We have demonstrated recently that Birbeck granule-positive Langerhans cells (LC) can be derived from CD34+ peripheral blood progenitor cells in the presence of a seven-cytokine cocktail (CC7–7). Here, we show that the sequential use of early-acting hematopoietic growth factors, stem cell factor, interleukin (IL)-3, and IL-6, followed on day 8 by differentiation in the two-factor combination IL-4 plus granulocytemacrophage colony-stimulating factor (GM-CSF) (CC4GM) is more efficient and allows the cells to be arrested in the LC stage for more than 1 week while continuous maturation occurs in CC7–7. Maturation of LC to interdigitating dendritic cells (DC) could specifically be induced within 60 hours by addition of tumor necrosis factor-alpha (20 ng/mL) or lipopolysaccharide (100 ng/mL). Using LC that had been enriched to greater than 90% CD1a+ cells by an immunoaffinity column, we were able to define clear-cut differences between LC and DC that corroborate data of the respective cells derived from epithelial borders (LC) or from lymph nodes (LN) and spleen (DC). Thus, molecules and functions involved in antigen (AG) uptake and processing were highly expressed in LC, while those involved in AG presentation were at maximum in DC. LC were CD1a+2 DR+2, CD23+, CD36+, CD80-, CD86-, and CD25-, while DC were CD1a+/- DR+3, CD23-, CD36-, CD80+, CD86+2, and CD25+, CD40 and CD32 were moderately expressed and nearly unchanged on maturation, in contrast to monocyte-derived DC. Macropinocytosis of fluorescein isothiocyanate-dextran was dominant in LC, as were multilamellar major histocompatibility complex (MHC) class II compartments (MIICs), which were detected by electron microscopy. The functional dichotomy of these cell types was finally supported by testing the AG-presenting cell function for tetanus toxoid to primed autologous T-cell lines, which was optimal when cells were loaded with AG as LC and subsequently induced to become DC.

Development of cell-based delivery systems that can release therapeutic levels of hematopoietic growth factors into the systemic circulation would facilitate treatment of patients requiring cytokine therapy. In this study, we have investigated the potential of granulocyte- macrophage colony-stimulating factor (GM-CSF)-secreting, irradiated syngeneic murine cells to accelerate hematopoietic recovery after cytotoxic chemotherapy. As a model, CMS-5 fibrosarcoma cells, were transduced with a retroviral vector containing the murine GM-CSF cDNA. Transduced cells secreted 38 ng GM-CSF/10(6) cells in 24 hours. After irradiation, in vitro GM-CSF production initially increased up to fivefold and was measurable for about 2 weeks. One and 2 days after injection of irradiated, GM-CSF-secreting CMS-5 cells (N2/CMVGM- CSF/CMS5 # 6 cells) into mice, GM-CSF serum levels of 405 +/- 58 pg/mL and 183 +/- 36 pg/mL were measured, respectively. Serum levels were comparable with levels detected 3 hours after injection of 100 ng recombinant murine GM-CSF (rmGM-CSF) subcutaneously (90 pg/mL). Injection of N2/CMVGM-CSF/CMS5 # 6 cells in cyclophosphamide-treated mice was as effective in accelerating neutrophil recovery as twice daily subcutaneous injections of rmGM-CSF. These data suggest that irradiated hematopoietic growth factor-secreting cells might offer an alternative to parenteral injections of recombinant cytokines in the treatment of neutropenic patients.

