Carboxyfluorescein Diacetate Succinimidyl Ester Staining Research Articles

Xuebijing enhances antitumor efficacy of anti-CD19 CAR-T cells

ObjectiveTo investigate the effects and mechanisms of Xuebijing injection (XBJ) on Chimeric antigen receptor-T (CAR-T) cell function and its therapeutic potential against CAR-T therapy-associated cytokine storms (CRS). MethodsAnti-CD19 CAR-T cells were established based on FMC63 antibodies. Different doses of XBJ (1 and 10 mg/mL) were added to the culture system. Untreated anti-CD19 CAR-T cells served as negative controls. After 48-h co-culture, the effects of XBJ on CAR-T cell function were assessed. Carboxyfluorescein diacetate succinimidyl ester staining was used to assess the effect of XBJ on CAR-T cell proliferation. Flow cytometry, luciferase reporter gene assays, and real time cellular analysis were employed to evaluate the effects of XBJ on CAR-T cell cytotoxicity in vitro. RNA-sequencing was performed to analyze the effects of XBJ on CAR-T cell gene expression. Network pharmacology predicted potential XBJ therapeutic targets for CRS, which were verified in a THP-1 macrophage inflammation model. ResultsXBJ enhanced both the proliferation and tumor killing capacities of CAR-T cells. Transcriptome analysis showed that XBJ treatment affects multiple genes and pathways in CAR-T cells, with differential gene enrichment in multiple cell proliferation and growth factor pathways. Potential targets for CRS control by XBJ were predicted using network pharmacology, and the inhibitory effect of XBJ on the expression of relevant genes was verified using a macrophage model. ConclusionThe results of this study indicate that XBJ can enhance the killing effect of CAR-T cells on tumor cells and that the mechanism is related to the regulation of T cell proliferation and activation. Moreover, XBJ inhibited excessive inflammation associated with CAR-T therapy. However, the current findings remain to be further validated through in vivo experiments.

FASCIA Method in the Assessment of Lymphocyte Mitogen Responses in the Laboratory Diagnostics of Primary Immunodeficiencies

Lymphocyte responses to mitogens constitute a key part of the diagnostics of combined immunodeficiency (CID). Currently, mostly radioactive thymidine incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution methods are used. Flow-cytometric assay for specific cell-mediated immune-response in activated whole blood (FASCIA) has been put forth as an easy-to-perform option for the measurement of lymphocyte responses with the advantage of recognizing different lymphocyte subtypes and avoiding the use of radioactive reagents. Our aim was to analyze retrospectively the usefulness of FASCIA in the diagnostics of CID. We included all lymphocyte stimulation tests done with FASCIA in HUSLAB (Helsinki, Finland) between February 2015 and September 2018 in our analysis. The cohort was divided into two groups according to the patients’ final diagnoses: CID (n = 30) or non-CID (n = 159). We evaluated the stimulation responses with a combined FASCIA score (the average of all mitogen responses). The FASCIA score was significantly lower among the CID group compared to the other patients (p = 0.002), and in the ROC analysis, the AUC was 0.75 (p < 0.001) for the FASCIA score. When the three mitogens were analyzed separately, phytohemagglutinin (PHA) was best in separating patients with CID from non-CID (in the ROC analysis AUC 0.71, p = 0.001). Immunosuppressive medication affected the FASCIA result significantly and needs to be considered when evaluating the results. In conclusion, FASCIA can reliably detect the CID patients in the absence of immunosuppressive medication. It emerges as a method with many benefits compared to tests requiring radioactive reagents or the complicated CFSE staining.

Hapalindoles from the Cyanobacterium Hapalosiphon sp. Inhibit T Cell Proliferation.

Novel immunomodulating agents are currently sought after for the treatment of autoimmune diseases and cancers. In this context, a screening campaign of a collection of 575 cyanobacteria extracts for immunomodulatory effects has been conducted. The screening resulted in several active extracts. Here we report the results of subsequent studies on an extract from the cyanobacterium Hapalosiphon sp. CBT1235. We identified 5 hapalindoles as the compounds responsible for the observed immunomodulatory effect. These indole alkaloids are produced by several strains of the cyanobacterial family Hapalosiphonaceae. They are known for their anti-infective, cytotoxic, and other bioactivities. Modulation of the activity of human immune cells has not yet been described. The immunomodulatory activity of the hapalindoles was characterized in vitro using flow cytometry-based measurements of T cell proliferation after carboxyfluorescein diacetate succinimidyl ester staining, and apoptosis and necrosis induction after annexin V/propidium iodide staining. The most potent compound, hapalindole A, reduced T cell proliferation with an IC50 of 1.56 µM, while relevant levels of apoptosis were measurable only at 10-fold higher concentrations. Hapalindole A-formamide and hapalindole J-formamide, isolated for the first time from a natural source, had much lower activity than the nonformylated derivatives while, at the same time, being less selective for antiproliferative over apoptotic effects.

