Purification Of Dendritic Cells Research Articles

An optimized method for the induction and purification of mouse bone marrow dendritic cells

Dendritic cells (DCs) play an essential role in the initiation of adaptive immune responses, but they are rare in all organs. The traditional methods used to increase the yield and purity of DCs are the early removal of granulocyte culture medium and the isolation of high-purity DCs by magnetic-activated cell sorting (MACS). This study provides a more rapid and economical optimization method to obtain more high-purity DCs. (i) We harvested 18% more bone marrow (BM) cells by using forceps to crack the epiphysis instead of cutting it with scissors during BM cell extraction. (ii) When the cells in the culture medium that is discarded on day 3 in the traditional method were centrifuged and then added back to the petri dish, the DC yield on day 5 increased by 61%. (iii) On the third day, the addition of fresh medium and the retention of the original medium rather than discarding it increased the number of DCs harvested on the fifth day by 137%. (i-iii) The improved method cost an average of 74% less than the conventional method and yielded the same number and function of cells. (iv) The initial number of BM cells was increased by 15% in 4-week-old mice compared with 8-week-old mice. (v) The Percoll density centrifugation (PDS) method was used to purify DCs on day 6 after induction, and the purity of the DCs was greater than 90%, which showed no significant difference from the MACS method. However, the yield of the PDS method increased by 21%. In addition, the PDS method has a lower cost, with an average purification cost of 4 CNY ($0.58) compared with 648 CNY ($93.25) for MACS, reducing the cost by 99%. Therefore, high-purity and high-yield DCs can be rapidly obtained through a five-step improvement in the process of BM cell extraction, induction and purification.

GM-CSF Culture Revisited: Preparation of Bulk Populations of Highly Pure Dendritic Cells from Mouse Bone Marrow.

Dendritic cells (DC) loaded with specific peptides are strongly immunogenic for T cells and can be used for cancer immunotherapy. For immunogenic tumors such as melanoma, injection of autologous DC loaded with tumor cell extracts or peptides can induce tumor regression but in only a small proportion of patients. Nevertheless, recent studies on the efficacy of checkpoint blockade for boosting antitumor immunity plus advances in defining tumor neoantigens are stimulating renewed interest in DC immunotherapy. Despite intensive investigation, however, preparation of bulk populations of mature DC has proved difficult, and most preparations contain a significant proportion of potentially tolerogenic immature DC. In this study, we have modified the well-established GM-CSF culture system to prepare substantial quantities of highly pure (>95%) mature DC from mouse bone marrow cells and defined their progenitors. We show that obtaining high yields and purity of DC are heavily dependent on cell density in the cultures and the tempo of addition of growth and maturation stimuli. When loaded with specific peptide, the DC are strongly immunogenic for CD4 and CD8 T cells in vivo and elicit effective antitumor immunity.

Dendritic cell chimerism in oral mucosa of transplanted patients affected by graft‐versus‐host disease

Graft-versus-host disease (GVHD) is one of the main complications after haematopoietic stem cell transplantation. Clinical features of GVHD include either an acute (aGVHD) or a chronic (cGVHD) condition that affects locations such as the oral mucosa. While the involvement of the host's dendritic cells (DCs) has been demonstrated in aGVHD, the origin (donor/host) and mechanisms underlying oral cGVHD have not been completely elucidated. In this study, we intend to determine the origin of DCs present in mucosal tissue biopsies from the oral cavity of transplanted patients affected by cGVHD. We purified DCs, from oral biopsies of three patients with cGVHD, through immunobeads and subsequently performed DNA extraction. The origin of the obtained DCs was determined by PCR amplification of 13 informative short tandem repeat (STR) alleles. We also characterised the DCs phenotype and the inflammatory infiltrate from biopsies of two patients by immunohistochemistry. Clinical and histological features of the biopsies were concordant with oral cGVHD. We identified CD11c-, CD207- and CD1a-positive cells in the epithelium and beneath the basal layer. Purification of DCs from the mucosa of patients affected by post-transplantation cGVHD was >95%. PCR-STR data analysis of DCs DNA showed that 100% of analysed cells were of donor origin in all of the evaluated patients. Our results demonstrate that resident DCs isolated from the oral tissue of allotransplanted patients affected by cGVHD are originated from the donor. Further research will clarify the role of DCs in the development and/or severity of oral cGVHD.

