Monocyte-derived Dendritic Cells In Patients Research Articles

In vitro impact of bisphenol A on maturation and function of monocyte-derived dendritic cells in patients with primary Sjögren’s syndrome

Background: Epidemiological studies have shown that environmental factors accelerate the progress of primary Sjögren’s syndrome (pSS). Bisphenol A (BPA), a classic endocrine disrupting chemical, affects the immune system. However, the impact of BPA on pSS has not yet been reported. The present study aimed to evaluate the potential relationship between BPA, estrogen receptor (ER), and pSS.Methods: We studied the impact of BPA on monocyte-derived dendritic cells (moDCs) from pSS patients and age-matched healthy controls (HCs). Morphological effects were observed under inverted microscope. Surface markers were analyzed by flow cytometry. ER and cytokine profiles were assessed using real-time polymerase chain reaction. The ability of moDCs to stimulate CD4+ T cells activation was assessed by mixed lymphocyte reaction (MLR).Results: moDCs from both pSS patients and HCs expressed ERα as well as ERβ. After BPA-exposure, expression of ERα increased significantly in pSS patients, while that of ERβ remained unchanged. moDCs from BPA-exposed pSS patients showed irregular morphology and reduction in cell aggregation. BPA increased HLA-DR on moDCs of pSS patients via ERα, and promoted the secretion of IL6 and IL12. When co-cultured with BPA-treated moDCs, cytokines (IFN-γ, IL4, IL17, IL10) and transcription factors (T-bet, Gata3, RoR-γt, Foxp3) of CD4+ T cells showed imbalance of Th1/Th2/Th17/Treg polarization, with Th1 and Th17 dominating.Conclusions: BPA altered the function of moDCs through ERα, including antigen capture, secretion of inflammatory factors, and ability to stimulate T cells, as well as accelerated the progression and further deterioration of pSS.

Different maturation pattern between myeloid dendritic cells and monocyte-derived dendritic cells in patients with immediate allergy reactions to betalactams

Different maturation pattern between myeloid dendritic cells and monocyte-derived dendritic cells in patients with immediate allergy reactions to betalactams

Decreased numbers and impaired function of dendritic cells in patients with hepatitis B-related acute-on-chronic liver failure

Objective To investigate the frequencies of circulating dendritic cell (DC) subsets and the function of monocyte-derived dendritic cells in patients with hepatitis B-related acute-on-chronic liver failure. Methods Peripheral blood was collected from hepatitis B-related acute-on-chronic liver failure patients (ACLF, n=40) and chronic hepatitis B (CHB, n=40) as well as normal controls (NCs, n=20). Circulating myeloid dendritic cell (Mdc) and plasmic dendritic cell (pDC) frequencies in peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometric analysis. Purified monocytes were isolated by combination of Histopaque-1.077 and CD14 Microbeads. Monocyte-derived dendritic cells (MoDCs) generated in vitro in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor upon activation by poly I∶C. Costimulatory molecule expression and allostimulatory mixed lymphocyte reaction (AMLR) of MoDCs were detected in patients with hepatitis B-related ACLF. Results The number of circulating mDC decreased only in patients with hepatitis B-related ACLF compared with that in normal controls. However, pDC numbers decreased in both CHB and ACLF patients. We observed a further decrease the pDC numbers in ACLF compared to CHB patients without statistical significance (P>0.05). MoDC from ACLF patients showed lower expression of costimulatory molecules CD80, CD86 and the mature marker CD83, as well as MHC Ⅱ molecule (HLA-DR) compared to CHB and NC group. Interestingly, MoDC impaired allostimulatory mixed lymphocyte reaction from ACLF patients compared to those in CHB patients and NCs. Conclusions Patients with hepatitis B-related ACLF have a significantly lower expression of surface markers and impaired AMLR of MoDC, as well as decreased number of circulating mDC and pDC, which may be partially related to HBV disease progression in these patients. Key words: Liver failure, acute/ME; Dendritic cells/ME

