Adherent Fraction Research Articles

Hippo Signaling Pathway Involvement in Osteopotential Regulation of Murine Bone Marrow Cells Under Simulated Microgravity.

The development of osteopenia is one of the most noticeable manifestations of the adverse effects of space factors on crew members. The Hippo signaling pathway has been shown to play a central role in regulating the functional activity of cells through their response to mechanical stimuli. In the present study, the components of the Hippo pathway and the protective properties of osteodifferentiation inducers were investigated under simulated microgravity (smg) using a heterotypic bone marrow cell culture model, which allows for the maintenance of the close interaction between the stromal and hematopoietic compartments, present in vivo and of great importance for both the fate of osteoprogenitors and hematopoiesis. After 14 days of smg, the osteopotential and osteodifferentiation of bone marrow stromal progenitor cells, the expression of Hippo cascade genes and the immunocytochemical status of the adherent fraction of bone marrow cells, as well as the paracrine profile in the conditioned medium and the localization of Yap1 and Runx2 in mechanosensitive cells of the bone marrow were obtained. Simulated microgravity negatively affects stromal and hematopoietic cells when interacting in a heterotypic murine bone marrow cell culture. This is evidenced by the decrease in cell proliferation and osteopotential. Changes in the production of pleiotropic cytokines IL-6, GROβ and MCP-1 were revealed. Fourteen days of simulated microgravity induced a decrease in the nuclear translocation of Yap1 and the transcription factor Runx2 in the stromal cells of the intact group. Exposure to osteogenic induction conditions partially compensated for the negative effect of simulated microgravity. The data obtained will be crucial for understanding the effects of spaceflight on osteoprogenitor cell growth and differentiation via Hippo-Yap signaling.

TIM-3 expression on monocyte-derived dendritic cells

The T cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM-3), an inhibitory checkpoint receptor, has been identified as a crucial regulator of cellular immune responses. TIM-3 has been discovered as a receptor involved in the negative regulation of T cells. Recent studies have demonstrated that TIM-3 is expressed on innate immune cells, including dendritic cells (DCs), even at a higher level than T cells. In the tumor microenvironment, the majority of DCs have a monocytic origin. Models for studying such DCs in vitro are DC cultures generated from monocytes in the presence of growth factors. The present study aimed to investigate the expression of TIM-3 in IFNα-induced monocyte-derived DCs (IFN-DCs) and the impact of DC activation on TIM-3 expression. DCs were obtained by culturing the adherent fraction of mononuclear cells from healthy donors for 4 days in the presence of GM-CSF and IFNα, followed by LPS addition for 24 hours. Human double-stranded DNA (dsDNA, 5 μg/mL) was added as an activation stimulus to intact IFN-DCs at the stage of maturation, along with LPS. Expression of the membrane TIM-3 molecule was determined by flow cytometry, and the level of expression of TIM-3 mRNA – by real-time RT-PCR with reverse transcription. Intact donor IFN-DCs expressed the membrane TIM-3 molecule at a high level (more than 70% of cells). The addition of LPS as a maturation stimulus almost halved the expression of TIM-3 (pW < 0.05) without affecting the expression of HAVCR2/TIM-3 mRNA. Exogenous dsDNA (along with LPS) increased the expression of HAVCR2/TIM-3 mRNA by more than three times (pW = 0.05) with a decrease in the number of TIM-3+DCs (pW = 0.003). Our findings indicate the presence of mechanisms that support expression of this inhibitory checkpoint receptor under conditions of DC activation. Further studies of the regulation of TIM-3 expression by monocyte-derived dendritic cells will expand the understanding of the biological significance of inhibitory receptors on DCs from the point of view of the immune response, as well as, in the future, increase the effectiveness of current approaches in cancer immunotherapy using IFN-DCs and inhibitors of checkpoint molecules.

RANKL-dependent osteoclast differentiation and gene expression in bone marrow-derived cells from adult mice is sexually dimorphic

Sex-specific differences in bone integrity and properties are associated with age as well as the number and activity of cells involved in bone remodeling. The aim of this study was to investigate sex-specific differences in adhesion, proliferation, and differentiation of mouse bone marrow derived cells into osteoclasts. The adherent fraction of bone marrow- derived cells from 12-week-old male and female C57BL/6J mice were assessed for their adhesion, proliferation, and receptor activator of nuclear factor κB (RANKL)-induced differentiation into osteoclasts. Female bone marrow derived macrophages (BMDMs) displayed higher adhesion and proliferation ratio upon macrophage colony stimulating factor (M-CSF) (day 0) and M-CSF + RANKL (day 4) treatment, respectively. On the contrary, male BMDMs differentiated more efficiently into osteoclasts upon RANKL-treatment compared to females (day 5). To further understand these sex-specific differences at the gene expression level, BMDMs treated with M-CSF (day 0) and M-CSF + RANKL (day 4), were assessed for their differential expression of genes through RNA sequencing. M-CSF treatment resulted in 1106 differentially expressed genes, while RANKL-treatment gave 473 differentially expressed genes. Integrin, adhesion, and proliferation-associated genes were elevated in the M-CSF-treated female BMDMs. RANKL-treatment further enhanced the expression of the proliferation- associated genes, and of genes associated with inhibition of osteoclast differentiation in the females, while RANK-signaling-associated genes were upregulated in males. In conclusion, BMDM adhesion, proliferation and differentiation into osteoclasts are sex-specific and may be directed by the PI3K-Akt signaling pathway for proliferation, and the colony stimulating factor 1-receptor and the RANKLsignaling pathway for the differentiation.

Comparative Analysis of Bacterial Diversity between the Liquid Phase and Adherent Fraction within the Donkey Caeco-Colic Ecosystem.

