Thy-1⁺ dendritic epidermal cells in mice: Precursor frequency analysis and cloning of concanavalin A-reactive cells

Bulk cultures of mouse Thy-1⁺ dendritic epidermal cells (Thy-1⁺ DEC) have been shown to proliferate in response to concanavalin A (Con A) and IL-2, to secrete IL-2-like growth factors, and to lyse target cells such as YAC-1. Limiting dilution microculture was utilized in order to determine the precursor frequency of Con A-responsive Thy-1⁺ DEC in suspensions of AKR/J epidermal cells as well as whether these several functional activities all reside within a single Thy-1⁺ DEC precursor. Precursor frequency analysis of cultures established with limiting numbers of FACS-purified Thy-1⁺ DEC, irradiated syngeneic splenic filler cells and exogenous IL-2 indicated that approximately 20% of Thy-1⁺ DEC proliferated in response to Con A. Parallel microcultures in which purified Thy-1⁺ DEC were plated at a density of 0.5 cells/well were used to establish clones. Twenty clones were characterized phenotypically, and ten of these were also tested for their capacities to proliferate in response to Con A or IL-2, to secrete IL-2-like growth factors, and to exhibit cytotoxicity. All clones were Thy-1⁺ and L3T4^-, but while most were also Lyt-2^-, several contained 3%-18% dull Lyt-2⁺ cells. Functional studies revealed that each clone displayed all of the above functional activities, albeit with substantial quantitative variation. Clones with the highest cytotoxic activity had relatively low responsiveness to Con A or IL-2 and included all clones containing dull Lyt-2⁺ cells; conversely, clones with the highest proliferative responses had relatively low cytotoxic activity and were all Lyt-2^-. This degree of functional and phenotypic heterogeneity among cloned Thy-1⁺ DEC may reflect their particular states of activation or differentiation; whether it reflects the biologically relevant in vivo activities of these cells must still be determined.