Abstract LB-230: SOCS5 mediates defective function of monocyte-derived dendritic cells in patients with chronic lymphocytic leukemia

Abstract Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries. This clinically heterogeneous disease is characterized by the progressive accumulation of monoclonal B cells co-expressing CD5, CD19, CD20 and CD23 in the peripheral blood and in the primary and secondary lymphoid organs. Additionally, a range of immune cells are altered in patients with CLL. The resulting immunologic defects predispose patients to severe recurrent infections, which is the major life-threatening complication associated with CLL. Functional impairment of dendritic cells (DCs) in CLL not only diminishes the response to microorganisms, but it also allows for tumor escape from immune control. Understanding the mechanism for this effect can provide insight into the molecular regulation of DC function and may also suggest therapeutic strategies to reverse the immunosuppressive effect of tumors on DCs. To elucidate the mechanism for DC dysfunction in patients with CLL, we focused on signal transduction pathways that regulate the expression of genes necessary for the immune response. We found that monocytes from CLL patients have a decrease in IL-4-induced activation of the transcription factor STAT6, which prevents the phenotypic and functional maturation of DCs. This does not reflect a generalized defect in cytokine-induced signal transduction, as the activation of the related transcription factor STAT5 in response to GM-CSF is unaffected. Monocyte-derived DCs from CLL patients display low levels of HLA-DR, costimulatory molecules and CD83, and reduced secretion of pro-inflammatory cytokines. These changes are associated with low expression of TLR4 and related molecules, which decrease the ability of these cells to respond to stimulus afforded by LPS, impairing their complete maturation. Consequently, monocyte-derived DCs from CLL patients have decreased ability to induce proliferation of T-cells and display an increased induction of immune-suppressive regulatory T cell. Although monocytes from CLL patients exhibit high IL-4R expression, activation of the downstream transcription factor STAT6 is inhibited because of increased expression of the negative regulator SOCS5. It is known that CLL cells produce IL-10, leading to elevated serum levels of this cytokine. IL-10-treatment of monocytes from healthy donors mimics the alteration in signaling observed in patients, through enhanced STAT3-dependent expression of SOCS5, which inhibits STAT6 activation and leads to defective DC differentiation. These findings suggest that SOCS5 can mediate the impaired function of DCs in CLL patients, and may be a new potential therapeutic target for reversing cancer-associated immune suppression. Note: This abstract was not presented at the meeting. Citation Format: Patricia A. Toniolo, Suhu Liu, Sarah R. Walker, Jose Alexandre M. Barbuto, David A. Frank. SOCS5 mediates defective function of monocyte-derived dendritic cells in patients with chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-230. doi:10.1158/1538-7445.AM2015-LB-230

IFN Regulatory Factor-1 Modulates the Function of Dendritic Cells in Patients with Acute Coronary Syndrome

Background: Atherosclerosis is widely recognized as a complex inflammatory disease involving pathogenic immune response of T cells and antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages. Accumulating evidence has revealed that mature DCs play critical roles in the differentiation of effector T cells into CD4+ T cells, which effectively participate in the onset of acute coronary syndrome (ACS). IFN regulatory factor (IRF)-1 has been shown to be involved in various immune processes. The role of IRF-1 in DCs in the pathogenesis of ACS has not been investigated. Methods and Results: We examined the relative mRNA and protein expression of IRF-1 in human monocyte-derived DCs in patients with coronary artery disease (CAD). The overexpression or silencing of IRF-1 expression in DCs in patients with ACS was performed to explore the possible role of IRF-1 in the maturation and function of DCs involved in ACS. The results showed that the relative expression of IRF-1 in DCs is obviously increased in patients with ACS. The overexpression or silencing of IRF-1 expression could effectively promote or attenuate the maturation and function of DCs. In addition, we revealed that the MAPK pathway (phosphorylation of JNK, p38 and ERK1/2) might be downstream of IRF-1 signalling pathway in activation of circulating DCs in ACS patients. Conclusion: The present data demonstrate that IRF-1 could effectively promote the immune maturation and function of DCs in ACS patients.

