γδ T cells mediate rapid tissue responses in murine skin and participate in cutaneous immune regulation including protection against cancer. The role of human γδ cells in cutaneous homeostasis and pathology is characterized poorly. In this study, we show in vivo evidence that human blood contains a distinct subset of proinflammatory cutaneous lymphocyte Ag and CCR6-positive Vγ9Vδ2 T cells, which is rapidly recruited into perturbed human skin. Vγ9Vδ2 T cells produced an array of proinflammatory mediators including IL-17A and activated keratinocytes in a TNF-α– and IFN-γ–dependent manner. Examination of the common inflammatory skin disease psoriasis revealed a striking reduction of circulating Vγ9Vδ2 T cells in psoriasis patients compared with healthy controls and atopic dermatitis patients. Decreased numbers of circulating Vγ9Vδ2 T cells normalized after successful treatment with psoriasis-targeted therapy. Taken together with the increased presence of Vγ9Vδ2 T cells in psoriatic skin, these data indicate redistribution of Vγ9Vδ2 T cells from the blood to the skin compartment in psoriasis. In summary, we report a novel human proinflammatory γδ T cell involved in skin immune surveillance with immediate response characteristics and with potential clinical relevance in inflammatory skin disease.
In murine skin, the function of γδ T cells has been investigated extensively. Mouse epidermis contains large numbers of dendritic Vγ5Vδ1+ T cells, appropriately termed dendritic epidermal T cells (DETC) (1). DETC have been shown to participate in immunoregulation of the skin (2, 3) and to protect against epithelial malignancies (4, 5). Furthermore, γδ T cells in murine skin may produce growth factors that maintain epidermal integrity (6, 7). In contrast to mouse skin, γδ T cells are rare in healthy human skin, and an equivalent of mouse DETC does not appear to exist. Approximately 2–9% of all dermal and 1–10% of all epidermal T cells are γδ T cells (8–14). Vδ1 T cells are considered to be the primary γδ T cell subset in skin playing a role in skin cancer immune surveillance (14) and wound healing (13), whereas a direct association of Vγ9Vδ2 T cells with cutaneous immunology has to date been lacking. Because γδ+ T cells are less frequent in human skin compared with the frequency of DETC in murine skin, a key question is whether they perform a similar lymphoid stress–surveillance role as their murine counterpart (15).
Psoriasis is a common chronic inflammatory skin disease with a significant genetic disease susceptibility component (16, 17). Although the contribution of effector cells of the adaptive immune system including T cells to disease pathogenesis is well established, recent interest has focused on effector cells of the innate immune system and their putative roles in the initiation and maintenance of psoriatic inflammation (18).
In this article, we characterize a novel proinflammatory human skin homing Vγ9Vδ2 T cell subset, which is characterized by early migration to perturbed human skin in vivo, suggesting a role in tissue immunosurveillance. In plaque-type psoriasis, this subset is preferentially decreased in peripheral blood and increased in psoriatic skin, indicating a potential clinical relevance in the pathogenesis of this major inflammatory skin disease.
Materials and Methods
Sixty-six patients (age, 25–56 y) with plaque-type psoriasis (Psoriasis Area and Severity Index [PASI] 1–33) were enrolled in this study. The PASI measures extent and severity of skin inflammation. Venous blood and in some cases skin biopsies of patients were taken. Thirty-two age-matched healthy volunteers (age, 28–50 y) and eight age-matched atopic dermatitis patients (age, 26–65 y) were used as control. The patients were gender matched (male-to-female ratio healthy, 0.56; psoriasis, 0.52; atopic dermatitis, 0.33). Discarded healthy skin from female donors aged between 19 and 59 y was obtained after plastic surgery procedures. Human studies were conducted in accordance with the Helsinki Declaration and approved by the Institutional Review Board of Guy’s and St. Thomas’ Hospital, and informed consent was obtained from all patients and control individuals.
