Comprehensive analysis of MHC-II expression in healthy human skin

Abstract

A number of antigen-presenting cells (APCs) expressing major histocompatibility complex class II (MHC-II) have been identified in healthy human skin including the Langerhans cells of the epidermis and the three recently defined dermal APC subsets. It is well documented that in other tissues HLA-DR expression is not exclusive to APCs. Following a comprehensive analysis of the cells in human skin using flow cytometry and fluorescence immunohistochemistry, we have identified additional cell subsets that express HLA-DR. Using markers exclusive for blood and lymphatic endothelium, we demonstrated that both of these cell populations have the capacity to express HLA-DR. In addition, a small subset of dermal T lymphocytes was found to express low-level HLA-DR suggesting an activated phenotype. Dermal T lymphocytes were often in intimate contact with either CD1a(+) CD207(-) dermal APCs or CD1a(+) CD207(+) dermal Langerhans cells, possibly explaining the activated phenotype of a subset of dermal T lymphocytes.

Figure 1. MHC-II is not exclusively expressed by the recognised cutaneous APC subsets in the human dermis

Single cell suspensions from human dermis and epidermis were analyzed using flow cytometry. In the dermal preparations CD14⁺ dermal APCs (a), CD1a⁺CD207^- dermal APCs and CD1a^highCD207⁺ dermal Langerhans cells (b and c) were detected, and each of these APC subsets expressed HLA-DR. In the epidermal preparations CD1a⁺CD207^- APCs and CD1a^highCD207⁺ Langerhans cells were detected, again each of these APC subsets expressed HLA-DR (d and e). All viable 7AAD^- cells are shown in these plots. Two-colour fluorescence immunohistochemistry was used to confirm HLA-DR expression by the cutaneous APC subsets in situ. CD14⁺ dermal APCs were distributed throughout the dermis and the majority expressed HLA-DR (f). CD1a⁺ APCs were located in the papillary area of the dermis and the epidermis, both expressed HLA-DR (g). CD207⁺ epidermal and dermal Langerhans cells expressed HLA-DR (h). Epidermis is orientated upwards and blue represents DAPI staining of cell nuclei. Scale bars represent 50μm. Data are representative of three independent experiments. Single cell suspensions from human dermis were analyzed using flow cytometry to show that cumulatively CD14⁺ dermal APCs, CD1a⁺ dermal APCs and CD1a⁺ dermal Langerhans cells do not account for all of the HLA-DR⁺ cells in the healthy human dermis (i). All viable 7AAD^- cells are shown in this plot. Three-color fluorescence immunohistochemistry also illustrated that HLA-DR expression was not restricted to the recognised cutaneous APC subsets in situ (j). Epidermis is orientated upwards and blue represents DAPI staining of cell nuclei. Scale bars represents 50μm. Data are representative of three independent experiments.

Figure 2. Identifying the non-APC subsets that express MHC class II in the human dermis and do these non-APCs in conjunction with the recognised cutaneous APC subsets account for all of the MHC-II positive cells in the human dermis?

Single cell suspensions from human dermis were analyzed using flow cytometry. A small proportion of the T-lymphocytes in the dermis expressed low level HLA-DR (a). In addition the majority of CD144⁺ blood endothelial cells (c) and a minor proportion of podoplanin⁺ lymphatic endothelial cells (d) expressed moderate to high levels of HLA-DR. The forward and side scatter profile of each of the subsets, are also shown. All viable 7AAD^- cells are shown in these plots. Two-colour fluorescence immunohistochemistry was used to confirm HLA-DR expression by the non-APC subsets in situ. CD3⁺ T-lymphocytes were often located close to dermal vessels and a proportion of these cells expressed HLA-DR (white arrow-head) (e). CD144 expression is restricted to the luminal surface of blood endothelial cells. CD144⁺ blood endothelial cells expressed low-level punctate HLA-DR (f-g); CD144^- cells expressing high levels of HLA-DR were often distributed around CD144⁺ capillaries (f-g). A few podoplanin⁺ lymphatic endothelial cells expressed HLA-DR in situ (h). Epidermis is orientated upwards and blue represents DAPI staining of cell nuclei. Scale bars represent 25μm (e), 10μm (f-g) and 20μm (h). Data are representative of three independent experiments. Flow cytometry data obtained using antibodies recognizing each of the cutaneous APC subsets, B and T-lymphocytes and blood and lymphatic endothelial cells confirms that nearly all HLA-DR⁺ cells in the healthy human dermis are accounted for using these cellular markers (i). All viable 7AAD^- cells are shown in this plot.

Figure 3. Dermal APCs form close associations with dermal T-lymphocytes

Single cell suspensions from human dermis were analyzed using flow cytometry. Aggregates of CD1a^mid dermal APCs and CD1a^high dermal Langerhans cells with T-lymphocytes were detected both by co-staining of markers specific for each cell type (a), and by shifts in the forward and side scatter profiles (e-f) compared with the single cell populations (b-d). CD14⁺ dermal APC were rarely detected aggregating with T-lymphocytes (g). All viable 7AAD^- cells are shown in these plots. Immunohistochemistry was used to confirm that both CD1a⁺ dermal APCs (h and j) and CD1a⁺CD207⁺ dermal Langerhans cells (i-j) formed close associations with dermal T-lymphocytes in situ. Epidermis is orientated upwards and blue represents DAPI staining of cell nuclei. Scale bars represent 10μm. Data are representative of three independent experiments.

Figure 4. Assessing the activation status of T-lymphocytes in the human dermis

Single cell suspensions from human dermis were analyzed using flow cytometry. A small subset of dermal T-lymphocytes expressed low-level surface CD25 (a). The activation markers CD38 and CD154 were not detected on the surface of dermal T-lymphocytes (b-c). Data are representative of three independent experiments.