Dendritic cell profile induced by Schistosoma mansoni antigen in cutaneous leishmaniasis patients.

The inflammatory response in cutaneous leishmaniasis (CL), although responsible for controlling the infection, is associated with the pathogenesis of disease. Conversely, the immune response induced by S. mansoni antigens is able to prevent immune-mediated diseases. The aim of this study was to evaluate the potential of the S. mansoni Sm29 antigen to change the profile of monocyte-derived dendritic cells (MoDCs) from subjects with cutaneous leishmaniasis (CL) in vitro. Monocytes derived from the peripheral blood mononuclear cells of twelve patients were cultured with GM-CSF and IL-4 for differentiation into dendritic cells and then stimulated with soluble Leishmania antigen (SLA) in the presence or absence of Sm29 antigen. The expression of surface molecules associated with maturation and activation (HLA-DR, CD40, CD83, CD80, and CD86), inflammation (IL-12, TNF), and downregulation (IL-10, IL-10R) was evaluated using flow cytometry. We observed that the frequencies of HLA-DR, CD83, CD80, and CD86 as well as of IL-10 and IL-10R on MoDCs were higher in cultures stimulated with Sm29, compared to the unstimulated cell cultures. Our results indicate that the Sm29 antigen is able to activate regulatory MoDCs in patients with cutaneous leishmaniasis. It might be useful to control the inflammatory process associated with this disease.

Interferon-α Abrogates the Suppressive Effect of Apoptotic Cells on Dendritic Cells in anIn VitroModel of Systemic Lupus Erythematosus Pathogenesis

An increased incidence of apoptotic cells and an increased activation of dendritic cells (DC) may be involved in the pathogenesis of systemic lupus erythematosus (SLE). We investigated the characteristics of apoptotic neutrophils and monocyte-derived DC of patients with SLE, their interaction, and the influence of autoantibodies and inflammatory cytokines on this interaction. Kinetics of neutrophil apoptosis and DC activation were studied by flow cytometry. To analyze the interaction of apoptotic cells with phagocytes, crossover coculture experiments were performed with DC from patients with SLE and apoptotic Jurkat T cells as well as with apoptotic neutrophils from patients with SLE and the monocytic cell line U937. SLE serum and cytokines were added to this coculture, and activation and suppression of DC were quantified by levels of inflammatory cytokine secretion. Apoptotic neutrophils and DC from patients with SLE showed no inherent defects compared to healthy controls, and the suppressive nature of their interaction was not affected. Autoantibodies as well as the inflammatory cytokines interleukin 17 (IL-17) and IL-1β had no influence on the interaction in this setup. Interferon (IFN)-α, however, substantially reduced the suppressive effect of apoptotic cells on DC. The data suggest that aberrant immune reactivity in SLE is not generally due to an intrinsic defect in apoptotic cells, their processing, or their interaction with DC, but likely arises from the milieu in which this interaction takes place. Our study highlights the importance of IFN-α during early stages of SLE and its potential as a therapeutic target.

Induction of Th17 Lymphocytes and Treg Cells by Monocyte-Derived Dendritic Cells in Patients with Rheumatoid Arthritis and Systemic Lupus Erythematosus

Dendritic cells (DCs) have a key role in the regulation of immune response. We herein explored, in patients with inflammatory diseases, the role of monocyte derived DC's (mo-DCs) on the generation of Th17 and T regulatory (Treg) lymphocytes. Peripheral blood was obtained from thirty-five patients with rheumatoid arthritis (RA), twelve with systemic lupus erythematosus (SLE), and twenty healthy subjects. Mo-DCs were generated under standard (IL-4/GM-CSF) or tolerogenic (IL-4/GM-CSF plus recombinant P-selectin or PD-1 or IL-10) conditions, and their ability to induce Th17 and Treg lymphocytes was tested. We detected that mo-DCs from patients with RA showed an enhanced release of IL-6 and IL-23 as well as an increased capability to induce Th17 cells. Although mo-DCs from SLE patients also released high levels of IL-6/IL-23, it did not show an increased ability to induce Th17 lymphocytes. In addition, mo-DCs, from patients with RA and SLE generated under the engagement of PSGL-1, showed a defective capability to induce Foxp3+ Treg cells. A similar phenomenon was observed in SLE, when DC's cells were generated under PDL-1 engagement. Our data indicate that DCs from patients with rheumatic inflammatory disease show an aberrant function that may have an important role in the pathogenesis of these conditions.

