Avery August

Vitamin D Receptor-Deficient Mice Fail to Develop Experimental Allergic Asthma

The active metabolite of vitamin D (1,25-dihydroxyvitamin D3 (1,25(OH)2D3)) is known to modulate the immune response in Th1 cell-directed diseases. To investigate the role of vitamin D in Th2 cell-directed diseases, experimental allergic asthma was induced in vitamin D receptor (VDR) knockout and in wild-type (WT) mice. As expected, WT mice developed symptoms of airway inflammation with an influx of eosinophils, elevated Th2 cytokine levels, mucous production, and airway hyperresponsiveness. The administration of 1,25(OH)2D3 had no effect on asthma severity. The only discernable effect of 1,25(OH)2D3 on experimental allergic asthma in WT mice was an increased expression of two Th2-related genes (soluble CD23 and GATA-3) in lungs of BALB/c mice exposed to Ag through the nasal route only. By contrast, asthma-induced VDR knockout mice failed to develop airway inflammation, eosinophilia, or airway hyperresponsiveness, despite high IgE concentrations and elevated Th2 cytokines. The data suggest that although 1,25(OH)2D3 induced these Th2-type genes, the treatment failed to have any affect on experimental asthma severity. However, VDR-deficient mice failed to develop experimental allergic asthma, suggesting an important role for the vitamin D endocrine system in the generation of Th2-driven inflammation in the lung.

Differential Expression of Interleukin-17A and -17F Is Coupled to T Cell Receptor Signaling via Inducible T Cell Kinase

T helper 17 (Th17) cells play major roles in autoimmunity and bacterial infections, yet how T cell receptor (TCR) signaling affects Th17 cell differentiation is relatively unknown. We demonstrate that CD4(+) T cells lacking Itk, a tyrosine kinase required for full TCR-induced phospholipase C-gamma (PLC-gamma1) activation, exhibit decreased interleukin-17A (IL-17A) expression in vitro and in vivo, despite relatively normal expression of retinoic acid receptor-related orphan receptor-gammaT (ROR-gammaT) and IL-17F. IL-17A expression was rescued by pharmacologically induced Ca(2+) influx or constitutively activated nuclear factor of activated T cells (NFAT). Conversely, decreased TCR stimulation or calcineurin inhibition preferentially reduced IL-17A expression. We further found that the promoter of Il17a but not Il17f has a conserved NFAT binding site that bound NFATc1 in wild-type but not Itk-deficient cells, even though both exhibited open chromatin conformations. Finally, Itk(-/-) mice also showed differential regulation of IL-17A and IL-17F in vivo. Our results suggest that Itk specifically couples TCR signaling to Il17a expression and the differential regulation of Th17 cell cytokines through NFATc1.

Attenuation of Immunological Symptoms of Allergic Asthma in Mice Lacking the Tyrosine Kinase ITK

Allergic asthma patients manifest airway inflammation and some show increases in eosinophils, TH2 cells, and cytokines, increased mucous production in the lung, and elevated serum IgE. This TH2-type response suggests a prominent role for TH2 cells and their cytokines in the pathology of this disease. The Tec family nonreceptor tyrosine kinase inducible T cell kinase (ITK) has been shown to play a role in the differentiation and/or function of TH2-type cells, suggesting that ITK may represent a good target for the control of asthma. Using a murine model of allergic asthma, we show here that ITK is involved in the development of immunological symptoms seen in this model. We show that mice lacking ITK have drastically reduced lung inflammation, eosinophil infiltration, and mucous production following induction of allergic asthma. Notably, T cell influx into the lung was reduced in mice lacking ITK. T cells from ITK−/− mice also exhibited reduced proliferation and cytokine secretion, in particular IL-5 and IL-13, in response to challenge with the allergen OVA, despite elevated levels of total IgE and increased OVA-specific IgE responses. Our results suggest that the tyrosine kinase ITK preferentially regulates the secretion of the TH2 cytokines IL-5 and IL-13 and may be an attractive target for antiasthmatic drugs.

