IL17A. Furthermore, Type I regulatory and Foxp3+ T regulatory (Tregs) cells, suppressive producers of IL10, are positively and negatively regulated by Itk respectively. Farmer’s lung is a subset of hypersensitivity pneumonitis caused by repeated exposure to the bacteria Saccharopolyspora rectivirgula (SR), and is dependent on IL17A and regulated by IL10. We find that surprisingly, exposure to SR drives robust IL17A response produced by CD4+ T cells even in the absence of Itk, accompanied by the development of pulmonary inflammation. SR also induced Itk independent development of a population of population of Foxp3+ Tregs cells that produce IL17A, accompanied by a significant decrease in Tr1 cells compared to WT mice. These studies suggest that TCR signaling through Itk differentially regulates the development of inflammatory Th17 cells and suppressive Tregs and Tr1 cells. Understanding how Itk modulates the development of Th17/Treg cytokine responses will allow us to better understand the precise role of Itk in the production of IL17A and IL10 in airway inflammation.

Itk independent development of natural Th17 cells and IL17A-driven hypersensitivity pneumonitis

Abstract The tyrosine kinase, Itk, plays a critical role in T cell receptor signaling, and we have previously shown that Itk is required for the development of Th17 cells and their production of the pro-inflammatory cytokine IL17A. IL17A has been implicated in numerous pulmonary inflammatory diseases. We have previously shown that Itk−/− mice do not develop allergic airway disease coupled with protected reduced production of IL17A in the lungs in a model of allergic asthma. Farmer’s lung is a subset of hypersensitivity pneumonitis caused by repeated exposure to the bacteria Saccharopolyspora rectivirgula (SR). This IL17A-driven disease is characterized by an influx of neutrophils and T lymphocytes. Here we show that a population of natural Th17 cell develop in the absence of Itk. Furthermore, exposure to SR drives robust IL17A response produced by CD4+ T cells even in the absence of Itk, accompanied by the development of pulmonary inflammation. These studies suggest that TCR signaling through Itk differentially regulates the development of conventional and natural Th17 cells. Understanding how Itk modulates the Th17 cytokine responses will allow us to better understand the precise role of Itk in the production of IL17A in airway inflammation.

ITK signaling via the Ras/IRF4 pathway regulates the development and function of type 1 regulatory T cells

Abstract Type 1 regulatory T (Tr1) cells differentiate in response to signals engaging T cell receptor (TCR), express high levels of the immunosuppressive cytokine IL-10 but not Foxp3, and can suppress inflammation and promote immune tolerance. Here, using IL-10GFP/Foxp3RFP dual reporter transgenic mouse system, we show that ITK, a key modulator of TCR signaling, is required for the development of Tr1 cells in various organs following TCR activation, and in the mucosal system during parasitic and viral infections. ITK kinase activity is required for mouse and human Tr1 cell fate programming and regulates the balance between IL-10 and IFN-γ production during Tr1 cell differentiation. The requirement for ITK function during Tr1 cell development and suppressive function can be restored by the expression of the transcription factor IRF4. Downstream of ITK, Ras, and MAP kinases ERK1/2, p38 and JNK, are required for Tr1 cell differentiation, but not Th17 cell differentiation. We have previously shown that Th17 differentiation is also dependent on ITK signaling. However, in the absence of ITK, expression of the constitutively active HRasG12V rescued IRF4 expression and Tr1 cell differentiation, but failed to restore Th17 cell differentiation. We conclude that the TCR/ITK signaling through the Ras/MAPK/IRF4 pathway is specifically required for functional development of Tr1 cells, and that targeting these signaling components may be of therapeutic benefit.

T-Bet independent development of IFN\u03b3 secreting natural T helper 1 cell population in the absence of Itk

Th1, Th2, Th9 and Th17 cells are conventional CD4+ effector T cells identified as secretors of prototypical cytokines IFNγ, IL4, IL9, and IL-17A respectively. Recently, populations of natural Th17 and Th1 cells (nTh17 and nTh1) with innate-like phenotype have been identified in the thymus that are distinct from conventional Th17 and Th1 cells. The absence of the Tec family kinase Interleukin-2 inducible T cell kinase (Itk) results in T cell immunodeficiency in mice and humans. Here we show that Itk negatively regulates the development of nTh1 cells that express IFNγ in a Tbet independent manner, and whose expansion can be enhanced by IL4. Furthermore, we show that robust induction of IL4 responses during Trichinella spiralis infection enhance the presence of nTh1 cells. We conclude T cell receptor signaling via Itk controls the development of natural Th1 cells, which are expanded by the presence of IL4.

