scholarly journals Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Rahul Ashok Gosavi ◽  
Sukeshani Salwe ◽  
Sandeepan Mukherjee ◽  
Ritwik Dahake ◽  
Sweta Kothari ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Positive Selection ◽  
Mhc Class Ii ◽  
Cell Population ◽  
Ex Vivo ◽  
Culture Method ◽  
Flask Culture ◽  
Suspended Cell ◽  
Significant Difference

12–14 days of culturing of bone marrow (BM) cells containing various growth factors is widely used method for generating dendritic cells (DCs) from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs’ purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P<0.05) between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.

scholarly journals A Live and Inactivated Chlamydia trachomatis Mouse Pneumonitis Strain Induces the Maturation of Dendritic Cells That Are Phenotypically and Immunologically Distinct

2005 ◽  
Vol 73 (3) ◽  
pp. 1568-1577 ◽  
Author(s):  
Jose Rey-Ladino ◽  
Kasra M. Koochesfahani ◽  
Michelle L. Zaharik ◽  
Caixia Shen ◽  
Robert C. Brunham
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Chlamydia Trachomatis ◽  
Mhc Class Ii ◽  
Protective Immunity ◽  
Ex Vivo ◽  
Natural Infection ◽  
Pulsed Dc ◽  
Low Levels ◽  
Dc Maturation

ABSTRACT The intracellular bacterial pathogen Chlamydia trachomatis is a major cause of sexually transmitted disease worldwide. While protective immunity does appear to develop following natural chlamydial infection in humans, early vaccine trials using heat-killed C. trachomatis resulted in limited and transient protection with possible enhanced disease during follow-up. Thus, immunity following natural infection with live chlamydia may differ from immune responses induced by immunization with inactivated chlamydia. To study this differing immunology, we used murine bone marrow-derived dendritic cells (DC) to examine DC maturation and immune effector function induced by live and UV-irradiated C. trachomatis elementary bodies (live EBs and UV-EB, respectively). DC exposed to live EBs acquired a mature DC morphology; expressed high levels of major histocompatibility complex (MHC) class II, CD80, CD86, CD40, and ICAM-1; produced elevated amounts of interleukin-12 and tumor necrosis factor alpha; and were efficiently recognized by Chlamydia-specific CD4+ T cells. In contrast, UV-EB-pulsed DC expressed low levels of CD40 and CD86 but displayed high levels of MHC class II, ICAM-1, and CD80; secreted low levels of proinflammatory cytokines; and exhibited reduced recognition by Chlamydia-specific CD4+ T cells. Adoptive transfer of live EB-pulsed DC was more effective than that of UV-EB-pulsed DC at protecting mice against challenge with live C. trachomatis. The expression of DC maturation markers and immune protection induced by UV-EB could be significantly enhanced by costimulation of DC ex vivo with UV-EB and oligodeoxynucleotides containing cytosine phosphate guanosine; however, the level of protection was significantly less than that achieved by using DC pulsed ex vivo with viable EBs. Thus, exposure of DC to live EBs results in a mature DC phenotype which is able to promote protective immunity, while exposure to UV-EB generates a semimature DC phenotype with less protective potential. This result may explain in part the differences in protective immunity induced by natural infection and immunization with whole inactivated organisms and is relevant to rational chlamydia vaccine design strategies.

Myeloma Cell-Derived Factors Retard the Differentiation and Function of Dendritic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2447-2447
Author(s):  
Qing Yi ◽  
Siqing Wang ◽  
Jin Xie ◽  
Jianfei Qian ◽  
Jin Yang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Growth Factor ◽  
P38 Mapk ◽  
Mhc Class Ii ◽  
Neutralizing Antibodies ◽  
Transforming Growth Factor ◽  
T Cell Response ◽  
Mitogen Activated Protein Kinase ◽  
And Function