Strain-specific requirement for eosinophils in the recruitment of T cells to the lung during the development of allergic asthma

Eosinophils have been implicated as playing a major role in allergic airway responses. However, the importance of these cells to the development of this disease has remained ambiguous despite many studies, partly because of lack of appropriate model systems. In this study, using transgenic murine models, we more clearly delineate a role for eosinophils in asthma. We report that, in contrast to results obtained on a BALB/c background, eosinophil-deficient C57BL/6 ΔdblGATA mice (eosinophil-null mice via the ΔDblGATA1 mutation) have reduced airway hyperresponsiveness, and cytokine production of interleukin (IL)-4, -5, and -13 in ovalbumin-induced allergic airway inflammation. This was caused by reduced T cell recruitment into the lung, as these mouse lungs had reduced expression of CCL7/MCP-3, CC11/eotaxin-1, and CCL24/eotaxin-2. Transferring eosinophils into these eosinophil-deficient mice and, more importantly, delivery of CCL11/eotaxin-1 into the lung during the development of this disease rescued lung T cell infiltration and airway inflammation when delivered together with allergen. These studies indicate that on the C57BL/6 background, eosinophils are integral to the development of airway allergic responses by modulating chemokine and/or cytokine production in the lung, leading to T cell recruitment.

A novel function for the Tec family tyrosine kinase Itk in activation of β1 integrins by the T-cell receptor

Stimulation of T cells via the CD3–T‐cell receptor (TCR) complex results in rapid increases in β1 integrin‐mediated adhesion via poorly defined intracellular signaling events. We demonstrate that TCR‐mediated activation of β1 integrins requires activation of the Tec family tyrosine kinase Itk and phosphatidylinositol 3‐kinase (PI 3‐K)‐dependent recruitment of Itk to detergent‐insoluble glycosphingolipid‐enriched microdomains (DIGs) via binding of the pleckstrin homology domain of Itk to the PI 3‐K product PI(3,4,5)‐P3. Activation of PI 3‐K and the src family kinase Lck, via stimulation of the CD4 co‐receptor, can initiate β1 integrin activation that is dependent on Itk function. Targeting of Itk specifically to DIGs, coupled with CD4 stimulation, can also activate β1 integrin function independently of TCR stimulation. Changes in β1 integrin function mediated by TCR activation of Itk are also accompanied by Itk‐dependent modulation of the actin cytoskeleton. Thus, TCR‐mediated activation of β1 integrins involves membrane relocalization and activation of Itk via coordinate action of PI 3‐K and a src family tyrosine kinase.

Enhanced development of CD4+ γδ T cells in the absence of Itk results in elevated IgE production

The Tec kinase Itk is critical for the development of alphabeta T cells as well as differentiation of CD4(+) T cells into Th2 cells. Itk null mice have defects in the production of Th2 cytokines; however, they paradoxically have significant elevations in serum IgE. Here we show that Itk null mice have increased numbers of gammadelta T cells in the thymus and spleen. This includes elevated numbers of CD4(+) gammadelta T cell, the majority of which carry the Vgamma1.1 and Vdelta6.2/3 gammadelta T-cell receptor with a distinct phenotype. The development of these CD4(+) gammadelta T cells is T cell intrinsic, independent of either major histocompatibility complex class I or class II, and is favored during development in the absence of Itk. Itk null CD4(+) gammadelta T cells secrete significant amounts of Th2 cytokines and can induce the secretion of IgE by wild-type B cells. Our data indicate that Itk plays important role in regulating gammadelta T-cell development and function. In addition, our data indicate that the elevated IgE observed in Itk-deficient mice is due in part to the enhanced development of CD4(+) gammadelta T cells in the absence of Itk.

