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Mg2+ is required at micromolar concentrations as a cofactor for ATP, enzymatic reactions, and other biological processes. We show that decreased extracellular Mg2+ reduced intracellular Mg2+ levels and impaired the Ca2+ flux, activation marker up-regulation, and proliferation after T cell receptor (TCR) stimulation. Reduced Mg2+ specifically impairs TCR signal transduction by IL-2–inducible T cell kinase (ITK) due to a requirement for a regulatory Mg2+ in the catalytic pocket of ITK. We also show that altered catalytic efficiency by millimolar changes in free basal Mg2+ is an unrecognized but conserved feature of other serine/threonine and tyrosine kinases, suggesting a Mg2+ regulatory paradigm of kinase function. Finally, a reduced serum Mg2+ concentration in mice causes an impaired CD8+ T cell response to influenza A virus infection, reduces T cell activation, and exacerbates morbidity. Thus, Mg2+ directly regulates the active site of specific kinases during T cell responses, and maintaining a high serum Mg2+ concentration is important for antiviral immunity in otherwise healthy animals.

Progenitor-like CD8+ T cells mediate long-term immunity to chronic infection and cancer and respond potently to immune checkpoint blockade. These cells share transcriptional regulators with memory precursor cells, including T cell-specific transcription factor 1 (TCF1), but it is unclear whether they adopt distinct programs to adapt to the immunosuppressive environment. By comparing the single-cell transcriptomes and epigenetic profiles of CD8+ T cells responding to acute and chronic viral infections, we found that progenitor-like CD8+ T cells became distinct from memory precursor cells before the peak of the T cell response. We discovered a coexpression gene module containing Tox that exhibited higher transcriptional activity associated with more abundant active histone marks in progenitor-like cells than memory precursor cells. Moreover, thymocyte selection-associated high mobility group box protein TOX (TOX) promoted the persistence of antiviral CD8+ T cells and was required for the programming of progenitor-like CD8+ T cells. Thus, long-term CD8+ T cell immunity to chronic viral infection requires unique transcriptional and epigenetic programs associated with the transcription factor TOX.

Th9 cells produce interleukin (IL)-9, a cytokine implicated in allergic asthma and autoimmunity. Here we show that Itk, a mediator of T cell receptor signalling required for Th2 immune responses and the development of asthma, is a positive regulator of Th9 differentiation. In a model of allergic lung disease, Itk-deficient mice show reduced pulmonary inflammation and IL-9 production by T cells and innate lymphoid type 2 cells (ILC2), despite normal early induction of ILC2s. In vitro, Itk−/− CD4+ T cells do not produce IL-9 and have reduced levels of IRF4 (Interferon Regulator Factor 4), a critical transcription factor for effector T cell function. Both IL-9 and IRF4 expression are rescued by either IL-2 or constitutively active STAT5, but not NFATc1. STAT5 binds the Irf4 promoter, demonstrating one mechanism by which IL-2 rescues weakly activated T cells. Itk inhibition also reduces IL-9 expression by human T cells, implicating ITK as a key regulator of Th9 induction., The Tec family tyrosine kinase, Itk, is a component of the T-cell receptor essential for optimal Th2 responses in vivo. Here the authors show in human cells and mouse models that Itk is also needed for the production of IL-9, an important contributor to allergic asthma.

SUMMARY Patients with activated phosphatidylinositol 3-kinase delta (PI3Kδ) syndrome (APDS) present with sinopulmonary infections, lymphadenopathy, and cytomegalvirus (CMV) and/or Epstein-Barr virus (EBV) viremia, yet why patients fail to clear certain chronic viral infections remains incompletely understood. Using patient samples and a mouse model (Pik3cdE1020K/+ mice), we demonstrate that, upon activation, Pik3cdE1020K/+ CD8+ T cells exhibit exaggerated features of effector populations both in vitro and after viral infection that are associated with increased Fas-mediated apoptosis due to sustained FoxO1 phosphorylation and Fasl derepression, enhanced mTORC1 and c-Myc signatures, metabolic perturbations, and an altered chromatin landscape. Conversely, Pik3cdE1020K/+ CD8+ cells fail to sustain expression of proteins critical for central memory, including TCF1. Strikingly, activated Pik3cdE1020K/+ CD8+ cells exhibit altered transcriptional and epigenetic circuits characterized by pronounced interleukin-2 (IL-2)/STAT5 signatures and heightened IL-2 responses that prevent differentiation to memory-like cells in IL-15. Our data position PI3Kδ as integrating multiple signaling nodes that promote CD8+ T cell effector differentiation, providing insight into phenotypes of patients with APDS., In brief Using T cells from patients and a mouse model of activated PI3Kδ syndrome (APDS), Cannons et al. provide evidence that activated PI3Kδ drives transcriptional, chromatin, and metabolic changes involving IL-2, mTOR, Myc, and TCF1 that promote the differentiation of terminal and long-lived effector populations at the expense of central memory cells., Graphical Abstract