Sema-3A Regulates the Expression of B7-H3 and B7-H4 on Murine DCs.

Objective To investigate the effection of Sema-3A on the expression of B7-H3 and B7-H4 on murine bone-marrow-derived dendritic cells (DCs). Method DCs were induced from bone-marrow monocytes of Balb/C mice. The expressions of B7-H3 and B7-H4 on DCs were examed by flow-cytometry after the stimulation of Sema-3A-Ab. Results 1. DCs was developed from bone-marrow-derived monocytes of BALB / c mice, the purity of DCs was 82.18%. 2.B7-H3(13.07±0.81%, 19.43±0.75%, P<0.05) and B7-H4 ( 8.24 ± 0.46%,17.38 ± 0.43%,p <0.05) were expressed on immature DCs and mature DCs. 3. Sema-3A antibody (1:500, 1:1000, 1:2000) inhibited B7-H3 expression on mature DCs (9.88±0.66%,10.24±0.17%,9.94±0.20 %, Compared to control group, p <0.05)Table: No Caption available.Sema-3A antibody (1:500, 1:1000, 1:2000) also inhibited B7-H4 expression on mature DCs (10.67±0.56%,11.88±0.47%,12.21±0.40%, Compared to control group, p <0.05)Table: No Caption available.Conclusion: Sema-3A antibody can inhibit B7-H4, B7-H3 expression on the matured bone marrow derived DCs, suggesting that Sema-3A may regulate DCs by promoting the expression of B7-H4, B7-H3 on mature DCs.This indicates that Sema-3A will be involved in immune recognization and immune regulation through co-stimulation moleculars B7-H3,B7-H4 on DCs.

Separation and culture of dendritic cells from regional lymph nodes of patients with breast cancer

Objective To investigate the separation and culture of dendritic cells (DC) from regional lymph nodes of patients with breast cancer,and the possibility of them used as immunotherapy for the tumor.Methods DC were isolated from fresh tumor-free lymph nodes of 21 cases of breast cancer undergoing surgical operations,by using mechanical crush,density gradient centrifugation and transient adherent culture.The isolated cells were divided into 3 groups,then cultured in vitro with recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) (1000 U/ml) + recombinant human interleukin (rhIL)-4 (500 U/ml),and rhGM-CSF (1000 U/ml) + rhIL-4 (500 U/ml) + tumor necrosis factor-α (TNF-α) (200 U/ml) in group B and group C,respectively.The morphology,size and changes of DC were observed under an inverted microscope,and the phenotype of DC was detected by using flow cytometry.Results (52.84 ± 20.87) × 105 DC were obtained from half of 5-12 lymph nodes of each patient with breast cancer,and the purity of isolated DC was (73.78 ± 13.31)％.These cells showed DC morphological characteristics after being cultured.After being cultured for 7 days,the purity and number of DC in three groups were greater than before,and there was significant difference between every two groups (P ＜0.05).The number of CD1 a + CD83 + cells and CD1 a-CD83 + cells in three groups was also greater than before,and there was significant difference between every two groups (P ＜ 0.05).The growth rate of CD1a+ CD83 + cells and CD1a-CD83 + cells in three groups was significantly higher than that of CD1a+CD83-cells (P ＜ 0.01).There was no significant difference in the number and growth rate of DC,CD1 a +CD83-cells,CD1a+ CD83+ cells and CD1a-CD83+ cells among three groups.Conclusion A large quantity and high purity of DC can be isolated from regional lymph nodes of patients with breast cancer.More mature DC with dendritic morphology characteristics can be obtained after these cells being cultured in vitro. Key words: Breast carcinoma; Lymph nodes; Dendritic cells; Cell culture