Simple SummaryThe bacteria residing in donkey hindgut are clearly divided into two distinct ecological sites: liquid phase (Lq) and adherent fraction (Ad). Though both the Lq and Ad bacteria play an important role in feed digestion, the Ad bacteria have not previously been specifically sampled or directly compared with the Lq bacteria. The present study was conducted to comparatively analyze the bacterial community composition between the Lq and Ad fraction within the donkey caeco-colic ecosystem. The results showed that the relative abundance of Bacteroidota, Spirochaetota, Fibrobacterota and Patescibacteria in the Ad fraction was greater than the Lq fraction, indicating that bacteria associated with plant biomass are mainly responsible for plant fiber degradation. Regarding the genus level, the liquid phase presented higher abundance in Lactobacillus compared to Ad fraction. The activities of enzymes related to fiber degradation were predicted by PICRUSt, and they were also higher in Ad bacteria than Lq. In addition, the bacterial community composition was also distinct within the donkey caecum, ventral colon and dorsal colon. The present study provides evidence that bacteria adherent to feed particles may be better at plant fiber degradation than Lq bacteria due to the greater cellulolytic populations and activities.Donkey hindgut is an enlarged fermentative chamber that harbors a highly complex and extremely abundant community of anaerobic bacteria. It can be divided into two different ecological sites: liquid (Lq) phase and adherent fraction (Ad) colonized by bacteria. However, the Ad bacteria have not previously been specifically collected or directly compared with the Lq bacteria. In the present study, the digesta collected from the caecum, ventral colon and dorsal colon of nine Dezhou donkeys was separated into Lq and Ad fractions. The bacterial community structure was comparatively determined using 16S rRNA gene sequences by Illumina MiSeq. The Ad bacteria had a higher bacterial diversity than Lq bacteria due to the higher Chao and ACE index (p < 0.05). The predominant bacteria at the phylum level were Firmicutes (55.4~74.3%) and Bacteroidota (13.7~32.2%) for both the Lq and Ad fraction. The relative abundance of Bacteroidota, Spirochaetota, Fibrobacterota and Patescibacteria in the Ad fraction was greater than Lq (p < 0.05), suggesting that bacteria associated with feed particles were mainly responsible for plant fiber degradation. At the genus level, the abundance of Lactobacillus in Lq was greater than that in the Ad fraction (p < 0.05), indicating that the bacteria in the Lq fraction were better at hydrolyzing readily fermentable carbohydrates. PICRUSt showed that the activities of enzymes related to fiber degradation in the Ad fraction were also greater than Lq. In addition, the hindgut region also had a significant effect on the bacterial community composition. The relative abundance of Rikenellaceae_RC9_gut_group, Clostridium_sensu_stricto_1, Christensenellaceae_R-7_group and norank_Bacteroidales_BS11_gut_group was increased (p < 0.05) along the donkey hindgut. In summary, the present study provides evidence that bacteria adherent to plant biomass were different to those in the liquid phase within the donkey caeco-colic digesta, and bacteria associated with feed particles may mainly be responsible for plant fiber degradation.

Neurogenesis of Adult Circulating Blood Mononuclears in Vitro

Cellular therapy of injuries of the nervous system with autologous cellular material of the patient requires the development of methods of neurodifferentiation of stem and progenitor cells of the adult organism. The use of autologous cells is the most acceptable from the point of view of biosafety and compatibility. In our work, we investigated the possibility of directed neurogenesis of circulating blood precursors adhered fraction, obtained from the patient with spinal cord injury. These cells were cultured for 2 weeks using neuroinducers (retinoic acid, BDNF, NGF). During the first 3 days of cultivation, the adherent fraction of cells consisted of round cells of small and medium size with a large nucleus and a narrow rim of the cytoplasm, which gradually stratified and formed an oval and granular shape. Subsequent cultivation showed that the cells acquire morphological characteristics of different types of cells of the nervous system. At the end of this period, the predominant cell population was Nest+. The patient’s own cells after stimulation of neurogenic differentiation in vitro were removed, washed from medium components and injected into the spinal canal. Intracisterial and paraventricular administration of this fraction to the patient significantly improved impaired function.

Effect of macrophage-activating factor (GcMAF-RF) upon ex vivo polarization of macrophages, activation of dendritic cells and production of cytokines by human whole blood cells

