ILT2 and ILT4 Drive Myeloid Suppression via Both Overlapping and Distinct Mechanisms.

Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.

Kaposi sarcoma presenting as small bowel obstruction.

Kaposi sarcoma (KS) is a low-grade tumor of the vascular endothelium. The majority of individuals affected have advanced human immunodeficiency virus (HIV) or acquired immunodeficiency syndrome (AIDS). The disease typically manifests as cutaneous lesions but reports have shown that systemic disease is not uncommon. Because gastrointestinal KS is often asymptomatic, it is likely underdiagnosed. Those with symptoms can present with vague abdominal pain, nausea/vomiting or anemia. Rarely the tumors can cause bowel obstruction or perforation. We present a case of small bowel obstruction cause by KS tumors in a young transgender male to female patient with poorly controlled AIDS, supported by literature review of the clinical presentation, diagnosis and treatment recommendations.

NAD+ augmentation with nicotinamide riboside improves lymphoid potential of Atm-/- and old mice HSCs.

NAD+ supplementation has significant benefits in compromised settings, acting largely through improved mitochondrial function and DNA repair. Elevating NAD+ to physiological levels has been shown to improve the function of some adult stem cells, with implications that these changes will lead to sustained improvement of the tissue or system. Here, we examined the effect of elevating NAD+ levels in models with reduced hematopoietic stem cell (HSC) potential, ATM-deficient and aged WT mice, and showed that supplementation of nicotinamide riboside (NR), a NAD+ precursor, improved lymphoid lineage potential during supplementation. In aged mice, this improved lymphoid potential was maintained in competitive transplants and was associated with transcriptional repression of myeloid gene signatures in stem and lineage-committed progenitor cells after NR treatment. However, the altered transcriptional priming of the stem cells toward lymphoid lineages was not sustained in the aged mice after NR removal. These data characterize significant alterations to the lineage potential of functionally compromised HSCs after short-term exposure to NR treatment.

Cell cycle-dependent phosphorylation regulates RECQL4 pathway choice and ubiquitination in DNA double-strand break repair.

Pathway choice within DNA double-strand break (DSB) repair is a tightly regulated process to maintain genome integrity. RECQL4, deficient in Rothmund-Thomson Syndrome, promotes the two major DSB repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR). Here we report that RECQL4 promotes and coordinates NHEJ and HR in different cell cycle phases. RECQL4 interacts with Ku70 to promote NHEJ in G1 when overall cyclin-dependent kinase (CDK) activity is low. During S/G2 phases, CDK1 and CDK2 (CDK1/2) phosphorylate RECQL4 on serines 89 and 251, enhancing MRE11/RECQL4 interaction and RECQL4 recruitment to DSBs. After phosphorylation, RECQL4 is ubiquitinated by the DDB1-CUL4A E3 ubiquitin ligase, which facilitates its accumulation at DSBs. Phosphorylation of RECQL4 stimulates its helicase activity, promotes DNA end resection, increases HR and cell survival after ionizing radiation, and prevents cellular senescence. Collectively, we propose that RECQL4 modulates the pathway choice of NHEJ and HR in a cell cycle-dependent manner.

WRN regulates pathway choice between classical and alternative non-homologous end joining.

Werner syndrome (WS) is an accelerated ageing disorder with genomic instability caused by WRN protein deficiency. Many features seen in WS can be explained by the diverse functions of WRN in DNA metabolism. However, the origin of the large genomic deletions and telomere fusions are not yet understood. Here, we report that WRN regulates the pathway choice between classical (c)- and alternative (alt)-nonhomologous end joining (NHEJ) during DNA double-strand break (DSB) repair. It promotes c-NHEJ via helicase and exonuclease activities and inhibits alt-NHEJ using non-enzymatic functions. When WRN is recruited to the DSBs it suppresses the recruitment of MRE11 and CtIP, and protects the DSBs from 5' end resection. Moreover, knockdown of Wrn, alone or in combination with Trf2 in mouse embryonic fibroblasts results in increased telomere fusions, which were ablated by Ctip knockdown. We show that WRN regulates alt-NHEJ and shields DSBs from MRE11/CtIP-mediated resection to prevent large deletions and telomere fusions.

Acid ceramidase deficiency associated with spinal muscular atrophy with progressive myoclonic epilepsy.

Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) is an extremely rare disorder related to the lysosomal storage disease, Farber lipogranulomatosis. Both disorders are autosomal recessive conditions caused by mutations in the ASAH1 gene encoding acid ceramidase. Farber disease is associated with joint deformities, lipomatous skin nodules, and often is fatal by 2-3 years of age; while SMA-PME is characterized by childhood-onset motor neuron disease and progressive myoclonic epilepsy. We report a case of SMA-PME with a novel mutation in the ASAH1 gene encoding acid ceramidase. The proband presented with childhood-onset of diffuse muscle atrophy and hypotonia. He also had diffuse weakness with greater proximal than distal involvement. Tongue fasciculations were present and his reflexes were either diminished or absent. He ambulated with an unsteady and hesitant gait. He subsequently developed myoclonic epilepsy along with other associated features including tremor, polymyoclonus, and sensorineural hearing loss. Neurophysiological studies revealed a motor neuron disorder and generalized epilepsy. Exome sequencing analysis identified compound heterozygous variants and biochemical analysis indicated acid ceramidase activity was approximately 12 percent of normal controls. Our proband was phenotypically similar to other cases of SMA-PME, albeit with somewhat lesser severity, slower progression, and greater longevity. As lysosomal disorders are sometimes amendable to early interventions, it is important to make early diagnoses in these cases. The combination of motor neuron disease and progressive myoclonic epilepsy should prompt genetic evaluation of ASAH1.

Protective properties of radio-chemoresistant glioblastoma stem cell clones are associated with metabolic adaptation to reduced glucose dependence.

Glioblastoma stem cells (GSC) are a significant cell model for explaining brain tumor recurrence. However, mechanisms underlying their radiochemoresistance remain obscure. Here we show that most clonogenic cells in GSC cultures are sensitive to radiation treatment (RT) with or without temozolomide (TMZ). Only a few single cells survive treatment and regain their self-repopulating capacity. Cells re-populated from treatment-resistant GSC clones contain more clonogenic cells compared to those grown from treatment-sensitive GSC clones, and repeated treatment cycles rapidly enriched clonogenic survival. When compared to sensitive clones, resistant clones exhibited slower tumor development in animals. Upregulated genes identified in resistant clones via comparative expression microarray analysis characterized cells under metabolic stress, including blocked glucose uptake, impaired insulin/Akt signaling, enhanced lipid catabolism and oxidative stress, and suppressed growth and inflammation. Moreover, many upregulated genes highlighted maintenance and repair activities, including detoxifying lipid peroxidation products, activating lysosomal autophagy/ubiquitin-proteasome pathways, and enhancing telomere maintenance and DNA repair, closely resembling the anti-aging effects of caloric/glucose restriction (CR/GR), a nutritional intervention that is known to increase lifespan and stress resistance in model organisms. Although treatment-introduced genetic mutations were detected in resistant clones, all resistant and sensitive clones were subclassified to either proneural (PN) or mesenchymal (MES) glioblastoma subtype based on their expression profiles. Functional assays demonstrated the association of treatment resistance with energy stress, including reduced glucose uptake, fatty acid oxidation (FAO)-dependent ATP maintenance, elevated reactive oxygen species (ROS) production and autophagic activity, and increased AMPK activity and NAD(+) levels accompanied by upregulated mRNA levels of SIRT1/PGC-1α axis and DNA repair genes. These data support the view that treatment resistance may arise from quiescent GSC exhibiting a GR-like phenotype, and suggest that targeting stress response pathways of resistant GSC may provide a novel strategy in combination with standard treatment for glioblastoma.

RAGE inhibits human respiratory syncytial virus syncytium formation by interfering with F-protein function.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.

RECQL4 localizes to mitochondria and preserves mitochondrial DNA integrity.

RECQL4 is associated with Rothmund-Thomson Syndrome (RTS), a rare autosomal recessive disorder characterized by premature aging, genomic instability, and cancer predisposition. RECQL4 is a member of the RecQ helicase family, and has many similarities to WRN protein, which is also implicated in premature aging. There is no information about whether any of the RecQ helicases play roles in mitochondrial biogenesis, which is strongly implicated in the aging process. Here, we used microscopy to visualize RECQL4 in mitochondria. Fractionation of human and mouse cells also showed that RECQL4 was present in mitochondria. Q-PCR amplification of mitochondrial DNA demonstrated that mtDNA damage accumulated in RECQL4-deficient cells. Microarray analysis suggested that mitochondrial bioenergetic pathways might be affected in RTS. Measurements of mitochondrial bioenergetics showed a reduction in the mitochondrial reserve capacity after lentiviral knockdown of RECQL4 in two different primary cell lines. Additionally, biochemical assays with RECQL4, mitochondrial transcription factor A, and mitochondrial DNA polymerase γ showed that the polymerase inhibited RECQL4's helicase activity. RECQL4 is the first 3'-5' RecQ helicase to be found in both human and mouse mitochondria, and the loss of RECQL4 alters mitochondrial integrity.