The non-receptor tyrosine kinase ITK is required for type 1 regulatory T cell development

Abstract Type 1 regulatory T (Tr1)cells lack the expression of Foxp3 but are potent producers of the immunosuppressive cytokine IL-10, with profound regulatory function in suppressing inflammation and promoting tolerance. Tr1 cells differentiate in response to signals engaging T cell receptor (TCR) and/or regulatory cytokine milieu. The non-receptor tyrosine kinase ITK is a key modulator downstream of TCR, playing critical role in T cell development and function. Using mouse models carrying Foxp3-RFP and IL-10-GFP dual reporters, we found that, in the absence of ITK, Foxp3− IL-10+ Tr1 cell development driven by TCR activation is severely impaired in various tissues (spleen, lymph nodes, lung, gut, and fat). Under Tr1 polarizing condition, naïve Foxp3− CD4+T cells isolated from WT mouse thymus and spleen can both give rise to Tr1cells, however, Itk−/− thymic and splenic naïve Foxp3− CD4+ T cells are deficient in Tr1 differentiation. Although Itk−/−CD4+ T cells proliferated under Tr1 differentiating conditions, they failed to up-regulate LAG3, CD49b, ICOS, PD-1, c-Maf, AHR, and IRF4 to levels observed in WT cells, suggesting that ITK is critical for Tr1 cell fate programming. Utilizing transgenic mouse model carrying an allele sensitive mutant of ITK that allows ITK kinase specific blockade by a small molecule 3MB-PP1, we also determined that the expression of the aforementioned markers is dependent on ITK kinas activity. Inhibiting ITK kinase activity also diminished Tr1 differentiation inhuman CD4+ T cells. We conclude that ITK is required for Tr1 cell development and targeting ITK signaling may be a strategy to modulate regulatory immunity for clinical benefit.

Nonreceptor tyrosine kinases ITK and BTK negatively regulate mast cell proinflammatory responses to lipopolysaccharide

Mast cells are indispensable for LPS-induced septic hypothermia, in which TNF-α plays an essential role to initiate septic responses. ITK and BTK regulate mast cell responses to allergens, but their roles in mast cell responses in LPS-induced sepsis are unclear. We sought to investigate the roles of ITK and BTK in mast cell responses during LPS-induced septic inflammation. Mice (genetically modified or bone marrow-derived mast cell-reconstituted Sash) were given LPS to induce septic hypothermia in the presence or absence of indicated inhibitors. Flow cytometry was used to determine LPS-induced cell influx and TNF-α production in peritoneal cells. Microarray was used for genomewide gene expression analysis on bone marrow-derived mast cells. Quantitative PCR and multiplex were used to determine transcribed and secreted proinflammatory cytokines. Microscopy and Western blotting were used to determine activation of signal transduction pathways. The absence of ITK and BTK leads to exacerbation of LPS-induced septic hypothermia and neutrophil influx. Itk(-/-)Btk(-/-) mast cells exhibit hyperactive preformed and LPS-induced TNF-α production, and lead to more severe LPS-induced septic hypothermia when reconstituted into mast cell-deficient Sash mice. LPS-induced nuclear factor kappa B, Akt, and p38 activation is enhanced in Itk(-/-)Btk(-/-) mast cells, and blockage of phosphatidylinositol-4,5-bisphosphate 3-kinase, Akt, or p38 downstream mitogen-activated protein kinase interacting serine/threonine kinase 1 activation significantly suppresses TNF-α hyperproduction and attenuates septic hypothermia. ITK and BTK regulate thermal homeostasis during septic response through mast cell function in mice. They share regulatory function downstream of Toll-like receptor 4/LPS in mast cells, through regulating the activation of canonical nuclear factor kappa B, phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt, and p38 signaling pathways.

