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Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture
Published on: April 8, 2016
Purification of functional active epidermal Langerhans cells: a simple and efficient new technique
1Unit of Dermatology, Royal Postgraduate Medical School, London, England.
Insights
Researchers developed a simple, reproducible method to purify functional epidermal Langerhans cells using immunomagnetic beads. This technique yields highly pure, active Langerhans cells for immunological studies.
Area of Science:
- Immunology
- Dermatology
- Cell Biology
Background:
- Langerhans cells are crucial for skin immunity.
- Previous methods for isolating Langerhans cells were inefficient and unreproducible.
- Understanding Langerhans cell function is vital for immunology and dermatology.
Purpose of the Study:
- To develop an efficient and reproducible method for purifying functional human epidermal Langerhans cells.
- To assess the functional activity of purified Langerhans cells.
- To investigate the influence of keratinocytes on Langerhans cell function.
Main Methods:
- Positive selection of human Langerhans cells using immunomagnetic beads conjugated to anti-HLA-DR antibodies.
- Detachment of immunomagnetic beads using a polyclonal anti-mouse immunoglobulin.
- Assessment of cell purity via CD1a positivity.
- Evaluation of functional activity using the allogeneic mixed epidermal cell-lymphocyte reaction.
Main Results:
- A technically simple method for isolating Langerhans cells was established.
- Recovery rates of Langerhans cells ranged from 40% to 80%.
- Purified cells achieved up to 99% CD1a positivity and demonstrated potent functional activity.
- Keratinocytes exhibited a significant inhibitory effect on Langerhans cell function, unaffected by indomethacin.
Conclusions:
- The developed immunomagnetic bead method provides a highly efficient and reproducible way to obtain pure, functionally active human epidermal Langerhans cells.
- This method overcomes previous limitations in Langerhans cell isolation, facilitating further research into their roles in immunity.
- The study highlights the inhibitory role of keratinocytes on Langerhans cell function, offering new insights into skin immune regulation.
Abstract:
Our knowledge of the functional activity of the epidermal Langerhans cell has been severely hampered by the lack of an easy method of purification of these cells that is both efficient and reproducible. In the present study we have used immunomagnetic beads directly conjugated to an IgM class mouse anti-human human leukocyte antigen DR monoclonal antibody to positively select human Langerhans cells from an epidermal cell suspension. Cells were then treated with a high-affinity polyclonal anti-mouse immunoglobulin that detached the beads by competing with the antigen for the antigen-binding site on the monoclonal antibody. This procedure allowed removal of the immunomagnetic beads, leaving Langerhans cells free from bound antibody. Recovery of Langerhans cells ranged from 40 to 80% of the starting number of Langerhans cells. The resulting cells were up to 99% CD1a positive and showed potent functional activity in the allogeneic mixed epidermal cell - lymphocyte reaction. Keratinocytes were shown to exert a profound inhibitory effect on Langerhans cell function that could not be prevented by indomethacin. This method is technically simple and allows good recovery of a highly purified population of Langerhans cells that are functionally active.
