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Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells have become attractive therapeutic targets for cancer immunotherapy because of their efficient mechanisms for killing cancerous cells. Both effector CTLs and mature NK cells are endowed with secretory granules containing pre-formed cytolytic proteins that, once released at the immunological synapse, initiate a cascade of reactions leading to the apoptosis of the target cell. CTLs and NK cells may also promote cell killing through death-receptor-mediated and cytokine-induced cell death mechanisms. Here we show a 16-color flow cytometry panel allowing clear identification of CTLs, NK cells and related cell subsets in whole blood. We also describe an optimized intracellular staining protocol for analysis of granules content, including granzyme B, granzyme K and perforin. Optimal resolution of cell populations was achieved by employing a selection of BD Horizon Brilliant™ Reagents and BD Horizon™ Red 718 Reagent, a new small molecule fluorochrome option for the red laser. Cell fixation and permeabilization were carried out directly in whole blood using the BD Phosflow™ Reagents, adding convenience to the workflow and practical analysis of cell frequencies. The data also showed the expression of selected activation and inhibitory surface receptors. These results combined with the analysis of intracellular proteins contribute to characterization of the functional status of cytotoxic cells. Class 1 Laser Product. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD 25443 (v1.0) 0121 BD, the BD Logo, FACSymphony, Horizon, Horizon Brilliant and Phosflow are trademarks of Becton, Dickinson and Company or its affiliates. © 2021 BD. All rights reserved.

An allelic variant of protein tyrosine phosphatase nonreceptor type 22 (PTPN22), PTPN22R620W, is strongly associated with type 1 diabetes (T1D) in humans and increases the risk of T1D by two- to fourfold. The NOD mouse is a spontaneous T1D model that shares with humans many genetic pathways contributing to T1D. We hypothesized that the introduction of the murine orthologous Ptpn22R619W mutation to the NOD genome would enhance the spontaneous development of T1D. We microinjected CRISPR-Cas9 and a homology-directed repair template into NOD single-cell zygotes to introduce the Ptpn22R619W mutation to its endogenous locus. The resulting Ptpn22R619W mice showed increased insulin autoantibodies and earlier onset and higher penetrance of T1D. This is the first report demonstrating enhanced T1D in a mouse modeling human PTPN22R620W and the utility of CRISPR-Cas9 for direct genetic alternation of NOD mice.

Tight regulation of hematopoietic stem cell (HSC) homeostasis ensures lifelong hematopoiesis and prevents blood cancers. The mechanisms balancing HSC quiescence with expansion and differentiation into hematopoietic progenitors are incompletely understood. Here, we identify Inositol-trisphosphate 3-kinase B (Itpkb) as an essential regulator of HSC homeostasis. Young Itpkb(-/-) mice accumulated phenotypic HSC, which were less quiescent and proliferated more than wild-type (WT) controls. Itpkb(-/-) HSC downregulated quiescence and stemness associated, but upregulated activation, oxidative metabolism, protein synthesis, and lineage associated messenger RNAs. Although they had normal-to-elevated viability and no significant homing defects, Itpkb(-/-) HSC had a severely reduced competitive long-term repopulating potential. Aging Itpkb(-/-) mice lost hematopoietic stem and progenitor cells and died with severe anemia. WT HSC normally repopulated Itpkb(-/-) hosts, indicating an HSC-intrinsic Itpkb requirement. Itpkb(-/-) HSC showed reduced colony-forming activity and increased stem-cell-factor activation of the phosphoinositide-3-kinase (PI3K) effectors Akt/mammalian/mechanistic target of rapamycin (mTOR). This was reversed by treatment with the Itpkb product and PI3K/Akt antagonist IP4. Transcriptome changes and biochemistry support mTOR hyperactivity in Itpkb(-/-) HSC. Treatment with the mTOR-inhibitor rapamycin reversed the excessive mTOR signaling and hyperproliferation of Itpkb(-/-) HSC without rescuing colony forming activity. Thus, we propose that Itpkb ensures HSC quiescence and function through limiting cytokine-induced PI3K/mTOR signaling and other mechanisms.