Abstract Multiple myeloma (MM) is a B-cell malignancy often associated with a suppressed immune system. The mechanisms for the immunosuppression are largely unknown. In this study, we examined, using the murine 5T33 myeloma model, the effects of tumor cell or its-derived factors on the differentiation and function of bone marrow-derived dendritic cells (BMDCs). Our results showed that differentiation of BMDCs was retarded in the presence of 10% of tumor-derived supernatant (TSN). This is evident by, compared with control cells, the downregulated expression of surface molecules including CD40, CD86 and MHC class II; secretion of higher levels of IL-10 and lower levels of IL-12; and a poor T-cell response in an allogeneic mixed lymphocyte reaction induced by TSN-treated cells. The same phenomenon was also observed when the bone marrow progenitor cells were cocultured, either in direct contact or separated by transwell membrane, with myeloma cells. The treatment downregulated the expression of phosphorylated extracellular signal-related kinase (ERK) and mitogen-induced extracellular kinase (MEK), and upregulated the expression of phosphorylated p38 mitogen-activated protein kinase (p38 MAPK) and signal transducer and activator of transcription-3 (STAT3) in the cells. As a high level of interleukin (IL)-10, IL-6, vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-b can be detected in TSN, we examined whether these cytokines were responsible. Our results showed that addition of TGF-b, IL-10 and/or IL-6 could largely replace TSN in retarding the differentiation of BMDCs, and neutralizing antibodies against these cytokines, especially in combination, were able to block the effects of TSN or tumor cells on BMDCs. Finally, our results showed that inhibiting p38 MAPK or STAT3 restored the differentiation and function of these cells. Hence, our study not only sheds light on the mechanisms of tumor-induced immune evasion in MM and but also provides one of the solutions to overcome the problems.

Myeloid Dendritic Cells Contribute to Hematopoietic Progenitor Mobilization by G-CSF.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2319-2319
Author(s):  
Teerawit Supakorndej ◽  
Mahil Rao ◽  
Daniel Link
Keyword(s):  
Bone Marrow ◽  
Dendritic Cells ◽  
Cell Population ◽  
Diphtheria Toxin ◽  
Myeloid Dendritic Cells ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
The Mean ◽  
Bone Marrow Chimeras

