Functional phenotyping of circulating human cytotoxic T cells and NK cells using a 16-color flow cytometry panel.

Cytotoxic T lymphocytes and natural killer (NK) cells are key effector cells in immune defenses against intracellular pathogens and cancer. In human blood, effector T and NK cytotoxic cells comprise a diverse and relatively rare group of cells. Herein, we describe a simplified intracellular staining workflow for classification of circulating human T and NK cells with cytolytic potential.

CRISPR-Cas9-Mediated Modification of the NOD Mouse Genome With Ptpn22R619W Mutation Increases Autoimmune Diabetes.

An allelic variant of protein tyrosine phosphatase nonreceptor type 22 (PTPN22), PTPN22(R620W), 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 Ptpn22(R619W) mutation to the NOD genome would enhance the spontaneous development of T1D.

Non-canonical antagonism of PI3K by the kinase Itpkb delays thymocyte β-selection and renders it Notch-dependent.

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.

IP3 3-kinase B controls hematopoietic stem cell homeostasis and prevents lethal hematopoietic failure in 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.

Themis sets the signal threshold for positive and negative selection in T-cell development.

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.

In silico modeling of Itk activation kinetics in thymocytes suggests competing positive and negative IP4 mediated feedbacks increase robustness.

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.

Inositol tetrakisphosphate limits NK cell effector functions by controlling PI3K signaling.

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.

A conserved motif in the ITK PH-domain is required for phosphoinositide binding and TCR signaling but dispensable for adaptor protein interactions.

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.

Protein kinase C η is required for T cell activation and homeostatic proliferation.

Protein kinase C η (PKCη) is abundant in T cells and is recruited to the immunological synapse that is formed between a T cell and an antigen-presenting cell; however, its function in T cells is unknown. We showed that PKCη was required for the activation of mature CD8+ T cells through the T cell receptor. Compared with wild-type T cells, PKCη-/- T cells showed poor proliferation in response to antigen stimulation, a trait shared with T cells deficient in PKCθ, which is the most abundant PKC isoform in T cells and was thought to be the only PKC isoform with a specific role in T cell activation.

Human X-linked variable immunodeficiency caused by a hypomorphic mutation in XIAP in association with a rare polymorphism in CD40LG.

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 (XIAP(G466X)) that cosegregated with a rare polymorphism in CD40LG (CD40 ligand(G219R)). These genes are involved in the X-linked lymphoproliferative syndrome 2 and the X-linked hyper-IgM syndrome, respectively.

Clinical similarities and differences of patients with X-linked lymphoproliferative syndrome type 1 (XLP-1/SAP deficiency) versus type 2 (XLP-2/XIAP deficiency).

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.

XIAP deficiency in humans causes an X-linked lymphoproliferative syndrome.

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).