The prognostic value of Tiam1 correlates with its roles in epithelial-mesenchymal transition progression and angiogenesis in lung adenocarcinoma.

BackgroundTiam1 has been identified as an oncogene and acts as an activator of GTPase Rac. Tiam1 was reported to be a promoter of cancer progression in various cancer types, while in lung adenocarcinoma, its mechanism of action is poorly understood.Materials and MethodsImmunohistochemistry staining and Western blot assay were used to determine Tiam1 expression in lung adenocarcinoma tissues, and its association with prognosis was determined by statistical analysis. We depleted Tiam1 in both A549 and H1975 cancer cell lines. Carboxyfluorescein diacetate succinimidyl ester staining and colony formation assays were used to evaluate its impact on cell proliferation ability after depletion. Transwell migration assay and wound healing assays were performed to determine its impact on migration ability of both cell lines. Western blot assay and immunofluorescence staining were used to analyze the association between Tiam1 and epithelial–mesenchymal transition (EMT) progression. Tube formation assay and vasculogenic mimicry assay were used to show the impact of Tiam1 depletion on cancer angiogenesis.ResultsIn this study, we demonstrated that Tiam1 overexpression in lung adenocarcinoma was significantly associated with advanced tumor grade and poor prognosis. In vitro assays indicated that Tiam1 depletion significantly inhibited cell proliferation, colony formation, and migration capacities in A549 and H1975 cells. Further investigations revealed that Tiam1 plays an important role in EMT program enhancement, angiogenesis, and accelerated tumor progression. Notably, Tiam1 depletion in cancer cells strongly inhibited human umbilical vein endothelial cell angiogenesis and vasculogenic mimicry capacities of both cancer cell lines.ConclusionTiam1 overexpression is associated with lung adenocarcinoma progression and may indicate poor prognosis. Tiam1 accelerated tumor progression due to EMT and angiogenesis enhancement. Our data may provide a novel therapeutic target for lung adenocarcinoma.

Effects of human umbilical cord-derived mesenchymal stem cells on hematologic malignancies.

Mesenchymal stem cells (MSCs) have been used in hematopoietic stem cell transplantation for years. However, the safety of MSCs applied in various types of hematologic malignancy has not been comprehensively explored. In the present study, the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on six representative hematologic malignancy cell lines were explored, including leukemia, multiple myeloma and lymphoma cells. Direct and indirect co-culture models were established, and cell proliferation was assessed by carboxyfluorescein diacetate succinimidyl ester staining. A cytometric bead array cytokine kit was used to quantify cytokines. The expression of interleukin (IL)-6 receptor elements on tumor cells was detected by reverse transcription-polymerase chain reaction and flow cytometry, and the effects of exogenous IL-6 on cell proliferation were determined using a Cell Counting kit-8 assay. The results demonstrated that hUC-MSCs inhibited the proliferation of most of the cell lines examined (THP-1, HL-60, K562 and RPMI-8226), but promoted the proliferation of Raji cells. In addition, hUC-MSCs secreted abundant IL-6, promoted the secretion of IL-10 by RPMI-8226 and Raji cells, and inhibited the secretion of tumor necrosis factor-α by THP-1 cells. These data indicate a varied effect of hUC-MSCs on various types of hematologic malignancy, including distinct mechanisms of cell-to-cell contact and cytokines. Researchers applying hUC-MSCs in lymphoma should be aware of a potential tumor growth-promoting effect.

Effects of Birch Polypore Mushroom, Piptoporus betulinus (Agaricomycetes), the "Iceman's Fungus", on Human Immune Cells.