Abstract Abstract 2319 Granulocyte-colony stimulating factor (G-CSF) is the prototypic agent used to mobilize hematopoietic stem and progenitor cells (HSPCs) into the blood where they can then be harvested for stem cell transplantation. G-CSF acts in a non-cell-intrinsic fashion to induce HSPC mobilization. We recently showed that G-CSF signaling in a CD68+ monocyte/macrophage lineage cell within the bone marrow initiates the HSPC mobilization cascade (Christopher et al., 2011). Consistent with this finding, two other groups showed that ablation of monocytes/macrophages induces HSPC mobilization (Winkler et al., 2010; Chow et al., 2011). CD68 marks a heterogeneous cell population that includes monocytes, macrophages, myeloid dendritic cells, and osteoclasts. To further define the relevant cell population(s) for HSPC mobilization by G-CSF, we first examined the role of osteoclasts. Receptor activator of NF-kappaB (RANK) signaling is required for osteoclast development. Osteoprotegerin (OPG) is a decoy receptor for RANK ligand, and treatment with OPG-Fc (a stabilized form of OPG) results in osteoclast ablation in mice. We treated mice with 100 μg of OPG-Fc and documented complete osteoclast ablation by histomorphometry. Osteoclast ablation did not result in constitutive HSPC mobilization, nor did it affect G-CSF-induced HSPC mobilization. To further assess the role of osteoclasts, we transplanted RANK−/− fetal liver cells into irradiated Csf3r−/− (G-CSF receptor deficient) recipients. Since RANK is required for osteoclast development, the osteoclasts in these bone marrow chimeras lack the G-CSFR, while other hematopoietic cells (including monocytes/macrophages) are G-CSFR sufficient. Again, G-CSF-induced HSPC mobilization in these mice was normal. Based on these data, we conclude that osteoclasts are dispensable for HSPC mobilization by G-CSF. We next quantified changes in monocytic/macrophage cell populations in the bone marrow after G-CSF treatment (250 μg/kg per day for 5 days) using a novel multi-color flow cytometry assay that includes CD115, F4/80, MHC class II, Gr-1, B220, and CD11c. Using this assay, we observed a significant decrease in macrophages (11.8 ± 3.6-fold) and, surprisingly, myeloid dendritic cells (MDCs; 5.5 ± 1.2-fold) in the bone marrow with G-CSF treatment. To further assess the role of MDCs, we used transgenic mice expressing the diphtheria toxin receptor under the control of the CD11c promoter (CD11c-DTR) to conditionally ablate MDCs. To avoid systemic toxicity, we transplanted CD11c-DTR bone marrow into congenic wild type recipients prior to MDC ablation. The resulting bone marrow chimeras were treated with diphtheria toxin (DT; 400 ng per day for 6 days), which resulted in a 92% reduction in MDCs. Ablation of MDCs resulted in a significant increase in colony-forming cells in the blood and spleen (figure 1A). Moreover, MDC ablation significantly increased mobilization of colony-forming cells and c-Kit+lineage−Sca-1+ (KLS) cells by G-CSF (figures 1B and 1C). Taken together, these data suggest that myeloid dendritic cells, but not osteoclasts, contribute to HSPC mobilization by G-CSF. Figure 1. HSPC mobilization in CD11c-DTR mice. CD11c-DTR bone marrow chimeras were treated with diphtheria toxin (DT) alone, G-CSF alone, or DT plus G-CSF. The number of CFU-C (A & B) or KLS cells (C) in the blood and spleen are shown. Data represent the mean ± SEM of 10–11 mice pooled from two independent experiments. *p < 0.05; **p < 0.001; ***p < 0.0001. Figure 1. HSPC mobilization in CD11c-DTR mice. CD11c-DTR bone marrow chimeras were treated with diphtheria toxin (DT) alone, G-CSF alone, or DT plus G-CSF. The number of CFU-C (A & B) or KLS cells (C) in the blood and spleen are shown. Data represent the mean ± SEM of 10–11 mice pooled from two independent experiments. *p < 0.05; **p < 0.001; ***p < 0.0001. Disclosures: No relevant conflicts of interest to declare.

Benfotiamine reduces dendritic cell inflammatory potency

2020 ◽  
Vol 20 ◽  
Author(s):  
Neda Djedovic ◽  
Iva Božić ◽  
Đorđe Miljković ◽  
Irena Lavrnja
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
Immune Response ◽  
Dendritic Cells ◽  
Mhc Class Ii ◽  
Morphological Changes ◽  
Marrow Cell ◽  
Class Ii ◽  
Immunofluorescent Staining ◽  
Mhc Class Ii Molecules

Background: Benfotiamine is a synthetic liposoluble derivative of vitamin B1 that has been shown to have antiinflammatory properties. Objective: To study the effects of benfotiamine on dendritic cells. Methods: Dendritic cells were obtained from murine bone marrow precursor cells in the presence of GM-CSF. Benfotiamine was applied to the cell culture during the process of bone marrow cell differentiation into dendritic cells. Dendritic cells were stimulated with lipopolysaccharide (LPS) and expression of MHC class II molecules and CD86 was determined by flow cytometry, while levels of tumor necrosis factor (TNF) and interleukin (IL)-1β in cell culture supernatants were measured by ELISA. F-Actin, NF-κB and Nrf2 were visualized by immunofluorescent staining and microscopy. Results: Benfotiamine potently reduced LPS-induced expression of MHC class II molecules and CD86, in addition to suppressing the release of pro-inflammatory cytokines TNF and IL-1β. It also prevented LPS-imposed morphological changes of dendritic cells, i.e. enlargement and intensified protrusions. The effects were paralleled with the reduction of NF-κB translocation to the nucleus, but not of Nrf2 activation inhibition. Conclusion: Having in mind the importance of dendritic cells for the configuration of the immune response, our results imply that benfotiamine has the ability to regulate the immune response through inhibition of inflammatory properties of dendritic cells.