Progenitor-like CD8

Gain-of-function mutations in the gene encoding the phosphatidylinositol-3-OH kinase catalytic subunit p110δ (PI3Kδ) result in a human primary immunodeficiency characterized by lymphoproliferation, respiratory infections and inefficient responses to vaccines. However, what promotes these immunological disturbances at the cellular and molecular level remains unknown. We generated a mouse model that recapitulated major features of this disease and used this model and patient samples to probe how hyperactive PI3Kδ fosters aberrant humoral immunity. We found that mutant PI3Kδ led to co-stimulatory receptor ICOS-independent increases in the abundance of follicular helper T cells (TFH cells) and germinal-center (GC) B cells, disorganized GCs and poor class-switched antigen-specific responses to immunization, associated with altered regulation of the transcription factor FOXO1 and pro-apoptotic and anti-apoptotic members of the BCL-2 family. Notably, aberrant responses were accompanied by increased reactivity to gut bacteria and a broad increase in autoantibodies that were dependent on stimulation by commensal microbes. Our findings suggest that proper regulation of PI3Kδ is critical for ensuring optimal host-protective humoral immunity despite tonic stimulation from the commensal microbiome.

Loss of the Tec family kinase Itk results in a bias to FoxP3+ Treg cell differentiation and reduced TCR-induced phosphorylation of mTOR targets., 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.

During chronic viral infections and in cancer, T cells become dysfunctional, a state known as T cell exhaustion. Although it is well recognized that memory CD8 T cells account for the persistence of CD8 T cell immunity after acute infection, how exhausted T cells persist remains less clear. Using chronic infection with lymphocytic choriomeningitis virus clone 13 and tumor samples, we demonstrate that CD8 T cells differentiate into a less exhausted TCF1high and a more exhausted TCF1low population. Virus-specific TCF1high CD8 T cells, which resemble T follicular helper (TFH) cells, persist and recall better than do TCF1low cells and act as progenitor cells to replenish TCF1low cells. We show that TCF1 is both necessary and sufficient to support this progenitor-like CD8 subset, whereas cell-intrinsic type I interferon signaling suppresses their differentiation. Accordingly, cell-intrinsic TCF1 deficiency led to a loss of these progenitor CD8 T cells, sharp contraction of virus-specific T cells, and uncontrolled viremia. Mechanistically, TCF1 repressed several pro-exhaustion factors and induced Bcl6 in CD8 T cells, which promoted the progenitor fate. We propose that the TCF1-Bcl6 axis counteracts type I interferon to repress T cell exhaustion and maintain T cell stemness, which is critical for persistent antiviral CD8 T cell responses in chronic infection. These findings provide insight into the requirements for persistence of T cell immune responses in the face of exhaustion and suggest mechanisms by which effective T cell–mediated immunity may be enhanced during chronic infections and cancer.

Mice deficient in the Tec kinase Itk develop a large population of CD8 cells with properties resembling NKT and other innate T cell lineages, including expression of memory markers, rapid production of cytokines and dependence on IL-15. Like NKT cells, these CD8 cells can be selected on hematopoietic cells. We demonstrate here that these CD8 cell phenotypes result from selection on hematopoietic cells—forcing selection on the thymic stroma reduces the number and innate cell phenotypes of mature CD8 SP cells associated with Itk-deficiency. We further demonstrate that, similar to NKT cells, selection of innate-type CD8 cells in Itk-deficient mice strictly requires the adaptor molecule SAP, whereas, acquisition of the innate characteristics requires CD28. Our results suggest that SAP and Itk reciprocally regulate selection of innate and conventional CD8 cells on hematopoietic cells and the thymic epithelium, respectively, whereas CD28 regulates development of innate phenotypes resulting from selection on hematopoietic cells.

