A system to maximize the in vitro immunological stability of human Langerhans cells

Langerhans cells (LC) are now known to be immunocompetent antigen-presenting cells (APC) that are involved with numerous immune reactions initiated in skin, including the induction of contact hypersensitivity and the rejection of skin grafts. Immortalized cells that closely resemble in vivo LC have not been developed and mature LC, in vitro, appear to only briefly pass through a stage of differentiation when immunological potential seems stable. The literature supports the concept that mature LC possess numerous ultrastructural and antigen (Ag) characteristics. These include suface ATPase activity, Fc receptors, MHC class II Ag, CD1a Ag and Birbeck granules. These studies were undertaken to determine the ideal conditions of LC isolation and enrichment for persistence of these characteristics, such that toxicological and immunoreactive studies could closely mimic the in vivo situation. Studies were carried out to evaluate sources of tissue, duration of manipulations, chemical perturbation, stored v. fresh tissue, methods of enrichment, donor serological information and cost. LC were evaluated for ultrastructural and Ag stability by light and electron microscopy utilizing monoclonal antibodies (Mab) to surface Ag and ultrastructural features. It was determined that the most critical conditions included duration of manipulations and method of enrichment. Differences between fresh-frozen and fresh tissue samples were negligible. Cells evaluated less than 18 hr post-collection and enriched by density gradient centrifugation consistently demonstrated stability while ATPase activity, MHC Ag and ultrastructural characteristics varied when other methods were used. Implications for immunological and toxicological testing of in vitro skin samples can be drawn.