Suppression of adaptive NK cell expansion by macrophage-mediated phagocytosis inhibited by 2B4-CD48.

Infection of mice by mouse cytomegalovirus (MCMV) triggers activation and expansion of Ly49H+ natural killer (NK) cells, which are virus specific and considered to be "adaptive" or "memory" NK cells. Here, we find that signaling lymphocytic activation molecule family receptors (SFRs), a group of hematopoietic cell-restricted receptors, are essential for the expansion of Ly49H+ NK cells after MCMV infection. This activity is largely mediated by CD48, an SFR broadly expressed on NK cells and displaying augmented expression after MCMV infection. It is also dependent on the CD48 counter-receptor, 2B4, expressed on host macrophages. The 2B4-CD48 axis promotes expansion of Ly49H+ NK cells by repressing their phagocytosis by virus-activated macrophages through inhibition of the pro-phagocytic integrin lymphocyte function-associated antigen-1 (LFA-1) on macrophages. These data identify key roles of macrophages and the 2B4-CD48 pathway in controlling the expansion of adaptive NK cells following MCMV infection. Stimulation of the 2B4-CD48 axis may be helpful in enhancing adaptive NK cell responses for therapeutic purposes.

CD47 masks pro-phagocytic ligands in cis on tumor cells to suppress antitumor immunity.

Cancer cells often overexpress CD47, which triggers the inhibitory receptor SIRPα expressed on macrophages, to elude phagocytosis and antitumor immunity. Pharmacological blockade of CD47 or SIRPα is showing promise as anticancer therapy, although CD47 blockade has been associated with hematological toxicities that may reflect its broad expression pattern on normal cells. Here we found that, in addition to triggering SIRPα, CD47 suppressed phagocytosis by a SIRPα-independent mechanism. This mechanism prevented phagocytosis initiated by the pro-phagocytic ligand, SLAMF7, on tumor cells, due to a cis interaction between CD47 and SLAMF7. The CD47-SLAMF7 interaction was disrupted by CD47 blockade and by a first-in-class agonist SLAMF7 antibody, but not by SIRPα blockade, thereby promoting antitumor immunity. Hence, CD47 suppresses phagocytosis not only by engaging SIRPα, but also by masking cell-intrinsic pro-phagocytic ligands on tumor cells and knowledge of this mechanism may influence the decision between CD47 blockade or SIRPα blockade for therapeutic purposes.

Integrated Analysis of Transcriptome and Secretome From Umbilical Cord Mesenchymal Stromal Cells Reveal New Mechanisms for the Modulation of Inflammation and Immune Activation.

Mesenchymal stromal cells (MSC) have been used in over 800 clinical trials with encouraging results in the field of transplant medicine and chronic inflammatory diseases. Today, Umbilical Cord (UC)-derived MSC are the second leading source used for clinical purposes, mainly due to its easy access and superior immune modulatory effects. Although the underlying molecular mechanisms of immune suppressive activities have not been fully understood, research over the last decade strongly suggests that MSC-mediated benefits are closely related to activation of secretome networks. Nevertheless, recent findings also point to cytokine-independent mechanisms as key players of MSC-mediated immune modulation. Here, we set up a robust in vitro immune assay using phytohemagglutinin- or anti-CD3/CD28-treated human peripheral blood mononuclear cells in cell-to-cell interaction or in cell-contact independent format with UC-MSC and conducted integrated transcriptome and secretome analyses to dissect molecular pathways driving UC-MSC-mediated immune modulation. Under inflammatory stimuli, multiparametric analyses of the secretome led us to identify cytokine/chemokine expression patterns associated with the induction of MSC-reprogrammed macrophages and T cell subsets ultimately leading to immune suppression. UC-MSC transcriptome analysis under inflammatory challenge allowed the identification of 47 differentially expressed genes, including chemokines, anti- and pro-inflammatory cytokines and adhesion molecules found also in UC-MSC-immunosupressive secretomes, including the novel candidate soluble IL-2R. This study enabled us to track functionally activated UC-MSC during immune suppression and opened an opportunity to explore new pathways involved in immunity control by UC-MSC. We propose that identified immunomodulatory molecules and pathways could potentially be translated into clinical settings in order to improve UC-MSC-therapy quality and efficacy.

