J. Hinrich Peters

Dendritic cells: from ontogenetic orphans to myelomonocytic descendants

Abstract Although dendritic cells (DCs) and macrophages share a bone marrow origin, these cells were long assumed to differentiate via discrete pathways. DCs have now been clearly shown to develop from myeloid lineage precursors, and recent evidence suggests that they may even differentiate from blood monocytes. Here, J. Hinrich Peters and colleagues assess current knowledge on the myelomonocytic origin and successive differentiation of human T-cell-directed DCs.

Signals Required for Differentiating Dendritic Cells from Human Monocytes in Vitro

Human peripheral blood monocytes (Mo) can quantitatively be differentiated into potent accessory cells which exhibit dendritic cell (DC) function and phenotype. This alternative differentiation of Mo into DC rather than into macrophages (M phi) will be triggered when signals leading to M phi differentiation are omitted from the culture. Serum contains such stimulatory signals and was therefore omitted from the cultures. The cells were cultured on solid agarose surfaces. This newly developed technique allows for the attachment-free differentiation of DC. In the absence of signals, Mo do not survive in culture. IL-1 and IL-6 are endogenously produced by Mo and create an autokrine stimulatory milieu which increases the accessory function. However, also mature Mph will respond by an increased accessory activity upon stimulation by these cytokines. Cyclic AMP is the most likely second messenger to trigger an increase in accessory activity. IL-4 plus GM-CSF further act to upregulate dendritic cell properties and function. By action of these mediators, virtually all markers and functions of Mo/M phi are lost, and the cells convert to the phenotype and function of dendritic cells.

IL-4 Decreases the Expression of the Monocyte Differentiation Marker CD14, Paralleled by an Increasing Accessory Potency

IL-4 has been found to affect the phenotype and a variety of functions of human monocytes and macrophages and has been discussed as a monocyte activating protein along with other cytokines, such as IL-1 and IL-6. In this study we compared the effects of the cytokines IL-1, IL-6, IL-4, and a combination of IL-1 and IL-6 on the expression of the CD14 antigen, the FcIIIg receptor molecule CD16 and the MHC-class II molecules HLA-DR and HLA-DP. These molecules represent characteristic monocyte surface markers. Furthermore, the CD14 molecule has been described as a surface antigen of high in vivo relevance representing an indirect receptor for LPS. We further analyzed the effect of IL-4 on monocytes and macrophages with respect to their accessory function to initiate T-lymphocyte proliferation. Human peripheral blood monocytes strongly express the antigen CD14 and maintain it as a stable surface molecule during their differentiation to macrophages. Flow cytometry analysis of cultured monocytes demonstrated that cells incubated in the presence of IL-4, but not IL-1 and/or IL-6 revealed a reduced expression of the CD14 antigen in a dose- and time-dependent manner. After 3 days IL-4 treated cells were virtually CD14-negative. At the same time the expression of the CD16 antigen (FcRIIIg) was also strongly reduced, whereas the treatment with IL-4 led to an increased expression of MHC class II antigens such as HLA-DR and HLA-DP. The spontaneous low expression of HLA-DQ antigen on monocytes was not affected by any of the cytokines. Functionally, IL-4 treated CD14-negative monocytes exhibited a more than 2-fold higher activity to stimulate an accessory cell-dependent T cell proliferation. This was found in a mitogenic assay and in MLC when compared to monocytes cultured in the absence of IL-4. These observations provide further evidence that IL-4 is a major modulator of monocyte surface antigen expression. Moreover, IL-4 has an enhancer-effect on monocytes as accessory cells and therefore may be of considerable importance as a regulatory factor during monocyte development to accessory cells. Inasmuch as the CD14 molecule functions as a receptor for LPS-binding protein, our results suggest that IL-4 might also play an important regulatory role in processes initiated by bacterial lipopolysaccharides during inflammation and sepsis.

The treatment of patients with disseminated malignant melanoma by vaccination with autologous cell hybrids of tumor cells and dendritic cells.