This article is the second communication in a series of articles devoted to the effects of a domestic preparation of macrophage-activating factor (GcMAF-RF) and assessment of its biological properties. The aim of this work was to study the effect of the GcMAF-RF upon M0 → M1 polarization of macrophages (Mph), and activation of the professional properties of ex vivo generated antigen-presenting dendritic cells (DC), as well as on ex vivo production of pro-inflammatory (TNFα, IL-1β, IL-6, IFNγ, IL-17, IL-18) and anti-inflammatory (TGF-β, IL-4, IL-10) cytokines, growth factors (IL-2, GM-CSF, G-CSF, VEGF) and chemokines (MCP, IL-8) by the whole blood cells from healthy donors. Mph and DC were generated from the monocytes (3 to 5×106 /ml) derived from adherent fraction of peripheral blood mononuclear cells (MNC) of healthy donors. Granulocyte/macrophage colony-stimulating factor (rhGM-CSF) was used to obtain Mph, whereas DC production was induced by GM-CSF and interferon-α. To provide M1 polarizing signals, bacterial lipopolysaccharide (LPS from E. coli 0114:B4) was used in controls. In experimental series, GcMAF-RF was added 48 h before the end of culture. The stimulating effect of the obtained Mph and DC upon cell proliferation was assessed in allogeneic mixed culture of leukocytes (alloMLC) using radiometric technique, by 3 H-thymidine incorporation. The influence index (IR) of Mph or DC upon allo-SCL was calculated as the ratio of the proliferative response of MNCs in the presence of Mph, or DC to the level of spontaneous MNC proliferation. To determine the cytokine production by human whole blood cells ex vivo, peripheral blood samples from 3 donors with two replicate GcMAF-RF preparations were used, at a total of 6 points. All variants of the study were carried out with mitogen-activated and non-activated blood cells. The cytokine content was determined by the ELISA assays. The effects of GcMAF-RF were quantified as a fold increase (FI), i.e., the ratio of cytokine production in the presence of GcMAF-RF to the level of their spontaneous production. It was shown that the GcMAF-RF preparation was as effective, as lipopolysaccharide (LPS), the standard Mph and DC activator which induces polarization of differentiated M0-macrophages into M1 cells and final maturation of DCs, manifesting by a significant increase in their allo-stimulatory activity in a mixed leukocyte culture (allo-MLC). Moreover, GcMAF-RF stimulates production of numerous cytokines and chemokines (TNFα, IL-1β, IL-6, IL-18, IL-4, IL-10, GM-CSF, G-CSF, VEGF, IL-8), by blood cells (granulocytes, lymphocytes, monocytes), thus indicating direct participation of the macrophage activator GcMAF-RF in various immune processes. The domestic GcMAF-RF drug induces polarization of macrophages M0 → M1, final maturation of DCs and allostimulating activity of Mf and DCs, and is also able to effectively stimulate circulating blood cells to synthesize cytokines/chemokines with pro-inflammatory and immunoregulatory activities.

Human type 2 macrophages biologically active soluble products in the editing of stress-induced depressive-like behavior

IntroductionIn the scientific world widely discussed phenomenon of “cytokine-induced depression”. Macrophages have high plasticity and are able to control the inflammatory response; in particular, anti-inflammatory type-2 macrophages have a pronounced potential due to complex soluble factors production.ObjectivesWe have developed an original method for the type-2 macrophages generation; the resulting macrophages are characterized by the high level of a whole range of neurotrophic, neuroprotective, proangiogenic and anti-inflammatory factors production. The aim of the study was to investigate effects of human type-2 macrophages soluble products on behavioral phenotype and brain cytokines synthesis in depressive-like animals.MethodsType-2 macrophages were generated by culturing an adherent fraction of mononuclear cells with 50 ng/ml recombinant human GM-CSF in serum deprivation conditions for 7 days. (CBA x C57Bl/6)F1 depressive-like male mice, developed under the long-term social stress, were undergoing the human type-2 macrophages conditioned medium intranasal administration (60 ml twice daily for one animal) for 6 days. Mice behavioral phenotyping was carried out using an automatic registration system (Noldus Information Technology). Cytokines were determined by ELISA.ResultsDepressive-like mice behavioral phenotyping after type-2 macrophages conditioned medium administration revealed anhedonia decrease, motor activity stimulation in the open field and forced swimming tests, anxiety reduction in elevated plus maze. Behavioral changes were recorded against the pro-inflammatory cytokines (TNF-α, IL-1β, IL- 6, INFγ) decrease in striatum and hippocampus, as well as anti-inflammatory IL- 10 increase in hippocampus and hypothalamus.ConclusionsResults demonstrated the effectiveness of human type-2 macrophages biologically active soluble products in relation to the stress-induced depressive-like behavior editingDisclosureNo significant relationships.

Bacillus Calmette–Guérin (BCG) vaccine generates immunoregulatory cells in the cervical lymph nodes in guinea pigs injected intra dermally

This work demonstrates the presence of immune regulatory cells in the cervical lymph nodes draining Bacillus Calmette-Guérin (BCG) vaccinated site on the dorsum of the ear in guinea pigs. It is shown that whole cervical lymph node cells did not proliferate in vitro in the presence of soluble mycobacterial antigens (PPD or leprosin) despite being responsive to whole mycobacteria. Besides, T cells from these lymph nodes separated as a non-adherent fraction on a nylon wool column, proliferated to PPD in the presence of autologous antigen presenting cells. Interestingly, addition of as low as 20% nylon wool adherent cells to these, sharply decreased the proliferation by 83%. Looking into what cells in the adherent fraction suppressed the proliferation, it was found that neither the T cell nor the macrophage enriched cell fractions of this population individually showed suppressive effect, indicating that their co-presence was necessary for the suppression. Since BCG induced granulomas resolve much faster than granulomas induced by other mycobacteria such as Mycobacterium leprae the present experimental findings add to the existing evidence that intradermal BCG vaccination influences subsequent immune responses in the host and may further stress upon its beneficial role seen in Covid-19 patients.

Involvement of Signaling Cascades in Granulocytopoiesis Regulation under Conditions of Cytostatic Treatment

Suppression of the production of granulocytic CSF under the effect of 5-fluorouracyl is related to disorders in the NF-κB-, cAMP-dependent signaling pathways and MAPK cascade. These secondary messengers are involved in the regulation of functional activity of nonadherent myelokaryocytes starting from day 10 of the experiment (initial period of the hemopoietic granulocytic stem regeneration after antimetabolite challenge). Granulocytic CSF does not play essential role in the formation of colony-stimulating activity of cells of the adherent and nonadherent fractions of the bone marrow. Only cAMP-dependent pathway is involved in the regulation of the realization of the granulocytic precursor growth potential in response to the challenge.