Defective IL-10 production in severe phenotypes of Crohn’s disease

Loss of tolerance toward commensal bacteria has been invoked as a mechanism for Crohn's disease. IL-10 is a key anti-inflammatory cytokine that plays a role in induction and maintenance of tolerance. The aim of this study is to determine IL-10 production in response to bacterial components in Crohn's disease patients, who were classified according to their phenotypes as stricturing, penetrating, or inflammatory. Peripheral blood was obtained from Crohn's disease patients and healthy controls. Cytokine production was measured in whole blood cultures, isolated CD4(+) cells, and monocyte-derived dendritic cells (MDDCs). Under unstimulated conditions, IL-10, but not IL-12, was down-regulated significantly in blood cultures of patients with severe phenotypes, compared with inflammatory, nonpenetrating, nonstricturing Crohn's disease patients. In response to LPS, IL-10 was up-regulated more significantly in patients with no fistulae or fibrosis. Study of IL-10 production by isolated cell subsets showed that DCs, but not CD4(+) T cells, from penetrating Crohn's disease produced significantly less IL-10 in response to LPS. Differences were not associated with the 1082A/G polymorphism in the IL-10 gene promoter. We show a defect in IL-10 production in whole blood cell cultures and MDDCs in patients with severe forms of Crohn's disease. This defect in IL-10 production by a group of Crohn's disease patients may represent a mechanism mediating more severe manifestations of the disease. We propose that treatment with IL-10 or IL-10-inducing therapies could be of particular benefit to these group of patients.

Editorial Comment on: Vaccine Therapy in Patients with Renal Cell Carcinoma

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Monocyte-derived dendritic cells of patients with coronary artery disease show an increased expression of costimulatory molecules CD40, CD80 and CD86 in vitro

Atherosclerosis is a disease triggered by diverse exogenous stimuli and sustained by chronic inflammatory processes. Dendritic cells (DCs) are key regulatory antigen-presenting cells and play a crucial role in regulating the adaptive and innate immune system in any chronic inflammatory process. DCs are present in atherosclerotic lesions in the areas of the highest T-cell density. So far, their role in atherosclerosis has not been fully elucidated. We investigated the phenotypic properties of DCs in patients with coronary artery disease (CAD) in comparison to healthy individuals. Peripheral blood monocytes were isolated from 50 patients with CAD and 19 healthy individuals and differentiated over 9 days to immature and mature DCs. Analysis of the distribution of important stimulatory and costimulatory molecules on the surface of immature and mature DCs was performed by flow cytometry. We observed no changes between the groups concerning cell numbers or expression of CD1a or HLA-DR on DCs. Patients with CAD, however, showed a significant upregulation of the costimulatory molecules CD80, CD86 and CD40 as compared with healthy controls. Expression of CD40, CD80 and CD86 on DCs partly correlated with smoking, family history of CAD, as well as with C-reactive protein levels. High-density lipoprotein cholesterol was inversely associated with the expression of CD40 and CD80 on mature DCs (P<0.05). Upregulation of important costimulatory molecules on monocyte-derived DCs of CAD patients, is influenced multifactorially. Our results show notable differences between CAD patients and healthy individuals, possibly contributing to the pathophysiological processes in atherogenesis.