Patients included in the study received different treatment regimes at the time of sampling: 32 had not received any systemic treatment for at least 4 wk prior to the time the blood sample was taken (“no systemic treatment”). Thirty-four patients received systemic therapies: 16 patients were treated with different biologic therapies such as etanercept (n = 12), adalimumab (n = 3), and efalizumab (n = 1), 4 patients received biologic therapies in combination with methotrexate (methotrexate + infliximab, n = 2; methotraxate + etanercept, n = 2), 9 patients were treated with methotrexate only, and 5 patients received other therapies such as fumaric esters (n = 2) and acitretin (n = 3). Skin biopsies were taken exclusively from patients who had not received any systemic treatment for at least 4 wk prior. Patients who were evaluated before and 4 wk after treatment initiation received etanercept (patients C and E) and adalimumab (patient D), respectively. For clinical correlation, only patients not receiving systemic treatment were used.
Cell isolation and culture
19). For isolation of primary human keratinocytes, skin was incubated overnight in dispase (Roche) on 4°C. Then, epidermis and dermis were separated, and epidermal sheets were immersed in trypsin for 10 min on 37°C. Resulting cell suspension was seeded in 75-cm2 flasks precoated with coating matrix (Life Technologies) in Epilife keratinocyte medium (Life Technologies) supplemented with human keratinocyte growth supplement (Life Technologies).
+Vδ2+ cells. The procedure of staining and gating was identical for each specimen.
Immunohistochemistry and immunofluorescence
Frozen 5-μm-thick sections of skin punch biopsies were fixed in ice-cold acetone for 10 min. For immunofluorescence staining, sections were incubated with 5% normal goat serum (DakoCytomation) for 20 min and were then immersed in Ab diluent (DakoCytomation) containing the primary Abs for 1 h, followed by extensive washing in PBS. Abs used for the identification of Vγ9Vδ2 T cells were polyclonal rabbit CD3 (1:100; DakoCytomation) and monoclonal mouse Vδ2 (1:50; Beckman Coulter). The secondary Abs (goat anti-mouse 488 and goat anti-rabbit 555) at a concentration of 1:200 were applied to the sections for 30 min. For nuclear staining, Topro-3 (1:500) was added to the secondary Ab solution. Immunofluorescence pictures were taken using a Zeiss LSM Confocal microscope and analyzed with LSM image browser software. For immunohistochemical staining with the Vγ9 Ab (1:50; Beckman Coulter), LSAB kit (DakoCytomation) was used according to the manufacturer’s instructions.
Determination of chemokine, cytokine, and growth factor levels
HMB-PP–expanded CLA+Vγ9Vδ2 T cells were sorted, seeded at 1.5 × 105 cells/well in 96-well plates, and cultured for 72 h with respective stimuli for analysis of supernatant. A total of 5–7.5 × 105 cells/well in 48-well plates were cultured for 24 h for subsequent RNA isolation. The following stimuli were used: 10 nM HMB-PP + 60 IU/ml IL-2, 10 μl/ml anti–CD3/CD28-coated beads + 60 IU/ml IL-2, 1000 IU/ml IFN-α, or 5 ng/ml PMA and 1 μM ionomycin. Medium only served as a negative control. Keratinocytes were activated as described in Keratinocyte activation assays
Keratinocyte activation assays
Primary human keratinocytes at passages 2–4 were incubated with a 1:1 mixture of Epilife medium and supernatant of resting or activated CLA+
Skin suction blisters were induced applying negative pressure of 25–40 kPa (200–300 mmHg) below atmospheric pressure via a suction chamber (Medical Engineering, Royal Free Hospital, London, U.K.) for 2–4 h using a clinical suction pump (VP25; Eschmann) until a unilocular blister measuring 10–15 mm in diameter was formed between dermis and epidermis at the level of the lamina lucida. Blisters were performed either on unperturbed skin or after skin perturbation (injection of 100 μl sterile saline 24 h prior to blister). Blister fluid was aspirated after 16–20 h using a sterile syringe. The amount of blister fluid was measured and then microcentrifuged at 650 × g for 4 min. Cells were stained for flow cytometry.