Piptoporus betulinus, the mushroom that has been carried by Ötzi the "Iceman", has a long tradition of use in medicinal practice for its antiseptic, anticancer, and immune-enhancing properties. With this study, we aimed to investigate the immunomodulatory effects of P. betulinus on primary human immunocompetent cells. The influence of P. betulinus water and methanol extract on apoptosis and necrosis induction of T cells and monocytes was analyzed using annexin V/propidium iodide staining and proliferation of T cells by carboxyfluorescein diacetate succinimidyl ester staining using flow cytometry. The effects on T-cell activation (CD69/CD25) and dendritic cell maturation (CD83, CD86, and CD14) were assessed using flow cytometric analysis of distinct marker expression. Alterations of the secretion of effector mediators interferon (IFN)-γ by T cells and interleukin (IL)-8 by monocytes and dendritic cells were detected by enzyme-linked immunosorbent assay. None of the P. betulinus extracts had a significant influence on apoptosis and necrosis induction, T-cell proliferation, or T-cell activation status, but P. betulinus water extract caused a strong increase in IFN-γ release. The same extract was slightly protective against apoptosis of monocytes and further triggered IL-8 secretion by monocytes and dendritic cells. Moreover, P. betulinus water extract induced dendritic cell maturation. Our results demonstrate the immune-enhancing properties of P. betulinus.

Human immune compartment comparisons: Optimization of proliferative assays for blood and gut T lymphocytes

The accumulation of peripheral blood late-differentiated memory CD8 T cells with features of replicative (cellular) senescence, including inability to proliferate in vitro, has been extensively studied. Importantly, the abundance of these cells is directly correlated with increased morbidity and mortality in older persons. Of note, peripheral blood contains only 2% of the total body lymphocyte population. By contrast, the gut-associated lymphoid tissue (GALT) is the most extensive lymphoid organ, housing up to 60% of total body lymphocytes, but has never been assessed with respect to senescence profiles. We report here the development of a method for measuring and comparing proliferative capacity of peripheral blood and gut colorectal mucosa-derived CD8 T cells. The protocol involves a 5-day culture of mononuclear leukocyte populations, from blood and gut colorectal mucosa respectively, labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and 5-bromo-2′-deoxyuridine (BrdU) and stimulated with anti-CD2/3/28-linked microbeads. Variables tested and optimized as part of the protocol development include: mode of T cell stimulation, CFSE concentration, inclusion of a second proliferation marker, BrdU, culture duration, initial culture concentration, and inclusion of autologous irradiated feeder cells. Moving forward, this protocol demonstrates a significant advance in the ability of researchers to study compartment-specific differences of in vitro proliferative dynamics of CD8 T cells, as an indicator of replicative senescence and immunological aging. The study's two main novel contributions are (1) Optimization and adaptation of standard proliferative dynamics blood T cell protocols for T cells within the mucosal immune system. (2) Introduction of the novel technique of combining CFSE and BrdU staining to do so.

Immunomodulatory effects of metal salts at sub-toxic concentrations.

Because different metals are used in complementary medicine for the treatment of diseases related to a dysfunction of the immune system, this study aimed at determining the immunomodulatory potential of Pb(NO3 )2 , AuCl3 , Cu(NO3 )2 , HgCl2 , AgNO3 , SnCl2 , AsCl3 and SbCl3 at sub-toxic concentrations and at assessing possible toxic side effects of low-concentrated metal preparations. The influence of the metal salts on primary human mononuclear cells was analyzed by measuring cell viability using the water-soluble tetrazolium salt assay, apoptosis and necrosis induction by annexin V/propidium iodide staining and proliferation by carboxyfluorescein diacetate succinimidyl ester staining and flow cytometry. Effects on T-cell activation were assessed with CD69 and CD25 expression using flow cytometry whereas CD83, CD86 and CD14 expression was measured to evaluate the influence on dendritic cell maturation. Alterations of interleukin-2 and interferon-γ secretion were detected by enzyme-linked immunosorbent assay and genotoxic effects were analyzed using the comet assay. At sub-toxic concentrations retardation of T-cell proliferation was caused by Pb(NO3 )2 , AuCl3 and Cu(NO3 )2 and inhibitory effects on interleukin-2 secretion were measured after incubation with Pb(NO3 )2 , AuCl3 , Cu(NO3 )2 , HgCl2 and AsCl3. Cu(NO3 )2 had immunosuppressive activity at dosages within the serum reference range for copper. All other metal salts showed effects at dosages above upper serum limits of normal. Therefore, only low-concentrated copper preparations are promising to have immunomodulatory potential. Toxic side effects of metal preparations used in complementary medicine are improbable because upper limits of metals set in the drinking water ordinance are either not exceeded or the duration of their application is limited. Copyright © 2016 John Wiley & Sons, Ltd.

