Functional Characteristics Of Dendritic Cells Research Articles

Dendritic Cells and Myeloid Derived Suppressor Cells Fully Responsive to Stimulation via Toll-Like Receptor 4 Are Rapidly Induced from Bone-Marrow Cells by Granulocyte-Macrophage Colony-Stimulating Factor

Dendritic cells (DCs) are commonly generated from bone marrow (BM) progenitor cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or in combination with interleukin 4 (IL-4). These cells are often harvested post day 5, when they acquire maturation markers and can stimulate T cells. Apart from DCs, myeloid derived suppressor cells (MDSCs) are also found within these cultures. However, little is known about the functional characteristics of DCs and MDSCs before day 5. Herein, using a murine model, it is shown that early DCs and MDSCs, even in cultures with GM-CSF alone, upregulate fully maturation and activation surface molecules in response to the toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) stimulation. Despite initially displaying lower marker expression levels, these cells efficiently induced T cell stimulation and cytokine production. Interestingly, Gr-1int MDSCs increased their T cell co-stimulatory activity upon TLR4 stimulation. Additionally, early DCs and MDSCs exhibited differential endocytic capacity for viral sized nanoparticles and bacterial sized microparticles. DCs internalized both particle sizes, whilst MDSCs only internalized the larger microparticles, with reduced endocytic activity over time in the culture. These findings have unveiled an important role for the rapid initiation of productive immunity by GM-CSF, with promising implications for future vaccine and DC immunotherapy developments.

THE ROLE OF INTRATUMORAL DENDRITIC CELLS IN THE PROGRESSION OF SQUAMOUS CELL CARCINOMAS

The aim of the studywas to summarize data on the role of tumor-associated dendritic cells (DC) in the formation of squamous cell carcinoma microenvironment, their participation in the development of immune inflammatory responses in the tumor stroma and relation to tumor progression.Material and Methods. We analyzed 79 publications available from Pubmed, Google Scholar, Elibrary databases from January 2000 to December 2017.Results. The characteristics of different types of DC, including Langerhans cells (CR), were presented. The different methods of DC identification were described. The information on the presence of DC in squamous cell carcinomas was analyzed. The influence of the tumor on DCs, as well as the relationship between the number and functional characteristics of DCs and invasive/metastatic tumor potentialities was described. The prognostic value of DCs and their effect on disease-free, metastasis-free and overall survival rates were analyzed. The data on the association between DCs and the response to chemoradiotherapy were presented. The analysis of the relationship between the DC characteristics and the development of immuno-inflammatory responses in the tumor microenvironment was carried out.Conclusion. The methodological approaches to the detection of DCs are variable, but the sensitivity of each method, as well as the comparison of different methods for estimating the number and functional characteristics of DCs, have been little studied. There is no data on the relationship between the length of DC dendrites and the parameters of invasive/metastatic tumor potentialities, disease-free, metastasis-free and overall survival rates. Numerous studies indicate the association between the number of DCs and the tumor progression, however these data are contradictory. There is no data about the relationship between the number of DCs and hematogenous metastasis of squamous cell carcinomas. The association of tumor-associated DC with the types of immunoinflammatory responses in the tumor microenvironment has been insufficiently studied.

The Effects of Immunosuppressive Factors on Primary Dendritic Cells from C57BL/6 and CBA Mice.

Introduction Dendritic cells (DCs) control immune responses by modulating T and B cells towards effector or tolerogenic responses. In this study, we evaluated the effects of different immunosuppressive molecules on the phenotypic and functional characteristics of primary dendritic cells from C57BL/6 and CBA mice. Methods DCs were derived from bone marrow cells in the presence of rmGM-CSF and rmIL-4. DCs were then treated with different types of immunosuppressive molecules (rmIL-10, rmTGF-β, and BAY 11-7082) and cocultured with syngeneic splenocytes. The amount of CD4+CD25hiFoxP3+ Tregs, IL-10 expression, and proliferation were evaluated. Results Tolerogenic factors were found to have different effects on DCs C57Bl/6 mice. In C57Bl/6 mice, BAY 11-7082 alone had no effect on the expression of DC maturation molecules (CD80, CD86). Transforming growth factor beta (TGF-β), alone and in combination with BAY 11-7082, reduced the expression of these molecules. Cocultivation of DCs with splenocytes in the presence of TGF-β and BAY 11-7082 favored regulatory T cell (CD4+CD25hiFoxP3+) differentiation and disfavored differentiation of CD4+ T cells producing IL-10. In CBA mice, we found that rmIL-10 and rmTGF-β have a weak effect on maturation of DCs and their functional properties to induce Treg cells and IL-10 production. Conclusion These results indicate that TGF-β and IL-10 have different effects on the phenotypic and functional characteristics of DCs and that the NF-κB inhibitor, BAY 11-7082, has no synergistic effect on these treatments. In mice with an opposite nature of the immune response, the effects of immunoregulatory cytokines (IL-10 and TGF-b) differ on maturation of dendritic cells.