Critical role of ITK in mouse γ-herpes virus 68 infection as a model for human immunodeficiency (VIR1P.1140)

Abstract IL-2-inducible T cell kinase (ITK) is a Tec family tyrosine kinase predominantly expressed in T cells. In the absence of ITK, the magnitude of T cell receptor (TCR) signaling is greatly reduced, but not abolished, leading to an overall decrease in TCR signaling strength. Recently, various mutations in ITK were identified in patients suffering from a fatal lymphoproliferation due to a failure to control Epstein-Barr virus (EBV) infection. Interestingly, Itk-/- mice have been shown to mount protective responses to several acute virus infections. Thus, we hypothesized that ITK plays an essential role in long-term anti-viral immunity against latent γ-herpes virus infection. To study this, we infected WT and Itk-/- mice with mouse γ-herpes virus 68 (MHV68). Despite a reduced response at D7 post-infection, Itk-/- mice ultimately controlled MHV68 replication in the spleen by D14. After the initial response, both WT and Itk-/- mice showed no difference in their viral titers in the spleen at later time points. However, Itk-/- mice developed severe gastrointestinal pathology following LPS-mediated reactivation of latent MHV68, accompanied by a high viral titer in the intestine. Furthermore, the number of lymphocytes in the intestinal epithelium and lamina propria in naïve Itk-/- mice was substantially less than WT mice. Overall, these data suggest that ITK is essential for anti-viral immunity against γ-herpes virus infection in the gut following reactivation from latency.

Innate PLZF+CD4+ αβ T cells develop and expand in the absence of Itk.

T cell development in the thymus produces multiple lineages of cells, including innate T cells. Studies in mice harboring alterations in TCR signaling proteins or transcriptional regulators have revealed an expanded population of CD4(+) innate T cells in the thymus that produce IL-4 and express the transcription factor promyelocytic leukemia zinc finger (PLZF). In these mice, IL-4 produced by the CD4(+)PLZF(+) T cell population leads to the conversion of conventional CD8(+) thymocytes into innate CD8(+) T cells resembling memory T cells expressing eomesodermin. The expression of PLZF, the signature invariant NKT cell transcription factor, in these innate CD4(+) T cells suggests that they might be a subset of αβ or γδ TCR(+) NKT cells or mucosal-associated invariant T (MAIT) cells. To address these possibilities, we characterized the CD4(+)PLZF(+) innate T cells in itk(-/-) mice. We show that itk(-/-) innate PLZF(+)CD4(+) T cells are not CD1d-dependent NKT cells, MR1-dependent MAIT cells, or γδ T cells. Furthermore, although the itk(-/-) innate PLZF(+)CD4(+) T cells express αβ TCRs, neither β2-microglobulin-dependent MHC class I nor any MHC class II molecules are required for their development. In contrast to invariant NKT cells and MAIT cells, this population has a highly diverse TCRα-chain repertoire. Analysis of peripheral tissues indicates that itk(-/-) innate PLZF(+)CD4(+) T cells preferentially home to spleen and mesenteric lymph nodes owing to increased expression of gut-homing receptors, and that their expansion is regulated by commensal gut flora. These data support the conclusion that itk(-/-) innate PLZF(+)CD4(+) T cells are a novel subset of innate T cells.

Itk-mediated integration of T cell receptor and cytokine signaling regulates the balance between Th17 and regulatory T cells

A proper balance between Th17 and T regulatory cells (Treg cells) is critical for generating protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk (IL2-inducible T cell kinase), a component of T cell receptor (TCR) signaling pathways, influences this balance by regulating cross talk between TCR and cytokine signaling. Under both Th17 and Treg cell differentiation conditions, Itk(-/-) CD4(+) T cells develop higher percentages of functional FoxP3(+) cells, associated with increased sensitivity to IL-2. Itk(-/-) CD4(+) T cells also preferentially develop into Treg cells in vivo. We find that Itk-deficient T cells exhibit reduced TCR-induced phosphorylation of mammalian target of rapamycin (mTOR) targets, accompanied by downstream metabolic alterations. Surprisingly, Itk(-/-) cells also exhibit reduced IL-2-induced mTOR activation, despite increased STAT5 phosphorylation. We demonstrate that in wild-type CD4(+) T cells, TCR stimulation leads to a dose-dependent repression of Pten. However, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and phosphoinositide-3-kinase (PI3K) pathways. Moreover, Itk-deficient CD4(+) T cells show impaired TCR-mediated induction of Myc and miR-19b, known repressors of Pten. Our results demonstrate that Itk helps orchestrate positive feedback loops integrating multiple T cell signaling pathways, suggesting Itk as a potential target for altering the balance between Th17 and Treg cells.

