Protein kinase N1 deficiency results in upregulation of cerebral energy metabolism and is highly protective in in vivo and in vitro stroke models.

Background And Aim: We recently identified protein kinase N1 (PKN1) as a master regulator of brain development. However, its function in the adult brain has not been clearly established. In this study, we assessed the cerebral energetic phenotype of wildtype (WT) and global Pkn1 knockout (Pkn1) animals under physiological and pathophysiological conditions.

Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake.

Objective: Glucose-1,6-bisphosphate (G-1,6-BP), a byproduct of glycolysis that is synthesized by phosphoglucomutase 2 like 1 (PGM2L1), is particularly abundant in neurons. G-1,6-BP is sensitive to the glycolytic flux, due to its dependence on 1,3-bisphosphoglycerate as phosphate donor, and the energy state, due to its degradation by inosine monophosphate-activated phosphomannomutase 1. Since the exact role of this metabolite remains unclear, our aim was to elucidate the specific function of G-1,6-BP in the brain.

PKN1 Exerts Neurodegenerative Effects in an In Vitro Model of Cerebellar Hypoxic-Ischemic Encephalopathy via Inhibition of AKT/GSK3β Signaling.

We recently identified protein kinase N1 (PKN1) as a negative gatekeeper of neuronal AKT protein kinase activity during postnatal cerebellar development. The developing cerebellum is specifically vulnerable to hypoxia-ischemia (HI), as it occurs during hypoxic-ischemic encephalopathy, a condition typically caused by oxygen deprivation during or shortly after birth. In that context, activation of the AKT cell survival pathway has emerged as a promising new target for neuroprotective interventions.

Melanoma-intrinsic NR2F6 activity regulates antitumor immunity.

Nuclear receptors (NRs) are implicated in the regulation of tumors and immune cells. We identify a tumor-intrinsic function of the orphan NR, NR2F6, regulating antitumor immunity. NR2F6 was selected from 48 candidate NRs based on an expression pattern in melanoma patient specimens (i.

New Insights into the Diverse Functions of the NR2F Nuclear Orphan Receptor Family.

Following gene expansion during evolution, today's phylogenetic tree of the NR2F family of nuclear orphan receptors in mammals is represented by three different isoforms: NR2F1, NR2F2, and NR2F6. Structural analysis of the NR2F family members has revealed that NR2F1 and NR2F2 are closely related and grouped together apart from NR2F6, which is more divergent in its biochemical characteristics. In this review, we highlight current knowledge on the cellular functions of NR2F family members.

A role for the nuclear receptor NR2F6 in peritoneal B cell homeostasis.

B cells are key mediators of humoral immunity. Mature B cells fall into various sub-classes that can be separated by their ontogeny, expression of cell surface markers, anatomical location, and function. B1 subsets play important roles in natural immunity and constitute the majority of B cells in newborns.

A MLR-Based Approach to Analyze Regulators of T Lymphocyte Activation In Vivo.

Depending on the context, robust and durable T lymphocyte activation is either desirable, as in the case of anti-tumor responses, or unwanted, in cases of autoimmunity when chronic stimulation leads to self-tissue damage. Therefore, reliable in vivo models are of great importance to identify and validate regulatory pathways of T lymphocyte activation. Here, we describe an in vivo mixed-lymphocyte-reaction (MLR) approach, which is based on the so-called parent-into-F1 (P → F1) mouse model in combination with the congenic marker CD45.

Cerebral Malaria: Current Clinical and Immunological Aspects.

This review focuses on current clinical and immunological aspects of cerebral malaria induced by infection. Albeit many issues concerning the inflammatory responses remain unresolved and need further investigations, current knowledge of the underlying molecular mechanisms is highlighted. Furthermore, and in the light of significant limitations in preventative diagnosis and treatment of cerebral malaria, this review mainly discusses our understanding of immune mechanisms in the light of the most recent research findings.

IFNγ Helps CBLB-Deficient CD8+ T Cells to Put Up Resistance to Tregs.

In this issue, Han and colleagues demonstrate in preclinical cancer models that genetic deletion of the E3 ubiquitin ligase Cbl proto-oncogene B (CBLB) in adoptively transferred CD8+ T cells induces resistance to regulatory T cells. CBLB deletion induces IFNγ and downmodulates TGFβ/SMAD signaling. This ultimately enforces these cells to be way more effective against various cancers.

Tumor rejection in mice depends on IL-9 and Th9 cells.

Background: Casitas B lymphoma-b (Cbl-b) is a central negative regulator of cytotoxic T and natural killer (NK) cells and functions as an intracellular checkpoint in cancer. In particular, Th9 cells support mast cell activation, promote dendritic cell recruitment, enhance the cytolytic function of cytotoxic T lymphocytes and NK cells, and directly kill tumor cells, thereby contributing to tumor immunity. However, the role of Cbl-b in the differentiation and antitumor function of Th9 cells is not sufficiently resolved.

