Characteristic Surface Markers Research Articles

Phenotypic profiling of human induced regulatory T cells at early differentiation: insights into distinct immunosuppressive potential

Regulatory T cells (Tregs) play a key role in suppressing systemic effector immune responses, thereby preventing autoimmune diseases but also potentially contributing to tumor progression. Thus, there is great interest in clinically manipulating Tregs, but the precise mechanisms governing in vitro-induced Treg (iTreg) differentiation are not yet fully understood. Here, we used multiparametric mass cytometry to phenotypically profile human iTregs during the early stages of in vitro differentiation at single-cell level. A panel of 25 metal-conjugated antibodies specific to markers associated with human Tregs was used to characterize these immunomodulatory cells. We found that iTregs highly express the transcription factor FOXP3, as well as characteristic Treg-associated surface markers (e.g. CD25, PD1, CD137, CCR4, CCR7, CXCR3, and CD103). Expression of co-inhibitory factors (e.g. TIM3, LAG3, and TIGIT) increased slightly at late stages of iTreg differentiation. Further, CD103 was upregulated on a subpopulation of iTregs with greater suppressive capacity than their CD103− counterparts. Using mass-spectrometry-based proteomics, we showed that sorted CD103+ iTregs express factors associated with immunosuppression. Overall, our study highlights that during early stages of differentiation, iTregs resemble memory-like Treg features with immunosuppressive activity, and provides opportunities for further investigation into the molecular mechanisms underlying Treg function.

Non-Cytotoxic Graphene Nanoplatelets Upregulate Cell Proliferation and Self-Renewal Genes of Mesenchymal Stem Cells.

Graphene nanoplatelets (UGZ-1004) are emerging as a promising biomaterial in regenerative medicine. This study comprehensively evaluates UGZ-1004, focusing on its physical properties, cytotoxicity, intracellular interactions, and, notably, its effects on mesenchymal stem cells (MSCs). UGZ-1004 was characterized by lateral dimensions and layer counts consistent with ISO standards and demonstrated a high carbon purity of 0.08%. Cytotoxicity assessments revealed that UGZ-1004 is non-toxic to various cell lines, including 3T3 fibroblasts, VERO kidney epithelial cells, BV-2 microglia, and MSCs, in accordance with ISO 10993-5:2020/2023 guidelines. The study focused on MSCs and revealed that UGZ-1004 supports their gene expression alterations related to self-renewal and proliferation. MSCs exposed to UGZ-1004 maintained their characteristic surface markers. Importantly, UGZ-1004 promoted significant upregulation of genes crucial for cell cycle regulation and DNA repair, such as CDK1, CDK2, and MDM2. This gene expression profile suggests that UGZ-1004 can enhance MSC self-renewal capabilities, ensuring robust cellular function and longevity. Moreover, UGZ-1004 exposure led to the downregulation of genes associated with tumor development, including CCND1 and TFDP1, mitigating potential tumorigenic risks. These findings underscore the potential of UGZ-1004 to not only bolster MSC proliferation but also enhance their self-renewal processes, which are critical for effective regenerative therapies. The study highlights the need for continued research into the long-term impacts of graphene nanoplatelets and their application in MSC-based regenerative medicine.

Hypoxia Promotes the Stemness of Mesangiogenic Progenitor Cells and Prevents Osteogenic but not Angiogenic Differentiation

The stem cell niche in the bone marrow is a hypoxic environment, where the low oxygen tension preserves the pluripotency of stem cells. We have identified mesangiogenic progenitor cells (MPC) exhibiting angiogenic and mesenchymal differentiation capabilities in vitro. The effect of hypoxia on MPC has not been previously explored. In this study, MPCs were isolated from volunteers' bone marrow and cultured under both normoxic and hypoxic conditions (3% O2). MPCs maintained their characteristic morphology and surface marker expression (CD18 + CD31 + CD90-CD73-) under hypoxia. However, hypoxic conditions led to reduced MPC proliferation in primary cultures and hindered their differentiation into mesenchymal stem cells (MSCs) upon exposure to differentiative medium. First passage MSCs derived from MPC appeared unaffected by hypoxia, exhibiting no discernible differences in proliferative potential or cell cycle. However, hypoxia impeded the subsequent osteogenic differentiation of MSCs, as evidenced by decreased hydroxyapatite deposition. Conversely, hypoxia did not impact the angiogenic differentiation potential of MPCs, as demonstrated by spheroid-based assays revealing comparable angiogenic sprouting and tube-like formation capabilities under both hypoxic and normoxic conditions. These findings indicate that hypoxia preserves the stemness phenotype of MPCs, inhibits their differentiation into MSCs, and hampers their osteogenic maturation while leaving their angiogenic potential unaffected. Our study sheds light on the intricate effects of hypoxia on bone marrow-derived MPCs and their differentiation pathways.Graphical