Malignant melanoma has been shown to be susceptible to T cell-mediated immunity and, therefore, is a candidate for vaccination approaches. Clinical trials using dendritic cells (DC) loaded with peptides corresponding to tumor antigens are ongoing in several institutions, and some promising results have already been published. However, every single peptide-based vaccine can only be used in a patient with a given single HLA type, and this strategy is not appropriate for patients with rare HLA types or with tumors without defined antigens. A clinical pilot study in patients with disseminated melanoma refractory to standard therapy was initiated using a different approach. The authors generated autologous monocyte-derived DC and fused these DC with gamma-irradiated primary autologous tumor cells by incubation in polyethylene glycol. In previous experiments, the authors had shown that these fused cell products are potent inducers of a T-cell response in a mixed lymphocyte tumor cell culture. Seventeen patients were immunized with the cell product by s.c. injection in monthly intervals without any serious side effects. Of these patients, one had a partial response with decrease in size of all evaluable tumor manifestations. In one patient, some of the metastases were regressing despite an overall progressive disease, and one patient achieved disease stabilization for six months. In the responding patient, in parallel to tumor regression, circumscript hair depigmentation occurred. These data show, that a hybrid vaccine of DC and tumor cells can be safely applied and can induce tumor regressions, however, the clinical efficacy of the approach in its present form is insufficient.

Dendritic Cells Differentiated from Human Monocytes through a Combination of IL-4, GM-CSF and IFN-γ Exhibit Phenotype and Function of Blood Dendritic Cells

Our previous studies demonstrated that monocytes, when cultured under certain conditions, are able to differentiate into DC-like cells (MoDC) presenting a high accessory activity and low phagocytic function. In the present study, we demonstrate that under the effect of a triple combination of IL-4, IFN-gamma and GM-CSF human blood monocytes are able to differentiate into the cells expressing an identical phenotype and functional features of blood dendritic cells. MoDC stimulated T cell proliferation 20-30 times higher than untreated monocytes, similar to blood DC. They expressed abundant HLA-DR molecules, but only trace amounts of the monocyte/macrophage markers CD16 (FcR III), CD32 (FcR II), and CD14. Phagocytosis of Ig- and complement-opsonized bacteria was reduced by 93%.

Down-regulation and release of CD14 on human monocytes by IL-4 depends on the presence of serum or GM-CSF.

IL-4 induces down-regulation of CD14 expression on human monocytes only when the cells are cultured with serum. In serum-free cultures we failed to down-regulate CD14 by IL-4. Instead of serum, GM-CSF was required as a co-factor to restore the regulatory effect of IL-4 on CD14-expression. After 4 days of culture human monocytes were quantitatively CD14-negative as determined by flow-cytometry. On day 6, high amounts of CD14 molecules were detected in the SUP of these cultures, whereas intracellular immunofluorescence staining revealed no detectable CD14 in cytokine-treated monocytes. Thus, CD14 is lost by down-regulation (as shown by others) as well as by delivery into the medium. We previously hypothesized that dendritic cells may originate from monocytes. Our present finding support that one of the key markers, distinguishing monocytes/macrophages from dendritic cells, can be lost upon physiological stimuli.

Alkaline phosphatase expression during monocyte differentiation. Overlapping markers as a link between monocytic cells, dendritic cells, osteoclasts and osteoblasts.

Human monocytes (Mo) in culture can be differentiated into macrophages (M phi), dendritic cells (DC) and osteoclasts. In addition, we have established a Mo-derived in vitro granuloma model which here was compared with ex-vivo isolated foreign body granuloma cells. In these models overlapping phenotypes developed between monocyte-derived dendritic cells (MoDC), osteoclasts, M phi, and osteoblasts. In Mo cultures granulomas were induced by immobilized particulate material. AP activity (osteoblast marker) was found to be co-expressed with cytoplasmic tartrate resistant acid phosphatase (TRAP) as a marker of osteoclasts. While proliferating, the number of AP+ cells decreased, being replaced by cells co-expressing the osteoclast markers vitronectin receptor (VNR) and TRAP. Coexpression of the Mo/M phi marker CD68 with AP or VNR confirmed the monocytic origin of the cells. When Mo were treated with interleukin-4 (IL-4), the number of AP+ cells markedly increased and remained stably expressed over 12 days. In explants from ex vivo granulomas obtained from endoprosthetic revisions the major cell type was the AP+ cell co-expressing CD68. The bone-specific alkaline phosphatase (BAP) as a marker of osteoblasts was detected by FACS analysis in the ex vivo granuloma cells. By RT-PCR the mRNA for osteocalcin, which is a highly specific marker for osteoblasts, was detected. From our results we conclude an ontogenetic relationship between macrophages, DC and osteoclasts. Furthermore, the data suggest a transdifferentiation between Mo and osteoblasts.

In-vitro differentiation of mature dendritic cells from human blood monocytes.

