Culture and characterization of murine dendritic Thy-1+ epidermal cells

Abstract Although numerous advances have been made in characterizing the phenotype, ontogeny, ultrastructure, and cytochemistry of the murine Thy-1 + dendritic epidermal cell (Thy-1 + EC), elucidation of its functional qualities has been hampered by the difficulty in preparing pure populations of these cells. We therefore sought to obtain expanded, purified populations of Thy-1 + EC using culture techniques. Since Thy-1 + EC are bone marrow-derived, density gradient enriched populations of freshly harvested epidermal cells (FH-EC) were placed in culture under conditions known or suspected to promote mitogenesis among leukocyte subsets. FH-EC prepared from truncal skin of C3H/HeN mice (Thy-1.2 + ) were cultured at 37°C in 5% CO 2 in complete medium (CM) of Eagle's Hanks' amino acid with 10% fetal calf serum, nutrients, and antibiotics at 10 6 FH-EC/well in 24-well culture plates. CM was supplemented with one or more of the following: concanavalin A (Con-A), interleukin-1/epidermal cell-derived thymocyte-activating factor (IL-1/ETAF), IL-2, IL-3, γ interferon, indomethacin (IM), and anti-Thy-1.2 antibody. Media with appropriate supplements were changed every 2–3 days. Freshly isolated, enriched FH-EC contained 7–20% Thy-1 + EC (defined as brightly fluorescing cells readily distinguishable from weakly fluorescing keratinocytes), which also stained with antibodies directed against asialo GM 1 , Ly 5.1, and vimentin but did not stain with antibodies to other T cell-, B cell- or macrophage phenotypic markers. Analysis of 10 separate cultures revealed a 3- to 10-fold expansion of nonkeratinocyte Thy-1 + cells after 21 ± 4 days in culture in CM supplemented with Con-A and IM, and 70–100% of viable cells after expansion were Thy-1 + . Phenotypic analysis of expanded cells revealed the emergence in 10 separate cultures of one of two mutually exclusive distinct populations: one Thy-1 + , asialo GM 1 + , L3T4 − (natural killer phenotype) and the other Thy-1 + , asialo GM 1 + , L3T4 − (T helper phenotype). Experiments designed to explain the emergence of an L3T4 + population suggest that phenotypic modulation occurred in vitro.