Type 1 regulatory T (Tr1) cells differentiate in response to signals engaging the 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 we show that ITK, an important modulator of TCR signalling, is required for the TCR-induced development of Tr1 cells in various organs, and in the mucosal system during parasitic and viral infections. ITK kinase activity is required for mouse and human Tr1 cell differentiation. Tr1 cell development and suppressive function of Itk deficient cells can be restored by the expression of the transcription factor interferon regulatory factor 4 (IRF4). Downstream of ITK, Ras activity is responsible for Tr1 cell induction, as expression of constitutively active HRas rescues IRF4 expression and Tr1 cell differentiation in Itk−/− cells. We conclude that TCR/ITK signalling through the Ras/IRF4 pathway is required for functional development of Tr1 cells.

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.

Following peripheral nerve injury, extracellular adenosine 5′-triphosphate (ATP)–mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury–induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain–like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury–induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.

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 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.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CD25hiFoxp3+CD4+ regulatory T cells (Tregs) are important immune regulators that promote immune tolerance, but can also form barriers to effective immunity against tumor and chronic infection. IL-2-inducible T cell kinase (ITK) is highly expressed in T cells and regulates T cell development and function. We find that ITK is required for full suppressive function of natural Tregs in preventing the expansion of pro-inflammatory CD4+ effector T cells and chronic pulmonary and colonic inflammation upon transfer into Rag-/- mice. In the absence of ITK, Treg homing to these inflamed mucosal systems is intact, however, a significant proportion is converted into pathogenic effector T cells with ability to produce IL-17A and/or IFN-γ in the inflamed lung and colon, but not in the spleen. Such loss of functional stability and gain of pathogenic reprograming are T cell intrinsic and can be rescued by re-expression of ITK in Itk-/- Tregs. Homeostatic expansion contributed to the initiation of Itk-/- Treg destabilization and pathogenic conversion but did not trigger pathogenesis. Stimulation through the T cell receptor in the presence or absence of various cytokines revealed that ITK is required for sustaining Foxp3 expression. We conclude that ITK signals suppress pathogenic conversion of Tregs by maintaining Foxp3 expression, and contributes to the functional stability of Tregs in the face of inflammation by limiting their reprograming towards the pathogenic effectors.

Following peripheral nerve injury, extracellular ATP-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.