Mature moDCs Research Articles

Altered monocyte-derived dendritic cell function in patients on hemodialysis: a culprit for underlying impaired immune responses

Patients with end-stage renal disease (ESRD) are known to have impaired immune function. Dendritic cells (DCs) are the major antigen-presenting cells that initiate primary immune responses, linking innate and adaptive immunity. Although suboptimal immune responses to vaccination, as frequently observed in ESRD patients, might suggest the presence of impaired DC function, the precise nature of altered DC function is not fully understood. In the current study, we compared the maturation status, viability, and function of monocyte-derived DCs (moDCs) of patients on hemodialysis (HD) with healthy controls. Surface expression of major histocompatibility complex class II, CD83, and CD86, and chemokine receptor CCR7 in moDCs was not different between HD patients and healthy controls. No significant difference was detected in the viability of moDCs determined by expression of annexin V and propidium iodide between two groups. However, moDCs from HD patients produced significantly higher amounts of IL-6 when stimulated by cytokine cocktails compared to healthy controls. In addition, mature moDCs from HD patients showed significantly enhanced allogeneic T-cell proliferation compared to healthy controls. Our data demonstrate aberrant DC function in HD patients and suggest that this might contribute to impaired immune responses.

The Effect of Gefitinib on Immune Response of Human Peripheral Blood Monocyte-Derived Dendritic Cells

Background: Synergistic antitumor effects of the combined chemoimmunotherapy based on dendritic cells have been reported recently. The aim of this study is to search new applicability of gefitinib into the combination treatment through the confirmation of gefitinib effects on the monocyte derived dendritic cells (moDCs); most potent antigen presenting cell (APC). Methods: Immature and mature monocyte-derived dendritic cell (im, mMoDC)s were generated from peripheral blood monocyte (PBMC) in Opti-MEM culture medium supplemented with IL-4, GM-CSF and cocktail, consisting of TNF-α (10 ng/mL), IL-1β (10 ng/mL), IL-6 (1,000 U/mL) and PGE(2) (1 μ/mL). Various concentrations of gefitinib also added on day 6 to see the influence on immature and mature MoDCs. Immunophenotyping of DCs under the gefitinib was performed by using monoclonal antibodies (CD14, CD80, CD83, CD86, HLA-ABC, HLA-DR). Supernatant IL-12 production and apoptosis of DCs was evaluated. And MLR assay with [3H]-thymidine uptake assay was done. Results: Expression of CD83, MHC I were decreased in mMoDCs and MHC I was decreased in imMoDCs under gefitinib. IL-12 production from mMoDCs was decreased under 10 μM of gefitinib sinificantly. Differences of cell proliferation capacity were not observed in each concentration of geftinib. Conclusion: In spite of decreased expressions of some dendritic cell surface molecules and IL-12 production under 10 μM of gefitinib, significant negative influences of gefitinib in antigen presenting capacity and T cell stimulation were not observed.

Indoleamine 2,3-dioxygenase–expressing mature human monocyte-derived dendritic cells expand potent autologous regulatory T cells

AbstractA comprehensive understanding of the complex, autologous cellular interactions and regulatory mechanisms that occur during normal dendritic cell (DC)–stimulated immune responses is critical to optimizing DC-based immunotherapy. We have found that mature, immunogenic human monocyte-derived DCs (moDCs) up-regulate the immune-inhibitory enzyme, indoleamine 2,3-dioxygenase (IDO). Under stringent autologous culture conditions without exogenous cytokines, mature moDCs expand regulatory T cells (Tregs) by an IDO-dependent mechanism. The priming of resting T cells with autologous, IDO-expressing, mature moDCs results in up to 10-fold expansion of CD4+CD25brightFoxp3+CD127neg Tregs. Treg expansion requires moDC contact, CD80/CD86 ligation, and endogenous interleukin-2. Cytofluorographically sorted CD4+ CD25brightFoxp3+ Tregs inhibit as much as 80% to 90% of DC-stimulated autologous and allogeneic T-cell proliferation, in a dose-dependent manner at Treg:T-cell ratios of 1:1, 1:5, and as low as 1:25. CD4+CD25brightFoxp3+ Tregs also suppress the generation of cytotoxic T lymphocytes specific for the Wilms tumor antigen 1, resulting in more than an 80% decrease in specific target cell lysis. Suppression by Tregs is both contact-dependent and transforming growth factor-β–mediated. Although mature moDCs can generate Tregs by this IDO-dependent mechanism to limit otherwise unrestrained immune responses, inhibition of this counter-regulatory pathway should also prove useful in sustaining responses stimulated by DC-based immunotherapy.