Evaluation of quality parameters in thawed dendritic cell vaccine

Background & Aim Manufacturing of Dendritic Cell (DC) vaccine for cancer, a type of Advanced Therapy Medicinal Products, is regulated by cGMP rules. Our experience of DC vaccine preparation started in 2001 and was tested in many clinical trials. Methods, Results & Conclusion DC vaccines were prepared from adherent fraction of Peripheral Blood Mononuclear Cells obtained from leukapheresis, by differentiation and maturation with standard cytokine cocktail after pulsing with autologous tumor homogenate. Aliquots of DC vaccine were frozen by automatic freezing in autologous plasma with 10% DMSO and stored in nitrogen vapors until their use. These aliquots were thawed and resuspended in sterile saline at a final concentration of 5 × 106 cells/ml, packed in two syringes for a total of 10 million that represent the thawed final product. To ensure the maintaining of compliance with release parameters of the thawed final product, three representative batches, stored at 2-8°C for 8 hours, were analyzed using the following tests: Mycoplasma Detection (Method EP. 2.6.7), Bacterial Endotoxin levels (Method EP 2.6.14), Sterility (Method EP 2.6.27) Viable Cell Count (Method EP 2.7.29) Phenotype analysis (internal method) Potency test (ELISPOT COSTIM assay was performed to establish the potency of DCs. We modified the COSTIM assay, which uses IFN-gamma ELISPOT as a read-out system. In this assay DCs provide responder allogeneic T cells, sub-optimally pre stimulated with OKT3, with the costimulatory signals required for full activation). The results and the acceptance criteria are listed in Table 1. Manufacturing of Dendritic Cell (DC) vaccine for cancer, a type of Advanced Therapy Medicinal Products, is regulated by cGMP rules. Our experience of DC vaccine preparation started in 2001 and was tested in many clinical trials. DC vaccines were prepared from adherent fraction of Peripheral Blood Mononuclear Cells obtained from leukapheresis, by differentiation and maturation with standard cytokine cocktail after pulsing with autologous tumor homogenate. Aliquots of DC vaccine were frozen by automatic freezing in autologous plasma with 10% DMSO and stored in nitrogen vapors until their use.

Development and Preliminary Validation of a Feasible Procedure for Isolating RNA from Fiber-Adherent Bacteria in Human Stool.

BackgroundIntestinal bacterial communities are not homogenous throughout the gastrointestinal tract. Human research on the gut microbiome often neglects intra-intestinal variability by relying on a single measurement from stool samples. One source of complexity is the adherence to undigested, residual fiber. Currently, no procedure exists to extract RNA from distinct bacterial subpopulations in stool samples.Material/MethodsA serial centrifugation procedure was developed in which bacterial RNA could be extracted from distinct stool-fractions – fiber-adherent and non-fiber-adherent bacteria. To test whether the separation procedure yielded distinct bacterial subpopulations, a set of RT-qPCR assays were developed for a fiber-adherent bacterial species, Bifidobacterium adolescentis, then a within-subject repeated-measures study was conducted with 3 human subjects undergoing 4 dietary regimens. At each timepoint, between-fraction differences in gene expression were evaluated.ResultsThe RNA isolation procedure was able to isolate intact RNA in 20 of 24 samples in the fiber-adherent fraction. PurB and sdh were identified as suitable reference genes for B. adolescentis RT-qPCR assays. When subjects were provided a high resistant starch diet, bacterial fractions exhibited different expression of the trp operon (p=0.031).ConclusionsOur study provides human gut microbiome researchers a novel tool for evaluating functional characteristics of bacterial subpopulations in human stool. Moreover, these experiments provide modest support for the existence of a functionally unique fiber-adherent subpopulation of B. adolescentis. Until a more thorough evaluation of the adherent and non-adherent fraction can be performed, researchers should be cautious when generalizing functional data derived solely from unfractionated stool samples.

Altered Structure and Function of Mesenchymal Stromal Cell-Derived Extracellular Matrix in MDS Can be Restored By Luspatercept