Effects of tumor treating fields (TTFields) on human mesenchymal stromal cells

PurposeMesenchymal stromal cells (MSCs) within the glioblastoma microenvironment have been shown to promote tumor progression. Tumor Treating Fields (TTFields) are alternating electric fields with low intensity and intermediate frequency that exhibit anti-tumorigenic effects. While the effects of TTFields on glioblastoma cells have been studied previously, nothing is known about the influence of TTFields on MSCs.MethodsSingle-cell RNA sequencing and immunofluorescence staining were employed to identify glioblastoma-associated MSCs in patient samples. Proliferation and clonogenic survival of human bone marrow-derived MSCs were assessed after TTFields in vitro. MSC’ characteristic surface marker expression was determined using flow cytometry, while multi-lineage differentiation potential was examined with immunohistochemistry. Apoptosis was quantified based on caspase-3 and annexin-V/7-AAD levels in flow cytometry, and senescence was assessed with ß-galactosidase staining. MSCs’ migratory potential was evaluated with Boyden chamber assays.ResultsSingle-cell RNA sequencing and immunofluorescence showed the presence of glioblastoma-associated MSCs in patient samples. TTFields significantly reduced proliferation and clonogenic survival of human bone marrow-derived MSCs by up to 60% and 90%, respectively. While the characteristic surface marker expression and differentiation capacity were intact after TTFields, treatment resulted in increased apoptosis and senescence. Furthermore, TTFields significantly reduced MSCs’ migratory capacity.ConclusionWe could demonstrate the presence of tumor-associated MSCs in glioblastoma patients, providing a rationale to study the impact of TTFields on MSCs. TTFields considerably increase apoptosis and senescence in MSCs, resulting in impaired survival and migration. The results provide a basis for further analyses on the role of MSCs in glioblastoma patients receiving TTFields.

Flow Cytometry as the Tool to Define Peripheral Blood Leukocyte Signatures in Acute EBV Infection.

Primary Epstein-Barr virus (EBV) infection which can manifest as infectious mononucleosis (IM) is commonly acquired during childhood. EBV primarily invades B cells leading to a lytic reaction; the control of the infection is handled by natural killer and T cells in immunocompetent individuals. The infection has a wide spectrum of clinical findings and can lead to serious complications in patients with certain underlying immunological dysfunctions. We retrospectively investigated peripheral white blood cell populations' surface marker characteristics in IM using a comprehensive flow cytometry marker panel. Twenty-one cases of IM and seventeen EBV-seropositive cases without IM serving as controls were included. We observed novel alterations in lymphocyte, neutrophil, and monocyte populations. In addition to increased activated cytotoxic T cells and low B cells, we demonstrated high T-large granular lymphocyte (T-LGL) populations in IM cases. Furthermore, despite T cells' increased HLA-DR expression, another activation marker, CD11b, was lower in T-LGL populations. Monocytes showed increased CD16 expression; CD64 was higher in neutrophils. Our findings point to monocyte and neutrophil activation which may account for acute clinical features and may contribute to the understanding of IM immunobiology. Furthermore, they may serve as a useful tool in investigating inherited and post-transplant conditions characterized by deficiencies in controlling EBV infection.

Key quality parameter comparison of mesenchymal stem cell product cryopreserved in different cryopreservation solutions for clinical applications.

Cryopreservation is a critical process of cell products for achieving a commercial viability through wide scale adoption. By preserving cells in a lower temperature, cryopreservation enables a product to be off-the-shelf and ready for infusion. An optimized cryopreservation strategy can maintain the viability, phenotype, and potency of thawed mesenchymal stromal/stem cells (MSCs) while being regulatory compliant. We compared three clinical-ready formulations with one research cryopreservation solutions and evaluated key quality parameters of post thawed MSCs. MSCs were cryopreserved at 3, 6, and 9 million cells/mL (M/mL) in four different cryopreservation solutions: NutriFreez (10% dimethyl sulfoxide [DMSO]), Plasmalyte A (PLA)/5% human albumin (HA)/10% DMSO (PHD10), CryoStor CS5 (5% DMSO), and CryoStor CS10 (10% DMSO). To establish post thaw viability, cells were evaluated with no dilution of DMSO (from 3M/mL), 1:1 dilution (from 6M/mL), or 1:2 dilution (from 9M/mL) with PLA/5% HA, to achieve uniform concentration at 3M/mL. Cell viability was measured at 0-, 2-, 4-, and 6-h post thaw with Trypan blue exclusion and Annexin V/PI staining. Dilution (1:2) of final cell products from 9M/mL resulted in an improvement of cell viability over 6h but showed a trend of decreased recovery. MSCs cryopreserved in solutions with 10% DMSO displayed comparable viabilities and recoveries up to 6h after thawing, whereas a decreasing trend was noted in cell viability and recovery with CS5. Cells from all groups exhibited surface marker characteristics of MSCs. We further evaluated cell proliferation after 6-day recovery in culture. While cells cryopreserved in NutriFreez and PHD10 presented similar cell growth post thaw, MSCs cryopreserved in CS5 and CS10 at 3M/mL and 6M/mL showed 10-fold less proliferative capacity. No significant differences were observed between MSCs cryopreserved in NutriFreez and PHD10 in their potency to inhibit T cell proliferation and improve monocytic phagocytosis. MSCs can be cryopreserved up to 9M/mL without losing notable viability and recovery, while exhibiting comparable post thaw potency with NutriFreez and PHD10. These results highlight the importance of key parameter testing for selecting the optimal cryopreservation solution for MSC-based therapy.