Pattern recognition receptor expression is not impaired in patients with chronic mucocutanous candidiasis with or without autoimmune polyendocrinopathy candidiasis ectodermal dystrophy.

Patients with chronic mucocutaneous candidiasis (CMC) have an unknown primary immune defect and are unable to clear infections with the yeast Candida. CMC includes patients with AIRE gene mutations who have autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), and patients without known mutations. CMC patients have dysregulated cytokine production, suggesting that defective expression of pattern recognition receptors (PRRs) may underlie disease pathogenesis. In 29 patients with CMC (13 with APECED) and controls, we assessed dendritic cell (DC) subsets and monocyte Toll-like receptor (TLR) expression in blood. We generated and stimulated monocyte-derived (mo)DCs with Candida albicans, TLR-2/6 ligand and lipopolysaccharide and assessed PRR mRNA expression by polymerase chain reaction [TLR-1-10, Dectin-1 and -2, spleen tyrosine kinase (Syk) and caspase recruitment domain (CARD) 9] in immature and mature moDCs. We demonstrate for the first time that CMC patients, with or without APECED, have normal blood levels of plasmocytoid and myeloid DCs and monocyte TLR-2/TLR-6 expression. We showed that in immature moDCs, expression levels of all PRRs involved in anti-Candida responses (TLR-1, -2, -4, -6, Dectin-1, Syk, CARD9) were comparable to controls, implying that defects in PRR expression are not responsible for the increased susceptibility to Candida infections seen in CMC patients. However, as opposed to healthy controls, both groups of CMC patients failed to down-regulate PRR mRNA expression in response to Candida, consistent with defective DC maturation, as we reported recently. Thus, impaired DC maturation and consequent altered regulation of PRR signalling pathways rather than defects in PRR expression may be responsible for inadequate Candida handling in CMC patients.

Regulatory T Cells Expanded by Autologous Mature Human Dendritic Cells Expressing Indoleamine 2,3-Dioxygenase Are Potent Suppressors of T Cell Proliferation.

Best characterized as initiators of immunity, dendritic cells (DCs) also play an integral role in immune modulation. Immature DCs, for example, process self-antigens to induce and maintain tolerance. The immunoregulatory effects of DCs, however, are not limited to immature subtypes. Immunogenic mature DCs can also induce T regs to curb immune responses. We have found that human monocyte-derived DCs (moDCs) upregulate the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO) with maturation and expand functionally active, naturally occurring as well as inducible regulatory T cells (T regs) in an IDO-dependent manner. Priming of resting bulk T cells with autologous, IDO-expressing, mature moDCs in the absence of exogenous cytokines results in up to 10-fold expansion of CD4+CD25hiFoxp3+CD127neg T cells that mediate significant dose-dependent suppression of both allogeneic and autologous T cells stimulated de novo by DCs. The expansion of T regs by IDO-expressing moDCs involves cell-to-cell contact, CD80/CD86 ligation, and IL-2. Autologous priming in the presence of a competitive inhibitor of IDO, 1-methyl-tryptophan, diminishes T reg expansion. Candidate T regs were further characterized after cytofluorographic sorting primed bulk T cells into CD4+CD25hi, CD4+CD25int, and CD4+CD25neg subpopulations. Post-sort analysis showed that >60% of the CD4+CD25hi cells coexpressed Foxp3, which was not present in the CD4+CD25neg cells. CD4+CD25hi T regs exerted dose-dependent inhibition of DC-stimulated allogeneic T cell proliferation, with >90% inhibition at a suppressor to responder T cell ratio of 1:1 and ~50% inhibition at a ratio of 1:25. CD4+CD25int cells produced intermediate suppression depending on dose, and CD4+CD25neg cells were not inhibitory. CD4+CD25hi T regs mediated similar suppression of autologous T cell responses to stimulation de novo by DCs. CD4+CD25hi T regs also inhibited the generation of cytotoxic T lymphocytes (CTLs) specific for the Wilms' tumor gene product (WT-1). The addition of CD4+CD25hi T regs to CTL-priming cultures resulted in a >80% decrease in specific target cell lysis of a WT-1-expressing cell line. Separate studies showed that T reg-mediated suppression is contact dependent and also requires TGF-beta, suggesting inhibition by naturally occurring and inducible T regs, respectively. Depletion of CD4+CD25hi T cells from bulk T cells by negative immunoselection with anti-CD25 magnetic beads at the outset of autologous priming significantly blunts T reg expansion, indicating a requirement for pre-existing T regs in the bulk T cell population. T reg expansion also occurs in priming cultures using cytofluorographically-sorted CD4+CD25neg T cells, indicating de novo generation of T regs from CD4+CD25neg precursors. In summary, our results demonstrate a mechanism by which mature, IDO-expressing, human moDCs expand autologous, naturally occurring as well as inducible T regs that functionally suppress the proliferation of both autologous and allogeneic T cells. Inhibition of this counter-regulatory pathway should result in more sustained benefit from active DC-based immunotherapy.

