We evaluated two bone marrow-derived dendritic cell (DC) populations from NOD mice, the murine model for type 1 human diabetes. DCs derived from GM-CSF [granulocyte/macrophage colony-stimulating factor] + interleukin (IL)-4 cultures expressed high levels of major histocompatibility complex (MHC) class II, CD40, CD80, and CD86 molecules and were efficient stimulators of naive allogeneic T-cells. In contrast, DCs derived from GM-CSF cultures had low levels of MHC class II costimulation/activation molecules, were able to take up mannosylated bovine serum albumin more efficiently than GM + IL-4 DCs, and were poor T-cell stimulators. The two DC populations migrated to the spleen and pancreas after intravenous injection. To determine the ability of the two DC populations to modulate diabetes development, DCs were pulsed with a mixture of three islet antigen-derived peptides or with medium before injection into prediabetic NOD mice. Despite phenotypic and functional differences in vitro, both populations prevented in vivo diabetes development. Pulsing of the DCs with peptide in vitro did not significantly improve the ability of DCs to prevent disease, which suggests that DCs may process and present antigen to T-cells in vivo. In addition, we detected GAD65 peptide-specific IgG1 antibody responses in DC-treated mice. Overall, these results suggest that a Th2 response was generated in DC-treated mice. This response was optimal when using GM + IL-4 DCs, which suggests that the balance between regulatory Th2 and effector Th1 cells may have been altered in these mice.

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Dendritic cells (DCs) function as antigen presenting cells in vivo and play a fundamental role in numerous diseases. New methods are described for high-efficiency intracellular labeling of DCs with superparamagnetic iron-oxide (SPIO) utilizing a receptor-mediated endocytosis (RME) mechanism. Bone marrow-derived DCs or a fetal skin-derived DC line were incubated with SPIO conjugated to anti-CD11c monoclonal antibody (mAb) under conditions favoring RME. These cells exhibited approximately a 50-fold increase in uptake relative to DCs incubated with SPIO without the mAb. Flow cytometry studies assaying cell surface markers showed a down-modulation of CD11c, but no other changes in phenotype. Immunological function of the DCs was unmodified by the labeling, as determined by cytokine secretion assays. The RME mechanism was confirmed using electron microscopy, endocytosis inhibition assays, and incubation experiments with SPIO conjugated to mAbs against accessory molecules that are not expressed on DCs. Labeled DCs were injected into murine quadriceps and monitored in vivo for several days using MR microimaging at 11.7 T. DCs were observed to remain within the muscle for >24 hr. The use of RME is an efficient way to label immune cells for in vivo MRI and can be applied to a wide variety of cell types.

Dendritic cells (DCs) activate T cells and regulate their differentiation into T helper cell type 1 (Th1) and/or Th2 cells. To identify DCs with differing abilities to direct Th1/Th2 cell differentiation, we cultured mouse bone marrow progenitors in granulocyte macrophage-colony stimulating factor (GM), GM + interleukin (IL)-4, or GM + IL-15 and generated three distinct DC populations. The GM + IL-4 DCs expressed high levels of CD80/CD86 and major histocompatibility complex (MHC) class II and produced low levels of IL-12p70. GM and GM + IL-15 DCs expressed low levels of CD80/CD86 and MHC class II. The GM + IL-15 DCs produced high levels of IL-12p70 and interferon (IFN)-gamma, whereas GM DCs produced only high levels of IL-12p70. Naive T cells stimulated with GM + IL-4 DCs secreted high levels of IL-4 and IL-5 in addition to IFN-gamma. In contrast, the GM + IL-15 DCs induced higher IFN-gamma production by T cells with little or no Th2 cytokines. GM DCs did not induce T cell polarization, despite producing large amounts of IL-12p70 following activation. A similar pattern of T cell activation was observed after in vivo administration of DCs. These data suggest that IL-12p70 production alone, although necessary for Th1 differentiation, is not sufficient to induce Th1 responses. These studies have implications for the use of DC-based vaccines in immunotherapy of cancer and other clinical conditions.

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