Increased Activated Protein C Response Rates to Thrombin Formation in Asymptomatic Factor V Leiden Carriers Are Driven By the Endothelium

Background The thrombophilic factor V Leiden (FVL) mutation shows a highly variable clinical expressivity. We have shown in vivo that asymptomatic FVL carriers respond to extrinsic coagulation activation with higher activated protein C (APC) formation rates than those with a history of venous thromboembolism (VTE). Recently we have established an ex vivo model for personalized assessment of the protein C (PC) pathway combining endothelial colony forming cells (ECFCs) and autologous plasma. Aim of this study was to investigate potential modulating factors of the APC response in the endothelium unraveling the enigma of the variable expressivity of the FVL phenotype. Methods ECFCs were isolated from FVL carriers with (FVL VTE+) or without (FVL VTE-) a history of unprovoked VTE and healthy controls (non-FVL) (n=7 each). Quality control of cultured cells included cobblestone morphology as examined by light microscopy, positive staining for CD31, CD309, CD201, and CD141, CD34, and negative staining for CD45 analyzed by flow cytometry. We either added autologous or pooled normal defibrinated plasma to the confluent cell culture and inititated thrombin formation by addition of tissue factor (1 pmol/L) and CaCl 2 (16.6 mmol/L) ( Fig. 1A). Thrombin and APC formation were measured over time using oligonucleotide-based enzyme capture assays (OECAs) and the ratio between the area under the curve (AUC) of APC formation and the AUC of thrombin formation was calculated as measure for APC response to thrombin. Moreover, we quantified thrombomodulin (TM) and endothelial protein C receptor (EPCR) expression in cell-based enzyme-linked immunosorbent assays (cell-ELISAs) and measured activation of purified PC on ECFCs in the APC-OECA. Results ECFC morphology in light microscopy and characteristic surface marker expression in flow cytometry did not differ between non-FVL, FVL VTE-, and FVL VTE+ subgroups. In the ECFC-based ex vivo model, the APC response in plasma on autologous cells was significantly higher in asymptomatic FVL VTE- carriers compared to non-FVL carriers (P = .0072) and compared to the FVL VTE+ subgroup (P = .0292) whereas it did not differ significantly between non-FVL and FVL VTE+ carriers. EPCR expression on ECFCs measured by cell-ELISA was higher in non-FVL carriers compared to FVL VTE+ subjects (P = .0374) but did not differ significantly when comparing non-FVL versus FVL VTE-, and FVL VTE+ versus FVL VTE-. Similarly, we showed that TM expression as well as activation of purified PC on ECFCs did not differ between cohorts. However, when the APC response was assessed on FVL VTE- and FVL VTE+ ECFCs using non-FVL plasma, the difference in the APC response between asymptomatic FVL carriers and FVL carriers with a history of VTE persisted (P = .0111) ( Fig. 1B). Conclusion Consistent with previous in vivo experiments, APC response rates to thrombin formation were increased on ECFCs obtained from asymptomatic FVL carriers compared to those from FVL carriers with a history of VTE in autologous plasma. Although TM and EPCR expression did not differ between both groups, we showed that the endothelial APC response in asymptomatic FVL carriers remained increased when using plasma from healthy controls. Our observations suggest that the increased APC response to thrombin in asymptomatic FVL carriers is driven by the endothelium. Further studies are warranted to elucidate the underlying mechanism for a better understanding of endogenous protective factors in FVL carriers. These findings may pave the way towards personalized, precision medicine approaches for managing the thrombotic risk in FVL and other disorders related to the PC pathway.