Cross-talk between T cell receptor and cytokine signaling is mediated by Itk: implications for the balance between Th17 and regulatory T cells (P1141)

Abstract A proper balance between Th17 and T regulatory cells (Tregs) is critical for the generation of immune protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk influences the balance between Th17 and T regulatory cells by regulating crosstalk between T Cell Receptor (TCR) and cytokine signaling. Under both Th17 and Tregs differentiation conditions, Itk-/- CD4 T cells develop higher percentages of functional Foxp3+ cells, associated with increased responsiveness to IL-2. Itk-deficient CD4 cells show reduced TCR-induced phosphorylation of mTOR targets, including ribosomal S6 and AKT, accompanied by changes in metabolic signatures that are affected by mTOR pathways. Surprisingly, iTreg Itk-/- cells also show reduced IL-2-induced mTOR pathways, despite increased STAT5 phosphorylation. We find that TCR stimulation in WT CD4+ T cells leads to a dose-dependent repression of expression of Pten; however, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and PI3K/mTOR pathways and increasing Foxp3 expression. Functional studies show that naïve Itk-/- CD4 T cells also preferentially develop into Tregs in an in vivo colitis model. Furthermore Itk-deficient Tregs are capable to inhibit the development of colitis demonstrating that Treg function is independent of Itk. Our results suggest that Itk is a potential target for altering the balance between Th17 and Treg cells.

Complex role for Interleukin-2 inducible T-cell kinase in T helper differentiation and effector function in vivo (P1204)

Abstract T helper responses are critical for a productive immune response but an inappropriate response results in inflammatory and autoimmune disorders. The tyrosine kinase ITK has been shown to regulate the development of Th2 responses. Here, we show that ITK regulates Th1/Th17 responses as well. We find that Itk-/- mice are more susceptible to infection by Th1 inducing intracellular pathogen T. gondii, with generation of fewer antigen specific Th1 cells in vivo and fewer effector cells ex vivo upon antigen re-challenge. Transfer of WT CD4+ T cells confers protection against infection in Itk-/- mice. Furthermore, WT but not Itk-/- CD4+ T cells can promote the survival of Rag-/- mice infected by T. gondii. Additionally, the absence of ITK is protective during Th1/Th17 mediated Experimental Autoimmune Encephalomyelitis (EAE) with fewer pathogenic Th1 and Th17 effector cells in the periphery. Furthermore, transfer of Itk-/- CD4+ T cells into Tcra-/- mice results in lower severity of EAE compared to mice that receive WT cells suggesting that Itk-/- CD4+ T cells are intrinsically less pathogenic. Finally, using mice that express an analog sensitive mutant of ITK (ITKas) we show that temporally inhibiting the kinase activity of ITK with a genetically selective small-molecule inhibitor impairs development of Th1, Th2, and Th17 lineages. This work has implications for understanding ITK as a therapeutic target for Th2 mediated inflammatory and Th1/Th17 mediated autoimmune disorders.

Abstract 492: Ibrutinib inhibits Interleukin-2 inducible kinase, driving a Th1 selective pressure in human leukemia patients that serves to alleviate tumor-induced immunosupression.

