Rapid purification of human Langerhans cells using paramagnetic microbeads

Abstract

Detailed studies on the biology of Langerhans cells (LC), which account for only 1-3% of all epidermal cells, require isolation from their cutaneous symbionts. Several techniques of LC isolation have been reported, including positive enrichment with mAb coupled to immunomagnetic beads. The disadvantage of this technique is the size of the beads (approximately 2-5 microns), which can interfere with subsequent phenotypic and functional analyses. This limitation prompted us to test whether paramagnetic microbeads (15 nm) employed by the MACS system could be used to purify LC from human skin. To isolate fresh LC (fLC), epidermal cell suspensions (EC) were stained with anti-CD1a mAb and with appropriate secondary reagents conjugated to microbeads and to FITC. They were then passed over a separation column and exposed to a strong magnetic field. Thereafter both CD1a-depleted and CD1a-enriched cells were collected. Cultured LC (cLC) were isolated by staining 72-h cultured EC with anti-HLA-DR mAb followed by the same isolation procedure. Using this technique, we could routinely isolate viable EC that were 45-88% CD1a+ or HLA-DR+ as determined by FACS. Two-color FACS analysis demonstrated the majority of MACS-purified cells to be CD1a+/HLA-DR+, indicating that they were indeed LC. By transmission electron microscopy (TEM), the MACS-purified CD1a+/HLA-DR+ cells showed typical ultrastructural characteristics of LC. Furthermore, MACS-purified fLC or cLC were functionally intact, because they stimulated the proliferation of alloreactive T cells in a primary, one-way, mixed epidermal cell leukocyte reaction (MECLR). We conclude that MACS-separation is an efficient and rapid method to isolate human fLC and cLC of high purity and unimpaired function.