Phenotypical and functional properties of generated dendritic cells in lung cancer patients

This study aimed to investigate phenotypical and functional characteristics of dendritic cells (DCs) generated from monocytes of peripheral blood of healthy donors and cancer patients.Material and methods. DCs were used as natural adjuvants with antitumor vaccine as a part of combined treatment scheme for lung cancer patients. Phenotypical and functional characteristics of DCs were study using flow cytometry and real-time PCR.Results. We have found that in lung cancer patients generated DCs had moderate level of maturity and demonstrated more pronounced tolerogenic features in contrast to DCs of healthy donors (patients DCs had higher mRNA expression levels of suppressive molecules TGF-β and IDO, and secreted lower amount of bioactive IL-12 protein). Expression of CCR7 gene was particularly on the normal level in DCs of cancer patients which indicates on saving of migratory properties of these cells. Expression level of DC maturity marker CD83 increased after each subsequent vaccine administration, while the levels of TGF-β, IL-10 mRNAs to the end of vaccine therapy course decreased to the level observed in healthy donors DCs.Conclusion. Thus, the study of biological characteristics of DCs will help to improve and develop the most effective protocols for rational use of DC vaccines. These data indicate the need for further optimization of technologies of DC generation in patients with lung cancer with emphasis on the stimulation of Th1-polarizing properties by increasing cytokine-secreting potential.

AB0450 Myeloid Dendritic Cells and Plasmacytoid Dendritic Cells as Possible Target for the Immunotherapy in Patients with Rheumatoid Arthritis

BackgroundRecently, dendritic cells (DC) are considered as an object to create a new therapeutic strategy in rheumatoid arthritis (RA), since they are involved in all stages of the inflammatory process....

Regulatory T cells form stable and long-lasting cell cluster with myeloid dendritic cells (DC)

Regulatory T cells (Treg) with the capacity to suppress T-cell proliferation exert various effects on T cell function. In addition, Treg have been shown to modulate the phenotype and function of antigen-presenting cells (APC) including dendritic cells (DC), B cells and monocytes/macrophages. However, the specific mechanism(s) of how Treg affect APC have not been entirely identified so far. In this study, we analyzed the interaction of human Treg and effector T cells (Teff) with peripheral blood myeloid and monocyte-derived dendritic cells in vitro. A strong tendency for cell cluster formation between Treg and DC was observed, which was dependent on the adhesion molecules ICAM-1, LFA-3 and ICAM-3. In addition, Treg were found to express higher levels of LFA-1, LFA-2, LFA-3 and ICAM-3 both before and after activation with anti-CD3 antibodies. Using in vitro live cell imaging, we were further able to show that Treg-DC cell clusters, in contrast to Teff-DC clusters, were stable and long lasting. Co-cultures of DC with Treg diminished the up-regulation of activation induced costimulatory molecule expression on DC, and further reduced the production of tumor necrosis factor alpha and stimulated the production of IL-4. In summary, our data indicate that Treg-DC cluster formation might enable Treg to modulate phenotypic and functional characteristics of DC and help to constrain Teff activation.