Generation of large numbers of highly purified dendritic cells from bone marrow progenitor cells after co-culture with syngeneic murine splenocytes

Dendritic cells (DCs) are called the sentinels of the human immune system because of their function as antigen presenting cells (APCs) that elicit a protective immune response. Given that DCs have been used for many years as target cells in a great number of experiments, it became essential to devise a new method for producing DCs in higher quantities and of greater purity. Here we report a novel technique for obtaining more dendritic cells, and with higher purity, from in-vitro co-culture of bone marrow (BM) cells with splenocytes.From a total of 20×106 BM cells and 120×106splenocytes, 3×106 BM cells along with 20×106splenocytes were co-cultured in petri dishes for DC generation; 120×106 splenocytes from one C57BL/6 mouse were also co-cultured in petri dishes for DC generation. BM cells were the control group cultured in the same conditions except for the presence of splenocytes. Purity and maturation state of DCs were checked by lineage surface markers (CD11c, CD11b, CD8α, and F4/80) and the expression levels of MHCII as well as co-stimulatory molecules (CD86, CD80, and CD40). Endocytosis and thymidine uptake capacity were also used to test the functionality of DCs. The levels of IL-12p70, IL-23, and IL-10 were also checked in the supernatant of cultured cells by ELISA.The number of DCs derived from co-culture of BM and splenocytes (DCsTME) was at least twice that of BM-derived DCs in the absence of splenocytes. In addition, the purity of DCs after co-culture of BM and splenocytes was greater than that of DCs in the control culture (90.2% and 77.2%, respectively; p<0.05). While functional assays showed no differences between co-culture and control groups, IL-10 levels were significantly lower in DCsTME compared to BM-derived DCs in the absence of splenocytes (193pg/ml and 630pg/ml, respectively; p<0.05).The results of the present study show that the generation of DCs from BM progenitors is accelerated in the presence of syngeneic splenocytes. Given the larger number of generated DCs, and with higher purity, in this technique, DCsTME could be more advantageous for DC-based immunotherapy and vaccination techniques.

367 COMPLEMENT FACTOR C5A MODULATES VEIN GRAFT REMODELING AND ACCELERATED ATHEROSCLEROSIS VIA INTERFERENCE IN MAST CELL ACTIVATION

Dendritic cells (DCs) play a pivotal role in linking the innate and adaptive immune response and have been implicated in a variety of pulmonary diseases. Currently, studies on the role of DCs are limited by difficulties in isolating DCs from the lung. Surgical lung specimens are not readily available and purification of DCs from digested lung tissue is likely to induce phenotypical and functional changes. DCs obtained from the alveolar spaces are thought to represent the local microenvironment and can be obtained using minimally invasive techniques. We developed a novel method of isolating DCs from bronchoalveolar lavage (BAL) fluid.After removal of macrophages, the remaining BAL cells were stained with a lineage mix (CD3-, CD14-, CD16-, CD19-, CD56-FITC), CD11c and HLA-DR and sorted with a FACS ARIA. DAPI was used as a dead-live marker. mDCs were low autofluorescent, lineage mix negative, CD11c+ and HLA-DR+ cells. pDCs were CD11c− but CD123+. Morphological assessment of sorted mDCs and pDCs was performed. Sorted mDCs were tested in a mixed leukocyte reaction (MLR) with naive CD4+ T cells and evaluated for T cell differentiation and cytokine production. With confocal microscopy DC–T cell interaction was assessed.Using our sorting strategy, mDCs and pDCs, with a high purity upon FACS analysis of the sorted fraction, were obtained. These cells showed the morphological characteristics of DCs. Most importantly, mDCs were able to induce T cell proliferation and differentiation in a MLR, and interact with T cells as assessed by confocal microscopy. These results indicate the presence of functional DCs. Freezing and thawing of the BAL cells did not affect phenotype or T cell stimulatory capacity of the isolated DCs.Using a novel sorting strategy, functional mDCs can be isolated from BAL fluid, enabling a detailed study in pulmonary disease.