Development of a self-tolerant T-cell receptor (TCR) repertoire with the potential to recognize the universe of infectious agents depends on proper regulation of TCR signalling. The repertoire is whittled down during T-cell development in the thymus by the ability of quasi-randomly generated TCRs to interact with self-peptides presented by major histocompatibility complex (MHC) proteins. Low-affinity TCR interactions with self-MHC proteins generate weak signals that initiate 'positive selection', causing maturation of CD4- or CD8αβ-expressing 'single-positive' thymocytes from CD4(+)CD8αβ(+) 'double-positive' precursors. These develop into mature naive T cells of the secondary lymphoid organs. TCR interaction with high-affinity agonist self-ligands results in 'negative selection' by activation-induced apoptosis or 'agonist selection' of functionally differentiated self-antigen-experienced T cells. Here we show that positive selection is enabled by the ability of the T-cell-specific protein Themis to specifically attenuate TCR signal strength via SHP1 recruitment and activation in response to low- but not high-affinity TCR engagement. Themis acts as an analog-to-digital converter translating graded TCR affinity into clear-cut selection outcome. By dampening mild TCR signals Themis increases the affinity threshold for activation, enabling positive selection of T cells with a naive phenotype in response to low-affinity self-antigens.

Natural killer (NK) cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections, tissue homeostasis, and reproduction. NK cell–based therapies are promising treatments for blood cancers. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize the loss of self-antigens or up-regulation of stress-induced ligands on pathogen-infected or tumor cells through invariant NK cell receptors (NKRs), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3 generation by PI3K and generation of diacylglycerol and IP3 by phospholipase-Cγ (PLCγ). In the present study, we identify a novel role for the phosphorylated IP3 metabolite inositol (1,3,4,5)tetrakisphosphate (IP4) in NK cells. IP4 promotes NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR-induced IFNγ secretion, granule exocytosis, and target-cell killing, in part by inhibiting the PIP3 effector-kinase Akt. This identifies IP4 as an important novel regulator of NK cell development and function and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.

The present study focuses on a large family with an X-linked immunodeficiency in which there are variable clinical and laboratory phenotypes, including recurrent viral and bacterial infections, hypogammaglobulinemia, Epstein-Barr virus–driven lymphoproliferation, splenomegaly, colitis, and liver disease. Molecular and genetic analyses revealed that affected males were carriers of a hypomorphic hemizygous mutation in XIAP (XIAPG466X) that cosegregated with a rare polymorphism in CD40LG (CD40 ligandG219R). These genes are involved in the X-linked lymphoproliferative syndrome 2 and the X-linked hyper-IgM syndrome, respectively. Single expression of XIAPG466X or CD40LG219R had no or minimal effect in vivo, although in vitro, they lead to altered functional activities of their gene products, which suggests that the combination of XIAP and CD40LG mutations contributed to the expression of clinical manifestations observed in affected individuals. Our report of a primary X-linked immunodeficiency of oligogenic origin emphasizes that primary immunodeficiencies are not caused by a single defective gene, which leads to restricted manifestations, but are likely to be the result of an interplay between several genetic determinants, which leads to more variable clinical phenotypes.

β-selection is the most pivotal event determining αβ T cell fate. Here, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. Besides the pre-TCR, β-selection also requires co-stimulatory signals from Notch receptors - key cell fate determinants in eukaryotes. Here, we show that this Notch-dependence is established through antagonistic signaling by the pre-TCR/Notch effector, phosphoinositide 3-kinase (PI3K), and by inositol-trisphosphate 3-kinase B (Itpkb). Canonically, PI3K is counteracted by the lipid-phosphatases Pten and Inpp5d/SHIP-1. In contrast, Itpkb dampens pre-TCR induced PI3K/Akt signaling by producing IP4, a soluble antagonist of the Akt-activating PI3K-product PIP3. Itpkb-/- thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4+CD8+ cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose metabolism. Thus, non-canonical PI3K-antagonism by Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement. DOI: http://dx.doi.org/10.7554/eLife.10786.001, eLife digest T cells defend our body against cancer and infectious agents such as viruses. However, they can also cause rheumatoid arthritis and other autoimmune diseases by attacking healthy tissue. T cells recognize target cells via receptor proteins on their surface. To maximize the variety of infections and cancers our immune system can recognize, we generate millions of T cells with different T cell receptors every day. To ensure T cells work correctly, T cell receptors are tested at various checkpoints. The first checkpoint involves a process called beta (β) selection, during which T cells produce their first T cell receptor – the so-called pre-T cell receptor. This receptor causes T cells to divide and mature, and sets their future identity or “fate”. To complete β-selection, T cells must also receive signals from another surface receptor – one that belongs to the Notch family, which determines cell fate in many different tissues. The Notch receptor and the pre-T cell receptor both activate an enzyme called PI3K – a key mediator of β-selection. But the pre-T cell receptor also activates another enzyme called Itpkb that is required for T cell development. Westernberg, Conche et al. have now investigated how these different proteins and signaling processes work and interact during β-selection, using mice that lack several immune genes, including the gene that produces Itpkb. The results of the experiments show that during β-selection, Itpkb limits the ability of PI3K to activate some of its key target proteins. This “dampened” PI3K signaling ensures that both the pre-T cell receptor and the Notch receptor must be activated to trigger T cell maturation. Without Itpkb, β-selection can occur in the absence of Notch signaling. As Notch signaling is important for determining the fate of many different cell types, Westernberg, Conche et al.’s findings raise the possibility that Itpkb might also regulate cell fate determination in other tissues. Moreover, Itpkb may suppress tumor development, because excessive PI3K signaling drives many cancers. DOI: http://dx.doi.org/10.7554/eLife.10786.002