Generation and characterization of U937-TR: a platform cell line for inducible gene expression in human macrophages.

Monocytes and macrophages are involved in a wide range of biological processes and parasitic diseases. The characterization of the molecular mechanisms governing such processes usually requires precise control of the expression of genes of interest. We implemented a tetracycline-controlled gene expression system in the U937 cell line, one of the most used in vitro models for the research of human monocytes and macrophages. Here we characterized U937-derived cell lines in terms of phenotypic (morphology and marker expression) and functional (capacity for phagocytosis and for Leishmania parasite hosting) changes induced by phorbol-12-myristate-13-acetate (PMA). Finally, we provide evidence of tetracycline-inducible and reversible Lamin-A gene silencing of the PMA-differentiated U937-derived cells.

Human Leukocyte Antigen and Red Blood Cells Impact Umbilical Cord Blood CD34+ Cell Viability after Thawing.

Hematopoietic progenitor cell (HPC) transplantation is a treatment option for malignant and nonmalignant diseases. Umbilical cord blood (UCB) is an important HPC source, mainly for pediatric patients. It has been demonstrated that human leukocyte antigen (HLA) matching and cell dose are the most important features impacting clinical outcomes. However, UCB matching is performed using low resolution HLA typing and it has been demonstrated that the unnoticed mismatches negatively impact the transplant. Since we found differences in CD34+ viability after thawing of UCB units matched for two different patients (p = 0.05), we presumed a possible association between CD34+ cell viability and HLA. We performed a multivariate linear model (n = 67), comprising pre-cryopreservation variables and high resolution HLA genotypes separately. We found that pre-cryopreservation red blood cells (RBC), granulocytes, and viable CD34+ cell count significantly impacted CD34+ viability after thawing, along with HLA-B or -C (R2 = 0.95, p = 0.01; R2 = 0.56, p = 0.007, respectively). Although HLA-B*40:02 may have a negative impact on CD34+ cell viability, RBC depletion significantly improves it.

A new strategy for umbilical cord blood collection developed at the first Colombian public cord blood bank increases total nucleated cell content.

BACKGROUND: The total nucleated cell dosage of umbilical cord blood (UCB) is an important factor in determining successful allogeneic hematopoietic stem cell transplantation after a minimum human leukocyte antigen donor-recipient match. The northern South American population is in need of a new-generation cord blood bank that cryopreserves only units with high total nucleated cell content, thereby increasing the likelihood of use. Colombia set up a public cord blood bank in 2014; and, as a result of its research for improving high total nucleated cell content, a new strategy for UCB collection was developed. STUDY DESIGN AND METHODS: Data from 2933 collected and 759 cryopreserved cord blood units between 2014 and 2015 were analyzed. The correlation of donor and collection variables with cellularity was evaluated. Moreover, blood volume, cell content, CD34+ count, clonogenic capacity, and microbial contamination were assessed comparing the new method, which combines in utero and ex utero techniques, with the conventional strategies. RESULTS: Multivariate analysis confirmed a correlation between neonatal birth weight and cell content. The new collection method increased total nucleated cell content in approximately 26% and did not alter pre-cryopreservation and post-thaw cell recovery, viability, or clonogenic ability. Furthermore, it showed a remarkably low microbial contamination rate (1.2%). CONCLUSION: The strategy for UCB collection developed at the first Colombian public cord blood bank increases total nucleated cell content and does not affect unit quality. The existence of this bank is a remarkable breakthrough for Latin-American patients in need of this kind of transplantation.