A novel method for isolating dendritic cells from human bronchoalveolar lavage fluid

Background Dendritic cells (DCs) play a pivotal role in linking the innate and adaptive immune response and have been implicated in a variety of pulmonary diseases. Currently, studies on the role of DCs are limited by difficulties in isolating DCs from the lung. Surgical lung specimens are not readily available and purification of DCs from digested lung tissue is likely to induce phenotypical and functional changes. DCs obtained from the alveolar spaces are thought to represent the local microenvironment and can be obtained using minimally invasive techniques. We developed a novel method of isolating DCs from bronchoalveolar lavage (BAL) fluid. Methods After removal of macrophages, the remaining BAL cells were stained with a lineage mix (CD3-, CD14-, CD16-, CD19-, CD56-FITC), CD11c and HLA-DR and sorted with a FACS ARIA. DAPI was used as a dead-live marker. mDCs were low autofluorescent, lineage mix negative, CD11c + and HLA-DR + cells. pDCs were CD11c − but CD123 +. Morphological assessment of sorted mDCs and pDCs was performed. Sorted mDCs were tested in a mixed leukocyte reaction (MLR) with naive CD4 + T cells and evaluated for T cell differentiation and cytokine production. With confocal microscopy DC–T cell interaction was assessed. Results Using our sorting strategy, mDCs and pDCs, with a high purity upon FACS analysis of the sorted fraction, were obtained. These cells showed the morphological characteristics of DCs. Most importantly, mDCs were able to induce T cell proliferation and differentiation in a MLR, and interact with T cells as assessed by confocal microscopy. These results indicate the presence of functional DCs. Freezing and thawing of the BAL cells did not affect phenotype or T cell stimulatory capacity of the isolated DCs. Conclusion Using a novel sorting strategy, functional mDCs can be isolated from BAL fluid, enabling a detailed study in pulmonary disease.

An improved enrichment method for functionally competent, highly purified peripheral blood dendritic cells and its application to HIV-infected blood samples.

Dendritic cells (DC) were purified from human peripheral blood using a rapid and simple method based on magnetic depletion of phagocytes with carbonyl iron, followed by centrifugation of nonphagocytic cells on a Percoll density gradient and depletion of lymphocytes and macrophages/monocytes with a panel of MoAbs and immunomagnetic beads. Enriched DC were obtained with > 99% purity as judged by non-specific esterase (NSE) staining. After isolation, these cells, representing 0.4% of the starting mononuclear cell population, still function as potent antigen-presenting cells for purified T lymphocytes. The present results confirm the ability of human peripheral blood DC to present soluble antigens to T cells including microbial antigens and show, further, that DC are more potent soluble antigen-presenting cells than monocytes. The method was successfully applied to the purification of DC from the blood of HIV-infected individuals. We could not detect decreased numbers of DC in four individuals with early HIV infection and no replicating HIV was detected by in situ hybridization in the DC.

Impact of transfection with total RNA of K562 cells upon antigen presenting, maturation, and function of human dendritic cells from peripheral blood mononuclear cells