Induction of high expression of CCR7 and high production of IL-12 in human monocyte-derived dendritic cells by a new bacterial component: LCOS 1013

Dendritic cells (DCs) are the most potent antigen presenting cells of the immune system as they can act as initiators, stimulators and regulators of the immune response. Human DCs are most commonly generated for clinical use by in vitro differentiation of monocytes with exogenous cytokines. Here, we investigate the effect of LCOS 1013 on the production of mature Mo-DCs. LCOS 1013 is a new bacterial component from walls of gram + Klebsellia pneumoniae bacteria that contain some OmpA glycoproteins. Purified peripheral blood monocytes were cultured for 6 days with IL-4 and GM-CSF in order to obtain immature dendritic cells (Im-MoDCs). On day six, Im-MoDCs were matured with either LCOS 1013, TNF alpha, LPS or CD40-Ligand. LCOS 1013 matured Mo-DCs (LCO-DCs) showed a higher expression of DC-LAMP, CD80, CD83, CD54 and CD40 than TNF alpha, LPS and CD40L matured Mo-DCs. Interestingly, LCO-DCs exhibited high expression of full competent CCR7 and high secretion of IL-12 during their maturation. Functionally, LCO-DCs have equivalent potency to trigger mixed leukocyte reaction and antigen-specific reaction and polarize immune response towards Th1 way. Moreover, we found that LCOS 1013 activates DCs through TLR2. LCOS 1013 represents an attractive therapeutic maturation agent of DCs allowing the production of Mo-DCs with high capacity to migrate and to induced Th1 immune responses.

Induction of recipient cell-specific donor T-cell anergy by UV-C-irradiated recipient immature monocyte-derived dendritic cells

The induction of donor T-cell anergy to recipient cells for reducing GVHD could be one way of expanding donor candidates for HLA-mismatched hematopoietic SCT. The present study was designed to clarify whether recipient cell-specific T-cell anergy could be induced by priming donor lymphocytes with recipient monocyte-derived DCs (mo-DCs) irradiated with ultraviolet-C (UV-C). By irradiation of mo-DCs with UV-C, the expression of DC-associated surface phenotypes such as CD83, CD80, CD86 and CD40 was reduced and the antigen-presenting ability of UV-C-irradiated mo-DCs was clearly decreased. By co-culturing normal donor 1 lymphocytes with UV-C-irradiated donor 2 immature mo-DCs, the response of the lymphocytes to donor 2 mature mo-DCs was markedly reduced as compared with that of the lymphocytes prestimulated with non-irradiated donor 2 immature mo-DCs or UV-C-irradiated mo-DCs derived from a different individual donor 3. The present study demonstrated that recipient cell-specific T-cell anergy could be induced by priming donor lymphocytes with UV-C-irradiated recipient immature mo-DCs in hematopoietic SCT. These data suggest the applicability of donor graft cells, which have been prestimulated with UV-C-irradiated recipient immature mo-DCs, for expanding donor candidates in HLA-mismatched hematopoietic SCT.

Induction of Recipient Cell-Specific Donor T Cell Anergy by UV-C-Irradiated Recipient Immature Monocyte-Derived Dendritic Cells.