Abstract Interleukin-2 Inducible Kinase (ITK) is an appealing therapeutic target given its critical role in polarizing T-cell immunity against various infectious, autoimmune, and neoplastic diseases. ITK activity promotes Th2 immunity, thereby subverting healthy Th1-based immune surveillance. Th2 CD4 T-cells depend on ITK for activation, whereas Th1 and CD8 T-cells have compensatory resting lymphocyte kinase (RLK) that conducts T-cell receptor activation in the absence of ITK. Similar to many other tumors, chronic lymphocytic leukemia (CLL) directs an aberrant Th2 bias that drives immune evasion, promotes a supportive microenvironment, and cripples adaptive immunity. In CLL, this immune suppression leads to severe infections which are a leading cause of death. Although ITK inhibitors have the promise of alleviating tumor-induced immune suppression, to date no clinically viable ITK inhibitor exists. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) with outstanding activity and tolerability in B-cell malignancies. The striking homology between BTK and ITK combined with our in silico docking studies and in vitro kinase inhibition profiles led us to identify ibrutinib as the first clinically viable irreversible ITK inhibitor. We further confirmed both molecular and functional outcomes using Jurkat T-cells and in vitro polarized Th1 and Th2 CD4 T-cells. We found that mutation of the ITK-Cys442 covalent binding residue for ibrutinib alleviated molecular inhibition. We also demonstrated that Th1 and CD8 T-cell restricted expression of RLK provides a compensatory platform for T-cell activation, offering a molecular explanation for the selective outgrowth of cytotoxic Th1-biased immunity. This effect was confirmed using T-cells directly derived from CLL patients. To clearly demonstrate this effect in vivo, we utilized the Leishmania major model of Th2-mediated infection. Ibrutinib enhanced resistance to the parasite by eliciting robust Th1 immunity. To demonstrate a direct clinical relevance in the setting of CLL we utilized a novel listeriosis/leukemia mouse model. In this model, we observed complete recovery of functional immunity, with all ibrutinib-treated mice surviving a potentially lethal Listeria monocytogenes infection. We further confirmed irreversible binding of ITK in patients currently receiving ibrutinib as part of a clinical trial. Finally, we found that ibrutinib inhibited phosphorlyation of ITK's direct downstream target PLCγ1 in CD4 T-cells. Together, our results provide novel molecular insights into the mechanism of action of ibrutinib in the context of Th2-biased immunosuppressive leukemia. We also postulate that ibrutinib's irreversible ITK inhibitory effects may prove beneficial in a number of other autoimmune, inflammatory, parasitic, and viral diseases. Citation Format: Jason A. Dubovsky, Kyle A. Beckwith, Gayathri Natarajan, Jennifer A. Woyach, Samantha Jaglowski, Joshua Hessler, Ta-Ming Liu, Betty Y. Chang, Karilyn M. Larkin, Matthew R. Stefanovski, Frank W. Frissora, Lisa L. Smith, Kelly A. Smucker, Joseph M. Flynn, Jeffrey A. Jones, Leslie A. Andritsos, Kami Maddocks, Amy M. Lehman, Richard Furman, Jeff Sharman, Anjali Mishra, Michael A. Caligiuri, Abhay R. Satoskar, Joseph J. Buggy, Natarajan Muthusamy, Amy J. Johnson, John C. Byrd. Ibrutinib inhibits Interleukin-2 inducible kinase, driving a Th1 selective pressure in human leukemia patients that serves to alleviate tumor-induced immunosupression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 492. doi:10.1158/1538-7445.AM2013-492

The Tec Kinase ITK Regulates Thymic Expansion, Emigration, and Maturation of γδ NKT Cells

The Tec family tyrosine kinase, Itk, regulates signaling downstream of the TCR. The absence of Itk in CD4(+) T cells results in impaired Th2 responses along with defects in maturation, cytokine production, and survival of iNKT cells. Paradoxically, Itk(-/-) mice have spontaneously elevated serum IgE levels, resulting from an expansion of the Vγ1.1(+)Vδ6.3(+) subset of γδ T cells, known as γδ NKT cells. Comparisons between γδ NKT cells and αβ iNKT cells showed convergence in the pattern of cell surface marker expression, cytokine profiles, and gene expression, suggesting that these two subsets of NKT cells undergo similar differentiation programs. Hepatic γδ NKT cells have an invariant TCR and are derived predominantly from fetal progenitors that expand in the thymus during the first weeks of life. The adult thymus contains these invariant γδ NKT cells plus a heterogeneous population of Vγ1.1(+)Vδ6.3(+) T cells with diverse CDR3 sequences. This latter population, normally excluded from the liver, escapes the thymus and homes to the liver when Itk is absent. In addition, Itk(-/-) γδ NKT cells persistently express high levels of Zbtb16 (PLZF) and Il4, genes that are normally downregulated in the most mature subsets of NKT cells. These data indicate that Itk signaling is required to prevent the expansion of γδ NKT cells in the adult thymus, to block their emigration, and to promote terminal NKT cell maturation.

