Original Article
Pro-inflammatory Vδ1+T-cells infiltrates are present in and around the hair bulbs of non-lesional and lesional alopecia areata hair follicles

https://doi.org/10.1016/j.jdermsci.2020.09.001Get rights and content

Highlights

  • Skin-resident γδT-cells are mostly Vδ1+, non-activated, and predominantly localized in/around distal hair follicles in human scalp skin.

  • Vδ1+T-cells are significantly increased in/around the hair bulb of lesional, and around non-lesional alopecia areata (AA) hair follicles.

  • Vδ1+T-cells infiltrating in/around AA hair follicles are pro-inflammatory.

  • Skin-resident Vδ1+T-cells represent previously overlooked, but potentially important, innate immunity protagonists in AA pathobiology.

  • Vδ1+T-cells may be involved in the early stages of human AA, and may thus deserve therapeutic targeting for optimal AA management.

Abstract

Background

It is widely accepted that NKG2D+cells are critically involved in alopecia areata (AA) pathogenesis. However, besides being expressed in CD8+T-cells and NK cells, NKG2D is also found in human γδT-cells. AA lesional hair follicles (HFs) overexpress NKG2D and γδTCR activating ligands, e.g. MICA and CD1d, and chemoattractants for γδT-cells, such as CXCL10.

Objective

To investigate whether abnormal activities of γδT-cells may be involved in AA pathogenesis.

Methods

We analyzed the number and activation status of γδT-cells in human healthy, lesional and non-lesional AA scalp biopsies by FACS and/or quantitative (immuno-)histomorphometry.

Results

In healthy human scalp skin, the few skin-resident γδT-cells were found to be mostly Vδ1+, non-activated (CD69NKG2Ddim) and positive for CXCL10, and CXCL12 receptors. These Vδ1+T-cells predominantly localized in/around the HF infundibulum. In striking contrast, the number of Vδ1+T-cells was significantly higher around and even inside the proximal (suprabulbar and bulbar) epithelium of lesional AA HFs. These cells also showed a pro-inflammatory phenotype, i.e. higher NKG2D, and IFN-γ and lower CD200R expression. Importantly, more pro-inflammatory Vδ1+T-cells were seen also around non-lesional AA HFs. Lesional AA HFs also showed significantly higher expression of CXCL12.

Conclusion

Our pilot study introduces skin-resident γδT-cells as a previously overlooked, but potentially important, mostly (auto-)antigen-independent, new innate immunity protagonist in AA pathobiology. The HF infiltration of these activated, IFN-γ-releasing cells already around non-lesional AA HFs suggest that Vδ1+T-cells are involved in the early stages of human AA pathobiology, and may thus deserve therapeutic targeting for optimal AA management.

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.

References (50)

  • T. Ito et al.

    Collapse and restoration of MHC class-I-dependent immune privilege: exploiting the human hair follicle as a model

    Am. J. Pathol.

    (2004)
  • S.J. Lalor et al.

    Memory γδ T Cells-Newly Appreciated Protagonists in Infection and Immunity

    Trends Immunol.

    (2016)
  • R. Woolf et al.

    Human skin-resident innate-like T cells in health and disease

    J. Invest. Dermatol.

    (2017)
  • A. Poggi et al.

    Migration of V delta 1 and V delta 2 T cells in response to CXCR3 and CXCR4 ligands in healthy donors and HIV-1-infected patients: competition by HIV-1 Tat

    Blood.

    (2004)
  • J.W. Oh et al.

    A guide to studying human hair follicle cycling in vivo

    J. Invest. Dermatol.

    (2016)
  • A. Gilhar et al.

    Alopecia areata: animal models illuminate autoimmune pathogenesis and novel immunotherapeutic strategies

    Autoimmun. Rev.

    (2016)
  • S. Tikoo et al.

    The lymphoid cell network in the skin

    Immunol. Cell Biol.

    (2018)
  • L.M. Ebert et al.