The induction of donor T cell anergy to recipient cells for reducing GVHD could be one way of expanding donor candidates for HLA-mismatched hematopoietic stem cell transplantation. The present study was designed to clarify whether recipient cell-specific T cell anergy could be induced by priming donor lymphocytes with recipient monocyte-derived dendritic cells (mo-DCs) irradiated with UV-C. By irradiation of mo-DCs with 100 J/m2 or more UV-C, the expression of DC-associated surface phenotypes such as CD1a, CD54, CD40, CD80, CD83 and CD86 was reduced in one day after irradiation and the effects of UV-C irradiation continued for at least 7 days. By irradiation of mo-DCs with 100 J/m2 or more UV-C, the antigen-presenting ability of both immature and mature mo-DCs, which was examined by 3H-thymidine incorporation assay, was clearly decreased at UV-C dose-dependent manner. Proliferation of CSFE-labeled lymphocytes by the stimulation with immature or mature Mo-DCs was suppressed by 300 J/m2 UV-C irradiation to immature or mature mo-DCs. The response of normal donor 1 lymphocytes, which had been co-cultured with 300–3,000 J/m2 UV-C-irradiated donor 2 immature mo-DCs for 7 days, against mature donor 2 mo-DCs in mixed leukocyte culture (MLC) for 7 days was markedly reduced, compared with the response of the donor 1 lymphocytes co-cultured with non-irradiated donor 2 mo-DCs or UV-C-irradiated mo-DCs derived from a different individual donor 3. CFSE-labeling analysis of donor 1 lymphocytes, which were co-cultured with 300 J/m2 UV-C irradiated donor 2 mo-DCs in the first MLC and then stimulated with donor 2 mature mo-DCs in the second MLC, showed that by stimulation with mature mo-DCs in the second MLC, the proliferation of donor 1 lymphocytes co-cultured with UV-C irradiated donor 2 mo-DCs in the first MLC was less than that of the lymphocytes co-cultured with non-irradiated mature mo-DCs. Flow cytometry analysis of the lymphocytes co-cultured with 300 J/m2 UV-C irradiated mo-DCs using surface CD4/CD25 and cytoplasmic Foxp3 monoclonal antibodies revealed that there was no increase of regulatory T cell population in the lymphocytes co-cultured with UV-C-irradiated immature mo-DCs, compared with the lymphocytes co-cultured with non-irradiated immature mo-DCs. Cell proliferation in allogeneic MLC consisting of lymphocytes as responder cells and mature mo-DCs as stimulator cells was not suppressed by the addition of the lymphocytes co-cultured with UV-C-irradiated immature mo-DCs. The present study demonstrated that recipient cell-specific T cell anergy could be induced by priming donor lymphocytes with UV-C-irradiated immature mo-DCs derived from a recipient and the T cell anergy was not associated with regulatory T cells. These data suggest the applicability of donor graft cells, which have been pre-stimulated with UV-C-irradiated recipient immature mo-DCs for expanding donor candidates for HLA-mismatched hematopoietic stem cell transplantation.

Anti-Tumor Cytotoxicity of γδ T Cells Expanded from Blood Cells of Myeloma and Leukemia Patients Against Self Tumor Cells — Enhancement of the Anti-Tumor Cytotoxicity by Type I IFN, Dendritic Cells, and Activated αβ T Cells.