Experiments were performed on fresh PBMCs and HMB-PP–expanded Vγ9Vδ2 T cell lines. A total of 1 × 105
RNA extraction, cDNA generation, and real-time PCR analysis
Total RNA was obtained using the RNAeasy Plus Mini Kit (Qiagen), according to the manufacturer’s instructions, and reverse transcribed into cDNA (Superscript II; Invitrogen). TNF, IFN-γ, IL-17A, IL-6, CXCL9, CXCL-10, CCL3, CCL4, CCL5, IL-8, CCL20, VEGF, FGF, KGF, IL-22, IL-4, S100A7, S100A8, β-defensin, and LL37 expression was assessed by multiplex real-time quantitative RT-PCR using TaqMan assays (Applied Biosystems), according to the manufacturer’s instructions. For each sample, mRNA abundance was normalized to the amount of human GAPDH (Vγ9Vδ2 T cells) or cyclophilin (keratinocytes). Data analysis was performed using the ΔΔCt method; results are expressed as fold change.
To calculate absolute numbers of CD3+ cells and Vγ9Vδ2 T cells in the blister, we used Trucount tubes (BD Biosciences) together with BD Tritest (CD3/CD16/CD56/CD45) (BD Biosciences), according to the manufacturer’s instructions. For absolute quantification of T cells in the skin, the method published by Clark et al. (20) was used. Pictures of four randomly selected fields (×20 magnification) were taken with a Zeiss LSM. Pictures were then analyzed with the LSM image browser software. For each picture, Vδ2 T cells were counted, the length of skin was recorded, and the number of Vδ2 T cells in 1-cm skin was calculated. For quantification of Vδ2 percentage of total CD3 T cells in skin, T cell numbers in skin were counted for CD3- and Vδ2/CD3-expressing cells using the ImageJ software.
Extrapolation of total absolute Vγ9Vδ2 T cell numbers in skin and blood
Absolute numbers of Vδ2 expressing cells in 1 cm skin were acquired as described above and the resulting number was multiplied by 2000 to calculate Vδ2 T cell numbers in 1 cm2 of skin. Patient body surface area (BSA) was calculated using the following formula by Mosteller: BSA (m2) = [(height [cm] × weight [kg])/3600]1/2. Clinical examination revealed the approximate percentage of BSA affected by psoriasis from which we extrapolated absolute Vδ2 T cell in psoriatic skin. For analysis of absolute numbers in blood, the absolute number of lymphocytes present in 1 μl blood was performed in the hematology laboratory at Guy’s and St. Thomas’ Hospital. Flow cytometry staining revealed the percentage of CD3 T cells and Vδ2 T cells in the lymphocyte population. Patient blood volume was estimated using the following formula: 0.3669 × height (m)3 + 0.03219 × weight (kg) + 0.6041, from which the absolute Vδ2 T cells in peripheral circulation was calculated.
Data were assayed for normal distribution by using the D’Agostino–Pearson test and analyzed using unpaired two-tailed student t test for determination of significant differences between two unpaired groups normally distributed and one-way ANOVA for more than two unpaired normally distributed groups. For comparison of two paired groups with normal distribution, a paired t test was used, and repeated measure one-way ANOVA was used for more than two matched groups. To compare not normally distributed samples, we used Mann–Whitney U test to compare two groups and Kruskal–Wallis test for comparison of three groups. As a nonparametric test for two paired groups, we used Wilcoxon matched pair test. Pearson correlation was used to correlate clinical variables and percentage/absolute numbers of γδ T cells, whereas one-way exponential decay was applied for calculation of R2. For all statistical tests, we considered p values <0.05 to be statistically significant. Results are expressed as mean ± SEM.