    Homing and function of human skin gammadelta T cells and NK cells: relevance for tumor surveillance

    J. Immunol.

    (2006)
  • D. Das et al.

    T helper type 1 polarizing γδ T cells and Scavenger receptors contribute to the pathogenesis of Pemphigus vulgaris

    Immunology

    (2018)
  • T.L. Hocker et al.

    Expression of T-cell receptor-γδ in normal human skin, inflammatory dermatoses and mycosis fungoides

    J. Cutan. Pathol.

    (2012)
  • E. Lo Presti et al.

    Squamous Cell Tumors Recruit γδ T Cells Producing either IL17 or IFNγ Depending on the Tumor Stage

    Cancer Immunol. Res.

    (2017)
  • U. Laggner et al.

    Identification of a novel proinflammatory human skin-homing Vγ9Vδ2 T cell subset with a potential role in psoriasis

    J. Immunol.

    (2011)
  • M.S. Cruz et al.

    Human αβ and γδ T Cells in Skin Immunity and Disease

    Front. Immunol.

    (2018)
  • D. Melandri et al.

    The γδTCR combines innate immunity with adaptive immunity by utilizing spatially distinct regions for agonist selection and antigen responsiveness

    Nat. Immunol.

    (2018)
  • A.E. Simões et al.

    Molecular Determinants of Target Cell Recognition by Human γδ T Cells

    Front. Immunol.

    (2018)
  • Cited by (23)

    • Advancements in the characterization of tissue resident memory T cells in skin disease

      2022, Clinical Immunology
      Citation Excerpt :

      γδT cells may contribute to the pathogenesis of alopecia areata by producing IFN-γ. Compared to those in normal scalp skin, significantly increased numbers of resident γδT cells have been found around the lesional hair follicles of alopecia areata patients, showing a proinflammatory phenotype, i.e., higher NKG2D and IFN-γ and lower CD200R expression [94]. In addition, IL-17-producing γδT cells have been shown to have antitumor activity.

    • CXCL12 inhibits hair growth through CXCR4

      2022, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      In alopecia areata (AA), an autoimmune disease that induces hair loss, human dermal γδ T cells are known to act as stress sentinels by directly eliciting autoimmune reactions. In addition, in AA, stressed hair follicles overexpress the γδ T cell chemoattractants CXCL10 and CXCL12 [20,21]. These findings suggest that CXCL12 and CXCR4 may play a role in hair loss in various disorders and that blocking CXCL12/CXCR4 pathway may be a therapeutic target for hair loss [22].

    • The current state of knowledge of the immune ecosystem in alopecia areata

      2022, Autoimmunity Reviews
      Citation Excerpt :

      Further studies are required to determine whether the Th2 profile that can be seen in the skin of AA patients is a contributing factor to the disease itself, or whether it is having an indirect effect and whether targeting Th2 cytokines may be an efficacious therapeutic strategy. Observations of healthy hair follicles have identified few γδ T cells to be present; however, in AA skin, the number of γδ T cells is significantly higher when compared to healthy skin, and they appear to be localized to the bulb and suprabulbar epithelium [45,131]. Because γδ T cells share some functional and phenotypic characteristics with conventional αβ T cells, including expression of NKG2D and secretion of cytotoxic molecules and pro-inflammatory cytokines, there is biological plausibility for their participation in AA pathogenesis [132].

    • Resident human dermal γδT-cells operate as stress-sentinels: Lessons from the hair follicle

      2021, Journal of Autoimmunity
      Citation Excerpt :

      One part was used for isolating the dermal cells, the other for microdissecting HFs. Human dermal cells were prepared exactly as described previously [10]. In brief, 40–60 × 4 mm full-thickness skin punches were immediately treated with 10 mg/mL Dispase® (Invitrogen-Gibco, Grand Island, NY) in a mixture of William's E (Biocrom KG, Seromed, Berlin) and RPMI1640 (Invitrogen-Gibco) medium (1:1 ratio) supplemented with penicillin/streptomycin and incubated overnight at 4 °C for epidermal-dermal separation.

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text