Análisis climático de la concentración de metales pesados asociados al sedimento depositado sobre vías urbanas / Climatic analysis of heavy metal concentration associated with urban road-deposited sediment

Objetivo Realizar una evaluación climática (diaria) de la concentración de metales pesados (Pb y Cu) asociada con el sedimento depositado sobre las superficies viales de las localidades de Kennedy y Puente Aranda (Bogotá D.C., Colombia). Adicionalmente, se evaluaran las concentraciones detectadas con respecto a la legislación seleccionada como de referencia para la protección de la salud humana en suelo urbano. Métodos La concentración se determinó mediante espectrometría de absorción atómica con llama; previamente las muestras fueron digeridas en una mezcla de ácido clorhídrico y nítrico (3:1; agua regia). Resultados Se evidencia con respecto a la fracción de tamaño del sedimento vial más cercana a la potencialmente inhalable desde el punto de vista de la salud pública (≤ 10 µm), que las concentraciones metálicas en promedio tienden a aumentar en tiempo seco (29 %). Al respecto, las concentraciones durante estos períodos de tiempo son en promedio 1,69 veces superiores al valor límite más bajo fijado por la normatividad de referencia. No obstante, se sugiere que las concentraciones metálicas están dominadas por el uso del suelo antes que por las condiciones climáticas. Conclusiones Los hallazgos se constituyen en un punto de referencia a nivel colombiano para la discusión y publicación de normatividad ambiental asociada con la protección de la salud humana por metales pesados, y para visualizar futuras líneas de investigación acerca del efecto del cambio climático sobre las concentraciones metálicas en ambientes urbanos.(AU)

Objective To climatically assess (daily) the concentration of heavy metals (Pb and Cu) in sediment deposited on road surfaces of the localities of Kennedy and Puente Aranda (Bogota, D.C., Colombia). Additionally, the detected concentrations are to be evaluated with respect to the legislation chosen as reference point for the protection of human health in urban areas. Methods The concentration was determined by flame atomic absorption spectrometry. The samples were previously digested in a mixture of hydrochloric and nitric acid (3:1; aqua regia). Results The results show, with respect to road sediment closest to the size that could be potentially inhaled from the point of view of public health (≤ 10 µm), that the metal concentrations on average tend to increase in dry weather (29 %). In this regard, the concentrations during these time periods are on average 1.69 times higher than the lowest limit value set by the reference legislation. However, it is suggested that the metal concentrations are mostly due to the use of the land rather than weather conditions. Conclusions The findings are a reference point in Colombia for the discussion and publication of environmental regulations associated with the protection of human health from heavy metals and for visualizing future lines of research about the effect of climate change on metal concentrations in urban environments.(AU)

[Climatic analysis of heavy metal concentration associated with urban road-deposited sediment]. / Análisis climático de la concentración de metales pesados asociados al sedimento depositado sobre vías urbanas.

Objective To climatically assess (daily) the concentration of heavy metals (Pb and Cu) in sediment deposited on road surfaces of the localities of Kennedy and Puente Aranda (Bogota, D.C., Colombia). Additionally, the detected concentrations are to be evaluated with respect to the legislation chosen as reference point for the protection of human health in urban areas. Methods The concentration was determined by flame atomic absorption spectrometry. The samples were previously digested in a mixture of hydrochloric and nitric acid (3:1; aqua regia). Results The results show, with respect to road sediment closest to the size that could be potentially inhaled from the point of view of public health (≤ 10 µm), that the metal concentrations on average tend to increase in dry weather (29 %). In this regard, the concentrations during these time periods are on average 1.69 times higher than the lowest limit value set by the reference legislation. However, it is suggested that the metal concentrations are mostly due to the use of the land rather than weather conditions. Conclusions The findings are a reference point in Colombia for the discussion and publication of environmental regulations associated with the protection of human health from heavy metals and for visualizing future lines of research about the effect of climate change on metal concentrations in urban environments.