Identification of a subset of circulating Vγ9Vδ2 T cells expressing CLA and skin homing chemokine receptors
We performed extensive phenotypic screening of innate and adaptive lymphocytes in peripheral blood from healthy volunteers and patients with psoriasis. The percentage of CD4 T cells, CD8 T cells, invariant NKT cells, CD3+CD161+ NK-like T cells, CD56+CD16+ NK cells, and CD56bright NK cells did not show any statistically significant difference between psoriasis patients and healthy individuals (Supplemental Fig. 1A–F). However, we found a significant decrease of circulating Vγ9Vδ2 T cells in psoriasis patients 2.16% (± 0.23%, n = 66) compared with healthy controls 4.21% (± 0.55%, n = 32; p = 0.01) (Fig. 1A). To investigate whether this observation was restricted to psoriasis, we assessed Vγ9Vδ2 T cells in atopic dermatitis, another common inflammatory skin disease with T cell involvement (21). Percentages of circulating Vγ9Vδ2 T cells in atopic dermatitis patients (n = 8) were significantly higher than in psoriasis patients (5.18% [± 1.75%]; p < 0.01) (Fig. 1A), indicating that the reduction of Vγ9Vδ2 T cells in psoriasis was not due to unspecific skin inflammation. Vδ1 T cells, the other main γδ T cell subset in humans, was unchanged in the peripheral blood of psoriasis compared with healthy controls and AD (Fig. 1B). We had absolute cell numbers for 44 of 66 psoriasis patients. Mean absolute Vγ9Vδ2 T cell numbers in 1 μl peripheral psoriatic blood was substantially lower (mean 29 [± 4.7]/μl) compared with what has been published for healthy controls (49 cells/μl (22) and 68.6 cells/μl (23)) (Supplemental Fig. 1G), further supporting a reduction of circulating Vγ9Vδ2 T cells in psoriasis.
We then assessed the skin homing phenotype of circulating Vγ9Vδ2 T cells in psoriasis patients and healthy volunteers using flow cytometry analysis of fresh PBMCs. A proportion of Vγ9Vδ2 T cells in patients and controls both expressed CLA, a marker for skin homing T cells (Fig. 2A). Only the CLA+Vγ9Vδ2 T cell subset was significantly decreased in patients (n = 32) compared with controls (n = 19) (0.94% [± 0.11%] versus 2.22% [± 0.38%]; p < 0.001), whereas the CLA− subset was not significantly changed (1.74% [± 0.29%] versus 2.67% [± 0.58%]) (Fig. 2B), demonstrating a preferential reduction of peripheral CLA+ Vγ9Vδ2 T cells in psoriasis. CLA+ total T cells were comparable in patients and controls (16.75% [± 1.39%] versus 18.76% [± 1.26%]) (data not shown).
Next, we investigated the expression of skin homing chemokine receptors. CCR6 expressing cells were the most represented within peripheral Vγ9Vδ2 T cells of patients and controls, whereas a variable Vγ9Vδ2 T cell subset expressed CCR4 and a small subset CCR10 (Fig. 2C). Comparing psoriasis patients and controls, we found that Vγ9Vδ2 T cells of patients showed decreased expression of CCR6 (16.5% [3.5–73%, n = 24] versus 30.61% [7.3–59%, n = 22]; p < 0.05) and CCR10 (1.6% [0–15%, n = 22] versus 5% [0–10%, n = 17]; p < 0.05) compared with healthy controls (Fig. 2D), whereas there was no difference in CCR4 expression (2.75% [0–14.9%, n = 22] versus 2.8% [1–17%, n = 17]; ns) (Fig. 2D). Vγ9Vδ2 T cells did not express CD103 or CCR9 associated with gut homing (Supplemental Fig. 2A, 2B).
