CD1a and CD1c cell sorting yields a homogeneous population of immature human Langerhans cells

There is increasing evidence that ex vivo generated Langerhans cells (LCs) cannot fully substitute for their physiological counterparts in normal epidermis when studying the immunobiology of this prototype of a tissue-residing immature dendritic cell (DC). Here, we present CD1-based magnetic-activated cell-sorting (MACS) protocols for the effective isolation of human epidermal LCs. CD1c selection yielded a homogeneous population of pure and viable HLA-DR(+)/CD1a(+) DCs, with the ultrastructural features, surface antigen expression and cytokine profile, characteristic of epidermis-resident immature LCs. The immature state and functional integrity were established by allogeneic mixed lymphocyte reactions showing a weak stimulatory capacity of freshly isolated cells and upregulation upon stimulation. Characterizing the cells in more detail, we could demonstrate for the first time that normal human LCs express CXCR4, CD40 ligand (CD40L), and Fas and Fas ligand (FasL). The observed constitutive transcription of TGF-beta suggests that the viability and immature state of epidermal LCs are maintained not only by the TGF-beta production from the microenvironment, but also in an autocrine or paracrine manner. LPS and IFN-omega stimulated the expression of the inflammatory cytokines TNF-alpha and IL-1beta, and there was secretion of IL-12p70 after CD40 ligation. Remarkably, the CD1-sorted LCs showed no loss of their Birbeck granules and CD1a expression upon culturing and no spontaneous phenotypic and functional maturation into potent antigen-presenting cells (APCs). We conclude that human epidermal LCs obtained by the CD1c cell-sorting protocol are optimal candidates with which to elucidate the properties and capabilities of immature cells and to develop immunotherapeutic vaccines.