Cell type-specific recognition of human metapneumoviruses (HMPVs) by retinoic acid-inducible gene I (RIG-I) and TLR7 and viral interference of RIG-I ligand recognition by HMPV-B1 phosphoprotein.

Human metapneumoviruses (HMPVs) are recently identified Paramyxoviridae that contribute to respiratory tract infections in children. No effective treatments or vaccines are available. Successful defense against virus infection relies on early detection by germ line-encoded pattern recognition receptors and activation of cytokine and type I IFN genes. Recently, the RNA helicase retinoic acid-inducible gene I (RIG-I) has been shown to sense HMPV. In this study, we investigated the abilities of two prototype strains of HMPV (A1 [NL\1\00] and B1 [NL\1\99]) to activate RIG-I and induce type I IFNs. Despite the abilities of both HMPV-A1 and HMPV-B1 to infect and replicate in cell lines and primary cells, only the HMPV-A1 strain triggered RIG-I to induce IFNA/B gene transcription. The failure of the HMPV-B1 strain to elicit type I IFN production was dependent on the B1 phosphoprotein, which specifically prevented RIG-I-mediated sensing of HMPV viral 5' triphosphate RNA. In contrast to most cell types, plasmacytoid dendritic cells displayed a unique ability to sense both HMPV-A1 and HMPV-B1 and in this case sensing was via TLR7 rather than RIG-I. Collectively, these data reveal differential mechanisms of sensing for two closely related viruses, which operate in cell type-specific manners.

I kappa B kinase 2 deficiency in T cells leads to defects in priming, B cell help, germinal center reactions, and homeostatic expansion.

Signal transduction from proinflammatory stimuli leading to NF-kappa B-dependent gene expression is mediated by the I kappa B kinase 2 (IKK2/IKK beta). Therefore, IKK2 has become an important drug target for treatment of inflammatory conditions. T cells, whose activation depends to a large extent on the activity of NF-kappa B transcription factors, play important roles in inflammation and autoimmunity. Ablation of IKK2 specifically in T cells in CD4cre/Ikk2(FL) mice allows their survival and activation by polyclonal stimuli in vitro, suggesting that IKK2 is dispensable for T cell activation. We report in this study that IKK2-deficient T cells expand efficiently in response to superantigen administration in vivo, but are completely deficient in recall responses, most likely due to inefficient priming. IKK2-deficient T cells provide suboptimal B cell help and fail to support germinal center reactions. Finally, IKK2 is essential for homeostatic expansion of naive T cells, reflected by the inability of IKK2-deficient T cells to induce colitis in lymphopenic hosts.

Differential dependence of CD4+CD25+ regulatory and natural killer-like T cells on signals leading to NF-kappaB activation.

Natural killer-like (NK) T, regulatory T (TR), and memory type T cells display surface phenotypes reminiscent of activated T cells. Previously, we reported that the generation of TR cells and, to a lesser extent, of memory type T cells, depends on IkappaB kinase 2. Here, we show that T cell-specific ablation of IkappaB kinase 2, in addition, completely precludes NKT cell development. T cell antigen receptor (TCR)-induced signals to activate NF-kappaB are essential for mature T cell activation, leading us to hypothesize that this pathway could play an important role in the generation of the antigen-driven T cell subsets comprising TR, memory type T, and NKT cells. TCR-mediated NF-kappaB activation critically depends on Bcl10 and PKCtheta. By using mice deficient for these proteins, we demonstrate that the generation of TR and, to a lesser extent, of memory type T cells, depends on Bcl10 and PKCtheta, and therefore, most likely on NF-kappaB activation initiated by TCR engagement. NKT cells, on the other hand, require PKCtheta for thymic development, whereas absence of Bcl10 leads primarily to the reduction of peripheral NKT cell numbers.