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Published on: April 8, 2016
Quantification of CD1a, HLA-DR, and HLA class I expression on viable human Langerhans cells and keratinocytes
L Meunier1, L Vian, C Lagoueyte
1Department of Dermatology, University of Montpellier, France.
Insights
Accurate quantification of epidermal cell surface molecules requires a viability marker. This study quantifies CD1 and MHC antigens on viable human Langerhans cells and keratinocytes using flow cytometry.
Area of Science:
- Immunology
- Dermatology
- Cell Biology
Background:
- Accurate quantification of epidermal cell surface molecules is crucial for understanding antigen presentation.
- Non-specific background fluorescence can interfere with accurate cell surface antigen quantification.
Purpose of the Study:
- To precisely determine the cellular density of surface molecules critical for antigen presentation in human epidermis.
- To establish reliable methods for quantifying cell surface antigens on viable human epidermal cells.
Main Methods:
- Quantitative immunofluorescence indirect assay and flow cytometric analysis of human epidermal cell (EC) suspensions.
- Utilized Tricolor-labeled streptavidin coupled to Cy-5 (SA-TC) as a marker for non-viable EC.
- Multicolor flow cytometry to quantify CD1 molecules and major histocompatibility complex (MHC) antigens on viable human Langerhans cells (LC) and keratinocytes (Kc).
Main Results:
- SA-TC is a reliable marker for non-viable EC and accounts for non-specific background fluorescence.
- Viable LC express significantly lower levels of MHC class I molecules (163 ± 19 x 10^3 molecules/cell) compared to viable Kc (785 ± 110 x 10^3 molecules/cell).
- Mean antigen density on viable LC: HLA-DR (579 ± 82 x 10^3 molecules/cell) and CD1a (1600 ± 133 x 10^3 molecules/cell).
Conclusions:
- Quantification of cell surface antigens on human EC necessitates the use of a viability marker.
- Quantitative flow cytometry of viable EC can evaluate membrane antigen expression related to cellular maturation or activation in skin diseases.
Abstract:
In order to determine precisely the cellular density of surface molecules that are critical for antigen presentation in human epidermis, we utilized a quantitative immunofluorescence indirect assay and performed flow cytometric analysis of human epidermal cell (EC) suspensions. We first demonstrated that Tricolor-labeled streptavidin coupled to Cy-5 (SA-TC) was a reliable marker for non viable EC and that SA-TC+ EC accounted for the frequent nonspecific background of fluorescence due to isotype controls binding, although Langerhans cells (LC) and Keratinocytes (Kc) express Fc receptors for IgG on their surfaces. These results indicate that quantification of cell surface antigens on human EC requires the concomitant use of a marker of viability. Multicolor flow cytometric analysis allowed us to quantify CD1 molecules and major histocompatibility complex (MHC) antigens on viable human LC and Kc. Our results demonstrated a weak expression of MHC class I molecules on viable LC (163 +/- 19 x 10(3) molecules/cell) compared to viable Kc (785 +/- 110 x 10(3) molecules/cell). Mean antigen density of HLA-DR and CD1a molecules on viable LC were 579 +/- 82 x 10(3) molecules/cell and 1600 +/- 133 x 10(3) molecules/cell, respectively. Quantitative flow cytometry of viable EC may be proposed to evaluate the number of membrane antigens whose level of expression is related to cellular maturation or activation that occurs in skin diseases.