Representing the most potent antigen-presenting cells, dendritic cells (DC) can now be generated from human blood monocytes. We recently presented a novel protocol employing GM-CSF, IL-4, and IFN-γ to differentiate monocyte-derived DC in vitro. Here, such cells are characterized in detail. Cells in culture exhibited both dendritic and veiled morphologies, the former being adherent and the latter suspended. Phenotypically, they were CD1a-/dim, CD11a+, CD11b++, CD11c+, CD14dim/-, CD16a-/dim, CD18+, CD32dim/-, CD33+, CD40+, CD45R0+, CD50+, CD54+, CD64-/dim, CD68+, CD71+, CD80dim, CD86+/++, MHC class I++/+++ HLA-DR++/+++ HLA-DP+, and HLA-DQ+. The DC stimulated a strong allogeneic T-cell response, and further evidence for their autologous antigen-specific stimulation is discussed. Although resembling a mature CD 11c+CD45R0+ blood DC subset identified earlier, their differentiation in the presence of the Thl and Th2 cytokines IFN-γ and IL-4 indicates that these DC may conform to mature mucosal DC.

Ex vivo generation of human anti-melanoma autologous cytolytic T cells by dendritic cell/melanoma cell hybridomas

Abstract Due to their central role in controlling immunity, dendritic cells are logical targets for priming naive cytotoxic T lymphocytes against tumour cells. In a strictly autologous system, we fused dendritic cells with melanoma cells, both of which were derived from patients with metastatic malignant melanoma. Hybridomas were positive for major histocompatibility complex (MHC) class II, CD40, CD54, CD83, CD86, and the pro-inflammatory cytokine interleukin-12. Autologous T lymphocytes were co-incubated with hybridomas. After 6 days, in-vitro-primed T lymphocytes revealed a strong proliferation activity and released Th-1-associated, but not Th-2-associated, cytokines. Furthermore they showed effective anti-melanoma activity, resulting in death of 70  ±  9% of autologous melanoma cells. After depletion of CD4+ cells from the mixed population of primed T lymphocytes, the remaining CD8+ cells were able to kill 63  ±  8% of autologous melanoma cells. Following depletion of CD8+ cells, however, the cytotoxic capacity of the remaining T lymphocytes caused death in only 32  ±  6% of autologous melanoma cells. Blocking of MHC class I, but not class II, molecules on hybridomas impaired T cell proliferation, secretion of Th-1-associated cytokines, as well as the cytotoxic activity of primed T cells. These findings strongly suggest that hybridomas deliver melanoma-associated antigens via MHC class I molecules to T lymphocytes, resulting in the generation of CD8+ cytotoxic T lymphocytes with effective anti-melanoma activity in vitro. The data may serve as a basis for the use of hybridomas in the immunotherapy of malignant melanoma in vivo.

Specific Autologous Anti-Melanoma T Cell Response in vitro Using Monocyte-Derived Dendritic Cells

Dendritic cells (DC) are antigen-presenting cells initiating primary and secondary immune responses. Since malignant tumors are able to escape immunologic control, DC might be prime candidates to activate the immune system against tumor cells. In an autologous system, a dynamic interaction among monocyte-derived DC (MoDC), T lymphocytes, and tumor cells obtained from melanoma patients could be noted. MoDC were generated from blood monocytes in the presence of GM-CSF, IL-4, and IFN-gamma. T cells were isolated either from peripheral blood or from lymph nodes. Melanoma cells were harvested from surgically removed tumor metastases. They were then gamma-irradiated and co-cultured with autologous MoDC and T lymphocytes. After 5 days, the lymphocytes showed a high proliferative activity and the majority of them were CD8-positive. In five cases tested, they revealed a high cytotoxic activity resulting in apoptosis of tumor cells. These findings suggest that MoDC are capable of initiating an effective specific anti-tumor response in a strictly autologous mixed lymphocyte tumor culture (MLTC), even though tumor-specific antigens had not been individually defined. Therefore (I) whole melanoma cells can serve as a source of antigen, (II) monocyte-derived dendritic cells may process and present melanoma-specific antigens resulting in a strong lymphocyte proliferation, (III) the majority of responding T lymphocytes are CD8-positive, and (IV) an acquired cytotoxic response eventually leads to apoptosis of the melanoma cells. The reaction demonstrated here permits to in vitro and quantitatively monitoring the effect of T cell directed immunotherapies such as the adoptive immunotherapy of tumors.