Because Vγ9Vδ2 T cells are rare in peripheral blood, we generated Vγ9Vδ2 T cell lines by culturing PBMCs with the Vγ9Vδ2 T cell-specific Ag HMB-PP and IL-2 as described previously (24). Using these Vγ9Vδ2 T cell lines, we examined the regulation of CLA on Vγ9Vδ2 T cells. Activation with HMB-PP or the superantigen staphylococcal enterotoxin B did not upregulate CLA expression on peripheral Vγ9Vδ2 T cells. However, incubation with the cytokine IL-12, which has been shown to induce CLA on αβ T cells (25), induced CLA expression also on Vγ9Vδ2 T cells independent of activation (n = 3) (Supplemental Fig. 2C). IL-12–dependent CLA upregulation was confirmed on fresh peripheral Vγ9Vδ2 T cells (n = 4) (Supplemental Fig. 2D).
Vγ9Vδ2 T cells are recruited to perturbed human skin in vivo
Expression of CLA on peripheral Vγ9Vδ2 T cells suggested the existence of a specialized skin homing Vγ9Vδ2 T cell subset possibly recruited to skin under conditions of skin perturbation. To assess in vivo dynamics of cutaneous Vγ9Vδ2 T cell homing, we performed skin suction blisters in healthy volunteers (Fig. 3A). The skin suction blister model allows for the ex vivo analysis of cells present within skin under conditions of tissue homeostasis and pathology (26, 27). We compared Vγ9Vδ2 T cells present in skin blisters induced on normal skin (“nonperturbed” condition) with those induced on “perturbed” skin previously injected with physiological saline. Within the CD3+ T cell population, perturbed skin contained significantly higher percentages of Vγ9Vδ2 T cells than nonperturbed skin (1.2% [± 0.16%] versus 0.7% [± 0.08%]; p < 0.05) (Fig. 3B). Absolute numbers of Vγ9Vδ2 T cells (4.6 × 102 [± 1.06 × 102] versus 1.02 × 103 [± 1.5 × 102]/ml blister fluid; p < 0.05) and of non-Vγ9Vδ2 T cells (7.3 × 104 [± 2.1 × 104] versus 1.04 × 105 [± 2.7 × 104]/ml blister fluid; p < 0.05) both increased in perturbed skin (Fig. 3C, 3D). However, the relative increase of Vγ9Vδ2 T cells between nonperturbed and perturbed skin of the same individual was significantly higher (125.9% [± 29.1%]) compared with the increase of non-Vγ9Vδ2 T cells (44.5% [± 11.1%]; p < 0.05)) (Fig. 3E). Vγ9Vδ2 T cells isolated from perturbed skin contained significantly more CLA+Vγ9Vδ2 T cells than circulating Vγ9Vδ2 T cells from the same individuals (perturbed, 63.8% [± 7.1%]; circulating, 28.8% [± 5.0%]; p < 0.01) (Fig. 3F). These data indicate migration of CLA+Vγ9Vδ2 T cells to skin and highlight the existence of a specialized circulating immune cell subset with the potential for immediate skin recruitment.
Vγ9Vδ2 T cells are increased in skin of psoriasis patients
Skin homing properties of peripheral Vγ9Vδ2 T cells suggested that the selective reduction of Vγ9Vδ2 T cells in the blood of psoriasis patients might be due to their redistribution into the skin. Therefore, we examined the presence of Vγ9Vδ2 T cells in psoriatic skin lesions. Vδ2+ T cells coexpressing CD3 were preferentially located in psoriatic dermis (Fig. 4A). Few Vδ2+ T cells were identified in clinically normal appearing psoriatic skin (“nonlesional psoriatic skin”) (Fig. 4B), whereas Vδ2+ T cells were rarely found in healthy skin (Fig. 4C). Immunohistochemical staining for Vγ9+ cells revealed positive cells around blood vessels in the dermis and scattered in the epidermis (Supplemental Fig. 2E). Vγ9+ T cells were also detected in nonlesional psoriatic skin (Supplemental Fig. 2F) while being rare in normal skin (Supplemental Fig. 2G). Vδ2 and CLA were commonly coexpressed in psoriatic skin confirming the infiltration of a skin homing Vγ9Vδ2 T cell subset (Fig. 4D).