Frequent occurrence of T cell–mediated late reactions revealed by atopy patch testing with hypoallergenic rBet v 1 fragments

BackgroundLate allergic reactions are common in the course of allergen-specific immunotherapy and even occur with allergy vaccines with reduced IgE reactivity.ObjectiveWe sought to study atopy patch test (APT) reactions and T-cell responses to the recombinant birch pollen allergen Bet v 1 and recombinant hypoallergenic T-cell epitope–containing Bet v 1 fragments in patients with birch pollen allergy with and without atopic dermatitis (AD).MethodsA clinical study was conducted in 15 patients with birch pollen allergy with AD (group 1), 5 patients with birch pollen allergy without AD (group 2), 5 allergic patients without birch pollen allergy (group 3), and 5 nonallergic subjects (group 4) by performing skin prick tests and APTs with rBet v 1 and hypoallergenic rBet v 1 fragments. T-cell, cutaneous lymphocyte antigen (CLA)+ and CCR4+ T-cell and cytokine responses were studied by thymidine uptake, carboxyfluorescein diacetate succinimidyl ester staining, and Luminex technology, respectively.ResultsrBet v 1 and hypoallergenic rBet v 1 fragments induced APT reactions in not only most of the patients with birch pollen allergy with AD (11/15) but also in most of those without AD (4/5). Patients with birch pollen allergy with AD had higher Bet v 1–specific proliferation of CLA+ and CCR4+ T cells compared with patients with birch pollen allergy without AD. There were no differences in Bet v 1–specific CLA+ and CCR4+ proliferation and cytokine secretion in patients with and without APT reactions.ConclusionHypoallergenic rBet v 1 fragments induce T cell–dependent late reactions not only in patients with birch pollen allergy with AD but also in those without AD, which can be determined based on APT results but not based on in vitro parameters.

Poor association of allergen-specific antibody, T- and B-cell responses revealed with recombinant allergens and a CFSE dilution-based assay.

BackgroundThe adaptive immunity underlying allergy comprises two components, the allergen‐specific antibody (i.e. IgE, IgG) and the T‐cell response. These two components are responsible for different disease manifestations and can be targeted by different therapeutic approaches. Here, we investigated the association of allergen‐specific antibody and T‐ as well as B‐cell responses in pollen‐allergic patients using recombinant (r) major birch pollen allergen rBet v 1 and major timothy grass pollen allergen rPhl p 5 as defined antigens.MethodsAllergen‐specific IgE and IgG antibody responses were determined by ELISA, and allergen‐specific T‐ and B‐cell responses were measured in peripheral blood mononuclear cells using a carboxyfluorescein‐diacetate‐succinimidylester (CFSE) dilution assay.ResultsCFSE staining in combination with T‐cell‐ and B‐cell‐specific gating allowed discriminating between allergen‐specific T‐cell and B‐cell responses. Interestingly, we identified patients where mainly T cells and others where mainly B cells proliferated in response to allergen stimulation. No association between the level of allergen‐specific Ig responses and B‐ or T‐cell proliferation was observed.ConclusionPurified recombinant allergens in conjunction with CFSE staining allow the dissection of allergen‐specific B‐ and T‐cell responses. The dissociation of allergen‐specific antibody, and B‐ and T‐cell responses may explain the occurrence of selective IgE‐ and T‐cell‐mediated manifestations of allergic inflammation and may be important for the development of diagnostic and therapeutic strategies selectively targeting B cells and T cells.

Simvastatin modulates mesenchymal stromal cell proliferation and gene expression.

Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway, responsible for the biosysnthesis of cholesterol. However, statins also have pleiotropic effects that interfere with several signaling pathways. Mesenchymal stromal cells (MSC) are a heterogeneous mixture of cells that can be isolated from a variety of tissues and are identified by the expression of a panel of surface markers and by their ability to differentiate in vitro into osteocytes, adipocytes and chondrocytes. MSC were isolated from amniotic membranes and bone marrows and characterized based on ISCT (International Society for Cell Therapy) minimal criteria. Simvastatin-treated cells and controls were directly assayed by CFSE (Carboxyfluorescein diacetate succinimidyl ester) staining to assess their cell proliferation and their RNA was used for microarray analyses and quantitative PCR (qPCR). These MSC were also evaluated for their ability to inhibit PBMC (peripheral blood mononuclear cells) proliferation. We show here that simvastatin negatively modulates MSC proliferation in a dose-dependent way and regulates the expression of proliferation-related genes. Importantly, we observed that simvastatin increased the percentage of a subset of smaller MSC, which also were actively proliferating. The association of MSC decreased size with increased pluripotency and the accumulating evidence that statins may prevent cellular senescence led us to hypothesize that simvastatin induces a smaller subpopulation that may have increased ability to maintain the entire pool of MSC and also to protect them from cellular senescence induced by long-term cultures/passages in vitro. These results may be important to better understand the pleiotropic effects of statins and its effects on the biology of cells with regenerative potential.

Estradiol enhances the proliferation and migration of Ishikawa cells by promotion of angiogenesis induced by activation of NF-κB via AKT pathway

The aim of this study was to explore whether estradiol induces the expression of VEGF and bFGF in the endometrial cancer Ishikawa cells by activation of NF-κB via AKT pathway, and its effect on cell proliferation. Western blot was used to detect the AKT protein expression in Ishikawa cells after stimulation with estradiol, and the effect of AKT inhibitor or ER inhibitor on the activation of AKT. TransAM kit was used to detect the NF-κB p65 activity. qPCR and Western blot were used to detect the expression of VEGF and bFGF mRNA and proteins in the Ishikawa cells after estradiol treatment (E2 group), and pretreated with AKT inhibitor (AKT group) or ER inhibitor (ER group) or NF-κB inhibitor (NF-κB group), following the estradiol treatment. Flow cytometry and CFSE (carboxyfluorescein diacetate, succinimidyl ester) staining were used to examine the cell proliferation. Transwell was used to detect the migration ability of Ishikawa cells. Expression of p-AKT protein in the Ishikawa cells was markedly higher than that in the control group (P < 0.05). Expressions of p-AKT protein in the AKT and ER groups were significantly decreased than that in the E2 group (P < 0.05). The NF-κB activity was highest after stimulation with 1×10(-6) mol/L estradiol for 30 min to 1 h. AKT inhibitor significantly reduced the NF-κB activity (P < 0.05). The expressions of VEGF and bFGF mRNA and proteins in the E2 group were significantly increased than that in the control group (P < 0.05), and their expression in the AKT, ER and NF-κB groups were significantly decreased than that in the E2 group (P < 0.05). The proliferation and migration abilities of the Ishikawa cells were significantly increased after estradiol stimulation. Estradiol induces the production of VEGF and bFGF through activating NF-κB via AKT pathway, and enhances the proliferation and migration ability of cancer cells.

Artesunate Enhances the Antiproliferative Effect of Temozolomide on U87MG and A172 Glioblastoma Cell Lines

As chemotherapy with temozolomide is far from providing satisfactory clinical outcomes for patients with glioblastoma, more efficient drugs and drug combinations are urgently needed. The anti-malarial artesunate was previously shown to exert a profound cytotoxic effect on various tumor cell lines including those derived from glioblastoma. In the current study, we sought to examine the antiproliferative effect of a combination of temozolomide and artesunate on two different established human glioblastoma cell lines. The IC50 and IC25 were determined for temozolomide and artesunate in U87MG and A172 glioblastoma cell lines after 144 h of continuous drug exposure. The antiproliferative effect of combining both agents at IC50/IC50 and IC25/IC25 was determined by a cell viability assay. Moreover, necrosis and apoptosis were analyzed by annexin V/PI staining and flow cytometric analysis. In addition, cytostatic effects were examined by carboxyfluorescein diacetate succinimidyl ester staining and subsequent flow cytometry. In both glioblastoma cell lines, artesunate was found to enhance the antiproliferative effect exerted by temozolomide. Moreover, artesunate acted in concert with temozolomide in terms of cytostatic and necrotizing effects. These observations suggest that a combination of artesunate and temozolomide might result in increased cytotoxicity in glioblastoma.