Introduction The involvement of the bone marrow microenvironment (BMME) into disease progression and therapeutic response of myelodysplastic syndromes (MDS) is indisputable. Hereby, mesenchymal stromal cells (MSCs) play an important role for both the support of the leukemic clone and the remaining healthy hematopoietic stem and progenitor cells (HSPCs). The extracellular matrix (ECM) secreted by MSCs regulates stem cell fate through the modulation of cytokine and growth factor delivery and may also be targeted by clinically available drugs such as luspatercept, a novel recombinant fusion protein containing modified extracellular domain of activin receptor IIB. Luspatercept is a first-in-class erythroid maturation agent with promising results in lower-risk MDS patients with red blood cell transfusion dependency. Aim To shed light on the largely unknown composition and function of the MSC-derived ECM, we have characterized ECM from MDS patients vs. healthy controls and elucidate how luspatercept may modulate their functional characteristics. Methods Bone marrow-derived MSCs from patients with lower-risk MDS and age-matched healthy donors (HD) were treated with RAP-536, a murine homologue of luspatercept harboring the same activin receptor IIB domain. MSCs of three patients were treated with RAP-536 and RNA sequencing was carried out. Gene expression and pathway analyses were performed using the Reactome tool (https://reactome.org). Candidate genes were validated by quantitative real-time PCR (qPCR). For the generation of ECM, MSCs were seeded on poly-octadecene-alt-maleic anhydride and human fibronectin coated glass slides in the presence or absence of RAP-536. To yield cell-free ECM structures, cultures were decellularized at day 10 and analyzed by scanning electron microscopy (SEM), sulfated glycosaminoglycan (GAG), fibronectin and collagen staining as well as GAG quantification (Blyscan assay). Moreover, purified HD CD34+ HSPCs were cultured on ECM scaffolds for 6 and 9 days, respectively. Subsequently, expansion of adherent and supernatant cells was determined and the phenotype was analyzed by flow cytometry. Results RNA sequencing of MDS MSCs after six days of RAP-536 treatment revealed a total of 58 significantly regulated genes, thereof 24 up- and 34 down-regulated genes. Gene enrichment and pathway analyses revealed a striking involvement in ECM organization, collagen biosynthesis and formation. Moreover, integrin cell surface interaction genes showed significantly differential expression. Focusing on collagens as important ECM components, we identified Col7A1 and Col4A2 to be down-regulated. Indeed, both collagen mRNAs were significantly decreased by 46% and 25%, respectively, in MSCs after RAP-536 treatment compared to untreated controls. SEM characterization and immunofluorescence staining of the ECM showed a more compact fiber network produced by MDS MSCs. Moreover, MDS ECM contained higher levels of collagen and GAGs. Blyscan assay confirmed the latter observation, showing significantly higher sulfated GAG concentrations in MDS ECM. Interestingly, trapping of TGFβ superfamily ligands, such as GDF-11, by RAP-536 clearly reduced Col4 staining intensity in MDS MSC ECM. Structural and compositional ECM differences had functional impact on the expansion of HSPCs cultured on the matrices. Significant higher total cell numbers were detected on healthy ECM (18.3-fold vs. 12.1-fold expansion, *p&lt; 0.05) but not on MDS ECM (12.9-fold) after 9 days of culture. The number of adherent cells increased 8.5-fold on healthy and 4.3-fold on MDS ECM and could be further increased after RAP-536 treatment of MSCs. Using flow cytometry, we found a 3.1-fold increased proportion of CD90+ HSPCs in the adherent fraction on healthy but only 1.8-fold on MDS MSC ECM. Integrin αIIb (CD41), αV (CD51) and β3 (CD61) were found to be significantly higher expressed in the adherent HSPC fraction. RAP-536 treatment resulted in up to 20% higher expression of both CD90 and integrin subunits. Summary We demonstrate an association between induced collagen abundance and reduced hematopoietic support in ECM derived from MDS MSCs and conclude that compact MDS ECM structure induced by TGFβ superfamily members may alter the cytokine environment for HSPCs. Consequently, TGFβ ligand trapping by RAP-536/luspatercept leads to ECM re-organization and thus an improved hematopoietic support. Disclosures Stoelzel: Shire: Consultancy, Other: Travel funding; Neovii: Other: Travel funding; JAZZ Pharmaceuticals: Consultancy. Platzbecker:Celgene: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Dendritic cells generated in the presence of interferon-α and modulated with dexamethasone as a novel tolerogenic vaccine platform.

Tolerogenic dendritic cells (tDCs) are considered a novel therapeutic tool in treating autoimmune diseases, allergies, and transplantation reactions. Among numerous pharmacological immune modulators, dexamethasone (Dex) is known to induce potent tolerogenicity in DCs generated from human monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), and these cells (IL-4-DCs/Dex) are being appraised as a tDC-based platform in clinical settings. Interferon-α (IFNα) represents another powerful inducer of monocyte-derived DCs, which possess higher migratory activity and stability. However, the functions of IFN-DCs/Dex have not been sufficiently analyzed and there are no comparative studies of the tolerogenicity of IFN-DCs/Dex and IL-4-DCs/Dex. This study aimed to investigate the properties of IFN-DCs/Dex in comparison with IL-4-DCs/Dex. DCs were obtained by cultivation of an adherent fraction of peripheral blood mononuclear cells (MNCs) in the presence of GM-CSF and IFNα or IL-4 with subsequent lipopolysaccharide-driven maturation. Dex (10-6M) was added to the cultures at day 3. We showed that generation of IFN-DCs with Dex resulted in decrease in percentage of CD83+ and CD86+ DCs and increase in numbers of CD14+, B7-H1+, and Toll-like receptor 2 (TLR2+) DCs. Treatment with Dex downregulated pro-inflammatory cytokine production, reduced DC allostimulatory activity, and inhibited DC capacity to stimulate Th1/pro-inflammatory cytokine production, altogether evidencing the induction of a tolerogenic phenotype. As compared to IL-4-DCs/Dex, IFN-DCs/Dex were characterized by larger proportion of TLR2+ and CD14+ cells, higher production of IL-10 and lower TNFα/IL-10 ratio, more potent capacity to induce T cell anergy, and more efficiently skewed T cell cytokine balance towards Th2/anti-inflammatory profile. The data obtained indicate that potent tDCs could be generated by treating IFN-DCs with dexamethasone. The tolerogenic properties of IFN-DCs/Dex are better than or at least equal to those of the IL-4-DCs/Dex, as assessed by in vitro phenotypic and functional assays, suggesting these cells as a new tolerogenic vaccine platform.

Functional flow cytometry of monocytes for routine diagnosis of innate primary immunodeficiencies

Functional flow cytometry of monocytes for routine diagnosis of innate primary immunodeficiencies

Local Administration of Caloric Restriction Mimetics to Promote the Immune Control of Lung Metastases.

Caloric restriction mimetics (CRMs), compounds that mimic the biochemical effects of nutrient deprivation, administered via systemic route promote antitumor effects through the induction of autophagy and the modulation of the immune microenvironment; however, collateral effects due to metabolic changes and the possible weight loss might potentially limit their administration at long term. Here, we investigated in mice local administration of CRMs via aerosol to reduce metastasis implantation in the lung, whose physiologic immunosuppressive status favors tumor growth. Hydroxycitrate, spermidine, and alpha-lipoic acid, CRMs that target different metabolic enzymes, administered by aerosol, strongly reduced implantation of intravenously injected B16 melanoma cells without overt signs of toxicity, such as weight loss and changes in lung structure. Cytofluorimetric analysis of lung immune infiltrates revealed a significant increase of alveolar macrophages and CD103+ dendritic cells in mice treated with CRMs that paralleled an increased recruitment and activation of both CD3 T lymphocytes and NK cells. These effects were associated with the upregulation of genes related to M1 phenotype, as IL-12 and STAT-1, and to the decrease of M2 genes, as IL-10 and STAT-6, in adherent fraction of lung immune infiltrate, as revealed by real-time PCR analysis. Thus, in this proof-of-principle study, we highlight the antitumor effect of CRM aerosol delivery as a new and noninvasive therapeutic approach to locally modulate immunosurveillance at the tumor site in the lung.