Immunomodulatory Effects of Vitamin D: Implications for the Prevention and Treatment of Graft Versus Host Disease

Allogeneic bone marrow transplantation is a uniquely curative therapy for a subset of patients with hematologic malignancies. However, morbidity and mortality related to graft versus host disease remain substantial. Dendritic cells (DCs) are highly potent antigen presenting cells that play a crucial role in maintaining the balance of immune activation and tolerance as well as the nature of immune reconstitution post-transplant. Persistence of host DCs in the early post transplant period has been shown to play an important role in the activation and expansion of alloreactive lymphocytes and the concomitant risk of GVHD. A major focus of research to enhance outcomes following allogeneic transplantation involves the manipulation of DC recovery post-transplant to minimize activation of alloreactive lymphocytes while preserving anti-tumor immunity responsible for the graft versus disease effect. Vitamin D is a hormone involved in bone metabolism and calcium homeostasis. More recently, vitamin D has been shown to have potent immunomodulatory effects. We have evaluated the effect of vitamin D on the phenotypic and functional characteristics of DC and T cell populations. We have demonstrated that vitamin D hinders the maturation of DCs such that cells that are differentiated in its presence exhibit decreased expression of costimulatory molecules. Peripheral blood mononuclear cells (PBMCs) were isolated from leukopaks obtained from normal donors by ficoll density centrifugation. DCs were generated by culturing the monocyte enriched adherent fraction with GM-CSF (1000 U/ml) and IL-4 (1000 U/ml) for 5 days, followed by maturation in the presence of TNFa (25 ng/ml) for 48 hours. DCs were generated in the presence and absence of 10nM of 1,25 hydroxyvitamin D. Mean expression of the costimulatory molecule CD80 and the maturation marker CD83 decreased from 60% to 37% and 53% to 27% respectively in the presence of vitamin D (N=3). To assess the effect of vitamin D on the functional potency of DCs as antigen presenting cells, the capacity of mature DCs to stimulate allogeneic T cell proliferation in the presence of vitamin D was determined. Mature DCs were generated from peripheral blood precursors and then cultured with allogeneic T cells isolated by T cell separation column at a ratio of 1:10. After 5 days, cocultures were pulsed with tritiated thymidine overnight and proliferation was determined by measuring the stimulation index (SI) as defined by uptake of tritiated thymidine of cocultured cells/uptake of tritiated thymidine of T cells alone. Results of 10 serial studies demonstrated the addition of vitamin D resulted in a blunted T cell proliferative response, with mean SI that decreased from 13 to 5. Similarly, the addition of vitamin D to a coculture of DCs and autologous T cells resulted in a 50% reduction in the T cell proliferative response to tetanus toxoid, a recall antigen. In addition, T cells stimulated by allogeneic DCs in the presence of vitamin D were polarized to secrete Th2 cytokines. In three experiments, the mean percentage of T cells secreting IL-10 increased from 2.7% to 4.4%, while expression of IFNγ decreased from a mean of 2.6% to 1.9% in the presence of vitamin D. The presence of vitamin D did not induce FOXP3 expressing regulatory T cell populations, which accounted for 8% and 7% of the T cell population following stimulation by allogeneic DCs in the presence and absence of vitamin D respectively. These data suggest that exposure to vitamin D exerts a tolerizing influence on T cells mediated by its impact on antigen presenting cells. Vitamin D may therefore have a role in the prevention and treatment of graft versus host disease. A clinical trial evaluating the use of vitamin D in the early post-transplant period for the prevention of GVHD is planned.

Adaptive functional differentiation of dendritic cells: integrating the network of extra- and intracellular signals

AbstractPhenotypic maturation, cytokine secretion, and migration are distinct functional characteristics of dendritic cells (DCs). These functions are independently regulated by a number of extracellular variables, such as type, strength, and persistence of an array of soluble and membrane-bound mediators. Since the exact composition of these variables in response to infection may differ between individuals, the intracellular signaling pathways activated by these extracellular networks may more closely correlate with DC function and predict the course of adaptive immunity. We found that activation of p38 kinase (p38K), extracellular signal–related kinase 1/2 (ERK1/2), and phosphatidylcholine-specific phospholipase C (PC-PLC) enhanced cytokine secretion, whereas p38K, cyclic adenosine monophosphate (cAMP), and PC-PLC enhanced migration. In contrast, phosphatidylinositol 3-kinase (PI3K)/Akt-1 and cAMP inhibited cytokine secretion while ERK1/2 inhibited migration. Migration and cytokine secretion further differed in their sensitivity to inhibition over time. However, although DCs could be manipulated to express migration, cytokine secretion, or both, the level of activation or persistence of intracellular pathway signaling was not predictive. Our results suggest a modular organization of function. We hypothesize that the expression of specific DC functions integrates a large variety of activating and inhibitory variables, and is represented by the formation of a functional unit of molecular networks—the signal response module (SRM). The combined activities of these modules define the functional outcome of DC activation.

