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IL ‐17A‐ and IFN γ ‐Producing T Cells in Healthy Skin

2016; Wiley; Volume: 83; Issue: 4 Linguagem: Inglês

10.1111/sji.12412

1365-3083

Beatrice Dyring‐Andersen, M. Nielsen, Carsten Geisler, Charlotte M. Bonefeld, Lone Skov,

Dermatology and Skin Diseases

Human skin is a complex immunological organ that comprises a multitude of T cell subsets, both migratory and resident. The first reports of lymphocytes in the skin (human, rabbit and rat) concluded that lymphocytes account for 1.4–3.9% of the epidermal cells 1, 2. Subsequent studies revealed a memory phenotype in the majority of these cells and the expression of the homing marker cutaneous lymphocyte-associated antigen (CLA) in 50% 3, 4. The total number of CD3+ cells in the epidermis and dermis has been estimated in two studies yielding a similar number, namely 448 and 590 cells/10 mm horizontal skin section, surprisingly adding up to twice as many T cells in the skin than in the blood 5, 6. In inflammatory skin diseases, such as psoriasis, atopic dermatitis and contact dermatitis, many more T cells are infiltrate the skin and contribute to the pathogenesis of inflammatory skin diseases through their production of cytokines such as IL-17A and IFNγ. The cytokine production of skin-derived T cells in healthy human skin has been investigated through explant studies and T cell clones 6. The aim of this study was to investigate the percentage of IL-17A- and IFNγ-positive T cell subsets in healthy skin. For this purpose, discarded skin from patients undergoing abdominoplastic surgery or mamma reduction was obtained, and after removing visible fat, the skin was minced and digested in collagenase D (1 mg/ml; Roche) for 45 min at 37 °C. As we experienced that many epitopes were damaged by this treatment, we incubated the cells for 36 h in 24-well plates in RPMI (R5886; Sigma, Brøndby, Denmark), 500 mIU/l penicillin (LEO Pharmaceuticals, Ballerup, Denmark), 500 mg/ml streptomycin (Merck KGaA, Darmstadt, Germany) with 8% inactivated human serum at 37 °C, 5% CO2 allowing the cells to re-express the collagenase-sensitive surface markers. The cells were subsequently stimulated with PMA/ionomycin/monensin for 4 h and analysed for expression of cell surface receptors and intracellular cytokines as previously described 7. The following antibodies were used for flow cytometric staining: CD45 BV510 (Biolegend, San Diego, CA, USA, 304035), TCR γδ FITC (BD Biosciences, 347903), CD4 PC5.5 (Biolegend, 317427, CD8 PeCy7 (BD Biosciences, 557746), IL-17A APC (eBioscience, San Diego, CA, USA, 12-7179-42) and IFN-γ BV605 (Biolegend, San Diego, CA, USA, 400162). We found that most CD45+ cells in healthy skin were either CD4+ (51.9 ± 11.2% of the CD45+ cells, mean ± STD, N = 6) or CD8+ (42.0 ± 12.5% of the CD45+ cells, N = 10) (Fig. 1). The high proportion of CD4+ T cells confirms the results of previous studies 8. Both CD4+IFNγ+ T cells (Th1 cells) (8.1 ± 6.6% of the CD4+ cells, N = 6) and CD4+IL-17+ T cells (Th17 cells) (4.2 ± 4.1% of the CD4+ cells, N = 6) were present in healthy skin (Fig. 1). The percentage of Th1 cells coincides with previous analysis of mitogen-stimulated skin-derived cells from explant cultures, where the authors found that approximately 10% of skin resident T cells were IFNγ+ (most CD4+) cells 6. We also found that CD8+IFNγ+ (Tc1) and CD8+IL-17+ (Tc17) T cells (2.8 ± 2.6% of the CD8+ cells, N = 6; 4.2 ± 4.1% of the CD8+ cells, N = 10, respectively) reside in normal skin (Fig. 1). Recently, the populations of CD4 and CD8 T cells within adult healthy skin have been subdivided in four functionally and phenotypically diverse populations (resident and recirculating cells). The study also shows that especially CD103+ resident memory T cells have cytokine effector function, including the production of IL-17 and IFNγ in both the epidermis and dermis 9. Lastly, we investigated the distribution of γδ T cells. γδ T cells were first described in murine epidermis as so-called Thy-1+ dendritic epidermal cells and later renamed dendritic epidermal T cells (DETC) 10, 11. These cells have been shown to play important roles in wound healing, tumour surveillance and in responses to contact allergens 12-14. No equivalent of DETC has been found in human skin where the prevalence of γδ T cells in previously publish studies is low (representing 2–5% of CD3+ cells) 15-17. The TCR δ repertoire of skin-derived γδ T cells seems to be restricted in healthy human skin dominated by Vδ1 cells 18, 19. An increased number of γδ T cells have been found in some inflammatory skin conditions (Langerhans cell histiocytosis, lichen planus, pityriasis lichenoides chronica and allergic contact dermatitis), but not in others (viral warts, graft-versus-host disease, discoid lupus erythematosus and non-melanoma skin cancer) 20, 21. As expected, we also found a low number of skin-derived γδ T cells (10.0 ± 7.1% of the CD45+ cells, N = 10) in our analysis (Fig. 1). Although the percentage of these cells was higher than expected, we were still not able to analyse their cytokine expression due to low absolute numbers. In conclusion, this study shows that Th1/Tc1 and Th17/Tc17 are present in healthy skin and at the same time it confirms previous immunohistochemical assessments of the distribution of γδ T cells. There are still many questions left unanswered including aspects of the biology, temporal development and role in the pathogenesis in inflammatory skin diseases of the resident and recirculating T cells found in human skin, production of other cytokines in healthy skin and the distribution and function of γδ T cell subsets in the human skin. The authors would like to thank The Department of Plastic Surgery, Herlev and Gentofte Hospital and Amalieklinikken, for discarded human skin and Anni Olsen and Mie Mouritzen for technical assistance.