Tracking in vivo migration and distribution of antigen-specific cytotoxic T lymphocytes by 5,6-carboxyfluorescein diacetate succinimidyl ester staining during cancer immunotherapy

Background Killing of targeted tumors during adoptive cell transfer therapy is associated with cytotoxic T lymphocyte (CTL) numbers, immunophenotype, tumor-specificity, and in vivo residence time, migration, and distribution. Therefore, tracing in vivo persistence, migration, and distribution of CTLs is important for cancer immunotherapy. Methods Optimal staining concentration for CTL proliferation was determined by cell counting kit-8 (CCK-8) assay and killing efficiencies of CTLs or carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled melanoma antigen-specific cytotoxic T lymphocytes (CFSE-CTLs) for malignant melanoma cells in vitro were compared. Additionally, CFSE-CTLs were intravenously transfused to mice receiving B16 melanoma, and their residence time, migration, and distribution in vivo were observed by measuring fluorescence intensities of CFSE-CTLs per gram of tissue (%FI/g) in various tissues and analyzing tumor/non-tumor (T/NT) values. Anti-tumor effects of transferred CTLs and correlation between %FI/g and D-value of tumor size were analyzed. Results Five-micromolar CFSE was optimal for labeling CTLs with minimal cytotoxicity. No significant difference occurred between CTLs and CFSE-CTLs for tumor cell killing (P=0.849) or interleukin-2 (P=0.318) and interferon-γ (P=0.201) levels. Distribution of CTLs in vivo varied with time. A negative correlation between %FI/g in tumors and D-value of tumor sizes by Spearman correlation analysis was observed. CTLs were recruited to and killed tumors from 6 hours to 3 days after cell infusion. CTLs were observed up to three weeks later in the tumor, liver, kidneys, and spleen; this was related to the abundant blood supply or the nature of immune organs. Conclusions CCK-8 assay is a novel method to select optimal CFSE staining concentrations. Fluorescence intensity of transferred CTLs reflects their killing efficiency of tumors. CFSE fluorescent markers can trace in vivo CTL persistence, migration, and distribution because of its stability, long half-life, and low toxicity.

The balance between two isoforms of LEF-1 regulates colon carcinoma growth

BackgroundColon cancer is one of the most aggressive human malignancies, with a very poor prognosis. Although it has been suggested that different isoforms of the lymphoid enhancer factor (LEF-1) have opposing biological activities, the biological outcome of aberrant LEF-1 activation in colon cancer is still unclear. The aim of this study was to evaluate the effect of the different LEF-1 phenotypes on the growth of colon carcinoma cell lines. A deeper understanding of these processes might improve the targeted therapies for colon cancer by regulating the expression of LEF-1.MethodsThe role of different isoforms of LEF-1 on the growth of human colon carcinoma cell lines (SW480 and HT-29) was studied using various in vitro and in vivo assays. In vitro proliferation, migration, adhesion and apoptosis of the cells stably transfected of different isoforms of LEF-1 were monitored by MTT assay, carboxyfluorescein diacetate–succinimidyl ester staining, annexin V staining, ECM adhesion assay and transwell assay, respectively. In nude mice, the formation of neovasculature in the tumors formed by our constructed cells was measured by immunohistochemistry. All the data were analyzed using a t test, and data were treated as significant when p < 0.05.ResultsOverexpression of truncated LEF-1 (LEF-1-ΔL) in the colon cell lines, SW480 and HT29, inhibited their growth significantly in vitro and in vivo, but the full-length LEF-1 (LEF-1-FL) promoted the proliferation of HT29. Inactivation of Wnt signaling by LEF-1-ΔL reduced the expression of CXCR4 in colon cell lines, which may lead to a decrease in activities such as migration, adhesion and survival. In nude mice, the formation of neovasculature as well as an increase in tumor volume were inhibited by the short isoform of LEF-1. LEF-1-FL, however, caused an increase in all these parameters compared with controls.ConclusionsThese findings suggest that LEF-1 might play an important role in colon carcinogenesis by acting as a regulator. Enhanced expression of LEF-1-FL, which occurs frequently in colon cancer, may be a new target for clinical therapy.