In order to establish an efficient gd T cell-mediated immunotherapy for hematological malignancies, we tried to clarify whether γδ T cells could be expanded from blood cells of patients with myeloma, lymphoma and acute leukemia by culture with zoledronate and a low dose of IL-2 and whether the expanded patients' γδ T cells could kill tumor cells including self tumor cells with sparing normal clone cells. In addition, we explored the methods to enhance the anti-tumor cytotoxicity of the expanded γδ T cells by activating them with type I IFN, monocyte-derived dendritic cells (mo-DCs), or ab T cells. Although γδ T cells could be expanded in patients with myeloma, lymphoma and leukemia as well as normal persons, the amplification rates of gd T cells before and after the culture were varied from patient to patient in the patients with hematological malignancies. γδ T cells generated in patients with myeloma and lymphoma showed a potent cytotoxic ability against myeloma/lymphoma cell lines (RPMI8226, Daudi) as shown in γδ T cells generated in normal persons. In addition, γδ T cells generated in a patient with myeloma and acute leukemia showed a cytotoxic ability against self myeloma or leukemia cells freshly prepared from bone marrow. However, the same γδ T cells were not cytotoxic to normal lymphocytes of the patients. Then the expanded γδ T cells were stimulated with type I IFN, mo-DCs, or αβ T cells and the activation (CD69 expression) and cytotoxicity against tumor cells were examined. By the stimulation with type I IFN, the expression of CD69 and Trail of γδ T cells was increased and the cytotoxic ability of γδ T cells was enhanced at dose-dependent manner of type I IFN. CD69 expression on γδ T cells was enhanced by co-culture with both immature and mature mo-DCs in a cell-number-dependent fashion. CD69 expression was enhanced after the addition of mo-DCs of either autologous or allogeneic origin. Activation of γδ T cells with mo-DCs enhanced anti-tumor cytotoxicity of γδ T cells against RPMI8226 and CML blastic crisis cell line (C2F8) in an effector-to-target ratio-dependent manner. Although CD69 expression of γδ T cells was enhanced by the co-culture with allogeneic ab T cells, autologous ab T cells couldn't activate γδ T cells. However, autologous ab T cells stimulated with IL-2 or PHA could induce the activation of γδ T cells. The activation of γδ T cells with stimulated αβ T cells required cell-to-cell interaction. These findings suggested that αβ T cells stimulated by allogeneic γδ T cells could activate the same allogeneic γδ T cells. The present data demonstrated that γδ T cells, which could be expanded in vitro from blood cells of the patients with myeloma, lymphoma and leukemia by culture with zoledronate and IL-2, possess an enough cytotoxic ability against tumor cells including self tumor cells with sparing normal cells. These findings suggested that in vitro generated patients' γδ T cells could be applied to γδ T cell-mediated immunotherapy for hematological malignancies. Besides, potent γδ T cells activated by type I IFN, mo-DCs or activated αβ T cells were considered to be applicable for γδ T cell-mediated immunotherapy.

Enhancement of Anti-tumor Cytotoxicity of Expanded .GAMMA..DELTA. T Cells by Stimulation with Monocyte-derived Dendritic Cells

In order to establish the method of generating powerful gammadelta T cells for anti-tumor immunotherapy, we investigated the effects of monocyte-derived dendritic cells (mo-DCs) on anti-tumor cytotoxicity of expanded gammadelta T cells. Activation of gammadelta T cells co-cultured for 2-3 days with immature or mature mo-DCs was evaluated by CD69 expression and anti-tumor cytotoxicity using two assays : the 5- (and 6-) carboxyfluorescein diacetate, succinimidyl ester-based cytotoxicity assay and the calcein-AM-based Terascan assay. gammadelta T cells were used as effector cells and myeloma cell line (RPMI8226) or chronic myelogenous leukemia blastic crisis cell line (C2F8) were used as target cells. CD69 expression on gammadelta T cells was enhanced by co-culture with both immature and mature mo-DCs in a cell-number-dependent fashion. CD69 expression was enhanced after addition of mo-DCs of either autologous or allogeneic origin. Activation of gammadelta T cells with mo-DCs enhanced anti-tumor cytotoxicity of gammadelta T cells against RPMI8226 and C2F8 in an effector-to-target ratio-dependent manner. Activation of gammadelta T cells by mo-DCs was associated with the enhancement of anti-tumor cytotoxicity of gammadelta T cells. Potent gammadelta T cells activated by mo-DCs were considered to be applicable to an efficient gammadelta T cell-mediated immunotherapy for tumors.

Design of bifunctional siRNAs: Combining immunostimulation and gene-silencing in one single siRNA molecule

Active suppression of T lymphocyte activation can limit the efficacy of immune surveillance and immunotherapy. Here we have explored the possibility of designing bifunctional small interfering RNAs (siRNAs) capable of inducing innate immunity through Toll-like receptors and simultaneously inhibiting the expression of immunosuppressive factors. Using interleukin (IL) 10 as a model, we found that liposomal delivery of IL10 siRNAs could efficiently activate the expression of cytokines (e.g. TNF-α, IL6, and IL12) and interferons (e.g. IFN-α) in peripheral blood mononuclear cells (PBMCs) and immature monocyte-derived dendritic cells (iMoDCs). Moreover, the designed siRNAs inhibited IL10 gene expression. Transfection of iMoDCs with either chemically or in vitro transcribed IL10 siRNAs induced their differentiation into mature MoDCs (mMoDCs) characterized by the expression of costimulatory molecules CD80/CD86 and the chemokine receptor CCR7. Lipid delivery of either chemically synthesized or T7-transcribed immunostimulatory siRNAs induced cytokine production. However, in contrast to chemically synthesized siRNAs, electroporation of in vitro transcribed siRNAs also induced cytokine production in iMoDCs. Interestingly, IL10 siRNA-transfected iMoDCs were capable for enhancing the response of allogeneic T cells, providing support for the rational design of bifunctional siRNAs as immune modulating therapy.