Specific Subpopulation Involved in Allergic Delayed Responses to Amoxicillin in an in Vitro System with Autologous Dendritic Cells

RATIONALE: Dendritic cells (DCs) are essential for initiating immune reactions mediated by T lymphocytes. Delayed type hypersensitivity reactions (DTHR) to amoxicillin (AX) are mediated by specific lymphocytes that recognize the haptenized drug. The aim of our work was to explore the immunological response generated by DCs in presence of AX compared to other penicillins in allergic subjects to AX by analyzing the specific proliferative response in vitro. The use of CFSE allowed us to test the proliferative response of different lymphocytes subpopulations. METHODS: Patients with a DTHR to betalactam and a positive delayed intracutaneous test to AX were included. Peripheral DCs were generated from monocytes by incubating with IL4 and GM-CSF. Lymphocytes were treated with 1μM CFSE prior the culture with autologous DCs and the corresponding drug (AX or other penicillin). The specific subpopulations proliferation was analyzed by flow cytometry. RESULTS: Proliferation was only detected when AX was used in the culture. We found a high proliferation of both CD4 and CD8 memory T-lymphocytes and also, although in less extent, of B-lymphocytes (CD19+) and NK (CD3-CD56+). We did not observed any proliferative response of naive T-lymphocyte or CLA+ cells in our system. CONCLUSIONS: The lymphocyte proliferation induced by monocyte-derived DCs in patients with a DTHR to AX was strictly related to this drug. Cells implicated were mostly CD4 and CD8 memory lymphocytes, as well as B and NK cells. The use of CFSE is a useful tool for discriminating the subpopulations that proliferate in an in vitro system.

Functional impairment of monocyte-derived dendritic cells in patients with severe chronic kidney disease

Dendritic cells (DCs) are antigen-presenting cells that are pivotal for the initiation of the primary immune response. Patients with chronic kidney disease (CKD) with or without chronic intermittent haemodialysis (CIHD) show an impaired immune response. Dysfunction of DCs may underlie this phenomenon. In this study, several different functions of monocyte-derived DCs (moDC) of patients with CKD class IV-V (glomerular filtration rate <30 ml/min) and patients on CIHD were studied in vitro and compared with age- and sex-matched healthy volunteers. We demonstrate that, independent of the maturation stimulus used, mature moDC from both groups of patients did not acquire the same level of terminal differentiation as moDC from controls, as shown by analysis of cell surface markers and the relative high macropinocytosis activity of moDC. The stimulation of allogeneic T-cells by immature moDC and mature moDC did not differ between patients and controls. However, in the presence of immature moDC or antigen-loaded maturated moDC from patients, less proliferation of autologous T-cells was observed in response to recall antigens. There was no difference between moDC from controls and patients in their ability to activate naive T-cells and to differentiate them into Th1 and Th2 cells. These results show that the terminal differentiation of moDC in patients with severe CKD is impaired. This impairment is not restricted to one maturation stimulus and is independent of treatment with haemodialysis.

Cancer therapy with tumor cell lysate pulsed monocyte-derived dendritic cells in patients with metastastic colon cancer: Improvement by danger signals

2563 Background: The prognosis of colon cancers with hematogenous spread remains poor at 12–20 months. We report the use of a dendritic cell based immunotherapy to improve patient survival, especially with cell culture conditions mimicking infection. Methods: After isolating monocytes from the blood of 39 patients with metastasized colon cancer, dendritic cells were generated ex vivo in the presence of recombinant cytokines (IL-4; GM-CSF) and autologous serum. The DC loaded with tumor cell lysate were administered to the patients intradermally. Culture conditions were tested for upregulation of costimulatory molecules, downregulation of IL-10 and upregulation of IL-12 secretion by ELISPOT and fluorescence cytometry. Results: DC vaccination induced a clinical response in 9 (23%) patients with a median overall survival after onset of DC-therapy of 12 months (11–44 months for responders and 1–20 months for non-responders) and 23 months after diagnosis of metastases (16–63 months for responders and 1–23 months for non-responders). These data show that a dendritic cell based immunotherapy may prolong the patients overall survival. However, complete remissions are rare. This can be due to weak stimulation of CTL response due to insufficient antigen presentation, lack of costimulatory molecules as well as secretion of IL-10 rather than IL-12 by the DC’s. Here we could show that Toll-like receptors (TLR) ligands like Poly-I:C or lipopeptides as so called danger signals in combination with interferons can induce an upregulation of costimulatory molecules accompanied by inhibition of IL-10 and induction of IL-12 secretion in vitro. By using these culture conditions we induced a clinical complete remission of liver metastases after failure of standard therapy in one patient (overall survival after onset of DC-therapy 13 months, after diagnosis 24 months). Conclusions: According to Matzinger’s hypothesis an effective immune response occurs only by responding to a danger signal associated with infection or stress. Thus, cell culture condition should be used with TLR ligands mimicking a bacterial or viral infection. In general, a dendritic cell based immunotherapy can be successful in advanced stages of colon cancer patients. [Table: see text]