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 nonconventional 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. In this study, we show hematopoietic MHC class I (MHCI)-dependent generation of Ag-specific IMP CD8(+) T cells using bone marrow chimeras. Wild-type 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, and independent of recipient thymus. In contrast, MHCI(-/-) bone marrow does not lead to IMP CD8(+) T cells in wild-type recipients. OTI IMP CD8(+) T cells generated via this method exhibited enhanced early response to Ag without prior primary stimulation. Our findings suggest a method to generate Ag-specific "naive" CD8(+) IMP T cells, as well as demonstrate that they are not homeostatic proliferation cells and can respond promptly in an Ag-specific fashion.

Development of CD4+ PLZF+ T Cells in the Absence of Itk (111.41)

Abstract Innate-like lymphocytes develop in the thymus and share characteristics of the innate and adaptive immune systems. These lymphocytes undergo antigen receptor rearrangement and are able to exert their effector function immediately upon ex vivo stimulation. Our lab is interested in how TCR signaling affects the development of conventional versus innate-like lymphocytes. Itk is a nonreceptor protein tyrosine kinase that signals downstream of the TCR. Previously, our lab has shown that in the absence of Itk, CD8+ Eomesodermin+ T cells develop that have a diverse TCR repertoire and produce IFNγ immediately upon ex vivo stimulation. Recently, several labs have demonstrated that the development of these CD8+ Eomesodermin+ T cells is dependent upon CD4+ PLZF+ thymocytes producing IL-4. We have determined that there are two CD4+ PLZF+ populations consisting of αβ and γδ T cells in Itk-/- thymi. In the absence of γδ T cells, Itk-/- CD4+ PLZF+ αβ T cells retain the ability to induce Eomesodermin expression in CD8+ T cells. Furthermore, Itk-/- CD4+ PLZF+ αβ T cells do not bind CD1d tetramer and develop independently of IL-15, class II MHC, and β2m. We have also found that Itk-/- CD4+ PLZF+ αβ T cells are found in the spleen and mesenteric lymph node in the periphery and have a restricted TCR repertoire. Thus, in the absence of Itk, a non-canonical innate-like population of CD4+ PLZF+ αβ T cells develop that influences the development of conventional T cells.

Mast cell specific negative regulation of LPS induced pro-inflammatory response by Itk and Btk (177.20)

Abstract Gram-negative bacterial endotoxin lipopolysaccharide (LPS) induces mast cell secretion of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, which is responsible for septic hypothermia. The non receptor tyrosine kinases Itk and Btk are both expressed in mast cells, and act as signaling regulators, but their roles in the response of mast cells to LPS are unclear. Here we show that Itk/Btk double knockout (DKO) mice suffered exacerbated and prolonged LPS induced hypothermia with increased TNF-α production by mast cells, compared to WT, Itk-/- and Btk-/- mice. This aggravated LPS induced hypothermia was recapitulated in mast cell deficient Sash mice reconstituted with DKO bone marrow derived mast cells (BMMCs). LPS (TLR4 ligand) stimulated DKO BMMCs exhibited hyperactive pro-inflammatory cytokine production with severely altered NF-κB signaling, which was not observed with Pam3CSK4 (TLR1/2 ligand). By contrast, DKO bone marrow derived macrophage and dendritic cells were not hyper-responsive to LPS as observed in DKO BMMCs, suggesting that Itk and Btk act as mast cell specific negative regulators in LPS/TLR4 signaling. Microarray analysis further revealed that the absence of Itk and Btk in mast cells significantly enhanced LPS induced pro-inflammatory program, which could potentially deteriorate endotoxin induced mast cell mediated inflammatory diseases.