Lyophilized human platelet lysate as a supplementation in the culture of umbilical cord derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are being used in clinical trials given their proliferative potential, immunomodulatory effects, and their pro-angiogenic, anti-apoptotic, and anti-fibrotic properties. Umbilical cord tissue is an excellent source of MSCs. Iron-fortified calf serum is being used to culture MSCs as an alternative to fetal bovine serum since it is relatively inexpensive. Fetal calf serum is fortified with iron since calves are often fed a low-iron diet. However, the use of iron-fortified calf serum is still problematic since it is xenogeneic. Lately, human platelet lysate is being used for the culture of human cells. To increase its shelf life, human platelet lysate was lyophilized and used for culturing human umbilical cord tissue mesenchymal stem cells (hUCT-MSCs). This study compares the culture of hUCT-MSCs with either iron-fortified calf serum or lyophilized human platelet lysate (LHPL). Trilineage differentiation potential (for chondrogenesis, adipogenesis, or osteogenesis) was assessed and immunomodulatory properties of hUCT-MSC were studied using the Mixed Lymphocyte Reaction (MLR) to detect inhibition of the proliferation of lymphocytes. This study concludes the potency of LHPL as the best alternative to Iron-Fortified Calf Serum (IFCS) for culture expansion of hUCT-MSC. hUCT-MSC cultured with LHPL display characteristic surface markers and have the capacity for trilineage differentiation.

A Novel Approach for Determining the Critical Quality Attributes of Mesenchymal Stem Cells by Specifying Cell Population With Replication Potential

We introduce a novel approach to determine the critical quality attributes (CQAs) of mesenchymal stem cells (MSCs) expected to exert immunosuppressive effects. MSCs retained homeostatic replication potentials, such as sustainable growth and consistent cell morphology as a population, in early passages, but lost them in late passages. Characteristic surface markers of MSCs (ie, CD73, CD90, and CD105) were no longer expressed at 2 weeks after subcutaneous transplantation into NOG mice when MSCs from late passages were transplanted, but not when MSCs from early passages were transplanted, suggesting that the biological effects of the MSCs differed according to the timing of cell harvesting and highlighting the importance of specifying MSCs that retained homeostatic features to define the CQAs. The homeostatic features of MSCs related to the balance of the redox system, nutrient requirements, and mitochondrial function were also observed until a certain passage. Therefore, we could define the CQAs of MSCs related to manufacturing by selecting process parameters (PPs) underlying the homeostatic features of MSCs and measuring these PPs quantitatively to specify the cell population with homeostatic features by limiting the passage number. The validity of the PPs stipulated in our pilot study was verified using an SKG murine arthritis model, and critical PPs (CPPs) were then selected among the PPs. Thus, CQAs related to manufacturing in the developmental phase could be defined by the CPPs in this manner, and the concept of CQAs could be refined continuously toward commercial manufacturing.

Nanomechanical Signatures in Glioma Cells Depend on CD44 Distribution in IDH1 Wild-Type but Not in IDH1R132H Mutant Early-Passage Cultures

Atomic force microscopy (AFM) recently burst into biomedicine, providing morphological and functional characteristics of cancer cells and their microenvironment responsible for tumor invasion and progression, although the novelty of this assay needs to coordinate the malignant profiles of patients' specimens to diagnostically valuable criteria. Applying high-resolution semi-contact AFM mapping on an extended number of cells, we analyzed the nanomechanical properties of glioma early-passage cell cultures with a different IDH1 R132H mutation status. Each cell culture was additionally clustered on CD44+/- cells to find possible nanomechanical signatures that differentiate cell phenotypes varying in proliferative activity and the characteristic surface marker. IDH1 R132H mutant cells compared to IDH1 wild-type ones (IDH1wt) characterized by two-fold increased stiffness and 1.5-fold elasticity modulus. CD44+/IDH1wt cells were two-fold more rigid and much stiffer than CD44-/IDH1wt ones. In contrast to IDH1 wild-type cells, CD44+/IDH1 R132H and CD44-/IDH1 R132H did not exhibit nanomechanical signatures providing statistically valuable differentiation of these subpopulations. The median stiffness depends on glioma cell types and decreases according to the following manner: IDH1 R132H mt (4.7 mN/m), CD44+/IDH1wt (3.7 mN/m), CD44-/IDH1wt (2.5 mN/m). This indicates that the quantitative nanomechanical mapping would be a promising assay for the quick cell population analysis suitable for detailed diagnostics and personalized treatment of glioma forms.