Microfluidic Shear Force Assay to Determine Cell Adhesion Forces.

Cell adhesion is implicated in many physiological settings such as the retention of hematopoietic stem cells (HSCs) in their bone marrow niches or their migration into the bloodstream. During HSC mobilization these adhesion sites are cleaved and have to be newly formed during HSC homing and engraftment. To determine the adhesive properties of HSCs on different extracellular matrix (ECM) molecules, we present a microfluidic shear force assay, where a laminar flow is used to detach a semi-adherent cell population, the HSC model cell line KG-1a, from an ECM protein-coated substrate. This technique combines the high throughput of population-based assays with the ability to observe cell detachment in real time. Additionally, it is suitable for weakly adherent cells, as the setup allows cell incubation on various substrates and application of shear stress ranging several orders of magnitude in one setup without additional washing or transfer steps. As a measure for the adhesion strength of the studied cell population on the substrate, the critical shear force τ50 is determined which is required to remove 50% of the initially adherent cell fraction.

Impairment of the Stromal SDF-1-Mediated Hematopoietic Support By GDF-11 in MDS Is Rescued By Luspatercept

Introduction:Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis due to genetic and functional abnormalities of hematopoietic stem and progenitor cells (HSPCs). Accumulating evidence now also points to the bone marrow microenvironment (BMME) as a key mediator of MDS pathophysiology.Increased levels of transforming growth factor beta (TGF-β) superfamily ligands, including growth differentiation factor 11 (GDF-11), in the bone marrow have been linked to ineffective erythropoiesis and activation of SMAD2/3 signalling in MDS. Luspatercept (ACE-536) is a novel recombinant fusion protein containing modified activin receptor type IIB linked to the fragment crystallisable (Fc) domain of human immunoglobulin G1 and is a first-in-class erythroid maturation agent. Luspatercept binds to selected TGF-β superfamily ligands, including GDF-11 and activin B, restoring late-stage erythropoiesis in MDS mice and patients. Whether luspatercept modulates the BMME is unknown.Aim:We investigated the potential impact of luspatercept on the biology of mesenchymal stromal cells (MSCs).Methods:MSCs from patients with either high-or low-risk MDS and age-matched healthy donors (HD) were treated with GDF-11 in the presence or absence of RAP-536, a homologue of luspatercept harboring the same activin receptor IIB domain. Subsequently, Smad signaling pathway activation was analyzed by Western blot. Moreover, MSC phenotype, stromal derived factor-1 (SDF-1/CXCL12) expression and secretion as well as the osteogenic differentiation potential were recorded. To study the impact on the hematopoietic support, MSCs were pre-treated for one week with GDF-11 ± RAP-536 before freshly isolated CD34+ HSPCs were seeded on the stromal layer. The colony formation (CAF-C) was analyzed weekly. After four weeks, the HSPCs were seeded in methylcellulose medium to perform a CFU-GEMM assay. Further, engraftment and migration potential of co-cultured HSPCs was analyzed in zebrafish embryos in vivo.Results:GDF-11/RAP-536 treatment did not impact viability, proliferation, and growth pattern of MSCs. The osteogenic differentiation was significantly improved by RAP-536 treatment which was shown by a 2.3-fold increase of ALP activity. GDF-11 induced Smad2/3 phosphorylation in MSCs which was inhibited by RAP-536 to about 50% of activity. The MSC phenotype characterized by the expression of CD73, CD90, CD105, CD146, CD29 and CD44 was not significantly influenced by RAP-536 treatment.Interestingly, the chemokine SDF-1 which plays an important role for the interaction and support of HSPCs was significantly up-regulated in MDS and HD MSCs by RAP-536 at both the mRNA (1.9-fold) and the protein level (2.1-fold; 313.0±35.0 vs. 649.7±76.8 pg/ml, *p< 0.05, n=3). This restored SDF-1 secretion of MSCs resulted in functional effects of HSPCs when co-cultured with MSCs. The number of CAF-C was significantly higher after two, three, and four weeks of HSPCs cultured on pre-treated MSC layers (12.0-fold / 3.4-fold / 1.75-fold). The phenotypical analysis of HSPCs demonstrated increased expression levels of CXCR4 in the adherent fraction. Addition of the CXCR4 antagonist AMD3100 blocked enhanced colony formation by RAP-536 confirming the previous observation on SDF-1 modulation. Clonal growth of CAF-C-derived HSPCs after four weeks of co-culture on un- or pre-treated MSC monolayers was analyzed in a secondary colony-forming cell assay (CFU) for an additional 14 days. The frequency of CFUs was increased in all lineages with significant differences in total colony numbers (15±5.8 vs. 35.8±14.7, *p< 0.05, n=6).These results were validated in vivo in a zebrafish model. HSPCs were co-cultured with GDF-11 and RAP-536 pre-treated MDS-MSCs for one week before injection into the zebrafish circulation. One and two days post-injection, a significantly higher cell proportion could be detected in the caudal hematopoietic (CHT) region as a result of RAP-536 pre-treatment (Fig.1).Conclusion:These data provide first evidence that RAP-536 has also the capacity to modulate MSCs which might contribute to the restoration of hematopoiesis in MDS. DisclosuresPlatzbecker:Celgene: Research Funding.