Emerging Next-Generation Target for Cancer Immunotherapy Research: The Orphan Nuclear Receptor NR2F6.

Additional therapeutic targets suitable for boosting anti-tumor effector responses have been found inside effector CD4 and CD8 T cells. It is likely that future treatment options will combine surface receptor and intracellular protein targets. Utilizing germline gene ablation as well as CRISPR/Cas9-mediated acute gene mutagenesis, the nuclear receptor NR2F6 (nuclear receptor subfamily 2 group F member 6, also called Ear-2) has been firmly characterized as such an intracellular immune checkpoint in effector T cells.

Loss of the orphan nuclear receptor NR2F6 enhances CD8 T-cell memory via IFN-γ.

Memory formation is a hallmark of T cell-mediated immunity, but how differentiation into either short-lived effector cells (SLECs, CD127KLRG1) or memory precursors cells (MPECs, CD127KLRG1) and subsequent regulation of long-term memory is adjusted is incompletely understood. Here, we show that loss of the nuclear orphan receptor NR2F6 in germ-line Nr2f6-deficient mice enhances antigen-specific CD8 memory formation up to 70 days after bacterial infection with Listeria monocytogenes (LmOVA) and boosts inflammatory IFN-γ, TNFα, and IL-2 cytokine recall responses. Adoptive transfer experiments using Nr2f6 OT-I T-cells showed that the augmented memory formation is CD8 T-cell intrinsic.

Chemically modified mRNA nucleofection of primary human T cells.

Here we show that an approach of in-vitro transcribed mRNA nucleofection expands the range of transfection of primary human T cells. It represents a reproducible and time-efficient technology, and is thus an ideal tool in basic research involving highly controlled in-vitro experiments with a gene of interest aiming at identifying its biological human T cell function.

Targeting the orphan nuclear receptor NR2F6 in T cells primes tumors for immune checkpoint therapy.

Background: NR2F6 has been proposed as an alternative cancer immune checkpoint in the effector T cell compartment. However, a realistic assessment of the in vivo therapeutic potential of NR2F6 requires acute depletion.

Methods: Employing primary T cells isolated from Cas9-transgenic mice for electroporation of chemically synthesized sgRNA, we established a CRISPR/Cas9-mediated acute knockout protocol of Nr2f6 in primary mouse T cells.

Loss-of-function phenotype of a PKCθ knockin mouse strain.

Background: Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line.

Methods: The present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain.

Orphan Nuclear Receptor NR2F6 Suppresses T Follicular Helper Cell Accumulation through Regulation of IL-21.

CD4 T follicular helper (Tfh) cells are specialized in helping B cells during the germinal center (GC) reaction and ultimately promote long-term humoral immunity. Here we report that loss of the nuclear orphan receptor NR2F6 causes enhanced survival and accumulation of Tfh cells, GC B cells, and plasma cells (PCs) following T cell-dependent immunization. Nr2f6-deficient CD4 T cell dysfunction is the primary cause of cell accumulation.

Nuclear Receptors Regulate Intestinal Inflammation in the Context of IBD.

Gastrointestinal (GI) homeostasis is strongly dependent on nuclear receptor (NR) functions. They play a variety of roles ranging from nutrient uptake, sensing of microbial metabolites, regulation of epithelial intestinal cell integrity to shaping of the intestinal immune cell repertoire. Several NRs are associated with GI pathologies; therefore, systematic analysis of NR biology, the underlying molecular mechanisms, and regulation of target genes can be expected to help greatly in uncovering the course of GI diseases.

Novel mutant mouse line emphasizes the importance of protein kinase C theta for CD4 T lymphocyte activation.

Background: The protein kinase C theta (PKCθ) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKCθ is not only essential for activation of NF-κB, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein.

Fcμ receptor as a Costimulatory Molecule for T Cells.

Fc receptor for IgM (FcμR)-deficient mice display dysregulated function of neutrophils, dendritic cells, and B cells. The relevance of FcμR to human T cells is still unknown. We show that FcμR is mostly stored inside the cell and that surface expression is tightly regulated.

Regulation of Lymphatic GM-CSF Expression by the E3 Ubiquitin Ligase Cbl-b.

Genome-wide association studies as well as lymphatic expression analyses have linked both Cbl-b and GM-CSF to human multiple sclerosis as well as other autoimmune diseases. Both Cbl-b and GM-CSF have been shown to play a prominent role in the development of murine encephalomyelitis; however, no functional connection between the two has yet been established. In this study, we show that knockout mice demonstrated significantly exacerbated severity of experimental autoimmune encephalomyelitis (EAE), augmented T cell infiltration into the central nervous system (CNS) and strongly increased production of GM-CSF in T cells and .

Microbial signals drive pre-leukaemic myeloproliferation in a Tet2-deficient host.

Somatic mutations in tet methylcytosine dioxygenase 2 (TET2), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies. In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage. However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2 mice and humans with TET2 mutations, suggesting that extrinsic non-cell-autonomous factors are required for disease onset.