Alexa Fluor® 647
Excitation: 650nm, Emission: 665nm
Dendritic cells (DCs) process and present bacterial and endogenous lipid antigens in complex with CD1 molecules to T cells and invariant natural killer T (NKT) cells. However, different types of DCs, such as blood myeloid DCs and skin Langerhans cells, exhibit distinct patterns of CD1a, CD1b, CD1c, and CD1d expression. The regulation of such differences is incompletely understood. Here, we initially observed that monocyte-derived DCs cultured in an immunoglobulin-rich milieu expressed CD1d but not CD1a, CD1b, and CD1c, whereas DCs cultured in the presence of low levels of immunoglobulins had an opposite CD1 profile. Based on this, we tested the possibility that immunoglobulins play a central role in determining these differences. IgG depletion and intravenous immunoglobulin (IVIg) add-in experiments strongly supported a role for IgG in directing the CD1 expression profile. Blocking experiments indicated that this effect was mediated by FcgammaRIIa (CD32a), and quantitative polymerase chain reaction data demonstrated that regulation of the CD1 profile occurred at the gene expression level. Finally, the ability of DCs to activate CD1-restricted NKT cells and T cells was determined by this regulatory effect of IgG. Our data demonstrate an important role for FcgammaRIIa in regulating the CD1 antigen presentation machinery of human DCs.
CD4+ CD25+ regulatory T cells (Tregs) have far-reaching immunotherapeutic applications, the realization of which will require a greater understanding of the factors influencing their function and phenotype during ex vivo manipulation. In murine models, IL-2 plays an important role in both the maintenance of a functional Treg population in vivo and the activation of suppression in vitro. We have found that IL-2 maintains optimal function of human CD4+ CD25+ Tregs in vitro and increases expression of both forkhead box protein 3, human nomenclature (FOXP3) and the distinctive markers CD25, cytotoxic T lymphocyte antigen-4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor superfamily member number 18 (GITR). Although IL-2 reduced spontaneous apoptosis of Tregs, this property alone could not account for the optimal maintenance of the regulatory phenotype. The inhibition of phosphatidylinositol 3-kinase (PI3K) signaling by LY294002, a chemical inhibitor of PI3K, abolished the maintenance of maximal suppressive potency by IL-2, yet had no effect on the up-regulation of FOXP3, CD25, CTLA-4 and GITR. Other common gamma chain (gammac) cytokines-IL-4, IL-7 and IL-15-had similar properties, although IL-4 showed a unique lack of effect on the expression of FOXP3 or Treg markers despite maintaining maximal regulatory function. Taken together, our data suggest a model in which the gammac cytokines IL-2, IL-4, IL-7 and IL-15 maintain the optimal regulatory function of human CD4+ CD25+ T cells in a PI3K-dependent manner, offering new insight into the effective manipulation of Tregs ex vivo.