Staining of Bartonella henselae with carboxyfluorescein diacetate succinimidyl ester for tracking infection in erythrocytes and epithelial cells

Bartonella infection (Bartonella henselae in particular) is responsible for a widening spectrum of human diseases. The persistent colonization of erythrocytes is a feature of Bartonella infection. Endothelial and epithelial cells are also widely used to study the pathogenesis of bartonellosis in vitro. Exploring a convenient method for visualizing the bacillus without affecting infectivity would be very interesting. Carboxyfluorescein diacetate succinimidyl ester (CFSE) has been previously used for staining several bacterial species to study their adhesion to host cells. The present study demonstrated the efficiency and safety of using CFSE in staining B. henselae. The staining of bacillus-invaded erythrocytes and epithelial cells in vitro successfully allowed for flow cytometry and confocol microscopy analyses. Parallel tests using untreated bacteria confirmed that CFSE staining did not result in side effects on the infectivity of B. henselae. Labeling Bartonella with CFSE is a valuable method for studying the bacteria–host interaction.

Vaccination leads to an aberrant FOXP3 T-cell response in non-remitting juvenile idiopathic arthritis

ObjectiveTo investigate how meningococcal C vaccination in patients with remitting (oligoarticular) or progressive (polyarticular) juvenile idiopathic arthritis (JIA) influences the specific T-cell response to both the vaccine and heat shock...

In Vivo divisional Tracking System Reveals Cycling Heterogeneity in Long-Term Hematopoietic Stem Cells.

Abstract 814Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and give rise to all mature cells of hemato-lymphoid system for the lifetime of an individual. To ensure this, HSCs are kept at homeostatic levels in adult bone marrow. Steady-state HSC cycling kinetics have been evaluated by in vivo labeling assay using 5-bromo-2-deoxyuridine (BrdU) (Cheshier et. al., PNAS 1999; Kiel et al., Nature 2007), biotin (Nygren et. al., PLoS ONE 2008) and histon 2B-green fluorescent protein (H2B-GFP) transgenic model systems (Wilson et. al., Cell 2008; Foudi et. al., Nat. Biotech. 2008). Based on the latter, it was suggested that one HSC pool turns over faster than another, dormant pool with very limited divisions during a lifetime. However, the fast cycling HSCs did not have long-term multilineage reconstitution capacity in lethally irradiated animals in contrast to dormant HSCs (Wilson et. al., Cell 2008; Foudi et.al., Nat. Biotech. 2008). From these experiments remained unclear, whether the faster cycling HSC loose long-term repopulation potential according to divisional history, or whether they represent progenitors with limited self-renewal potential, sharing a long-term HSC phenotype. Therefore, the dynamics of steady-state long-term HSC homeostasis and blood production remains to be determined.To address this directly, we set up an in vivo HSC divisional tracking assay. Here we show i.v. transfer of CFSE (carboxyfluorescein diacetate succinimidyl ester) -labeled HSCs into non-conditioned CD45.1/2 congenic F1 recipient mice that allows evaluation of steady-state HSC dynamics as CFSE distributes equally to daughter cells upon each cellular division. Sorted naïve CD4+CD62L+ T cells were used as non-dividing control cell population to determine the zero division CFSE staining level over time. Upon transfer of Lin-c-kit+Sca-1+ cells (LKS) into sublethally irradiated mice, all donor derived Lin-c-kit+ cells had divided >5 times after 3 weeks. However, transfer of LKS cells into non-irradiated mice revealed non-divided LKS cells in recipient bone marrow over 20 weeks. FACS analysis with HSC or progenitor specific marker expression showed that most of 0-2 time-divided and few of >5x divided LKS cells maintained a long-term HSC phenotype (CD150+, c-mpl+, CD34-). In order to test HSC potential, non- or >5x divided cells were sorted based on divisional history from primary recipients at different time points after transplantation, and competitively transplanted into lethally irradiated secondary recipients. At 3 weeks post primary transfer, single non-divided LKS cell was able to multi-lineage repopulate recipients, while 50 of >5x divided LKS cells showed no engraftment. Interestingly, both non- and >5x divided LKS cells at 7 or 12-14 weeks after primary transfer had long-term multilineage repopulating potential. Limiting dilution transplantation experiments demonstrated that HSC with long-term multilineage capacity (LT-HSC) were maintained at constant numbers that fit the numbers of free bone marrow niche space, with non-divided LT-HSC decreasing and >5x divided LT-HSC increasing with a constant division rate. We next tested the effects of hemato-immunological challenge on HSC cycling dynamics. Upon i.p. LPS injection into mice, previously transplanted with CFSE-labeled LKS, almost all LT-HSCs entered cell cycle within one week after challenge.These findings directly demonstrate that some LT-HSCs are quiescent for up to one fifth of the life-time of a mouse, while other LT-HSCs divide more actively, thus proving asynchronous LT-HSC division and contribution to hematopoiesis in steady-state. In addition, the results demonstrate that quiescent LT-HSCs are driven into division in response to naturally-occurring hematopoietic challenges, such as systemic bacterial infection. The CFSE-tracking model established here now allows to directly test the role of intrinsic versus environmental cues on cycling-dynamics of HSCs as well as leukemia initiating cells in steady-state and upon challenge on multiple genetic and different species background. Disclosures:No relevant conflicts of interest to declare.