Phenotypic and functional characteristics of dendritic cells derived from human peripheral blood monocytes

This study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro. PBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFalpha) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFalpha or lipopolysaccharide (LPS) stimulations for 24 h. After six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD1a, CD80 and CD86, features of DCs. TNFalpha treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity. This study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.

Dendritic cells acquire tolerogenic properties at the site of sterile granulomatous inflammation

Subcutaneous implantation of polyvinyl sponges represents a suitable model for studying the mechanisms of acute and chronic inflammation, granulomatous foreign-body reaction, as well as wound healing. Using such a model in rats, we studied the phenotypic and functional characteristics of dendritic cells (DC). DC were purified from the sponge exudate using a combination of separation gradients, adherence to plastics, and immunomagnetic sorting. We have shown that the number of DC progressively increased in the sponges, reaching maximal values at day 10 after implantation, followed by their decrease thereafter. Inflammatory DC expressed MHC class II molecules and myeloid markers CD11b, CD11c, and CD68. A subset of DC expressed CD4, R-MC46, DEC-205, R-MC17, and CCR1. Compared to DC isolated in the early phase of inflammation (day 6 DC), DC in the late stage of inflammation (day 14 DC) had a lower capability to stimulate the proliferation of allogeneic lymphocytes and CD4+ T cells. This finding correlated with the downregulation of CD80, CD86, and CD54 expression and the increased proportion of plasmacytoid MHC class II+ His 24+ His 48+ DC. The suppression of allogeneic lymphocyte proliferation was abrogated by the treatment of DC with lipopolysaccharide. In addition, day 14 DC exerted tolerogenic capability in co-culture with allogenic CD4+ T cells. These results correlated with the increased levels of IL-10 and TGF-β in culture supernatants and the sponge exudate.

Transdifferentiation of polymorphonuclear neutrophils: acquisition of CD83 and other functional characteristics of dendritic cells.

Polymorphonuclear neutrophils (PMN) are in the first line of defense against bacterial infections. They are considered to be end-differentiated cells undergoing constitutive apoptosis within hours after release from the bone marrow. During pathological events, however, their life span is extended in conjunction with morphological and functional alterations indicative of a transdifferentiation of mature PMN. To further characterize differentiated PMN, the alterations seen in vivo were reproduced by cultivating PMN of healthy donors with either gamma-interferon, granulocyte/macrophage colony stimulating factor, or a combination thereof. Thus cultivated cells escaped from apoptosis, and protein synthesis was induced, notably of the major histocompatibility complex (MHC) class II antigens, CD80 and CD86. Moreover, CD83, thought to be specific for dendritic cells was synthesized, while typical markers of PMN, including CD66b, CD11a/CD11b/CD11c, CD15, CD18 were preserved. A profound alteration of both cellular morphology and of function was seen: the cultivated PMN lost their chemotactic activity but had acquired the ability to present to T-cells a peptide antigen in a MHC class II restricted manner. The data lead to the conclusion that mature PMN can differentiate further to cells with characteristics of DCs, thereby connecting PMN to the specific T-cell response.

Development of mouse dendritic cells from lineage-negative c-kit(low) pluripotent hemopoietic stem cells in vitro.