Abstract 3156: Tumor Treating Fields reduce cellular survival of human mesenchymal stromal cells via apoptosis and senescence induction

Abstract Background Tumor Treating Fields (TTFields), alternating electric fields with intermediate field intensity, have shown clinical efficacy in several tumor entities, leading to clinical approval for glioblastoma and mesothelioma treatment. As mesenchymal stromal cells (MSCs) belong to the tumor microenvironment and exhibit - depending on the tumor type and microenvironment - either pro-tumorigenic or anti-tumorigenic abilities, we aimed to study the effects of TTFields on human MSCs. Methods Human MSCs were isolated from the bone marrow of three different healthy donors and were exposed to TTFields for 72 hours. MSCs’ clonogenic survival, proliferation and viability was analyzed after different TTFields frequencies. Boyden chamber assays were carried out to investigate the influence of TTFields on the migratory potential of MSCs. Flow cytometry analyses of caspase-3 and annexin V were conducted to determine MSCs’ apoptosis rates after TTFields treatment. TTFields-induced senescence was quantified using beta-galactosidase stainings. MSCs’ characteristic surface marker expression (CD73, CD90 and CD105) was studied after TTFields treatment. Result TTFields led to significantly reduced clonogenic survival (70-90% reduction compared to controls) and cellular proliferation (approximately 50% reduction) in all analyzed MSC specimens. However, MSCs’ metabolic viability was largely unaffected after TTFields exposure. Within a range between 100 and 500 kHz, there was no frequency dependence of TTFields’ anti-proliferative effect on human MSCs. As determined by Bodyen Chamber assays, TTFields resulted in a considerable reduction of MSCs’ migratory potential. TTFields increased both cellular apoptosis and senescence in all MSC samples, whereas there was no alteration of MSCs’ characteristic surface marker pattern. Conclusion As TTFields considerably reduce MSCs’ survival and migratory potential, TTFields could attenuate the pro-tumorigenic effects of tumor-associated MSCs that were recently described in glioblastoma patients. Co-culture experiments of MSCs and tumor cells after TTFields treatment as well as in vivo experiments are planned in the future. Citation Format: Alexander Rühle, Maren Strack, Anca-Ligia Grosu, Nils H. Nicolay. Tumor Treating Fields reduce cellular survival of human mesenchymal stromal cells via apoptosis and senescence induction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3156.

Establishment of PLAFMCi007-A, an induced pluripotent stem cell line, from peripheral blood mononuclear cells (PBMCs) of a healthy adult woman

Induced pluripotent stem cell (iPSC) lines for studies investigating many diseases can be established from peripheral blood mononuclear cells; here, an iPSC line was established from CD34+ cells isolated from the peripheral blood of a healthy woman. The cells were electrotransfected with three different recombinant plasmids to generate a normal-karyotype iPSC line that expresses characteristic surface markers and other pluripotent stem cell genes and can differentiate into all three germ layers in vivo. These newly established iPSC lines, a normal human cell line, can serve as a control line in studies investigating the pathogenesis of various diseases and meet the conditions for organoid studies.

Cannabidiol Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in the Inflammatory Microenvironment via the CB2-dependent p38 MAPK Signaling Pathway.

Chronic inflammation of bone tissue often results in bone defects and hazards to tissue repair and regeneration. Cannabidiol (CBD) is a natural cannabinoid with multiple biological activities, including anti-inflammatory and osteogenic potential. This study aimed to investigate the efficacy and mechanisms of CBD in the promotion of bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation in the inflammatory microenvironment. BMSCs isolated from C57BL/6 mice, expressed stem cell characteristic surface markers and presented multidirectional differentiation potential. The CCK-8 assay was applied to evaluate the effects of CBD on BMSCs' vitality, and demonstrating the safety of CBD on BMSCs. Then, BMSCs were stimulated with lipopolysaccharide (LPS) to induce inflammatory microenvironment. We found that CBD intervention down-regulated mRNA expression levels of inflammatory cytokines and promoted cells proliferation in LPS-treated BMSCs, also reversed the protein and mRNA levels downregulation of osteogenic markers caused by LPS treatment. Moreover, CBD intervention activated the cannabinoid receptor 2 (CB2) and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. While AM630, a selective CB2 inhibitor, reduced phosphorylated (p)-p38 levels. In addition, AM630 and SB530689, a selective p38 MAPK inhibitor, attenuated the enhancement of osteogenic markers expression levels by CBD in inflammatory microenvironment, respectively. CBD promoted osteogenic differentiation of BMSCs via the CB2/p38 MAPK signaling pathway in the inflammatory microenvironment.

Human mesenchymal stromal cells maintain their stem cell traits after high-LET particle irradiation - Potential implications for particle radiotherapy and manned space missions.

The influence of high-linear energy transfer (LET) particle radiation on the functionalities of mesenchymal stromal cells (MSCs) is largely unknown. Here, we analyzed the effects of proton (1H), helium (4He), carbon (12C) and oxygen (16O) ions on human bone marrow-MSCs. Cell cycle distribution and apoptosis induction were examined by flow cytometry, and DNA damage was quantified using γH2AX immunofluorescence and Western blots. Relative biological effectiveness values of MSCs amounted to 1.0-1.1 for 1H, 1.7-2.3 for 4He, 2.9-3.4 for 12C and 2.6-3.3 for 16O. Particle radiation did not alter the MSCs' characteristic surface marker pattern, and MSCs maintained their multi-lineage differentiation capabilities. Apoptosis rates ranged low for all radiation modalities. At 24h after irradiation, particle radiation-induced ATM and CHK2 phosphorylation as well as γH2AX foci numbers returned to baseline levels. The resistance of human MSCs to high-LET irradiation suggests that MSCs remain functional after exposure to moderate doses of particle radiation as seen in normal tissues after particle radiotherapy or during manned space flights. In the future, in vivo models focusing on long-term consequences of particle irradiation on the bone marrow niche and MSCs are needed.