Vaccination of dendritic cells (DCs) with tumor antigens is a potential strategy to induce tumor-specific immunity in tumor-bearing patients. The purpose of this study was to investigate whether human monocyte-derived DCs were able to present P210(Bcr-Ab1) protein and induce antigen-specific cytotoxic T lymphocyte (CTL) responses in vitro after transfected with total RNA of K562 cells (K562-RNA). DCs derived from human peripheral blood mononuclear cells were transfected with K562-RNA with electroporation or DOTAP lipofection. The successful transfection was determined by reverse transcription-polymerase chain reaction and Western blot. The phenotypes of the DCs were analyzed by flow cytometry (FCM), and cytotoxicity of CTL was assessed by propidium iodide staining followed by FCM analysis. The CD1a expression and purity of DCs were measured by FCM. The Bcr-Abl fusion gene was detected in the DCs with 24 hours after the transfection. The transfected cell expressed increased levels of CD80, CD83, CD86, and HLA-DR. Moreover, the transfected DCs strongly stimulated the T lymphocytes to gain cytotoxic activity against K562 cells. Culture medium containing 1 percent human plasma was the most effective for DC growth. Human DCs transfected with K562-RNA effectively induce specific immune responses. This method can be used to induce tumor-specific immune response and may have potential application in immunotherapy of tumors.

CMRF44+ Dendritic Cells from Peripheral Blood Stem Cell Harvests of Patients with Myeloma as Potential Cellular Vectors for Idiotype Vaccination

The optimal conditions required to harvest dendritic cells (DC) for immunotherapy were investigated in a series of preliminary investigations using peripheral blood stem cell (PBSC) harvests and blood from patients with myeloma. There was no difference in the number of DC (CMRF44+, CD19-, CD14-) in PBSC mobilized with G-CSF (mean 0.28%, n = 7) compared with GM-CSF (mean 0.24%, n = 6) and apheresis itself did not concentrate DC. In longitudinal studies (n = 10), the peak DC count (day 12 post PBSC harvest) did not correlate with the peak CD34+ cell count or white cell count. A simple affinity purification of DC resulted in a mean 63-fold purification. Affinity enriched suspensions from normal blood contained more DC (mean = 18.8%; n = 5) than those from patients with myeloma (mean = 9.9%; n = 13). The percentage of DC with a lymphoid phenotype (CD11c-, CDw123hi+) was significantly higher in G-CSF mobilized PBSC harvests (22.7%; n = 6) than in peripheral blood samples from patients with myeloma (7.0%; n = 13; p = 0.01). DC endocytosis was normal and did not change throughout the course of the disease. Neither DC numbers nor subsets changed significantly between days 1 and 3 of culture. Current mobilization procedures, optimized for PBSC, need to be altered when harvesting DC.

Unimpaired dendritic cells can be derived from monocytes in old age and can mobilize residual function in senescent T cells

Dendritic cells (DC) are powerful antigen presenting cells, which have the unique capacity to stimulate naive T cells. In spite of the well-known decline of T cell function in old age, little information is available on whether DC are also affected by the aging process. This is mainly due to problems with the isolation and purification of DC. Rapid progress in the characterization of DC has been made in recent years, as simple methods to generate large numbers of DC from precursors have been developed. It was the aim of the present study to compare monocyte derived DC from old and young healthy persons. The generation of DC from blood monocytes in response to GM-CSF and IL-4 treatment was similar in cells from young and old persons. The DC population thus obtained had a typical dendritic morphology and expressed DC surface markers, such as HLA class II, CD1a, CD11c, CD54, CD80 and CD86, but not CD14 for a period of up to three weeks in culture. DC from young and old persons produced IL-12 and TNF-α and responded equally well to maturation-inducing stimuli. DC maturation was stimulated by purified protein derivative (PPD) of Mycobacterium tuberculosis, whole inactivated influenza virus and by influenza split vaccine, but not by purified viral RNA. When tested for their antigen-presenting capacity, DC from young and old persons were capable of stimulating the proliferation and the cytokine production of T cells. It was of particular interest that CD45RA + as well as CD45RO + T cells from aged donors were unable to respond to stimulation with influenza proteins presented by monocytes, but were triggered to proliferate and to produce cytokines when antigen was presented by DC. The results demonstrate that DC from old persons (a) may still function as powerful antigen-presenting cells provided the right differentiation and maturation stimuli are present; (b) are capable of mobilizing residual capacity in senescent T cells and (c) may therefore represent a potent tool for immunotherapy and vaccines in old age.