Fig. 3

DC-dependent and independent T cell proliferation and programming. a SlanDCs or CD1+ DCs of healthy donors were pretreated with different concentrations of FTY or FTYP and co-cultured with allogeneic CFSE-labeled CD4+ or CD8+ T cells. DC-depending proliferation was calculated by CFSE-incorporation by flow cytometry and quantified by CDI. Proliferation after pretreatment is depicted in proportion to proliferation without pretreatment (A–D). SlanDCs or CD1+ DCs of healthy donors were pretreated with different concentrations of FTY or FTYP and co-cultured with allogeneic naïve CD4+ T cells. T cells were then analyzed regard their differentiation into pro-inflammatory IFN-gamma-expressing Th1 cells (E/F), IL-17-expressing Th17 cells (G/H). Mean values ± SEM of eight individual experiments are presented. b In addition slanDCs of FTY-treated MS patients (DC FTY) and of healthy controls (DC HC) were co-cultured with allogeneic CFSE labeled CD4+ or CD8+ T cells of the same donor. DC-dependent proliferation was calculated by CFSE-incorporation by flow cytometry and quantified by CDI (A/B). SlanDCs of FTY-treated RRMS patients (DC FTY) and healthy controls (DC HC) were co-cultured with allogeneic naïve CD4+ T cells of the same donor. T cells were then analyzed regard their differentiation into Th1 cells (C) and Th17 cells (D). Mean values ± SEM of five different donors are presented. c DC-independent T cell proliferation and polarization. FTY- and FTYP-treated CFSE-labeled CD4+ or CD8+ T cells of healthy donors were stimulated with human anti-CD3/CD28. Proliferation was calculated by CFSE-incorporation by flow cytometry and quantified by CDI (A–B). Polarization of FTY- and FTYP-treated naïve CD4+ T cells of healthy donors into IFN-gamma-expressing Th1 cells or IL-17-expressing Th17 cells are depicted (G–H). Mean values ± SEM of six individual experiments are presented. d In addition, proliferation of CD4+ or C8+ T cells of FTY-treated patients (CD4 FTY, CD8 FTY) compared to healthy controls (CD4 HC, CD8 HC) after anti-CD3/CD28 stimulation was evaluated (A/B). Asterisks indicate a statistically significant difference (*p < 0.05, **p < 0.01, ***p < 0.001)