X-linked lymphoproliferative syndromes (XLP) are primary immunodeficiencies characterized by a particular vulnerability toward Epstein-Barr virus infection, frequently resulting in hemophagocytic lymphohistiocytosis (HLH). XLP type 1 (XLP-1) is caused by mutations in the gene SH2D1A (also named SAP), whereas mutations in the gene XIAP underlie XLP type 2 (XLP-2). Here, a comparison of the clinical phenotypes associated with XLP-1 and XLP-2 was performed in cohorts of 33 and 30 patients, respectively. HLH (XLP-1, 55%; XLP-2, 76%) and hypogammaglobulinemia (XLP-1, 67%; XLP-2, 33%) occurred in both groups. Epstein-Barr virus infection in XLP-1 and XLP-2 was the common trigger of HLH (XLP-1, 92%; XLP-2, 83%). Survival rates and mean ages at the first HLH episode did not differ for both groups, but HLH was more severe with lethal outcome in XLP-1 (XLP-1, 61%; XLP-2, 23%). Although only XLP-1 patients developed lymphomas (30%), XLP-2 patients (17%) had chronic hemorrhagic colitis as documented by histopathology. Recurrent splenomegaly often associated with cytopenia and fever was preferentially observed in XLP-2 (XLP-1, 7%; XLP-2, 87%) and probably represents minimal forms of HLH as documented by histopathology. This first phenotypic comparison of XLP subtypes should help to improve the diagnosis and the care of patients with XLP conditions.

The homeostasis of the immune response requires tight regulation of the proliferation and apoptosis of activated lymphocytes. In humans, defects in immune homeostasis result in lymphoproliferation disorders including autoimmunity, haemophagocytic lymphohystiocytosis and lymphomas. The X-linked lymphoproliferative syndrome (XLP) is a rare, inherited immunodeficiency that is characterized by lymphohystiocytosis, hypogammaglobulinaemia and lymphomas, and that usually develops in response to infection with Epstein-Barr virus (EBV). Mutations in the signalling lymphocyte activation molecule (SLAM)-associated protein SAP, a signalling adaptor molecule, underlie 60% of cases of familial XLP. Here, we identify mutations in the gene that encodes the X-linked inhibitor-of-apoptosis XIAP (also termed BIRC4) in patients with XLP from three families without mutations in SAP. These mutations lead to defective expression of XIAP. We show that apoptosis of lymphocytes from XIAP-deficient patients is enhanced in response to various stimuli including the T-cell antigen receptor (TCR)-CD3 complex, the death receptor CD95 (also termed Fas or Apo-1) and the TNF-associated apoptosis-inducing ligand receptor (TRAIL-R). We also found that XIAP-deficient patients, like SAP-deficient patients, have low numbers of natural killer T-lymphocytes (NKT cells), indicating that XIAP is required for the survival and/or differentiation of NKT cells. The observation that XIAP-deficiency and SAP-deficiency are both associated with a defect in NKT cells strengthens the hypothesis that NKT cells have a key role in the immune response to EBV. Furthermore, by identifying an XLP immunodeficiency that is caused by mutations in XIAP, we show that XIAP is a potent regulator of lymphocyte homeostasis in vivo.

> L’homeostasie des lymphocytes au cours de la reponse immunitaire est un mecanisme soumis a une regulation fine. C’est un equilibre permanent entre la proliferation des cellules activees et leur apoptose (ou mort cellulaire programmee). Chez l’homme, des defauts de l’homeostasie lymphocytaire peuvent conduire a de nombreuses maladies comme les lymphomes, les syndromes d’activation lymphocytaire et macrophagique (ou syndrome hemophagocytaire) ou les maladies auto-immunes. Le syndrome de Purtilo, ou syndrome lymphoproliferatif lie a l’X (XLP), est une immunodeficience primaire rare, caracterisee par une sensibilite accrue des patients a l’infection par le virus Epstein-Barr [1]. Cette maladie atteint environ un garcon pour un million. La plupart du temps, les patients souffrent d’une mononucleose infectieuse severe qui est frequemment associee a un syndrome hemophagocytaire (SH) similaire a celui observe dans les formes hereditaires causees par des defauts des fonctions cytotoxiques des lymphocytes [2]. Le SH est caracterise par une reponse immunitaire exacerbee au cours de laquelle les lymphocytes sont fortement actives, proliferent et secretent de grandes quantites d’IFN-γ qui stimulent secondairement les macrophages. Ceuxci phagocytent les elements figures du sang et infiltrent les organes en produisant des quantites importantes de cytokines pro-inflammatoires. Dans ce contexte, on observe une necrose massive des tissus qui peut entrainer la mort du patient. Chez certains patients, on note aussi l’apparition d’hypogammaglobulinemies et de lymphomes. Le gene responsable du XLP a ete identifie en 1998, il s’agit du gene SH2D1A (ou SAP) codant une petite proteine adaptatrice de signalisation SAP (SLAM-associated protein) qui regule les fonctions des NOUVELLE