Dendritic cells (DCs) are essential for the presentation of antigens in the primary immune response. To examine the generation of DCs from hemopoietic stem cells in the bone marrow (BM), lineage-negative (Lin-)/CD71- bone marrow cells (BMCs) from C57BL/6 mice were separated into major histocompatibility complex (MHC) class Ihigh/ c-kit(low) and MHC class Ihigh/c-kit(low)(phenotypically c-kit-negative, but c-kit message only detected by reverse transcriptase-polymerase chain reaction) populations. A large number of cells with the morphological, phenotypical, and functional characteristics of DCs was generated from both c-kit(low) and c-kit(low) populations when cultured with a combination of cytokines (GM-CSF, tumor necrosis factor-a [TNF-a], interleukin 7 [IL-7], IL-3, stem cell factor [SCF], and flt3 ligand); the cytokine combination studies revealed that SCF and IL-3 in addition to GM-CSF and TNF-a are essential for DCs to be generated from these primitive populations. To our surprise most (>80%) generated cells expressed high levels of DC surface markers such as DEC205 and MHC class II, and they were potent stimulators in the primary allogeneic T cell activation. The development of DCs from c-kit(<low) cells was slower than that from c-kit(low) cells. These results indicate that c-kit(<low) cells are more primitive than c-kit(low) cells, although both c-kit*(low) cells and c-kit(<low) cells can differentiate into DCs. It should be noted that the combination of these cytokines selectively induces DCs from both c-kit(<low) and c-kit(low) cells in vitro, suggesting that the ex vivo expansion of DCs using these primitive cells would be applicable to immunotherapy.

Phenotypic and functional characteristics of monocyte-derived dendritic cells from patients with myelodysplastic syndromes.

We have compared the phenotypic and functional characteristics of dendritic cells (DC) generated in vitro from the peripheral blood mononuclear fraction of myelodysplastic syndrome (MDS) patients (four refractory anaemia, four refractory anaemia with excess of blasts) with DCs generated in a similar way from eight healthy donors. After 10 d of culture in the presence of GM-CSF and IL-4, reduced numbers and percentages of DCs were obtained in MDS subjects. MDS DCs exhibited significantly lower expression of CD1a, CD54, CD80 and MHC class II molecules. Their ability to stimulate T lymphocytes in an allogeneic mixed leucocyte reaction was reduced in comparison to normal subjects. Furthermore, MDS DCs also showed a reduced receptor-mediated endocytosis as demonstrated by FITC-dextran uptake. Simultaneous fluorescence in situ hybridization (FISH) and immunophenotypic analysis demonstrated that MDS DCs have the same cytogenetic abnormality of the malignant clone. Taken together these findings indicate that, in MDS, DCs are part of the malignant clone and exhibit a deficient antigen uptake and presentation.

Effects of UV on the migration and function of epidermal antigen presenting cells

Depletion of epidermal Langerhans cells (LC) and the concomitant depression of the skin immune system after excessive exposure to ultraviolet B light (UVB) has been established in the international literature for some time. Our investigations were intended to determine whether or not these phenomena occurred as a direct result of increased LC migration being triggered by the UVB exposure. To test this hypothesis, a sheep model was established in which the lymphatic vessels draining a defined region of skin were cannulated and the cells migrating towards the regional lymph node continuously collected. Cell populations in these collections were identified and enumerated by indirect immunofluorescence and flow cytometry. These experiments showed there was a significant, dose-dependent increase in the rate of LC migration from sheep skin after exposure to doses of UVB light exceeding 1 minimal erythemal dose (MED). In a series of parallel experiments, the functional characteristics of dendritic cells (DC) migrating from normal or UVB irradiated sheep were studied. To assay them, enriched preparations of DC were collected via cannulated afferent lymphatic vessels and pulsed with antigen prior to incubation with autologous peripheral blood lymphocytes. The relative efficiency of antigen presentation was determined by the ability of DC to induce T cell proliferation. Our data clearly demonstrate that there is a profound loss of normal antigen-presenting cell function after exposure to UVB light. Various experiments were undertaken to determine the mechanism(s) associated with these changes in migration kinetics and cellular function. Electron microscopic examinations of LC migrating from normal or UVB irradiated skin have demonstrated a profound loss of dendritic processes after UVB exposure. This provides a possible explanation for the changes in skin immunity after UVB exposure.

MHC class II and invariant chain biosynthesis and transport during maturation of human precursor dendritic cells.