Binding of Thymoglobulin to Natural Killer Cells Leads to Cell Activation and Interferon-γ Production

Thymoglobulin is an antithymocyte globulin preparation used in hematopoietic stem cell transplantation (HSCT) to prevent rejection and graft-versus-host disease. Because natural killer (NK)-cell alloreactivity improves HSCT outcome, but only in patients receiving thymoglobulin, we investigated the in vitro effects of thymoglobulin on purified NK cells. Thymoglobulin binding to NK cells and NK-cell activation were assessed by flow cytometry. NK surface targets for thymoglobulin were determined by competition inhibition assays using monoclonal antibodies. Chromium 51 (Cr) release assay, Annexin V combined with 7-amino-actinomycin D staining, and carboxyfluorescein diacetate succinimidyl ester staining were used to study cytotoxic activity, apoptosis/cell death, and NK-cell proliferation, respectively. Interferon (IFN)-gamma production was determined by ELISA. Thymoglobulin, thymoglobulin derived-F(ab')2 fragments as well as rabbit IgG bound NK cells, and competed strongly with anti-CD16. Thymoglobulin enhanced the expression of activation (CD69 and NKG2D) and degranulation (CD107a) markers on NK cells. It competed with CD18 binding and decreased NK activity, but not interleukin-15-induced killer activity. Effects on apoptosis/cell death and proliferation were minimal. F(ab')2 fragments and rabbit IgG strongly induced IFN-gamma production by NK cells. Thymoglobulin binds to NK cells by CD16 by its variable and constant regions. The decrease in NK-cell cytotoxic activity is restored by interleukin-15, and contrasts sharply with the induction of activation, degranulation, and IFN-gamma production. These data support the hypothesis that thymoglobulin treatment is required to observe the improvement in HSCT outcome by NK-cell alloreactivity.

Elifacs: A Multiplex Assay to Detect Antigen Specific T Cells and to Monitor the Immune Response

The accumulation of peripheral blood late-differentiated memory CD8 T cells with features of replicative (cellular) senescence, including inability to proliferate in vitro, has been extensively studied. Importantly, the abundance of these cells is directly correlated with increased morbidity and mortality in older persons. Of note, peripheral blood contains only 2% of the total body lymphocyte population. By contrast, the gut-associated lymphoid tissue (GALT) is the most extensive lymphoid organ, housing up to 60% of total body lymphocytes, but has never been assessed with respect to senescence profiles. We report here the development of a method for measuring and comparing proliferative capacity of peripheral blood and gut colorectal mucosa-derived CD8 T cells. The protocol involves a 5-day culture of mononuclear leukocyte populations, from blood and gut colorectal mucosa respectively, labeled with 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and 5-bromo-2′-deoxyuridine (BrdU) and stimulated with anti-CD2/3/28-linked microbeads. Variables tested and optimized as part of the protocol development include: mode of T cell stimulation, CFSE concentration, inclusion of a second proliferation marker, BrdU, culture duration, initial culture concentration, and inclusion of autologous irradiated feeder cells. Moving forward, this protocol demonstrates a significant advance in the ability of researchers to study compartment-specific differences of in vitro proliferative dynamics of CD8 T cells, as an indicator of replicative senescence and immunological aging. The study's two main novel contributions are (1) Optimization and adaptation of standard proliferative dynamics blood T cell protocols for T cells within the mucosal immune system. (2) Introduction of the novel technique of combining CFSE and BrdU staining to do so.