Gerold Schuler

An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow.

As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.

Generation of mature dendritic cells from human blood An improved method with special regard to clinical applicability

Two methods to generate human dendritic cells from hematopoietic precursor cells in peripheral blood have recently been published. One approach utilizes the rare CD34+ precursors and GM-CSF plus TNF-α. The other method makes use of the more abundant CD34− precursor population and GM-CSF plus IL-4. Here we report a method that is based on the latter approach. However, the GM-CSF and IL-4 treated cells are not stable mature dendritic cells, e.g., the characteristic morphology and nonadherence of dendritic cells is lost if the cytokines are removed. We describe the need for a monocyte-conditioned medium to generate fully mature and stable dendritic cells. This is achieved by adding a 3 day ‘maturation culture’ to the initial 6–7 day culture in the presence of GM-CSF and IL-4. Macrophage-conditioned medium contains the critical maturation factors. Mature dendritic cells are defined by their pronounced display of motile cytoplasmic processes (‘veils’), their high capacity to induce proliferative responses in resting T cells, particularly in naive umbilical cord T cells, their down-regulated antigen processing ability, and their characteristic phenotype: expression of CD83, high levels of MHC molecules and CD86, lack of CD115 and perinuclear dot-like CD68 staining. These features are stable for at least 3 days upon withdrawal of cytokines and conditioned media. IL-4 can be replaced by IL-13. When CD34+ progenitors are depleted from blood, there is only a minor reduction in the yield of dendritic cells by this method. We have adapted the method to consider several variables that are pertinent to clinical use, including a change from fetal calf serum to human plasma and to media approved for clinical use like X-VIVO or AIM-V. 1% plasma and RPMI 1640 are currently optimal. Additional reagents used for cell culture (Ig, cytokines) and cell separation (immunomagnetic beads) are approved for or already used in clinical applications. For 40 ml blood, the yield is 0.8–3.3 × 106 mature dendritic cells as defined by the expression of the new dendritic cell-restricted marker CD83. CD83+ cells constitute between 30 and 80% of all cells recovered at the end of the culture period. Yields can be enhanced up to six-fold if the blood donors are pretreated with G-CSF. Stable, mature dendritic cells generated by this method should be a powerful tool for active immunotherapy.

Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?

Dendritic cells (DCs) are currently divided into tolerogenic immature and immunogenic mature differentiation stages. However, recent findings challenge this model by reporting mature DCs as inducers of regulatory CD4+ T cells in vivo. This implies that decisive tolerogenic and immunogenic maturation signals for DCs might exist. Closer inspection reveals that tolerance is observed when partial- or semi-maturation of DCs occurs, whereas only full DC maturation is immunogenic. The decisive immunogenic signal seems to be the release of proinflammatory cytokines from the DCs. Moreover, the semi-mature DC phenotype is comparable to steady-state migratory veiled DCs within the lymphatics, which seem to continuously tolerize lymph node T cells against tissue-derived self-antigens or apoptotic cells.

Repetitive Injections of Dendritic Cells Matured with Tumor Necrosis Factor α Induce Antigen-specific Protection of Mice from Autoimmunity

Mature dendritic cells (DCs) are believed to induce T cell immunity, whereas immature DCs induce T cell tolerance. Here we describe that injections of DCs matured with tumor necrosis factor (TNF)-α (TNF/DCs) induce antigen-specific protection from experimental autoimmune encephalomyelitis (EAE) in mice. Maturation by TNF-α induced high levels of major histocompatibility complex class II and costimulatory molecules on DCs, but they remained weak producers of proinflammatory cytokines. One injection of such TNF/DCs pulsed with auto-antigenic peptide ameliorated the disease score of EAE. This could not be observed with immature DCs or DCs matured with lipopolysaccharide (LPS) plus anti-CD40. Three consecutive injections of peptide-pulsed TNF/DCs derived from wild-type led to the induction of peptide-specific predominantly interleukin (IL)-10–producing CD4+ T cells and complete protection from EAE. Blocking of IL-10 in vivo could only partially restore the susceptibility to EAE, suggesting an important but not exclusive role of IL-10 for EAE prevention. Notably, the protection was peptide specific, as TNF/DCs pulsed with unrelated peptide could not prevent EAE. In conclusion, this study describes that stimulation by TNF-α results in incompletely matured DCs (semi-mature DCs) which induce peptide-specific IL-10–producing T cells in vivo and prevent EAE.

Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application.

Dendritic Cell (DC)-based vaccination approaches in man require a reproducible DC generation method that can be performed in conformity with GMP (Good Manufacturing Practice) guidelines and that circumvents the need for multiple blood drawings to generate DC. To this end we modified our previously described method to generate mature DC from CD14 + monocytes by a two step method (priming in GM-SF + IL-4 followed by maturation in monocyte conditioned medium) for use with leukapheresis products as a starting population. Several adaptations were necessary. We established, for example, a modified adherence step to reliably enrich CD14 + DC precursors from apheresis mononuclear cells. The addition of GM-CSF + IL-4 at the onset of culture proved disadvantageous and was, therefore, delayed for 24 h. DC development from apheresis cells occurred faster than from fresh blood or buffy coat, and was complete after 7 days. Monocyte conditioned medium when added on day 6 resulted in fully mature and stable DC (veiled, highly migratory and T cell sensitizing cells with a characteristic phenotype such as 85% CD83 + , p55/fascin + , CD115/M-CSF-R - , CD86 + ) already after 24 h. The mature DC progeny were shown to remain stable and viable if cultured for another 1-2 days in the absence of cytokines, and to be resistant to inhibitory effects of IL-10. Freezing conditions were established to generate DC from frozen aliquots of PBMC or to freeze mature DC themselves for later use. The approach yields large numbers of standardized DC (5-10 x 10(8) mature CD83 + DC/leukapheresis) that are suitable for performing sound DC-based vaccination trials that can be compared with each other.

Human CD4+CD25+ Regulatory, Contact-dependent T Cells Induce Interleukin 10–producing, Contact-independent Type 1-like Regulatory T Cells

It has been recently demonstrated that regulatory CD4+CD25+ CD45RO+ T cells are present in the peripheral blood of healthy adults and exert regulatory function similar to their rodent counterparts. It remains difficult to understand how the small fraction of these T cells that regulate via direct cell-to-cell contact and not via secretion of immunosuppressive cytokines could mediate strong immune suppression. Here we show that human CD4+CD25+ T cells induce long-lasting anergy and production of interleukin (IL)-10 in CD4+CD25− T cells. These anergized CD4+CD25− T cells then suppress proliferation of syngenic CD4+ T cells via IL-10 but independent of direct cell contact, similar to the so-called type 1 regulatory T (Tr1) cells. This ‘catalytic’ function of CD4+CD25+ T cells to induce Tr1-like cells helps to explain their central role for the maintenance of immune homeostasis.

Rapid Induction of Tumor-specific Type 1 T Helper Cells in Metastatic Melanoma Patients by Vaccination with Mature, Cryopreserved, Peptide-loaded Monocyte-derived Dendritic Cells

There is consensus that an optimized cancer vaccine will have to induce not only CD8+ cytotoxic but also CD4+ T helper (Th) cells, particularly interferon (IFN)-γ–producing, type 1 Th cells. The induction of strong, ex vivo detectable type 1 Th cell responses has not been reported to date. We demonstrate now that the subcutaneous injection of cryopreserved, mature, antigen-loaded, monocyte-derived dendritic cells (DCs) rapidly induces unequivocal Th1 responses (ex vivo detectable IFN-γ–producing effectors as well as proliferating precursors) both to the control antigen KLH and to major histocompatibility complex (MHC) class II–restricted tumor peptides (melanoma-antigen [Mage]-3.DP4 and Mage-3.DR13) in the majority of 16 evaluable patients with metastatic melanoma. These Th1 cells recognized not only peptides, but also DCs loaded with Mage-3 protein, and in case of Mage-3DP4–specific Th1 cells IFN-γ was released even after direct recognition of viable, Mage-3–expressing HLA-DP4+ melanoma cells. The capacity of DCs to rapidly induce Th1 cells should be valuable to evaluate whether Th1 cells are instrumental in targeting human cancer and chronic infections.