Azacitidine Directly Acts on the Mesenchymal Stromal Cell Compartment in Myelodysplastic Syndromes to Alter Niche Function and Suppress Malignant Hematopoeitic Stem/Progenitor Cells

Background: Hematopoietic stem/progenitor cells (HSPC) are highly dependent on interaction with the specific bone marrow microenvironment (niche) which supports their survival and directs their differentiation. Increasing evidence suggests that the niche plays a central role in the development of clonal stem cell disorders such as myelodysplastic syndromes (MDS). Malignant clonal HSPC in MDS have been shown to reprogram mesenchymal stromal cells (MSC) to promote disease progression. In turn, the altered stromal microenvironment in MDS may directly impair healthy hematopoiesis. Accordingly, MSC isolated from MDS patients have shown phenotypical and functional abnormalities, which were not observed in patients successfully treated with azacitidine (AZA), currently the standard of care for higher-risk MDS patients. Although AZA is a potent inhibitor of DNA methyltransferases and is thus considered a hypomethylating agent, the exact anti-leukemic mechanisms are not understood and a clear correlation between DNA demethylation and response to AZA has not been demonstrated thus far. Hence, we looked beyond the current focus of AZA effects on clonal hematopoiesis and hypothesized that AZA may also act through direct modulation of the MSC compartment. To answer this question, we examined primary MSC treated in vitro with AZA as well as the functional interaction between MSC and HSPC after AZA treatment.Methods: Primary MSC were isolated from bone marrow of untreated MDS patients or age-matched healthy controls, expanded by plastic adherence, cultured until confluency under defined conditions and used for up to 4 passages. Identity of MSC was confirmed by presence of characteristic cell surface markers CD73, CD271, CD105 and CD90 and absence of CD45 and CD34 by flow cytometry. MSC were differentiated into the adipogenic or osteogenic lineage after in vitro treatment with AZA (10 uM). Co-culture experiments with CD34+ HSPC from MDS patients or healthy controls were performed on AZA-treated or untreated MDS-MSC or healthy MSC, respectively. After four days, CD34+ cells were harvested and assessed by colony-forming unit assays in methylcellulose. Single colonies were genotyped for MDS-associated mutations known to be present in the bone marrow sample. Each colony was genotyped for one or two known MDS-associated mutations and an input patient specimen was used as a control. Finally, RNA sequencing was performed on a mesenchymal stromal cell line (EL08-1D2), either untreated or treated with AZA for 4 days in vitro to uncover pathways affected by AZA in stromal cells.Results: MDS-MSC showed a decreased proliferative capacity as well as markedly impaired differentiation into the osteoblastic lineage compared to healthy MSC. Treatment with AZA had no effect on cell surface marker expression or viability of either healthy or MDS-MSC. AZA treatment significantly reduced the ability of MDS-MSC to differentiate towards the adipogenic lineage while osteogenic differentiation was severely impaired with or without AZA treatment. In contrast, osteogenic differentiation of healthy MSC was enhanced after AZA treatment. In co-culture experiments pretreatment with AZA significantly improved stromal support of healthy CD34+ HSPC while malignant MDS-HSPC were suppressed. RNA sequencing analysis revealed differential gene expression in AZA-treated vs untreated stroma with significantly more genes upregulated in AZA-treated samples. Specific changes in pathways involved in ECM-receptor interaction, cell adhesion molecules (CAMs), cytokine-cytokine receptor interaction and metabolic pathways in AZA treated EL08-1D2 stromal cells were determined and validated in primary MDS MSC samples.Conclusion: We show here that AZA directly modulates altered MSC function in MDS, thus influencing interaction with HSPC and leading to suppression of malignant hematopoiesis. Our data suggest an additional mode of action by which AZA exerts its therapeutic activity. To our knowledge this analysis is the first to examine the direct effect of AZA treatment on MSC. DisclosuresGötze:BMS: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Abbvie: Honoraria.