Therapeutic models using gene transfer into tumor cells have pursued three objectives: (1) to induce rejection of the tumor transduced with therapeutic genes, (2) to induce immune-mediated regression of metastatic disease, and (3) to induce long-lasting immunity to protect against challenge with tumor cells or clinical regrowth of micrometastatic disease. Because in vivo therapy for patients with cancer using gene transfer would, as a first step, attempt to eliminate the existing tumor, we have investigated whether antitumor immunity induced by tumor cells secreting a single cytokine could be increased by cotransfer of a second cytokine gene. To test this approach, CMS-5, a murine fibrosarcoma, was transduced with retroviral vectors carrying interleukin-2 (IL-2), interferon-gamma (IFN-gamma), or granulocyte- macrophage-colony-stimulating factor (GM-CSF) cDNA alone or IL-2 cDNA in combination with IFN-gamma or GM-CSF cDNA. Single cytokine-secreting clones were selected to match levels of cytokine production by double cytokine-secreting clones so that similar amounts of cytokine were secreted. IFN-gamma- and IL-2/IFN-gamma-secreting CMS-5 cells showed increased levels of major histocompatability complex class I expression compared with IL-2- and GM-CSF-secreting or parental CMS-5 cells, IL- 2/IFN-gamma-secreting CMS-5 cells were always rejected by syngeneic mice, whereas the same number of CMS-5 cells secreting only one of these cytokines or mixtures of single cytokine-secreting CMS-5 cells were not rejected. In vivo depletion of CD4+, CD8+, or natural-killer effector cell subpopulations showed that CD8+ cytotoxic T cells were primarily responsible for rejection of IL-2/IFN-gamma-transduced tumor cells. Our data show the successful use of a single retroviral vector to stably transduce two cytokine genes into the same tumor cell, leading to an increased effect on the in vivo induction of antitumor immunity.

It is well established by in vivo and in vitro studies that dendritic cells (DCs) originate from hematopoietic progenitor cells. However, the presumed intermediate of Birbeck granule (BG)+ Langerhans cells (LCs) has not been detected in cultures derived from bone marrow or peripheral blood progenitor cells (PBPCs), thus contrasting with the data obtained with cord blood. We show here that large numbers of BG+ LCs can be generated from human CD34+ PBPCs in vitro, when granulocyte-macrophage colony-stimulating factor and interleukin-4, potent promotors of LC/DC differentiation, are combined with a cocktail of early acting hematopoietic growth factors. LCs were found to emerge from CD33+CD11b+CD14-progenitor cells that they share with the monocytic lineage. During culture, these cells exhibited a sequence of dramatic morphologic changes, starting with a major increase in granularity followed by an increase in size herein exceeding that of all peripheral blood cells. At the same time, CD1a and major histocompatibility complex class II expression were upregulated and virtually all CD1a++ cells were BG+ by electron microscopy. With prolonged culture, CD1a was downregulated on a major population of cells, paralleled by a loss of BG and an increase of CD4, CD25, and CD80 expression that may correspond to the maturation of epidermal LC in vitro. However, these cells were consistently CD5- and did not exhibit changes in the CD45-isoform expression during culture. The availability of large numbers of these highly purified BG+ LCs and mature DCs allows for specific analysis of these subpopulations and provides a source of potent antigen-presenting cells from individual patients for vaccination protocols against infectious or tumor-associated antigens.

Development of cell-based delivery systems that can release therapeutic levels of hematopoietic growth factors into the systemic circulation would facilitate treatment of patients requiring cytokine therapy. In this study, we have investigated the potential of granulocyte- macrophage colony-stimulating factor (GM-CSF)-secreting, irradiated syngeneic murine cells to accelerate hematopoietic recovery after cytotoxic chemotherapy. As a model, CMS-5 fibrosarcoma cells, were transduced with a retroviral vector containing the murine GM-CSF cDNA. Transduced cells secreted 38 ng GM-CSF/10(6) cells in 24 hours. After irradiation, in vitro GM-CSF production initially increased up to fivefold and was measurable for about 2 weeks. One and 2 days after injection of irradiated, GM-CSF-secreting CMS-5 cells (N2/CMVGM- CSF/CMS5 # 6 cells) into mice, GM-CSF serum levels of 405 +/- 58 pg/mL and 183 +/- 36 pg/mL were measured, respectively. Serum levels were comparable with levels detected 3 hours after injection of 100 ng recombinant murine GM-CSF (rmGM-CSF) subcutaneously (90 pg/mL). Injection of N2/CMVGM-CSF/CMS5 # 6 cells in cyclophosphamide-treated mice was as effective in accelerating neutrophil recovery as twice daily subcutaneous injections of rmGM-CSF. These data suggest that irradiated hematopoietic growth factor-secreting cells might offer an alternative to parenteral injections of recombinant cytokines in the treatment of neutropenic patients.