Selective targeting of ITK blocks multiple steps of HIV replication

Treatment for HIV has relied on the use of antiretroviral agents that can be subject to the development of resistant viruses. The study of inhibitors directed against cellular proteins required for HIV replication is therefore of growing interest. Inducible T cell kinase (ITK) is a Tec family tyrosine kinase that regulates T cell receptor (TCR)-induced activation of PLCγ-1, Ca2+ mobilization and transcription factor activation, and actin rearrangement downstream of both TCR and chemokine receptors. Because productive infection of T cells with HIV requires T cell activation, chemokine receptors and actin reorganization, we asked whether ITK affects HIV infection using ITK-specific siRNA, a kinase-inactive ITK mutant or an ITK inhibitor. We demonstrate that loss of ITK function resulted in marked reductions in intracellular p24 levels upon HIV infection. Loss of ITK function after establishment of HIV infection also decreased virus spread within the culture. Inhibition of ITK did not affect expression of the HIV coreceptors CD4 or CXCR4 but partially blocked HIV viral entry, an effect that correlated with decreased actin polarization to gp120. Additionally, ITK was required for efficient HIV transcription, and overexpression of ITK increased both viral transcription and virus-like particle formation. Our data suggest that inhibition of ITK blocks HIV infection by affecting multiple steps of HIV replication.

Memory phenotype CD8+ T cells with innate function selectively develop in the absence of active itk

T cells with a memory‐like phenotype and possessing innate immune function have been previously identified as CD8+CD44hi cells. These cells rapidly secrete IFN‐γ upon stimulation with IL‐12/IL‐18 and are involved in innate responses to infection with Listeria monocytogenes. The signals regulating these cells are unclear. The Tec kinase Itk regulates T cell activation and we report here that a majority of the CD8+ T cells in Itk null mice have a phenotype of CD44hi similar to memory‐like innate T cells. These cells are observed in mice carrying an Itk mutant lacking the kinase domain, indicating that active Tec kinase signaling suppresses their presence. These cells carry preformed message for and are able to rapidly produce IFN‐γ upon stimulation in vitro with IL‐12/IL‐18, and endow Itk null mice the ability to effectively respond to infection with L. monocytogenes or exposure to lipopolysaccharides by secretion of IFN‐γ. Transfer of these cells rescues the ability of IFN‐γ null mice to reduce bacterial burden following L. monocytogenes infection, indicating that these cells are functional CD8+CD44hi T cells previously detected in vivo. These results indicate that active signals from Tec kinases regulate the development of memory‐like CD8+ T cells with innate function.

Naive 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 in this study that Itk null mice have increased percentage of CD62LlowCD44high memory phenotype CD4+ T cells compared with wild-type mice. These cells arise directly in the thymus, express high levels of transcripts for the T-bet and IFN-γ and are able to produce IFN-γ directly ex vivo in response to stimulation. Itk deficiency greatly decreases the number of CD4+ T cells with CD62LhighCD44low naive 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 naive phenotype CD4+ T cells is dependent on active Itk signals and can be rescued by expression of Itk specifically in T cells. Our data also show that Itk is required for functional TCR signaling in these cells, but not for the innate function in response to IL-12/IL-18 or Listeria monocytogenes stimulation. These results indicate that CD62LhighCD44low “naive” CD4+ and CD62LlowCD44high “innate memory phenotype” CD4+ T cells may be independent populations that differ in their requirement for Itk signals for development. Our data also suggest that CD4+CD62LlowCD44high memory phenotype T cells have innate immune function.

Differential regulation of NFAT and SRF by the B cell receptor via a PLCγ–Ca2+-dependent pathway

NFAT and SRF are important in the regulation of proliferation and cytokine production in lymphocytes. NFAT activation by the B cell receptor (BCR) occurs via the PLCγ–Ca2+–calcineurin pathway, however how the BCR activates SRF is unclear. We show here that like NFAT, BCR regulation of SRF occurs via an Src–Syk–Tec–PLCγ–Ca2+ (Lyn–Syk–Btk–PLCγ–Ca2+) pathway. However, SRF responds to lower Ca2+ and is less dependent on IP3R expression than NFAT. Ca2+‐regulated calcineurin plays a partial role in SRF activation, in combination with diacylglycerol (DAG), while is fully required for NFAT activation. Signals from the DAG effectors protein kinase C, Ras and Rap1, and the downstream MEK–ERK pathway are required for both SRF and NFAT; however, NFAT but not SRF is dependent on JNK signals. Both SRF and NFAT were also dependent on Rac, Rho, CDC42 and actin. Finally, we show that Ca2+ is not required for ERK activation, but instead for its association with nuclear areas of the cell. These data suggest that combinatorial assembly of signaling pathways emanating from the BCR differentially regulate NFAT and SRF, to activate gene expression.