Cancer therapy with immature monocyte-derived dendritic cells in patients with advanced breast cancer

2528 Background: Recent studies have shown that immunotherapy with monocyte-derived dendritic cells can induce clinical antitumor response in various types of solid tumors. Methods: After isolating monocytes from the peripheral blood of n=143 patients with advanced breast cancer, immature dendritic cells (iMoDC) were generated ex vivo in the presence of IL-4, GM-CSF and autologous serum. The iMoDC loaded with tumor cell lysate, known peptides or with tumor proteins from autologous serum were administered to patients intradermally or intravenously. Using flow cytometry and fluorescence microscopy, the phagocytic activity and surface markers on the iMoDC and on tumor cells of select treated patients were investigated. Results: MoDC vaccination induced a clinical response in n=56 (37%) patients producing 7 complete remissions, 14 partial or minor remissions, 33 stable diseases and 3 mixed responses with a median overall survival after onset of the MoDC-therapy of 25 months (2–59 months) and a 3-years surviva...

Defective tumor necrosis factor alpha-induced maturation of monocyte-derived dendritic cells in patients with myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective and dysplastic hematopoiesis. MDS patients have a defective immune response manifested by increased susceptibility to bacterial infections, autoimmune phenomena, and high incidence of lymphoid malignancies. Presently, we investigated the phenotype and function of monocyte-derived dendritic cells (MoDC) in 23 MDS patients and 15 controls at different stages of differentiation using the maturation stimuli tumor necrosis factor-alpha (TNF-α) and LPS. Monocytes from MDS patients showed low potential to differentiate into dendritic cells (DC), as determined by low cell yield and CD1a expression. MDS-MoDCs exhibited low expression of mannose receptor and reduced endocytic capacity. MDS-MoDCs showed a diminished response to TNF-α with low CD83, CD80, and CD54 expression and allostimulatory capacity. In patients with 5q syndrome, monocytes and MoDCs were positive for the 5q deletion, suggesting their origin from the malignant clone. Our data indicate that MoDCs in MDS display quantitative and functional abnormalities that may contribute to the defective immune response of these patients.

Cancer therapy with unloaded monocyte-derived dendritic cells in patients with inoperable pancreatic and gall bladder cancer