The Greek philosopher Plato emphasized the importance of moderating personal desires for the functioning of society. The virtue of moderation is also critical for the live-long function of hematopoietic stem cells (HSC), the origin of all blood cells. To prevent undue activation, which can deplete HSC and increase the risk of blood cancer, HSC need to restrain signaling by phosphoinositide 3-kinases (PI3K) - a pivotal pathway whose hyperactivation contributes to many diseases [1]. Yet, the precise mechanisms limiting PI3K-signaling within HSC remain ill understood. We recently presented evidence that they include non-canonical PI3K-regulation by the little-studied enzyme inositoltrisphosphate 3-kinase B (IP3 3-kinase B/Itpkb) in HSC [2]. Itpkb−/− mice died with HSC/hematopoietic-progenitor-cell (HPC) depletion and anemia. Thus, we speculate that defective PI3K-dampening by Itpkb or other moderators contributes to the frequent HSC-defects in bone marrow (BM) failure syndromes [3]. HSC are pluripotent and long-lived. Their longevity relies on their relative metabolic quiescence and only rare division for self-renewal. This dormancy is facilitated by HSC-residence in hypoxic BM niches. Metabolic adaptation and interactions with niche adhesion-molecules and cyto-/chemokines including stem-cell-factor (SCF) keep HSC quiescent and self-renewing [2, 4]. Hematopoietic stress causes the release of cytokines or other factors which mobilize and activate HSC to proliferate and generate shorter-lived HPC. These give rise to the different blood cell lineages. Once blood cell homeostasis is re-established, HSC re-enter dormancy. This is critical, because continuous HSC-activation reduces self-renewal and ultimately depletes HSC, causing BM failure and immunodeficiencies, and promoting certain blood cancers [1, 2, 4]. Increasing evidence suggests that signaling from cytokines like SCF in dormant HSC must be tuned into an intensity-range that ensures self-renewal, but avoids activation. The underlying mechanisms are incompletely understood [1, 2]. Activated by cytokines and other signals, PI3K and its effectors Akt and mTORC1 are required for HSC self-renewal and function. But excessive PI3K/Akt activity transiently expands HSC, followed by depletion and reduced long-term engraftment associated with variable myeloproliferative disease, T-cell-acute-lymphoblastic (T-ALL) or acute-myeloblastic-leukemia (AML) [1, 2]. PI3K produce the membrane-lipid phosphatidylinositol(3,4,5)trisphosphate (PIP3), a recruiting and activating ligand for Akt and other effectors. To prevent excessive PI3K-signaling, PIP3-levels in many cell types are limited through its removal by the lipid-phosphatases PTEN and SHIP (references in [1, 2]). But the relative importance of HSC in- versus extrinsic PTEN functions remains controversial, and SHIP-1 may primarily control HSC-homeostasis cell-extrinsically (references in [2]). Could there be another HSC-intrinsic PI3K-moderator? Itpkb phosphorylates the Ca2+-mobilizing second-messenger IP3 into inositol(1,3,4,5)tetrakisphosphate (IP4). We and others have identified receptor-induced IP4 production by Itpkb as essential for signaling in lymphocytes, granulocyte-monocyte-progenitors (GMP) and neutrophils (references in [2, 5]). IP4 is identical with the protein-binding moiety of PIP3. In NK cells, GMP and neutrophils, IP4 competitively limits PIP3-binding to and activation of Akt (references in [2]). ItpkB is expressed in HSC. To thus determine if IP4/PIP3-antagonism moderates PI3K-signaling in HSC, we studied Itpkb−/− mice. Young Itpkb−/− mice accumulated phenotypic HSC which were less quiescent and proliferated more than Itpkb+/+ controls [2]. Transcriptome-analyses showed downregulation of stemness- and quiescence-associated, but upregulation of activation- and differentiation-associated genes in Itpkb−/− vs. wt HSC. Itpkb−/− HSC showed intact BM-homing, but reduced in vitro persistence and colony-forming-unit (CFU) activity. They had severely reduced competitive long-term-repopulating potential. Aging ItpkB−/− mice lost HSC and HPC and died with anemia. Supporting an Itpkb-role in moderating PI3K-signaling in HSC, Itpkb−/− HSC had elevated mTORC1 activity in vivo, and showed increased SCF-activation of Akt and mTORC1 in vitro. This was prevented by treatment with cell-permeable IP4 or an Akt-inhibitor. Transcriptome-analysis suggested Akt/mTORC1-hyperactivity and increased oxidative phosphorylation and protein biosynthesis in Itpkb−/− HSC. A recent study suggests that HSC-quiescence requires restrained protein biosynthesis and implies moderation of mTOR signaling [6]. Injection of the mTOR-inhibitor Rapamycin reversed the HSC-hyperproliferation in Itpkb−/− mice [2]. Thus, we propose that Itpkb limits cytokine and PI3K/Akt/mTOR signaling in HSC to ensure quiescence and longevity (Figure ​(Figure11). Figure 1 Symmetric signaling by PI3K and Itpkb through the antagonistic second messengers PIP3 and IP4 restricts signaling by Akt and downstream mTORC1 in HSC into a window that ensures self-renewal, quiescence and longevity but prevents activation, differentiation ... The HSC transient expansion but later depletion in Itpkb−/− mice resembles the phenotypes of PTEN-inactivation or dominant-active Akt expression. But Itpkb−/− mice have not yet shown T-ALL or AML. Possible reasons include differential effects of PTEN-loss, Akt-activation or Itpkb-loss on PI3K-signaling in HSC, or premature death of Itpkb−/− mice from anemia or infections [2]. Itpkb-loss might also impair PI3K-unrelated signaling events that promote neoplasia. Clarifying whether Itpkb-loss transforms hematopoietic cells will require conditional knockouts and larger aged cohorts. This will also rule out that Itpkb-loss in other cells indirectly contributes to the HSC-activation in Itpkb−/− mice [5]. Interestingly, Rapamycin reversed the HSC-hyperproliferation in Itpkb−/− mice but did not rescue their CFU-activity. This contrasts with PTEN−/− or myrAkt-expressing HSC and may suggest that mTORC1-unrelated mechanisms contribute to HSC-control by Itpkb [2]. But Rapamycin also reduced wt HSC CFU-activity, and genetic studies suggest mTORC1-requirements for HSC-regeneration and -function (references in [2]). This might explain the difficulty of rescuing Itpkb−/− HSC-function with mTORC1-inhibitors, and raises concerns that long-term therapeutic mTORC1-inhibition to prevent aging [7] might damage HSC. Wrapping up, Itpkb-identification as a moderator of cytokine- and PI3K-signaling in HSC advances our understanding of the HSC-intrinsic mechanisms balancing self-renewal with activation and broadens the importance of IP4/PIP3-antagonism as a non-canonical mechanism regulating PI3K-function. It will now be important to assess the human relevance of this mechanism, and to determine if human BM failure patients show disease-driving mutations in Itpkb. Moreover, it will be interesting o explore if transient specific and selective Itpkb-inhibition can mobilize or expand human HSC without impairing their function.