Expression of Cytoplasmic and Surface VEGF by Plasma Cells in Myeloma and Related Disorders.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3655-3655
Author(s):  
Teresa K. Kimlinger ◽  
Thomas E. Witzig ◽  
S. Vincent Rajkumar
Keyword(s):  
Bone Marrow ◽  
Cell Population ◽  
Plasma Cell ◽  
Plasma Cells ◽  
The Other ◽  
Cell Fraction ◽  
Vegf Expression ◽  
Cell Compartment ◽  
Significant Difference ◽  
Normal Controls

Abstract Background: Previous studies quantitating VEGF in plasma cells from multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), and smoldering myeloma (SMM) have found no significant difference in expression between the groups. These studies have been done using immunohistochemistry, quantitative RT-PCR, and Western Blots using material from isolated CD138+ plasma cells. These studies may have been limited by specificity of reagents, low numbers of plasma cells, or heterogeneity within the plasma cell population. Asosingh et al have shown that 5T2MM murine myeloma cells show greater VEGF expression in the CD45- compared to the CD45+ plasma cell compartment (Asosingh K, Blood2004;103:3131–7). Quantitative flow cytometric methods allow for the quantitation of antigen expression with high sensitivity and allow discrimination among cell subsets. Flow studies have suggested that myeloma patients have both CD45+ and CD45 - plasma cell subsets and that each compartment may contain phenotypic and functionally different types of clonal plasma cells. This two part study evaluated bone marrow plasma cells for intracellular and surface levels of VEGF in MM, MGUS, SMM, amyloid, and normal controls. Methods: Cytoplasmic VEGF expression (cVEGF) was measured on lysed and permeabilized (FACSTM Permeabilizing Solution, BD Biosciences, San Jose, CA) whole bone marrow samples using anti-VEGF PE (R and D Systems, Minneapolis, MN). With each run, QuantumTM Simply Cellular Beads (Bangs Laboratories, Fishers, IN) were stained with the same VEGF antibody to create a standard curve for calculation of MESF/ABC (antibody binding capacity) values. A median VEGF MESF value was calculated for the plasma cells, lymphocytes, and granulocytes from each patient sample. Surface expression of VEGF (sVEGF) was determined using CD138 FITC/ VEGF PE/ CD45 Percp/ CD38 APC staining on ACK lysed whole bone marrow cells. sVEGF expression was determined on the plasma cell population as a whole and on the individual CD45 fractions. Median expression and proportion of cases with plasma cells expressing >20% sVEGF were calculated. Cytoplasmic kappa and lambda staining was also done to determine clonality. Results: cVEGF MESF differences within the plasma cell populations were greater than in the other cell types. The MESF was higher in MM (n=14 patients, median MESF 247,866) compared to MGUS/SMM (n=14, median MESF 89,794) and amyloid (n=5, median MESF 134,429)(p=0.038). The lymphocytes and granulocytes in each group had similar MESF values, but the median MESF was lower than in the plasma cells (median 41,485 for lymphocytes, and 24,227 for granulocytes). sVEGF was expressed by less than 20% of plasma cells in all groups studied. Similar results were also seen in the CD45- plasma cell fraction. In the CD45+ plasma cell fraction, all groups showed varying levels of positive expression: MM (n=15 patients; median, 52% cells positive), normal controls (n=4; 51%), MGUS/SMM (n=11; 34%). Conclusions: cVEGF expression was significantly higher in MM compared to SMM/MGUS in this study. On the other hand, sVEGF expression was confined to the CD45+ plasma cell compartment in all groups studied, with a trend to higher % positive cells in the MM group compared to MGUS. We are working on studying levels of secreted VEGF in various human plasma cell subsets, both alone and in conjunction with stromal cell contact.

