Nm23-H1 induces apoptosis in primary effusion lymphoma cells via inhibition of NF-κB signaling through interaction with oncogenic latent protein vFLIP K13 of Kaposi's sarcoma-associated herpes virus.

BACKGROUND: Primary effusion lymphoma (PEL) is an aggressive form of non-Hodgkin lymphoma of B cells caused by Kaposi's Sarcoma-associated Herpes Virus (KSHV). KSHV encoded latent and lytic antigens promote oncogenic transformation and evade apoptosis through the modulation of various host cellular signaling pathways. Nm23-H1 is a known metastatic suppressor whose expression inversely correlates with the metastatic potential of different cancers. Here, we set out to assess the role of Nm23-H1 in PEL development. METHODS: Flow cytometry and real-time PCR assays were performed for Nm23-H1 expression analysis. Induction of apoptosis was assessed using Western blotting and flow cytometry-based assays in Nm23-H1 overexpressing cells. Co-immunoprecipitation assays, confocal microscopy and imaging flow cytometry were performed to determine Nm23-H1 and vFLIP K13 protein-protein interaction. A PEL cell-induced xenograft model was established in non-obese diabetic/severely combined immunodeficient (NOD/SCID) mice to validate the effect of Nm23-H1 overexpression. RESULTS: We found that Nm23-H1 expression was significantly downregulated both at transcriptional and protein levels in PEL cell lines and that its overexpression triggered mitochondrial-mediated caspase-dependent apoptosis. We revealed Nm23-H1 interacts with the latent protein vFLIP K13 and that Nm23-H1 overexpression leads to inhibition of vFLIP K13 driven nuclear factor-kappa B (NF-κB) signaling with concurrent inhibition of autocrine and paracrine growth factors and downregulation of latent KSHV antigens without induction of active lytic reactivation. We also confirmed the effects of Nm23-H1 overexpression in a PEL cell-induced xenograft model in NOD/SCID mice. CONCLUSION: Downregulation of Nm23-H1 expression in KSHV-infected PEL cells and its overexpression trigger apoptosis by impairing vFLIP K13-driven NF-κB signaling, suggesting therapeutic implications of Nm23-H1 for primary effusion lymphomas.

Reactive arthritis and undifferentiated peripheral spondyloarthritis share human leucocyte antigen B27 subtypes and serum and synovial fluid cytokine profiles.

OBJECTIVES: Peripheral SpA (pSpA) is comprised of ReA, PsA, enteritis-associated arthritis and undifferentiated pSpA (upSpA). ReA and upSpA share T cell oligotypes and metabolomics in serum and SF. We investigated HLA-B27 subtypes and cytokines in serum and SF that were compared between ReA and upSpA. METHODS: ReA and upSpA were compared in two cohorts. In cohort I (44 ReA and 56 upSpA), HLA-B27 subtyping was carried out. In cohort II (17 ReA and 21 upSpA), serum and SF cytokines were compared using a multiplex cytokine bead assay (27 cytokines). A total of 28 healthy controls with similar age and sex to cohort II were included for comparison of serum cytokine levels. RESULTS: In cohort I, HLA-B27 was positive in 81.8% (36/44) of ReA and 85.71% (48/56) of upSpA patients. HLA-B27 typing was successful in 70 patients (30 ReA and 40 uSpA). HLA-B*2705 was the most common, followed by HLA-B*2704 and HLA-B*2707. Frequencies were the same between ReA and upSpA. In cohort II, 14 cytokines were detectable in the serum of patients. The levels of eight cytokines were higher than in the controls. The cytokine levels of ReA and upSpA were similar. Sixteen cytokines were detectable in the SF of patients. There was no statistical difference in the levels between ReA and upSpA. The cytokine profiles in sera and SF were also similar among HLA-B27-positive and negative patients. CONCLUSION: ReA and upSpA have similar HLA-B27 subtype associations and similar cytokine profiles. They should be considered as a single entity during studies as well as clinical management.

Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer.

Adoptive T cell therapy (ACT) using ex vivo-expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate complete regression of certain human cancers. The impact of TIL phenotypes on clinical success of TIL-ACT is currently unclear. Using high-dimensional analysis of human ACT products, we identified a memory-progenitor CD39-negative stem-like phenotype (CD39-CD69-) associated with complete cancer regression and TIL persistence and a terminally differentiated CD39-positive state (CD39+CD69+) associated with poor TIL persistence. Most antitumor neoantigen-reactive TILs were found in the differentiated CD39+ state. However, ACT responders retained a pool of CD39- stem-like neoantigen-specific TILs that was lacking in ACT nonresponders. Tumor-reactive stem-like TILs were capable of self-renewal, expansion, persistence, and superior antitumor response in vivo. These data suggest that TIL subsets mediating ACT response are distinct from TIL subsets enriched for antitumor reactivity.

Defective glycosylation and multisystem abnormalities characterize the primary immunodeficiency XMEN disease.

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαß+ T cells (αßDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

Quantifying the Dynamics of Hematopoiesis by In Vivo IdU Pulse-Chase, Mass Cytometry, and Mathematical Modeling.

We present a new method to directly quantify the dynamics of differentiation of multiple cellular subsets in unperturbed mice. We combine a pulse-chase protocol of 5-iodo-2'-deoxyuridine (IdU) injections with subsequent analysis by mass cytometry (CyTOF) and mathematical modeling of the IdU dynamics. Measurements by CyTOF allow for a wide range of cells to be analyzed at once, due to the availability of a large staining panel without the complication of fluorescence spillover. These are also compatible with direct detection of integrated iodine signal, with minimal impact on immunophenotyping based on the surface markers. Mathematical modeling beyond a binary classification of surface marker abundance allows for a continuum of cellular states as the cells transition from one state to another. Thus, we present a complete and robust method for directly quantifying differentiation at the systemic level, allowing for system-wide comparisons between different mouse strains and/or experimental conditions. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Cytokine-mediated communication: a quantitative appraisal of immune complexity.

Intercellular communication mediated by cytokines is the main mechanism by which cells of the immune system talk to each other. Many aspects of cytokine signalling in the immune system have been explored in great detail at the structural, biophysical, biochemical and cellular levels. However, a systematic understanding of the quantitative rules that govern cytokine-mediated cell-to-cell communication is still lacking. Here, we discuss recent efforts in the field of systems immunology to bring about a quantitative understanding of cytokine-mediated communication between leukocytes and to provide novel insights into the orchestration of immune responses and inflammation.

Non-Classical monocytes display inflammatory features: Validation in Sepsis and Systemic Lupus Erythematous.

Given the importance of monocytes in pathogenesis of infectious and other inflammatory disorders, delineating functional and phenotypic characterization of monocyte subsets has emerged as a critical requirement. Although human monocytes have been subdivided into three different populations based on surface expression of CD14 and CD16, published reports suffer from contradictions with respect to subset phenotypes and function. This has been attributed to discrepancies in reliable gating strategies for flow cytometric characterization and purification protocols contributing to significant changes in receptor expression. By using a combination of multicolour flow cytometry and a high-dimensional automated clustering algorithm to confirm robustness of gating strategy and analysis of ex-vivo activation of whole blood with LPS we demonstrate the following: a. 'Classical' monocytes are phagocytic with no inflammatory attributes, b. 'Non-classical' subtype display 'inflammatory' characteristics on activation and display properties for antigen presentation and c. 'Intermediate' subtype that constitutes a very small percentage in circulation (under physiological conditions) appear to be transitional monocytes that display both phagocytic and inflammatory function. Analysis of blood from patients with Sepsis, a pathogen driven acute inflammatory disease and Systemic Lupus Erythmatosus (SLE), a chronic inflammatory disorder validated the broad conclusions drawn in the study.