The inositol-phosphate messenger inositol(1,3,4,5)tetrakisphosphate (IP4) is essential for thymocyte positive selection by regulating plasma-membrane association of the protein tyrosine kinase Itk downstream of the T cell receptor (TCR). IP4 can act as a soluble analog of the phosphoinositide 3-kinase (PI3K) membrane lipid product phosphatidylinositol(3,4,5)trisphosphate (PIP3). PIP3 recruits signaling proteins such as Itk to cellular membranes by binding to PH and other domains. In thymocytes, low-dose IP4 binding to the Itk PH domain surprisingly promoted and high-dose IP4 inhibited PIP3 binding of Itk PH domains. However, the mechanisms that underlie the regulation of membrane recruitment of Itk by IP4 and PIP3 remain unclear. The distinct Itk PH domain ability to oligomerize is consistent with a cooperative-allosteric mode of IP4 action. However, other possibilities cannot be ruled out due to difficulties in quantitatively measuring the interactions between Itk, IP4 and PIP3, and in generating non-oligomerizing Itk PH domain mutants. This has hindered a full mechanistic understanding of how IP4 controls Itk function. By combining experimentally measured kinetics of PLC{\gamma}1 phosphorylation by Itk with in silico modeling of multiple Itk signaling circuits and a maximum entropy (MaxEnt) based computational approach, we show that those in silico models which are most robust against variations of protein and lipid expression levels and kinetic rates at the single cell level share a cooperative-allosteric mode of Itk regulation by IP4 involving oligomeric Itk PH domains at the plasma membrane. This identifies MaxEnt as an excellent tool for quantifying robustness for complex TCR signaling circuits and provides testable predictions to further elucidate a controversial mechanism of PIP3 signaling.

Accounting for ∼4% of all cancers in the US, Non-Hodgkin Lymphoma (NHL) is the most prevalent blood cancer. Diffuse large B-cell Lymphoma (DLBCL) is the most common and aggressive type of NHL. Here, we identify inositol-trisphosphate (IP3) 3-kinase B (Itpkb) as a novel tumor suppressor whose deficiency triggers DLBCL in mice. We found that aging Itpkb-/- mice die prematurely with anemia and splenomegaly. 15% of these mice showed multiorgan-infiltration with neoplastic germinal-center phenotype B cells reminiscent of DLBCL. Itpkb-/- B cell lymphomas are mono-or oligoclonal, transplantable, and constitutively hyperactivate the oncogenic phosphoinositide 3-kinase (PI3K) signaling pathway. Growth of Itpkb-/- B cell lymphoma xenografts in immunodeficient mice is sensitive to treatment with PI3K or mTOR inhibitors. In humans, subsets of DLBCL patients carry Itpkb missense mutations, deletions or copy number reductions. Additionally, Itpkb expression is altered in several other hematopoietic and non-hematopoietic human cancers. We propose that Itpkb suppresses tumors by producing inositol-tetrakisphosphate (IP4), a soluble analog of the PI3K product phosphatidylinositol-trisphosphate (PIP3). We and others have previously shown that IP4 antagonism with PIP3 for Akt effector kinase binding prevents excessive Akt/mTORC1 signaling in hematopoietic cells. Our new results suggest that this mechanism prevents oncogenic PI3K signaling in the B cell lineage and possibly in other cell types. Thus, Itpkb might be a novel biomarker for tumor aggressiveness or prognosis, and Itpkb activating drugs or IP4 might have therapeutic potential. This work was supported by NIH grants AI070845 and GM100785 to KS, The Leukemia & Lymphoma Society Scholar Award 1440-11 to KS, the AAI Careers in Immunology Fellowship Program to SR, DFG Fellowship SI 1547/1-1 to SS, and an ARC fellowship to CC. Citation Format: Karsten Sauer, Claire Conche, Hyun-Yong Jin, Kelly Bethel, Stephanie Rigaud, Luise Westernberg, Sabine Siegemund, Changchun Xiao. IP3 3-kinase B suppresses B-cell lymphoma by antagonizing PI3K/mTOR in B cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-020.