2569 Background: Several studies could show that immunotherapy with monocyte-derived dendritic cells (MoDC) loaded with tumor-cell-lysate or known peptides induce clinical antitumor response in certain patients with various types of solid tumors. Given that tumor proteins in the serum of cancer patients may be sufficient to internalize and presented by immature dendritic cells, we investigated whether immature dendritic cells incubated with the serum of cancer patients could induce an antitumor response in vivo. Methods: After isolating monocytes from peripheral blood of patients with pancreatic (n=17) and gall bladder cancer (n=6), dendritic cells were generated ex vivo in the presence of recombinant cytokines (IL-4;GMC-SF) and autologous serum. After 7 days of culture, the autologous MoDC were administered to the cancer patients (intradermal or intratumoral). Results: Flow cytometric analysis of the surface markers revealed that the generated MoDC were of immature type with expression of CD1a and MHC-II but not monocyte specific marker CD14. The generated MoDC could induce an antigen specific lymphocyte proliferation in vitro. Using a phagocytosis assay it could be demonstrated by flow cytometry and electron microscopy that immature dendritic cells are in general able to phagocytose different sized particles. The vaccination induced a clinical response in n=5 patients with pancreatic cancer including 2 stable diseases, 2 minor remissions and 1 mixed response (overall survival 4–32 months; 2 patients died of their disease after 10 and 21 months, respectively) as well as in n=1 patient with gall bladder cancer (stable disease after 18 months using intratumoral administration). A biopsy taken from the patient with the gall bladder cancer within the treatment period demonstrated unusually low proliferative activity. Conclusions: The results suggest that immature dendritic cells could be loaded by soluble tumor antigens in the serum of cancer patients as well as able to uptake tumor antigens in vivo. No significant financial relationships to disclose.

Cancer therapy with unloaded monocyte-derived dendritic cells in patients with inoperable pancreatic and gall bladder cancer

2569 Background: Several studies could show that immunotherapy with monocyte-derived dendritic cells (MoDC) loaded with tumor-cell-lysate or known peptides induce clinical antitumor response in certain patients with various types of solid tumors. Given that tumor proteins in the serum of cancer patients may be sufficient to internalize and presented by immature dendritic cells, we investigated whether immature dendritic cells incubated with the serum of cancer patients could induce an antitumor response in vivo. Methods: After isolating monocytes from peripheral blood of patients with pancreatic (n=17) and gall bladder cancer (n=6), dendritic cells were generated ex vivo in the presence of recombinant cytokines (IL-4;GMC-SF) and autologous serum. After 7 days of culture, the autologous MoDC were administered to the cancer patients (intradermal or intratumoral). Results: Flow cytometric analysis of the surface markers revealed that the generated MoDC were of immature type with expression of CD1a and MHC-II but not monocyte specific marker CD14. The generated MoDC could induce an antigen specific lymphocyte proliferation in vitro. Using a phagocytosis assay it could be demonstrated by flow cytometry and electron microscopy that immature dendritic cells are in general able to phagocytose different sized particles. The vaccination induced a clinical response in n=5 patients with pancreatic cancer including 2 stable diseases, 2 minor remissions and 1 mixed response (overall survival 4–32 months; 2 patients died of their disease after 10 and 21 months, respectively) as well as in n=1 patient with gall bladder cancer (stable disease after 18 months using intratumoral administration). A biopsy taken from the patient with the gall bladder cancer within the treatment period demonstrated unusually low proliferative activity. Conclusions: The results suggest that immature dendritic cells could be loaded by soluble tumor antigens in the serum of cancer patients as well as able to uptake tumor antigens in vivo. No significant financial relationships to disclose.

Generation of Monocyte-Derived Dendritic Cells in Patients with Hereditary Hemochromatosis

Hereditary hemochromatosis (HH) is a common genetic disease with autosomal recessive transmission and is characterized by a dysregulation of iron metabolism, leading to serum iron overload and its progressive accumulation in most body tissues. The effects of HH on the immune system include altered lymphocytosis and functions of monocytes. Moreover, monocytes can differentiate into dendritic cells (DCs), which play crucial roles in the immune response (capture, processing, and presentation of antigen to effector T cells) and this process was shown to be impaired in several pathologies. The aim of this study was to determine whether the monocytes from HH patients still displayed the ability to differentiate into DCs. To that purpose, purified monocytes from healthy donors and HH patients were cultured in the appropriate medium. The results showed no phenotypic and functional differences, at both the immature and the mature stages. Furthermore, our work reports altered lymphocytosis with expanded CD8 +CD28 − T cell subset. These monocyte-derived DCs could therefore be a solid vector for DC-based immunotherapy and a powerful tool for investigating the immune regulatory loops and especially the biological relevance of the expanded CD8 +CD28 − T cells since this population has also been described as suppressor T cells.