Dendritic cells (DC) are highly potent activators of the immune response. The precise mechanisms that give rise to the DC phenotype are not known. To investigate the mechanisms that contribute to the generation of the DC phenotype, precursor DC were freshly isolated from human blood and allowed to mature in vitro. These matured DC showed the phenotypical and functional characteristics of DC. Analysis of the MHC class II and invariant chain (li) biosynthesis revealed that upon maturation, class II synthesis was induced whereas li synthesis was significantly up-regulated. In mature DC, despite the presence of large amounts of li, export of MHC class II molecules from the endoplasmic reticulum was incomplete, up to 4 h after biosynthesis. Thus, MHC class II-li synthesis and transport in DC is highly regulated during maturation of DC. Analysis of the regulatory mechanisms may contribute to a better understanding of antigen-presenting capacities during the differentiation of DC.

Generation of dendritic cells from bone marrow progenitors using GM-CSF, TNF-alpha, and additional cytokines: antagonistic effects of IL-4 and IFN-gamma and selective involvement of TNF-alpha receptor-1.

We report the generation of dendritic cells (DC) starting from CD34+ bone marrow (BM) progenitor cells, using a two-stage culture system in which, besides granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha), stem-cell factor (SCF) was added during the first 5 days, while interleukin-4 (IL-4) and/or interferon-gamma (IFN-gamma) were added during the secondary culture period of 9 days. Addition of IL-4 favoured the outgrowth of CD1a+, HLA-DR+, CD4+, CD40+, CD80+ but CD14- cells with dendritic morphology and strong antigen-presenting capacity. Addition of IFN-gamma selectively induced HLA-DR and CD86 but did not up-regulate CD1a expression or antigen-presenting capacity of the differentiated cells. An antagonism between IL-4 and IFN-gamma could further be confirmed in that, as compared with IL-4 alone, the simultaneous addition of IL-4 and IFN-gamma to GM-CSF plus TNF-alpha during maturation reduced both the phenotypical (CD1a, CD4, CD40) and functional characteristics of DC. Using receptor-specific TNF-alpha mutants, we investigated the relative involvement of TNF-alpha receptors R1 and R2 in the generation of DC. The induction of CD1a and HLA-DR, as well as the increase in allostimulatory capacity were dependent on TNF-R1 triggering, whereas triggering through TNF-R2 had no measurable effect. We conclude first, that the expansion of DC from BM progenitors could most effectively be enhanced in a two-stage culture assay using SCF, GM-CSF, TNF-alpha and IL-4; second, that the effect of TNF-alpha in DC generation involves signalling via the TNF-R1 receptor; and third, that IFN-gamma counteracts some of the effects of IL-4 in DC generation.

Molecular and functional characteristics of dendritic cells generated from highly purified CD14+ peripheral blood monocytes.

Dendritic cells (DC) are the most potent APCs within the immune system. We show here that highly purified CD14(bright) peripheral blood monocytes supplemented with granulocyte-monocyte (GM)-CSF plus IL-4 develop with high efficacy (>95% of input cells) into DC. They neo-expressed CD1a, CD1b, CD1c, CD80, and CD5; they massively up-regulated CD40 (109-fold) and HLA-DQ and DP (125- and 87-fold); and significantly (>5-fold) up-regulated HLA-DR, CD4, CD11b, CD11c, CD43, CD45, CD45R0, CD54, CD58, and CD59. CD14, CD15s, CD64, and CDw65 molecules were down-regulated to background levels, and no major changes were observed for HLA class I, CD11a, CD32, CD33, CD48, CD50, CD86, CDw92, CD93, or CD97. Monocytes cultured in parallel with GM-CSF plus TNF-alpha were more heterogeneous in expression densities but otherwise similar in their surface molecule repertoire. They clearly differed, however, in their accessory cell capacity. Only GM-CSF plus IL-4-cultured cells were found to be potent stimulators in allogeneic and autologous MLR and they presented tetanus toxoid 100- to 1000-fold more efficiently than other cell populations tested. Furthermore, only cytokine-treated monocytes formed clusters with resting T cells. At variance from all these similarities between in vitro-generated monocyte-derived DC and in vivo-developing DC, the DC populations generated by us contained significant amounts of myeloperoxidase and also expressed lysozyme. At least in this respect they, thus, differ from "classical" DC types.