T cells, key orchestrators of adaptive immunity, sense pathogen-derived antigen peptides through T cell receptors (TCRs). Developing T cells express TCRs of random antigen specificity that interact with self-peptides with a wide range of affinity. A strict selection process warrants generation of a functional, protective but self-tolerant T cell repertoire by removing T cell precursors failing to interact or stimulated strongly by self-peptides, and inducing survival and maturation for low-affinity/mild TCR signals. How different TCR signals can have such vastly different outcomes is ill understood. Among crucial TCR effectors, the oligomeric enzyme Interleukin-2 inducible T cell kinase (Itk) controls early (min scale) TCR signaling. Transient Itk activation is controlled by a positive feedback feeding into a negative feedback. Both are mediated by the soluble small messenger molecule inositol(1,3,4,5)tetrakisphosphate (IP4) generated via signal-dependent metabolism of membrane lipids (Huang et al, Science 2007). We combine computational modeling and biochemical experiments to elucidate the role of antigen affinity and Itk oligomerization in regulating duration and amplitude of Itk and T cell activation. Our results suggest that high affinity peptides cause strong but short-lived Itk activation necessary to induce downstream Ras and MAPK activation. Low affinity antigens cause prolonged Itk activation with smaller amplitudes. This is sufficient to activate Erk, an essential mediator for survival in developing T cells. Our findings also suggest that certain modes of Itk oligomerization can inhibit signaling by low-affinity peptides. Regulation of transient Itk activation by IP4 may point to a novel mechanism used by different cell signaling networks to generate specific functional decisions. In developing T cells, it may contribute to an enigmatic TCR signal splitter that determines whether TCR engagement causes death or survival and maturation.

In β-selection, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. This pivotal αβ T cell fate-determining event requires co-stimulatory Notch signals. Here, we show that this Notch dependence is enforced through antagonistic signaling by inositol-trisphosphate 3-kinase B (Itpkb) and phosphoinositide 3-kinase (PI3K). Itpkb produces soluble inositol-tetrakisphosphate which competes with the PI3K lipid-product PIP3 for binding to the effector-kinase Akt. Itpkb-/- thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4+CD8+ cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose-metabolism. Thus, Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement, and to prevent premature thymocyte maturation.

Tight regulation of HSC homeostasis ensures life-long hematopoiesis and prevents blood cancers. The mechanisms balancing HSC quiescence with expansion and differentiation into hematopoietic progenitors are incompletely understood. Here, we identify inositoltrisphosphate (IP3) 3-kinase B (Itpkb) as a novel essential regulator of HSC quiescence and function. Young Itpkb-/- mice accumulated phenotypic HSC which were less quiescent and proliferated more than wildtype controls. Itpkb-/- HSC downregulated quiescence associated mRNAs, but upregulated activation, oxidative metabolism, protein synthesis and lineage associated transcripts. Although they showed no significant homing defects and had normal to elevated viability, Itpkb-/- HSC had a severely reduced competitive long-term repopulating potential. Aging Itpkb-/- mice lost hematopoietic stem and progenitor cells and died with severe anemia. Wildtype HSC normally repopulated Itpkb-/- hosts, indicating a HSC-intrinsic Itpkb requirement. In vitro, Itpkb-/- HSC had reduced cobblestone-area forming cell activity and showed increased stem cell factor activation of the phosphoinositide 3-kinase (PI3K) effector Akt. This was reversed by exogenous provision of the Itpkb product IP4, a known PI3K/Akt antagonist. Itpkb-/- HSC also showed transcriptome changes consistent with hyperactive Akt/mTOR signaling. Thus, we propose that Itpkb ensures HSC quiescence and function in part by limiting cytokine-induced PI3K signaling in HSC.

NK cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections and reproduction. NK cell-based therapies are promising blood cancer treatments. Overcoming their limited efficacy requires a better understanding of the molecular mechanisms dampening their effector functions. NK cells recognize and kill pathogen-infected or tumor cells through invariant NK cell receptors (NKR). Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3-generation by PI3K, and generation of DAG and IP3 by PLCγ. IP3 plays a key signaling role by mobilizing Calcium, but can also be converted into soluble IP4. We and others previously showed that IP4 can act as a soluble analog of the PI3K lipid-product PIP3 and control PIP3-mediated protein membrane recruitment and activation in thymocytes and granulocytes. Here, we show data which suggest a novel IP4 function in promoting NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR induced IFNγ secretion, granule exocytosis and target-cell clearance, in part by inhibiting the PIP3 effector Akt. This identifies IP4 as an important novel regulator of NK cell development and function, and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.