Dendritic cell maturation by CD11c− T cells and Vα24+ natural killer T‐cell activation by α‐Galactosylceramide

Human invariant Valpha24+ natural killer T (NKT) cells display potent antitumor activity upon stimulation. Activation of endogenous Valpha24+ NKT cells would be one strategy for the treatment of cancer patients. For example, dendritic cells (DCs) loaded with a glycolipid NKT cell ligand, alpha-galactosylceramide (alphaGalCer, KRN7000), are a possible tool for the activation and expansion of functional Valpha24+ NKT cells in vivo. In this report, we demonstrate that the levels of expansion and the ability to produce IFN-gamma of Valpha24+ NKT cells induced by alphaGalCer-loaded whole PBMCs cultured with IL-2 and GM-CSF (IL-2/GM-CSF-cultured PBMCs) were superior to those of cells induced by monocyte-derived CD11c+ DCs (moDCs) developed with IL-4 and GM-CSF. Interestingly, CD11c+ cells in the IL-2/GM-CSF-cultured PBMCs showed a mature phenotype without further stimulation and exerted potent stimulatory activity on Valpha24+ NKT cells to enable them to produce IFN-gamma preferentially at an extent equivalent to mature moDCs induced by stimulation with LPS or a cytokine cocktail. Cocultivation with CD11c- cells in the IL-2/GM-CSF-cultured PBMCs induced maturation of moDCs. In particular, CD11c-CD3+ T cells appeared to play important roles in DC maturation. In addition, TNF-alpha was preferentially produced by CD11c-CD3+ T cells in IL-2/GM-CSF-cultured PBMCs and was involved in the maturation of moDCs. Thus, the maturation of DCs induced by CD11c- T cells through TNF-alpha production appears to result in the efficient expansion and activation of Valpha24+ NKT cells to produce IFN-gamma preferentially.

Migration of polymorphonuclear leucocytes is influenced by dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells and populate many tissues where they may participate in inflammatory reactions. The infiltration of polymorphonuclear leucocytes (PMNLs) into tissues is a prominent feature of inflammation. The mechanisms of PMNL recruitment depend on chemotactic factors and adhesion molecules expressed on endothelial cells. The aim of the present study was to determine whether DCs participate in the early recruitment of PMNLs. Dendritic cells derived from peripheral blood monocytes were used for this study. PMNLs incubated with culture supernatant (CS) from untreated or from tumour necrosis factor-alpha (TNF-alpha)-treated (1 hr, 100 U/ml, 37 degrees ) monocyte-derived DCs (moDCs) had increased surface expression of both CD11b and CD18. Moreover, both untreated and TNF-alpha-treated moDCs induced PMNL chemotaxis. By blocking CXCL8, CXCL5, CXCL7 and Pan GRO (CXCL1, CXCL2, CXCL3), we observed that CXCL8/interleukin-8 might be the chemokine that induced the PMNL chemotactic activity in the CS of untreated and TNF-alpha-treated moDC. Furthermore, we investigated the regulation of CXCL8 production in moDCs by adhesion molecule engagement. Our data demonstrated that CD31, CD18, CD29 and CD49d participated in the adhesion of immature moDCs to endothelium. Moreover, engagement of domains 1-3 of CD31, but not of CD29 or CD18, decreased the production of CXCL8 by immature but not mature moDCs (which display lower CD31 levels than immature moDCs). Overall, these results suggest that DCs not only trigger a specific immune response, but also the innate immune response by recruiting PMNLs. Furthermore, our results also suggest that CXCL8 production by immature DCs might be regulated by signalling through CD31 during their migration through the vascular endothelium.