Survivin Is Required for Proliferation, but Not Polyploidization of Megakaryocytes.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1328-1328
Author(s):  
Jeremy Q Wen ◽  
Cindy Leung ◽  
Zan Huang ◽  
Sara Small ◽  
John Crispino
Keyword(s):  
Bone Marrow ◽  
Ex Vivo ◽  
Survivin Expression ◽  
Annexin V ◽  
Retroviral Infection ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Megakaryocyte Progenitors

Abstract Survivin is a member of chromosome passenger complex, which plays an important role in chromosome alignment, separation and cytokinesis. We recently reported that survivin is necessary for the proliferation and survival of hematopoietic stem and progenitor cells. Furthermore, we previously showed that reduced levels of survivin expression facilitates megakaryocyte development, whereas elevated levels of survivin inhibit their maturation and polyploidization. However, the extent to which survivin is necessary for polyploidization and terminal differentiation of committed megakaryocytes remains unclear. To determine whether survivin is required for megakaryocyte and platelet biogenesis, we mated mice with floxed alleles of survivin (sur fl/fl) to mice that express Cre recombinase under the control of the PF4 promoter. Compound mutant animals appeared grossly normal and harbored normal platelet counts. Furthermore, survivin deleted and control littermates displayed similar expression of CD41 and CD42, as well as similar DNA content within the CD41+ population. The only significant difference detected was an increase in annexin V staining of CD41+ cells within the bone marrow of the mice with survivin deletion. Analysis of DNA extracted from these bone marrows showed no evidence of the survivin deletion, indicating that the surviving cells all escaped excision. These in vivo findings are consistent with a requirement for survivin in the survival or proliferation of megakaryocyte progenitors. Next, to induce megakaryocyte development ex vivo, we cultured bone marrow from surv fl/fl mice in vitro in the presence of TPO. Using this approach, we were able to induce survivin deletion in 75% of the cells as evidenced by PCR. Despite the deletion of survivin, polyploidization of the ex vivo generated megakaryocytes was unaffected. Finally, we induced deletion of survivin by retroviral infection of surv fl/fl progenitors with MSCV-Cre and found that megakaryocyte polyploidization was actually increased in the excised population. Taken together, our results suggest that survivin is not required for polyploidization, but is necessary for proliferation of megakaryocyte progenitors.

scholarly journals Antigen-dependent and -independent Ca2+ Responses Triggered in T Cells by Dendritic Cells Compared with B Cells

1998 ◽  
Vol 188 (8) ◽  
pp. 1473-1484 ◽  
Author(s):  
Jérôme Delon ◽  
Nadège Bercovici ◽  
Graça Raposo ◽  
Roland Liblau ◽  
Alain Trautmann
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
Mhc Class Ii ◽  
Ex Vivo ◽  
Cell Formation ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Survival Signal

Dendritic cells (DCs) are much more potent antigen (Ag)-presenting cells than resting B cells for the activation of naive T cells. The mechanisms underlying this difference have been analyzed under conditions where ex vivo DCs or B cells presented known numbers of specific Ag–major histocompatibility complex (MHC) complexes to naive CD4+ T cells from T cell antigen receptor (TCR) transgenic mice. Several hundred Ag–MHC complexes presented by B cells were necessary to elicit the formation of a few T–B conjugates with small contact zones, and the resulting individual T cell Ca2+ responses were all-or-none. In contrast, Ag-specific T cell Ca2+ responses can be triggered by DCs bearing an average of 30 Ag–MHC complexes per cell. Formation of T–DC conjugates is Ag-independent, but in the presence of the Ag, the surface of the contact zone increases and so does the amplitude of the T cell Ca2+ responses. These results suggest that Ag is better recognized by T cells on DCs essentially because T–DC adhesion precedes Ag recognition, whereas T–B adhesion requires Ag recognition. Surprisingly, we also recorded small Ca2+ responses in T cells interacting with unpulsed DCs. Using DCs purified from MHC class II knockout mice, we provide evidence that this signal is mostly due to MHC–TCR interactions. Such an Ag-independent, MHC-triggered calcium response could be a survival signal that DCs but not B cells are able to deliver to naive T cells.

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