Bone Marrow Stromal Cells Mediate Adhesion Based Drug Resistance in Acute Myeloid Leukaemia through Reciprocal Feedback of the β-Catenin/CD44 Signalling Axis

Eradication of minimal residual disease is a key goal in AML treatment. It has been found that interaction between leukaemic blasts and different cells of the bone marrow niche contributes to AML drug resistance. Previously we have demonstrated that β-catenin may mediate drug resistance in both short and long-term stromal co-culture assays. β-catenin is known to correlate with poor prognosis in AML. β-catenin has dual roles as both a central effector molecule of the canonical Wnt signalling pathway and a component of adherens junction. The role of Wnt/β-catenin in AML has been well established, but the mechanism through which it mediates adhesion in AML remains unclear.Using a human:human co-culture adhesion model, β-catenin was found to be significantly up-regulated on HS5 and AML derived mesenchymal stromal cell (MSC). Lentivirus mediated β-catenin knock-down in AML cells significantly reduced their adhesion on HS5 and primary normal MSC and AML MSC (n=3, P<0.001). β-catenin knock-down also significantly reduced the proliferation of AML cells either on their own or in suspension from co-culture with primary normal MSC and AML MSC (P<0.001, n=3).In order to dissect the mechanisms underlying β-catenin mediated blast cell adhesion phenotype, KG-1a cells were chosen based on their primitive phenotype, high adhesion to human stroma and β-catenin nuclear translocation ability.KG-1a β-catenin knock-down and control cells were co-cultured with HS5 cells for 24hr and soluble factors in supernatants were harvested and analysed by Luminex bead arrays. β-catenin knock down induced reciprocal feedback signals in both KG-1a cells and HS5 cells. KG-1a catenin knockdown cells displayed increased levels of pro-inflammatory and adhesion pathways including: Galectin-3, N-CAM, NCAM1-1, CEACAM-1, osteonectin, CCL3, C5a, CCL4 and TNFα. Other soluble factors, including S100A8, TIMP-1, CD44, IL-2 and MMP3, were found to be down-regulated in KG-1a cells with β-catenin knock down and co-culture with HS5 abrogated these changes, clearly demonstrating stromal compensation in response to β-catenin knockdown.KG-1a β-catenin knock down and control cells were co-cultured with HS5 cells and suspension and adherent fractions harvested for nuclear and cytoplasmic protein extraction. Proteomic analysis of protein lysates using TMT labelling revealed that significant knock down of catenin target and adhesion molecule CD44 was observed in both cytoplasmic and nuclear fractions in both suspension and adherent KG-1a cells with beta-catenin knock down, suggesting canonical Wnt signalling is universally affected regardless of cellular context. Reduced CD44 production in catenin knockout cell lines was also confirmed in the soluble secretory factors from supernatants analysed by Luminex bead arrays, which also showed a significant reduction in soluble CD44 in β-catenin knock down lines (p<0.01). CD44 was also found to be significantly downregulated on KG-1a cell surface by flow cytometry in both suspension and adherent cells with β-catenin knock down(P<0.01), although adherent KG-1a cells showed considerably higher reduction in CD44 compared with suspension KG-1a cells (P<0.001). Proteomic data further demonstrated the upregulation of nuclear CD44 in adherent fractions, compared with suspension fractions, in both β-catenin knock down cells and control cells, suggesting activated and internalized CD44. Previous publications have shown that nuclear CD44 activates stemness factors, such as Nanog, SOX2 and Oct-4 and also PI3K/AKT and MAPK/ERK signal pathways which might contribute to the stroma mediated drug resistance we observed before.In conclusion, stromal feedback drives an AML blast adhesion phenotype through the Catenin/CD44 signalling axis, which is a contributing mechanism underlying the β-catenin-mediated stromal drug protection in AML. This finding indicates the recent development of novel Wnt inhibitors may be insufficient as a monotherapy to eradicate minimal residual disease in AML and further investigation of stromal-specific Wnt/CD44 targeting combinations is required to tackle this resistance mechanism. DisclosuresOttmann:Fusion Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Incyte: Consultancy, Research Funding; Takeda: Consultancy; Pfizer: Consultancy; Amgen: Consultancy.

Adipogeneis in the Bone Marrow Microenvironment Confers a Proliferative Advantage in Residual Leukaemic Cell Populations in Acute Myeloid Leukaemia after Allogeneic Stem Cell Transplantation