Clonal T-Lymphocyte Populations in Healthy Donors

IntroductionT-cell clonality analysis based on T-cell receptor gamma chain (TCRG) genes rearrangements in peripheral blood could be useful for T-cell neoplasms diagnosis. Monoclonal products that exceed polyclonal background more than 3 times are often seen in lymphoid malignancies. Doubtful clonal results might be a possible feature of a relatively low tumor burden as well as a sign of a benign reactive process or reduction of the repertoire of lymphocytes associated with age. T-cell clonality in peripheral blood has been described in autoimmune diseases and viral infections. Examination of T-cell clonality in healthy people is necessary to define normal values of clonal variation and to develop algorithms for differential diagnosis of reactive and neoplastic conditions. Cytotoxic T-lymphocytes are the intended substrate of reactive processes. CD8 and CD57 are the characteristic surface markers of the cell population. Selection of CD8+ and CD57+ cells followed by determination of clonality based on TCRG genes rearrangements used to correlate clonal products with a specific functional population of lymphocytes and thereby to understand the basis of clonal expansion.Thus the aim of the research was to study the frequency of T-cell clonality and possible features of the clonal expansion of T-lymphocytes in healthy adults.Materials and methodsThe study included 25 healthy donors with a median age of 40 years (from 19 to 62 years). Samples of peripheral blood were obtained at the blood transfusion facility of the National Research Center for Hematology. All donors gave informed consent for participation in the research.CD8+ and CD57+ lymphocytes were isolated by means of immunomagnetic selection on Miltenyi Biotec beads. T-cell clonality was assessed by TCRG gene rearrangements using multiplex PCR with BIOMED-2 primer system and subsequent GeneScan analysis.ResultsBulk lymphocyte population in 2 of 25 donors (8%) was monoclonal, in 5 (20%) donors -doubtful monoclonal, in 2 donors (8%) - oligoclonal and in 16 of 25 donors (64%) - polyclonal (table 1). Thus there was the predominance of reactive clonal populations in the bulk population of cells in 28% (7/25) of donors.No correlation of the presence of clonal products with the age of donors has been demonstrated (Spearman test). According to the literature the frequency of clonal expansion of lymphocytes increases after 60 years. On the contrary, all participants in our study with the sole exception were younger. The median age of donors was 40 years. We suggest than at least in our donor sample T-cell clonality might be associated with individual characteristics of the donors rather than age related T-cell repertoire narrowing.Clonal products dominant in bulk population of lymphocytes, were attributed to CD8+ and CD57+ cells by means of magnetic selection. Therefore, the association of reactive clonal products with cytotoxic lymphocytes (figure 1, #8, #13) might be assumed.Additionally, in the case of donor #7 doubtful clonal product was discovered in the CD8- population, not previously dominant in the bulk population of lymphocytes and not identified in the CD8+ population (figure 1, #7).Since clear monoclonality of donor case #12 (figure 2) additional blood tests were run. No deviations in blood formula were found. Clinical evaluation confirmed the woman to be healthy, without any symptoms or history of chronic viral or autoimmune diseases.ConclusionsThe presence of clonal expansion of T-lymphocytes in peripheral blood should not be taken as a specific feature of lymphoid malignancy when seen in the absence of clinical or pathological signs of the disease. The typical cause of such conditions is the accumulation of clonal cytotoxic CD8+ CD57+ cells. The nature of clonal products in CD8- populations of lymphocytes are currently not clear and requires more extensive investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

T-cell Exhaustion in Organ Transplantation.

Exhaustion of T cells occurs in response to long-term exposure to self and foreign antigens. It limits T cell capacity to proliferate and produce cytokines, leading to an impaired ability to clear chronic infections or eradicate tumors. T-cell exhaustion is associated with a specific transcriptional, epigenetic, and metabolic program and characteristic cell surface markers' expression. Recent studies have begun to elucidate the role of T-cell exhaustion in transplant. Higher levels of exhausted T cells have been associated with better graft function in kidney transplant recipients. In contrast, reinvigorating exhausted T cells by immune checkpoint blockade therapies, while promoting tumor clearance, increases the risk of acute rejection. Lymphocyte depletion and high alloantigen load have been identified as major drivers of T-cell exhaustion. This could account, at least in part, for the reduced rates of acute rejection in organ transplant recipients induced with thymoglobulin and for the pro-tolerogenic effects of a large organ such as the liver. Among the drugs that are widely used for maintenance immunosuppression, calcineurin inhibitors have a contrasting inhibitory effect on exhaustion of T cells, while the influence of mTOR inhibitors is still unclear. Harnessing or encouraging the natural processes of exhaustion may provide a novel strategy to promote graft survival and transplantation tolerance.

Cancer stem cells in colorectal cancer and the association with chemotherapy resistance.