Infection of mature monocyte-derived dendritic cells with human cytomegalovirus inhibits stimulation of T-cell proliferation via the release of soluble CD83

AbstractWe have studied the mechanisms by which human cytomegalovirus (HCMV) infection of monocyte-derived dendritic cells (moDCs) contribute to immune suppression. Unlike infection of immature moDCs, infection of mature moDCs is not lytic and results in minimally decreased surface major histocompatibility complex (MHC) and costimulatory molecule expression. The presence of a small percentage of CMV-infected mature moDCs, or the transfer of supernatant from infected moDCs depleted of infectious virions, is nevertheless sufficient to cause marked inhibition of immunostimulation by normal uninfected moDCs. Neither viral nor human interleukin 10 (IL-10) nor transforming growth factor-β-1 (TGF-β-1) could account for this inhibition. In contrast, we show that infected mature moDCs lose surface CD83 while maintaining intracellular protein expression. Soluble CD83 accumulates in the supernatants of CMV-infected mature moDCs, and CD83 immunodepletion removes the inhibitory effect of these supernatants on normal DC immunostimulation. We have thus discovered a new mechanism by which HCMV infection may establish a nonlytic reservoir in mature moDCs that inhibits DC-mediated T-cell responses. (Blood. 2004;103:4207-4215)

Phenotype, function and chimaerism of monocyte-derived blood dendritic cells after allogeneic haematopoietic stem cell transplantation.

Dendritic cells (DCs) are essential for initiating T-cell responses against either host- or leukaemia-specific antigens. We analysed phenotype, allostimulatory capacity and chimaerism of monocyte-derived DCs (moDCs) serially in 28 patients receiving allogeneic stem cell grafts after conventional myeloablative (n = 14) or reduced-intensity conditioning (RIC, n = 14). Although the recovery of phenotype and function of moDCs after myeloablative stem cell transplantation (SCT) was prompt, there was a trend to a lower expression of co-stimulatory molecules and major histocompatibility antigen class II antigens on mature moDCs in patients who had received RIC transplants. Similarly, the allostimulatory capacity of mature moDCs after RIC transplants was reduced for up to 6 months. Six out of 14 (43%) RIC transplant patients showed a pattern of mixed chimaerism within the first 3 months after transplant. RIC transplant patients with a mixed donor DC chimaerism had a significantly higher risk of relapse (75% versus 35% for patients with full donor DC chimaerism, P = 0.03) but a lower incidence of acute graft-versus-host disease (25% versus 56% for patients with full donor DC chimaerism, P = 0.157). These data, although preliminary, provide evidence that DC function is impaired after RIC transplants and that DC chimaerism may have an impact on graft-versus-host and graft-versus-leukaemia reactions.

Efficacy of dendritic cell generation for clinical use: recovery and purity of monocytes and mature dendritic cells after immunomagnetic sorting or adherence selection of CD14+ starting populations.

Immunotherapy with monocyte-derived dendritic cells (Mo-DCs) is applied to an increasing number of patients requiring large-scale production of clinical-grade dendritic cells with standardized Mo-DC generation protocols. In many countries, e.g., in Germany, Mo-DCs are legally considered medicinal products, which must be produced under Good Manufacturing Practice (GMP) conditions by an institution holding an official production license. Plastic adherence, immunomagnetic selection of CD14(+) monocytes and depletion of CD2(+) and CD19(+) cells are used to enrich monocytes for Mo-DC culture. The latter two have received approval of the European Union (CE). However, enrichment by plastic adherence is well-established and commonly used for clinical and research Mo-DC applications. The various plastic materials, nevertheless, have not been officially approved for monocyte selection for clinical use. In the present study therefore, we compared three methods for enrichment of CD14(+) monocytes with regard to efficiency of enrichment, yield of monocyte-derived functional mature dendritic cells, cost effectiveness, and handling. We demonstrate that CD14 selection and CD2 and CD19 depletion yield similar results regarding purity of mature DEs MoDCs (97-99% vs. 64-97%) and their immunostimulatory capacity. However, cell preparations cultured after CD14 selection possessed 91% to 97% CD14(+) cells, whereas CD2-/and DC19-depleted preparations contained only 8% to 57% CD14(+) cells. Thus, positive selection requires smaller culture volumes to generate equal numbers of Mo-DCs. Both methods gave better results than plastic adherence. In conclusion, of the techniques examined, CD14 selection of monocytes gave the best results regarding reproducibility, yield, and purity of the resulting monocytes and mature Mo-DCs.