IMMATURE DENDRITIC CELLS GENERATED WITH LOW DOSES OF GM-CSF IN THE ABSENCE OF IL-4 ARE MATURATION RESISTANT AND PROLONG ALLOGRAFT SURVIVAL IN VIVO

Dendritic cells (DC) were cultured from mouse bone marrow (BM) progenitors in low concentrations of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) (GMlo DC) by two different protocols. The phenotype and functional properties of these GMlo DC were compared to those of standard BM‐DC cultures generated in high concentrations of GM‐CSF (GMhi DC) or in low GM‐CSF plus IL‐4 (GMlo/IL‐4 DC). An effect of IL‐4 on maturation was observed only at low but not high doses of GM‐CSF. Compared to mature DC, GMlo DC were phenotypically immature, weak stimulators of allogeneic and peptide‐specific T cell responses, but substantially more potent in presentation of native protein. Immature GMlo DC were resistant to maturation by lipopolysaccharide, TNF‐α or anti‐CD40 monoclonal antibodies, as the expression of co‐stimulatory molecules was not increased, and stimulatory activity in oxidative mitogenesis was not enhanced. These maturation‐resistant immature GMlo DC induced T cell unresponsiveness in vitro and in vivo. GMlo DC also prolonged haplotype‐specific cardiac allograft survival (from 8 days to >100 days median survival time) when they were administered 7 days (but not 3, 14 or 28 days) before transplantation. Our findings may have important implications for future studies in T cell tolerance induction in vivo.

A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T-cell responses in humans following intranodal injection

Dendritic cells (DCs) elicit potent anti‐tumoral T‐cell responses in vitro and in vivo. However, different types of DC have yet to be compared for their capacity to induce anti‐tumor responses in vivo at different developmental stages. Herein, we correlated the efficiencies of different types of monocyte‐derived DC as vaccines on the resulting anti‐tumor immune responses in vivo. Immature and mature DCs were separately pulsed with a peptide derived from tyrosinase, MelanA/MART‐1 or MAGE‐1 and a recall antigen. Both DC populations were injected every 2 weeks in different lymph nodes of the same patient. Immune responses were monitored before, during and after vaccination. Mature DCs induced increased recall antigen‐specific CD4+ T‐cell responses in 7/8 patients, while immature DCs did so in only 3/8. Expansion of peptide‐specific IFN‐γ–producing CD8+ T cells was observed in 5/7 patients vaccinated with mature DCs but in only 1/7 using immature DCs. However, these functional data did not correlate with the tetramer staining. Herein, immature DCs also showed expansion of peptide‐specific T cells. In 2/4 patients vaccinated with mature DCs, we observed induction of peptide‐specific cytotoxic T cells, as monitored by chromium‐release assays, whereas immature DCs failed to induce peptide‐specific cytotoxic T cells in the same patients. Instead, FCS‐cultured immature DCs induced FCS‐specific IgE responses in 1 patient. Our data demonstrate that this novel vaccination protocol is an efficient approach to compare different immunization strategies within the same patient. Thus, our data define FCS‐free cultured mature DCs as superior inducers of T‐cell responses in melanoma patients.

Human and murine dermis contain dendritic cells. Isolation by means of a novel method and phenotypical and functional characterization.

Dendritic cells (DC) comprise a system of cells in lymphoid and nonlymphoid organs that are specialized to present antigens and to initiate primary T cell responses. The Langerhans cell of the epidermis is used as a prototype for studies of DC in the skin. We have characterized a population of DC in human dermis, one of the first examples of these cells in nonlymphoid organs other than epidermis. To identify their distinct functions and phenotype, we relied upon the preparation of enriched populations that emigrate from organ explants of dermis. The dermal cells have the following key features of mature DC: (a) sheet-like processes, or veils, that are constantly moving; (b) very high levels of surface MHC products; (c) absence of markers for macrophages, lymphocytes, and endothelium; (d) substantial expression of adhesion/costimulatory molecules such as CD11/CD18, CD54 (ICAM-1), B7/BB1, CD40; and (e) powerful stimulatory function for resting T cells. Dermal DC are fully comparable to epidermis-derived DC, except for the lack of Birbeck granules, lower levels of CD1a, and higher levels of CD36. DC were also detected in explants of mouse dermis. We conclude that cutaneous DC include both epidermal and dermal components, and suggest that other human nonlymphoid tissues may also serve as sources of typical immunostimulatory DC.