Up to 40% of poor risk Acute Myeloid Leukaemia (AML) patients receiving an allogeneic bone marrow transplant (BMT) from a donor will suffer disease recurrence/relapse within a year, significantly shortening their overall survival. Drug development to date has focused on clearing the bulk of leukaemic cells and how to target residual ‘Leukaemic stem cells,‘ which may give rise to relapse, however little work has been undertaken to discover the mechanisms by which the bone marrow niche may be unable to support normal haematopoietic stem cell growth and this may have a significant impact on the re-emergence of the patient's leukaemia. With the rise in the use of reduced intensity conditioning (RIC) regimes prior to transplant, the role of the patient microenvironment into which donor HSCs are engrafted warrants closer investigation.Stromal layers (AML-MSC) were derived from a cohort of 45 serial diagnostic, early (1-3 month), mid (6-9 month) and late (12 month+) post-transplant AML patients. Uniformity of stromal immunophenotyping markers (CD29, CD73, CD90, CD105, CD166, CD44, CD146) was present from passage 2 with concomitant loss of CD45 and other myeloid lineage markers (CD14, CD13, CD33, CD34). Diagnostic AML-MSCs showed a significant increase in stromal multipotency (CFU-F colony assays and CD146 expression, p<0.001) compared to both NBM and post-transplant MSCs. In comparison, early post-transplant colony formation was significantly reduced compared to other timepoints.AML-MSC supportive capacity for matched allogeneic donor (normal HSC) or autologous (malignant) blast populations was investigated using 14 day co-culture assays. Flow cytometric analysis of suspension and adherent co-culture fractions revealed AML blast cell numbers were consistently higher than comparative donor cell cultures at all post-transplant stages (p<0.001), suggesting patient AML-MSCs retain a preference for supporting their own blasts throughout different treatment stages. This observation was also confirmed in long-term LTC-IC assays in the stroma-adherent cobblestone fraction. Throughout serial samples, supportive capacity increased in later transplant AML-MSCs samples, confirming previous multipotency observations that stromal fitness to support stem cell growth may be compromised early post-transplant.Cytokine induced differentiation of AML-MSCs to osteoblast and adipocyte lineages resulted in a significant enhancement of adhesion and supportive capacity in the adipocyte lineage for AML blasts at all timepoints (p<0.002), which was absent in donor cell cultures. Osteoblast cultures gave results similar to that of the undifferentiated stromal layers for both AML and donor blast support. Parallel experiments using donor NBM-MSC layers revealed reduced support for AML blasts compared to that of matched AML-MSC stroma, however the same predisposition for adipogenic support of AML blasts was also observed.Luminex secretome profiling of serial AML-MSC supernatants using a panel of 105 pro-survival, inflammatory, migratory and immunomodulation cytokines revealed significant alterations in a number of targets associated with the diagnostic and post-transplant setting. Of the 80/105 targets detectable (>100pg/ml), high expression levels were seen in adhesion and ECM remodelling targets highlighting the ability of stromal secretory molecules to modify the surrounding environment. Hierarchical clustering analysis revealed post-transplant AML profiles correlated more closely with that of NBM-MSC at later time points, however significant increases in inflammatory/metabolic stress signalling molecules including IGFBP-3, Protein S, DKK1, VEGF, MMP1,MMP3, TPO (p,0.001), which have all been implicated HSC quiescence were associated with diagnostic and early post-transplant secretory profiles.In conclusion we identified several changes in the molecular and functional behaviour of the patient bone marrow microenvironment that may promote pro-leukaemic cell survival in post-transplant AML. Alterations in stromal fitness, adipogenic preference and metabolic stress signalling may contribute to a failing ability to support normal donor blood cells. Further exploration of therapies that target these alterations and prime the niche in favour of donor cells may provide a critical window for early clinical intervention and improved patient outcome. DisclosuresOttmann:Novartis: Consultancy; Celgene: Consultancy, Research Funding; Pfizer: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Fusion Pharma: Consultancy, Research Funding; Incyte: Consultancy, Research Funding.

Induced Pluripotent Stem Cells to Model Blood Diseases

Our group is developing induced pluripotent stem cell (iPSC) models of myeloid malignancies, including MDS, MPN and AML. We are generating iPSCs from the bone marrow or blood of patients, which can be maintained indefinitely as pluripotent cell lines and, upon in vitro differentiation along hematopoietic lineages, exhibit hallmark features of these diseases. By integrating mutational analyses with cell reprogramming we can derive iPSCs capturing dominant clones, subclones and normal cells from the same patient and thus have established a collection of iPSC lines representing distinct disease stages along the spectrum of myeloid transformation: predisposition syndromes/preleukemic cells/clonal hematopoiesis; low risk MDS; high risk MDS; and MDS/AML. In parallel, we are using the CRISPR/Cas9 system to introduce or correct mutations in normal or malignant iPSCs, respectively, in isogenic settings and sequential CRISPR gene editing to model mutational cooperation. We recently reported that iPSC lines derived from patients with AML re-establish upon differentiation a leukemic phenotype characterized by extensive proliferation of immature myeloid cells that serially transplant a lethal leukemia into NSG mice (Kotini et al. Cell Stem Cell 2017). Strikingly, we observed that the AML-iPSC-derived hematopoietic stem/progenitor cells (HSPCs) contain two morphologically and immunophenotypically distinct cell subpopulations: a cell fraction (adherent, A) exhibiting adherent growth and containing immature cells with an HSC immunophenotype (CD34+/CD38-/CD90+/CD45RA-/CD49f+); and a non-adherent fraction (suspension, S) of more differentiated cells. Fate-tracking experiments revealed a hierarchical organization, with the A cells renewing themselves and continuously giving rise to the S cells through symmetric and asymmetric divisions. The NSG engraftment potential was largely contained within the adherent cell fraction. Thus, AML-iPSCs exhibit the hallmarks of a leukemia stem cell (LSC) model, namely phenotypic and functional heterogeneity and hierarchical organization, with the A fraction containing LSCs that serially transplant leukemia and give rise to more differentiated cells (S fraction) without engraftment potential. LSCs are believed to be a prominent source of AML relapse, but their rarity and the unavailability of universal and specific immunophenotypic markers prohibits their prospective isolation and makes the study of their properties challenging. This new iPSC-based AML-LSC model enables us for the first time to prospectively obtain large numbers of genetically clonal human LSCs and perform genome-wide integrative molecular analyses and large-scale screening to identify key molecular mechanisms sustaining the properties of LSCs as potential new therapeutic targets. Using this model we characterized the effects of previously proposed compounds with LSC selectivity in self-renewal vs differentiation of LSCs. We also screened a small molecule library of 1280 compounds in the A and S cells in parallel to identify compounds with selectivity for the former as candidates for LSC-specific targeting. DisclosuresNo relevant conflicts of interest to declare.