In β-selection, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. This pivotal αβ T cell fate-determining event requires co-stimulatory Notch signals. Here, we show that this Notch dependence is enforced through antagonistic signaling by inositol-trisphosphate 3-kinase B (Itpkb) and phosphoinositide 3-kinase (PI3K). Itpkb produces soluble inositol-tetrakisphosphate which competes with the PI3K lipid-product PIP3 for binding to the effector-kinase Akt. Itpkb-/- thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4+CD8+ cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose-metabolism. Thus, Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement, and to prevent premature thymocyte maturation.

NK cells have important functions in cancer immunosurveillance, bone marrow allograft rejection, fighting infections and reproduction. NK cell-based therapies are promising blood cancer treatments. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize pathogen-infected or tumor cells through invariant NK cell receptors (NKR), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3-generation by PI3K, and generation of DAG and IP3 by PLCγ. IP3 plays a key signaling role by mobilizing Calcium, but can also be converted into soluble inositol(1,3,4,5)tetrakisphosphate (IP4). We and others previously showed that IP4 can act as a soluble analog of the PI3K lipid-product phosphatidylinositol(3,4,5) trisphosphate (PIP3) and control PIP3-mediated signaling protein membrane recruitment and activation in thymocytes and granulocytes. Here, we show data which suggest a novel IP4 function in promoting NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR induced IFNγ secretion, granule exocytosis and target-cell clearance, in part by inhibiting the PIP3 effector-kinase Akt. This identifies IP4 as an important novel regulator of NK cell development and function, and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.

Binding of the membrane phospholipid phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) to the Pleckstrin Homology (PH) domain of the Tec family protein tyrosine kinase, Inducible T cell Kinase (ITK), is critical for the recruitment of the kinase to the plasma membrane and its co-localization with the TCR-CD3 molecular complex. Three aromatic residues, termed the FYF motif, located in the inner walls of the phospholipid-binding pocket of the ITK PH domain, are conserved in the PH domains of all Tec kinases, but not in other PH-domain containing proteins, suggesting an important function of the FYF motif in the Tec kinase family. However, the biological significance of the FYF amino acid motif in the ITK-PH domain is unknown. To elucidate it, we have tested the effects of a FYF triple mutant (F26S, Y90F, F92S), henceforth termed FYF-ITK mutant, on ITK function. We found that FYF triple mutation inhibits the TCR-induced production of IL-4 by impairing ITK binding to PIP(3), reducing ITK membrane recruitment, inducing conformational changes at the T cell-APC contact site, and compromising phosphorylation of ITK and subsequent phosphorylation of PLCγ(1). Interestingly, however, the FYF motif is dispensable for the interaction of ITK with two of its signaling partners, SLP-76 and LAT. Thus, the FYF mutation uncouples PIP(3)-mediated ITK membrane recruitment from the interactions of the kinase with key components of the TCR signalosome and abrogates ITK function in T cells.

Inositol phosphate second messengers often regulate crosstalk between receptor signaling and lipid metabolic networks critically affecting cell decision processes, however, molecular mechanisms underlying such cross-regulation are poorly understood. Pairing mathematical modeling and experiments we elucidate these mechanisms in thymocyte activation initiated by T cell receptor (TCR) and antigen interactions. Thymocyte activation is carefully controlled by transient activation kinetics of the Tec-family protein tyrosine kinase Itk generated by TCR signaling, and, production of the membrane lipid phosphatidylinositol(3,4,5)trisphosphate (PIP3) and soluble inositol(1,3,4,5) tetrakisphosphate (IP4). By combining PLC-g (substrate of active Itk) activation kinetics in experiments with maximum entropy based computational approaches we show multiple possible in-silico models describing different modes of molecular interactions between Itk, PIP3, and IP4, can be distinguished. We show that models displaying maximum robustness share a cooperative-allosteric mode of Itk regulation by IP4 involving oligomeric Itk PH domains which induces dueling positive and negative feedbacks in Itk activation. Models lacking the feedbacks or containing monomeric Itk are significantly less robust. We also elucidate key mechanisms regulating the "shape" of the transient Itk kinetics that can be manipulated in experiments for developing therapeutic strategies targeting TCR signaling.