Original ArticlePro-inflammatory Vδ1+T-cells infiltrates are present in and around the hair bulbs of non-lesional and lesional alopecia areata hair follicles
Introduction
Human skin-resident lymphocytes are composed of αβT-cells, NK cells, and γδT-cells [1]. γδT-cells represent a minor T-cell population in human skin (<5% in healthy skin) [2], where they are thought to preferentially reside in the epidermis and papillary dermis and to express mainly Vδ1 [[1], [2], [3]]. Although intracutaneous γδT-cells represent less than 5% of the lymphocytic infiltrate, and are undetectable in more than 70 % cases [4], their count is increased in most cutaneous inflammatory conditions and are known producers of pathological cytokines, e.g. IFN-γ or IL-17 [2,5,6]. So far, in clinical dermatology, they have been mainly associated with rare cases of γδT-cell cutaneous lymphoma [6], and some skin diseases [1,3,7], including autoimmune disorders, such as psoriasis [3,5,7,8]. Despite this evidence, overall, the functional roles of γδT-cells in human skin physiology and pathology remains quite unclear [9].
These evolutionarily ancient, mostly antigen-non-specific lymphocytes recognize groups of conserved antigens (e.g., certain lipids as well as cell surface molecules shared by different bacteria) and are thought to represent protagonists of so-called “transitional immunity” [1,3,[10], [11], [12], [13], [14]]. In mice, γδT-cells are well-known to execute stress, tumor immunosurveillance, immunoregulatory, and infection-defense functions [1,10,11]. In addition, γδT-cells play key roles in wounding-induced hair follicle (HF) neogenesis [15] and in regulating HF cycling [16] in mice, further suggesting intimate bidirectional communication between HFs and γδT-cells in murine skin. Instead, even though we have previously shown that very few γδT-cells can also be found in and around healthy human HFs [17], the role of γδT-cells in the physiology and pathology of human scalp skin and its appendages remains quite obscure.
Alopecia areata (AA) is one of the most frequent human autoimmune disorders and is characterized by HF immune privilege collapse, abnormal HF cycling and HF dystrophy [18,19]. It is well appreciated that the number of αβT-cells and NK cells is significantly increased around AA lesional HFs [[20], [21], [22], [23]], and that IFN-γ releasing [24,25] CD8+NKG2D+ cells represent key effector cells in disease pathogenesis [19,22]. Given that AA affected human HFs overexpress CD1d [26], key molecule for presenting antigens (e.g., certain lipids) to the γδTCR [1,11], as well as the distress ligands (MICA, ULBP3/6) [21,23,27] that activate NKG2D receptors, which are expressed also on γδT-cells [1,11,28], and CXCL10 [29,30], a chemoattract for γδT-cells [6], we hypothesized that also γδT-cells may play a role in the pathogenesis of this human autoimmune disease.
In the current pilot study, we have begun to probe this novel working hypothesis in AA research by evaluating the distribution, subset, and activation status of γδT-cells in human scalp skin in vivo by FACS analysis, and by comparing relevant γδT-cells subpopulations in healthy human scalp skin with those in lesional and non-lesional human AA scalp biopsies via quantitative (immuno-)histomorphometry. In addition, we also asked if γδ+T-cells infiltrating in/around non-lesional and/or lesional AA HFs show pro-inflammatory phenotype by evaluating the expression of NKG2D, IFN-γ, i.e. key cytokine involved in AA pathology [18,19,31,32], and CD200R, a key immunoinhibitory receptor expressed by γδT-cells [14,33], in these cells.
The observations reported in this initial pilot study strongly suggest that pro-inflammatory HF-infiltrating Vδ1+T-cells were indeed previously overlooked, but are potentially important new players in the pathobiology of human AA.
Section snippets
Human samples
This study was conducted according to the Declaration of Helsinki principles. Human lesional and non-lesional skin biopsies were obtained from AA patients showing mainly active hair loss of the AA totalis or universalis phenotype, while one patient showed only a stable hair loss patch, after written consent and ethic committee approval from the University “La Sapienza” of Rome (n. 2973, 28−11-13). Clinically healthy human skin scalp specimens were obtained after informed written patient consent
γδT-cells in human scalp skin are mainly Vδ1+
First, we investigated the subtype of freshly isolated human scalp skin γδT-cells by flow cytometry in human scalp skin from 3 different clinically “healthy” donors undergoing face lift surgery. Our results showed that in human scalp skin around 0.2–1 % of CD3+T-cells were indeed γδT-cells, i.e. a lower percentage than reported for other human body locations [35,36]. Interestingly, high inter-individual variations were detected for the preferential intracutaneous localization of these cells,
Discussion
Our pilot study provides the first evidence that lesional HFs in scalp biopsies from AA patients display a greatly increased number of both peri- and even intra-follicular γδT-cells. These are mostly Vδ1+, i.e. skin-resident γδT-cells and not circulating Vδ2+ T-cells [9,38,39], and infiltrate the proximal HF epithelium, just like the key effector cells NKG2D+CD8+T-cells, and NK cells in acute human AA [[20], [21], [22], [23],48].
These γδT-cells are responsive to HF-derived chemokines known to
Funding sources
The study was supported in part by research grants from National Alopecia Areata Foundation (NAAF, USA) to R.P. and M.B, from Associazione Nazionale Mediterranea Alopecia Areata (ANMAA, Italy) to M.B., by the NIHR Manchester Biomedical Research Centre, “Inflammatory Hair Diseases” program, and by the German Research Foundation (DFG; GR3946/3−1 and SFB/Transregio 128 A09) to C.C.G.. Monasterium Laboratory supported data analyses and the writing of this manuscript.
Declaration of Competing Interest
The authors have declared that no conflict of interest exists.
Acknowledgements
Most experiments for this study were performed when Y.U., J.G. M.A., M.B. and R.P. worked together at the Department of Dermatology, University of Muenster, Germany, while the study was finalized at Monasterium Laboratory, Münster, after they had left the former institution. The support of Drs. Mathias Sulk, Leslie Ponce, Janin Edelkamp, Ana Monteiro, and of our technicians is gratefully acknowledged. We thank Prof. Karin Loser for access to MACS facilities.
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These authors contributed equally to this work.