Fetal Calf Serum-Free Generation of Functionally Active Murine Dendritic Cells Suitable for In Vivo Therapeutic Approaches  Gabriele Müller, Anke Müller, Helmut Jonuleit, Kerstin Steinbrink, Claudia Szalma, Lydia Paragnik, Jürgen Knop, Alexander H. Enk  Journal of Investigative Dermatology  Volume 114, Issue 1, Pages 142-148 (January 2000) DOI: 10.1046/j.1523-1747.2000.00832.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Effect of different culture conditions on the morphology of bone marrow-derived mouse DC. Depleted bone marrow progenitor cells were cultured in RPMI containing 5 ng GM-CSF per ml and 10 ng IL-4 per ml. Dendritic cells on day 7 of culture are shown. Sera used in the cultures were (a) RPMI/10% fetal calf serum and (b) RPMI/1.5% MS. Journal of Investigative Dermatology 2000 114, 142-148DOI: (10.1046/j.1523-1747.2000.00832.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effect of different culture conditions on the phenotype of DC at day 7 of culture. DC progenitors were cultured in the presence of 5 ng GM-CSF per ml and 10 ng IL-4 per ml. Sera used are given at the left. At day 5 nonadherent cells were transferred to fresh culture plates and cultured for an additional 2 d. Mature DC were harvested at day 7. DC were stained as described in the Materials and Methods. Prior to FACS analysis dead cells and debris were excluded by software gating. Dot plots of remaining cells are shown. Journal of Investigative Dermatology 2000 114, 142-148DOI: (10.1046/j.1523-1747.2000.00832.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Phenotype of fetal calf serum-free DC after stimulation with proinflammatory cytokines – comparison with the standard culture. DC progenitors were cultured in the presence of 5 ng GM-CSF per ml and 10 ng IL-4 per ml under fetal calf serum-free and fetal calf serum-conditions as described. At day 2 of culture 10 ng TNF-α per ml and 10 ng IL-1β per ml were added to the cultures alone or in combination. At day 5 nonadherent cells were transferred to fresh culture plates and cultured for another 2 d. DC were harvested at day 7 and stained as described. Prior to FACS analysis dead cells and debris were excluded by software gating. Dot plots of remaining cells are shown. Journal of Investigative Dermatology 2000 114, 142-148DOI: (10.1046/j.1523-1747.2000.00832.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Allostimulatory properties of fetal calf serum-DC and MS-DC-stimulation with proinflammatory cytokines. Allogeneic naïve CD4+ T cells were prepared as described. DC cultured in RPMI containing 1.5% MS or 10% fetal calf serum were stimulated with the proinflammatory cytokines TNF-α and IL-1β alone or in combination at day 2 of culture. Such DC were used as stimulators of 2 × 105 T cells in an allogeneic MLR. The figure shows the results of five independent experiments. Journal of Investigative Dermatology 2000 114, 142-148DOI: (10.1046/j.1523-1747.2000.00832.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Migratory activity of DC cultured fetal calf serum-free. DC were labeled with red dye PKH26 as describe in the Materials and Methods and 10 × 106 DC were injected intravenously into BALB/C mice on day 0. Twenty-four hours later mice were killed and spleens removed. Cryosections of spleens were immunostained and analysed by fluorescence microscopy. T cell areas were stained with a monoclonal antibody against TCR-β chains. (a) Staining of T cells with TCR-β chain monoclonal antibody-FITC; (b) detection of PKH 26-stained, injected DC in red in T cell areas. Journal of Investigative Dermatology 2000 114, 142-148DOI: (10.1046/j.1523-1747.2000.00832.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Haptenized fetal calf serum-free cultured DC mediate contact sensitivity. DC were grown in RPMI/1.5% MS and haptenized with 10 mM TNBS for 10 min. After three washes with MS-containing medium DC were resuspended in 0.9% NaCl and 5 × 105 cells per mouse were injected subcutanously. 5 d later, mice were challenged, and 24 h later ear swelling responses were measured. The experiment was performed four times showing equal results. Journal of Investigative Dermatology 2000 114, 142-148DOI: (10.1046/j.1523-1747.2000.00832.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 OVA-pulsed fetal calf serum-free cultured DC prime naïve OVA TCR transgenic T cells in vivo. DC were grown in RPMI/1.5% MS and pulsed with 50 μg OVA protein per ml at day 5. Five × 105 DC were injected per OVA TCR transgenic animal. After 10 d spleen cells were restimulated with 5 μg per ml of the OVA peptide (+OVApep) or control peptide (+contr.) in vitro. After 48 h [3H]Tdr was added. Proliferation was reported as mean cpm from triplicate samples. This experiment was repeated five times with similar results. Journal of Investigative Dermatology 2000 114, 142-148DOI: (10.1046/j.1523-1747.2000.00832.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions