Viral transduction of primary human lymphoma B cells reveals mechanisms of NOTCH-mediated immune escape

Hotspot mutations in the PEST-domain of NOTCH1 and NOTCH2 are recurrently identified in B cell malignancies. To address how NOTCH-mutations contribute to a dismal prognosis, we have generated isogenic primary human tumor cells from patients with Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL), differing only in their expression of the intracellular domain (ICD) of NOTCH1 or NOTCH2. Our data demonstrate that both NOTCH-paralogs facilitate immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-? signaling. In addition, NOTCH-activation causes silencing of the entire HLA-class II locus via epigenetic regulation of the transcriptional co-activator CIITA. Notably, while NOTCH1 and NOTCH2 govern similar transcriptional programs, disease-specific differences in their expression levels can favor paralog-specific selection. Importantly, NOTCH-ICD also strongly down-regulates the expression of CD19, possibly limiting the effectiveness of immune-therapies. These NOTCH-mediated immune escape mechanisms are associated with the expansion of exhausted CD8+ T cells in vivo.© 2022. The Author(s)

Phospholipases in the nucleus

There is a well established role for various phospholipases involved in the production of intracellular signals at the plasma membrane. In contrast much less is known of their role in other intracellular compartments, however, emerging evidence would suggest that some of these enzymes are also involved in the production of signals within the nucleus. Translocation to and activation of protein kinase C (PKC) within the nucleus has been suggested to be important in a number of diverse cellular processes suggesting the requirement for the intranuclear production of diacylglycerol (DAG), a known physiological activator of this enzyme. As the activation of a number of phospholipases leads to the production of DAG this review will consider the notion that these enzymes are present within the nucleus and that their activities can be stimulated to produce this important regulator of PKC

Phospholipid signalling in the nucleus. Een DAG uit het leven van de inositide signalering in de nucleus

Diverse methodologies, ranging from activity measurements in various nuclear subfractions to electron microscopy, have been used to demonstrate and establish that many of the key lipids and enzymes responsible for the metabolism of inositol lipids are resident in nuclei. PtdIns(4)P, PtdIns(4,5)P2 and PtdOH are all present in nuclei, as well as the corresponding enzyme activities required to synthesise and metabolise these compounds. In addition other non-inositol containing phospholipids such as phosphatidylcholine constitute a significant percentage of the total nuclear phospholipid content. We feel that it is pertinent to include this lipid in our discussion as it provides an alternative source of 1, 2-diacylglycerol (DAG) in addition to the hydrolysis of PtdIns(4, 5)P2. We discuss at length data related to the sources and possible consequences of nuclear DAG production as this lipid appears to be increasingly central to a number of general physiological functions. Data relating to the existence of alternative pathways of inositol phospholipid synthesis, the role of 3-phosphorylated inositol lipids and lipid compartmentalisation and transport are reviewed. The field has also expanded to a point where we can now also begin to address what role these lipids play in cellular proliferation and differentiation and hopefully provide avenues for further research

Tissue and cell specific expression of Ins(1,4,5)P3 3-kinase isoenzymes

The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

T lymphocyte nuclear diacylglycerol is derived from both de novo synthesis and phosphoinositide hydrolysis

Novel phospholipid metabolism in T lymphocytes and the generation of biologically active lipid second messengers (LSMs) has attracted much attention in recent years. Despite this interest, no reports have attempted to characterise such events in the nuclei of these cells. In order to gain insight into the structural relationships between the lipids diglyceride (DG) and phosphatidic acid (PtdOH) and their structural precursors phosphatidylcholine (PtdCho) and phosphatidylinositides (PtdIns) in the nuclei of CTLL-2 T lymphocytes, an analysis of their molecular species was performed. The results clearly indicated that there were two pools of DG. The major pool consisted primarily of saturated and monunsaturated structures whereas the minor pool consisted of more unsaturated species, most likely derived from PtdIns. Only the latter pool was found to be accessible to endogenous nuclear diacylglycerol kinase (DGK) activity which showed partial inhibition with the recognised DGK inhibitor R59949. Molecular species analysis of the endogenous nuclear PtdOH revealed it to be distinct from that generated by the endogenous DGK, but instead resembled that of PtdCho species. We were unable to detect enzymatic activities which targeted PtdCho (PtdCho-phospholipase C (PtdCho-PLC), PtdCho-phospholipase D (PtdCho-PLD) and sphingomyelin synthase (SMS)) but instead a detectable PtdOH phosphatase (PAP) activity. We propose that, in exponentially growing CTLL-2 cells, synthesis de novo represents one of the routes for the biosynthesis of structural phospholipids which may be the source of biologically active LSMs in the nucleus