The incidence and mortality of colorectal cancer (CRC) have always been among the highest in the world, although the diagnosis and treatment are becoming more and more advanced. At present, the main reason is that patients have acquired drug resistance after long-term conventional drug treatment. An increasing number of evidences confirm the existence of cancer stem cells (CSCs), which are a group of special cells in cancer, only a small part of cancer cells. These special cell populations are not eliminated by chemotherapeutic drugs and result in tumor recurrence and metastasis after drug treatment. CSCs have the ability of self-renewal and multidirectional differentiation, which is associated with the occurrence and development of cancer. CSCs can be screened and identified by related surface markers. In this paper, the characteristic surface markers of CSCs in CRC and the related mechanism of drug resistance will be discussed in detail. A better understanding of the mechanism of CSCs resistance to chemotherapy may lead to better targeted therapy.

Endoscopic atomization of mesenchymal stromal cells: in vitro study for local cell therapy of the lungs

Background aimsCell-based therapies of pulmonary diseases with mesenchymal stromal cells (MSCs) are increasingly under experimental investigation. In most of these, MSCs are administered intravenously or by direct intratracheal instillation. A parallel approach is to administer the cells into the lung by endoscopic atomization (spraying). In a previous study, the authors developed a flexible endoscopic atomization device that allows administration of respiratory epithelial cells in the lungs with high survival. MethodsIn this study, the authors evaluated the feasibility of spraying MSCs with two different endoscopic atomization devices (air and pressure atomization). Following atomization, cell viability was evaluated with live/dead staining. Subsequent effects on cytotoxicity, trilineage differentiation and expression of MSC-specific markers as well as on MSC metabolic activity and morphology were analyzed for up to 7 days. ResultsMSC viability immediately after spraying and subsequent metabolic activity for 7 days was not influenced by either of the devices. Slightly higher cytotoxicity rates could be observed for pressure-atomized compared with control and air-atomized MSCs over 7 days. Flow cytometry revealed no changes in characteristic MSC cell surface marker expression, and morphology remained unchanged. Standard differentiation into osteocytes, chondrocytes and adipocytes was inducible after atomization. ConclusionsIn the literature, a minimal survival of 50% was previously defined as the cutoff value for successful cell atomization. This is easily met with both of the authors’ devices, with more than 90% survival. Thus, there is a potential role for atomization in intrapulmonary MSC-based cell therapies, as it is a feasible and easily utilizable approach based on clinically available equipment.

Group 2 innate lymphoid cells contribute to IL-33-mediated alleviation of cardiac fibrosis.

Rationale: The major cause of heart failure is myocardium death consequent to detrimental cardiac remodeling and fibrosis following myocardial infarction. The cardiac protective cytokine interleukin (IL)-33, which signals by ST2 receptor binding, is associated with group 2 innate lymphoid cell (ILC2) activation and regulates tissue homeostasis and repair following tissue injury in various tissues. However, the distribution and role of IL-33-responsive ILC2s in cardiac fibrosis remain unclear. In this study, we elucidated the roles of IL-33-responsive cardiac-resident ILC2s and IL-33-mediated immunomodulatory functions in cardiac fibrosis.Methods: We examined the distribution of cardiac ILC2s by using flow cytometry. The roles of IL-33-mediated ILC2 expansion in cardiac fibrosis was evaluated in the mouse model of catecholamine-induced cardiac fibrosis. ILC-deficient Rag2‒/‒IL2Rγc‒/‒ mice were implemented to determine the contribution of endogenous ILC in the progression of cardiac fibrosis. Histopathological assessments, speckle tracking echocardiography, and transcriptome profile analysis were performed to determine the effects of IL-33-mediated cardiac protective functions.Results: We identified the resident cardiac ILC2s, which share similar cell surface marker and transcriptional factor expression characteristics as peripheral blood and lung tissue ILC2s. IL-33 treatment induced ILC2 expansion via ST2. In vivo, ILC-deficient Rag2‒/‒IL2Rγc‒/‒ mice developed exacerbated cardiac fibrosis following catecholamine-induced stress cardiac injury. IL-33 treatment expanded cardiac ILC2s and revealed protective effects against cardiac tissue damage with reduced cardiomyocyte death, immune cell infiltration, tissue fibrosis, and improved myocardial function. Transcriptome analysis revealed that IL-33 attenuated extracellular matrix synthesis- and fibroblast activation-associated gene expressions. IL13-knockout or epidermal growth factor receptor (EGFR) inhibition abolished IL-33-mediated cardiac protective function, confirming IL-13 and EGFR signaling as crucial for IL-33-mediated cardioprotective responses. Moreover, ILC2-produced BMP-7 served as a novel anti-fibrotic factor to inhibit TGF-β1-induced cardiac fibroblast activation.Conclusion: Our findings indicate the presence of IL-33-responsive ILC2s in cardiac tissue and that IL-33-mediated ILC2 expansion affords optimal cardioprotective function via ILC2-derived factors. IL-33-mediated immunomodulation is thus a promising strategy to promote tissue repair and alleviate cardiac fibrosis following acute cardiac injury.