The catalytic p110δ subunit of PI3K drives AKT pathways orchestrating activation and differentiation of T and B cells. Patients with gain of function mutations in Pik3cd (encoding for p110δ) exhibit a primary immunodeficiency characterized by lymphopenia, lymphadenopathy, recurrent and chronic infections, and occasionally lymphoma. They have reduced circulating naïve and increased effector T cell numbers, fewer class switched memory B cells and inefficient responses to vaccination. Nonetheless, in patient peripheral blood we find increased frequencies of T follicular helper (Tfh) cells. We have generated a mouse model expressing constitutively active p110δ that recapitulates many features of the human disease. Pik3cdMut mice, both in steady state condition and after immunization, display increased Tfh and germinal center (GC) B cell numbers in secondary lymphoid organs. Moreover, naïve mutant transgenic T cells (OT-II) preferentially acquire Tfh cell phenotype when adoptively transferred to WT mice, indicative of a T cell intrinsic defect. Nonetheless, despite increased Tfh and GC B cells, antigen specific B cell responses are reduced, particularly antigen-specific IgG. In contrast, we find the appearance of autoantibodies in Pik3cdMut mice. We further find that mutant OT-2 cells differentiate to Tfh cells independently from ICOS-ICOSL interactions. In line with this, mutant T cells show higher phosphorylation and inactivation of FoxO1, potent inhibitor of Tfh cell differentiation, that is phosphorylated and inhibited by AKT activation downstream of ICOS. Our results demonstrate that hyperactivated PI3Kδ leads to increased generation of Tfh cells and germinal centers, yet with defective in vivo function.

The p110d subunit of phosphatidylinositol-3-OH kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. We previously reported patients heterozygous for three different germline, gain-of-function mutations in PIK3CD (encoding p110d). Patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and CMV and/or EBV viremia, suggestive of immune dysfunction. We established a mouse model (PIK3cd E1024K) that recapitulates features of this disease including lymphopenia, yet enlarged secondary lymphoid organs, as well as increased Akt phosphorylation and hyperactivation of the metabolic checkpoint kinase mTOR. In response to TCR stimulation, mutant T cells proliferate, but rapidly die via a Fas/FasL-mediated apoptosis. The few surviving mutant CD8 T cells exhibit features of short-lived effectors, expressing abundant cytokines and elevated granzymes, and kill target cells more efficiently than WT counterparts. This phenotype is mirrored at early time points following viral infection. However, at late time-points post-viral infection, mutant CD8 T cells fail to sustain expression of proteins critical for maintenance of long-lived memory cells. Thus, our data suggests that activated p110d limits effective CD8 T cell memory. Our mouse model therefore recapitulates facets of the human primary immunodeficiency, revealing insight into the pathogenesis of this disease.

T helper 9 (Th9) cells produce IL-9, a cytokine implicated in allergic asthma and autoimmunity. We show that Itk, a mediator of T-cell receptor signalling in Th2 immune responses and the development of asthma, is a positive regulator of Th9 differentiation in vivo and in vitro. In a papain-induced model of allergic lung disease, Itk-deficient mice have reduced pulmonary inflammation and IL-9 production by T cells and innate lymphoid type 2 cells (ILC2), despite normal induction of ILC2s. In vitro, naïve Itk−/− CD4+ T cells do not produce IL-9 and have reduced levels of IRF4, a critical transcription factor for effector T-cell function. Both IL-9 and IRF4 expression are rescued by either IL-2 or expression of constitutively-active STAT5, but not NFATc1. STAT5 binds the Irf4 promoter, indicating a mechanism by which IL-2 rescues weakly-activated T cells. Itk inhibition also reduced IL-9 expression by human CD4 T cells, thereby implicating ITK as a possible target to prevent Th9-mediated inflammatory diseases.

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.

One of the main functions of the adaptive immune system is to mount specific responses to pathogens. These effector CD4 T helper cells, include Th1, Th2, Th9 and Th17 cells, produce distinct cytokines important in the eradication of different infectious pathogens--however excessive activation of these cells can be harmful to the host, contributing to immunopathology, including autoimmunity and asthma. ITK-deficient mice exhibit reduced responses in models of allergic asthma, a disease attributed to increased Th2 responses. Polymorphisms in the Itk promoter in humans have also been linked to increased asthma incidence. Th9 cells are a recently appreciated T helper cell population that produce IL-9, a cytokine implicated in allergic asthma, responses to microbial infections, inflammatory gut diseases and autoimmunity. However, the factors contributing to IL-9 regulation are still poorly understood. We have found that ITK-deficient CD4 cells completely fail to produce IL-9 in culture. This defect is one of the most profound phenotypes we have observed for Itk-/- cells to date, and is associate with reduced levels of IRF4 during Th9 differentiation. Furthermore, in preliminary experiments, we found decreased IL-9 and IL-13 expression in vivo using a papain hypersensitivity model. Our studies demonstrate important roles for Itk in Th9 differentiation, suggesting Itk as a therapeutic candidate for treatment of diseases involving Th9-mediated inflammation.

Intrathymic injection has traditionally been performed using surgical procedures to open the chest cavity for direct visualization and injection into mice thymus. This technique subjects the mice to significant pain and distress, which can alter T cell development and potentially increase mortality. Intrathymic injection can also be performed by blind injection into the thoracic cavity. However, the success rate for this technique is usually low. In order to improve success rates with non-surgical intrathymic injection, our lab has preformed intrathymic injections in isoflurane-anesthetized mice using ultrasound (US)-mediated guidance. The success rate using this technique has been high as the needle and injectate can both be visualized by US as they enter the thymus. Using this technique, we have been able to label thymocytes using either FITC or Biotin to trace their fates. Complications have been very rare and were limited to the accidental laceration of an intrathoracic vessel that required in the euthanasia of one mouse out of approximately 85 injected to date. Moreover, it is important to note, that we were able to see the immediate hemorrhage on US and euthanized the mouse while it was still anesthetized, thereby avoiding any pain or distress. Our results suggest that use of US-mediated guidance is a refinement that can help reduce morbidity, distress and animal usage in experiment involving intrathymic injection.

Th17 CD4+ effector T cells play important roles in autoimmunity and responses to bacterial infections. The roles of cytokines in regulating the transcription factors STAT3 and RORγT in Th17 differentiation have been extensively studied. However, the role of T cell receptor (TCR) signaling in Th17 differentiation is less appreciated. To examine this issue, we evaluated IL17 production from T cells lacking Itk, a tyrosine kinase required for full TCR-induced activation of PLC-γ and downstream pathways. We find that Itk-/- CD4+T cells exhibit specific defects in the expression of IL-17A despite relatively normal expression of RORγT, RORα and the other Th17 cytokines, including the closely related IL-17F. Defects in IL-17A expression and differential regulation of IL-17A and IL-17F were also observed in vivo in Itk-/- mice challenged with an allergic asthma model. Although Itk-deficient cells have slightly depressed phosphorylation of STAT-3 in response to IL-6, expression of constitutively activated STAT-3 failed to rescue their IL-17A production. In contrast, expression of IL-17A could be rescued by pharmacologically-induced Ca2+ influx or by a constitutively active NFATc1. The effects of Itk on transcriptional complexes and chromatin at the IL-17 locus are under further investigation. Our results suggest that Itk specifically couples TCR signaling strength to IL-17A expression through NFATc1 and that TCR signaling pathways differentially influence Th17 cytokine production.

Mutations affecting the Tec kinases Itk and Rlk impair both positive and negative selection and alter CD4 and CD8 T cell development. We previously demonstrated that mature CD8 SP thymocytes and peripheral CD8 T cells in Itk−/− and Rlk−/−Itk−/− mice do not resemble conventional CD8 T cells. Instead, these cells express memory markers and can be selected on MHC class I expressed on hematopoietic cells, suggesting these cells resemble non-conventional MHC class Ib-selected “innate-type” cells. Indeed, Itk-deficiency increases positive selection on MHC class Ib in the Kb−/−Db−/− background. We further find that either improving TCR signaling with stronger signals orblocking CD28 costimulation, partially corrects memory phenotypes of Itk−/− CD8 T cells, arguing that different strength or quality of signals may lead to development of this distinct CD8 T cell population. Our results suggest that Tec kinases differentially regulate development of conventional versus non-conventional lymphocytes. The requirement for signals from hematopoietic cells for selection of these “innate-type” CD8 T cells in Itk−/− mice will be discussed.

Summary Follicular T helper (Tfh) cells provide critical help to B cells for germinal center (GC) formation. Mutations affecting SLAM-associated protein (SAP) prevent GC formation because of defective T cell-B cell interactions, yet effects on Tfh cell differentiation remain unclear. We describe the in vitro differentiation of functionally competent "Tfh-like" cells that expressed interleukin-21, Tfh cell markers, and Bcl6 and rescued GC formation in SAP-deficient hosts better than other T helper (Th) cells. SAP-deficient Tfh-like cells appeared virtually indistinguishable from wild-type, yet failed to support GCs in vivo. Interestingly, both Tfh-like and in vivo-derived Tfh cells could produce effector cytokines in response to polarizing conditions. Moreover, Th1, Th2, and Th17 cells could be reprogrammed to obtain Tfh cell characteristics. ChIP-Seq analyses revealed positive epigenetic markings on Tbx21 , Gata3 , and Rorc in Tfh-like and ex vivo Tfh cells and on Bcl6 in non-Tfh cells, supporting the concept of plasticity between Tfh and other Th cell populations.