Absolute numbers of Vγ9Vδ2 T cells were significantly higher in psoriatic lesions (23.62 [± 4.95]/cm) than in nonlesional psoriatic skin (8.00 [± 2.55]/cm; p < 0.01) and healthy skin (0.63 [± 0.63]/cm; p < 0.001) (Fig. 4E). Quantifying Vγ9Vδ2 T cells in skin sections as percentage of total CD3+ cells, we found a significantly higher percentage of Vγ9Vδ2 T cells in lesional (1.31% [± 0.19%]; n = 13) as well as nonlesional (1.23% [± 0.32%], n = 8) psoriatic skin compared with healthy skin (0.12% [+0.12%], n = 8; both p < 0.01) (Fig. 4F). Higher proportions of Vγ9Vδ2 T cells already strategically positioned in nonlesional psoriatic skin indicate a potential role in development of early lesions.
Flow cytometry analysis of T cell lines from lesional (n = 12), nonlesional (n = 11) and healthy (n = 8) dermis revealed significantly higher percentages of Vγ9Vδ2 T cells in lesional psoriatic skin compared with healthy skin (1.03% [± 0.31%] versus 0.21% [± 0.18%]; p < 0.05), whereas the percentage of Vγ9Vδ2 T cells in nonlesional psoriatic skin was also slightly increased (0.39% [± 0.16%]; ns) (Fig. 4G).
Correlation between severity of psoriatic disease and percentage of circulating Vγ9Vδ2 T cells
To evaluate the clinical relevance of Vγ9Vδ2 T cells, we analyzed circulating Vγ9Vδ2 T cells in various disease severity states as reflected by the PASI. We found a significant correlation between severe clinical disease (higher PASI) and lower percentages of circulating Vγ9Vδ2 T cells (R2 = 0.53, p < 0.001, n = 30) (Fig. 5A). Absolute Vγ9Vδ2 T cell numbers were obtained in a subpopulation of patients (n = 16) and also negatively correlated with disease severity (R2 = 0.92, n = 16; p = 0.05) (Fig. 5B). In addition, there was no correlation between age or gender with levels of peripheral Vγ9Vδ2 T cells (data not shown). None of the other immune cell subsets investigated (total CD3+, CD4+, CD8+, Vδ1+, CD56+, and CD161+ NK-like T cells, CD56+CD16+ NK cells, and CD56bright NK cells) showed a significant positive or negative correlation with disease severity as indicated by the PASI index (data not shown).
We selected two patients with a PASI >20 for detailed calculation of Vγ9Vδ2 T cell numbers. We took advantage of a method developed for quantification of T cell numbers in skin by the group of Kupper and Clark (20). On the basis of this method, calculations in patient A (with 50% of the body surface involved) showed a total of 7.7 × 108 Vγ9Vδ2 T cells present in inflamed skin. This patient had ∼14 times more Vγ9Vδ2 T cells in inflamed psoriatic skin than in peripheral blood (0.53 × 108 Vγ9Vδ2 T cells) (Supplemental Fig. 3A). Similar numbers were obtained for an additional patient B with a PASI score > 20 (Supplemental Fig. 3B). These data are in line with our hypothesis that Vγ9Vδ2 T cells redistribute from blood to skin during skin inflammation.
Next, we sought to investigate whether treatment would affect peripheral Vγ9Vδ2 T cells. We found that Vγ9Vδ2 T cells percentage (2.44% [± 0.35%] versus 1.93 [± 0.31%]; ns) (Fig. 5C) and absolute number (data not shown) were similar in peripheral blood of untreated patients and patients on systemic therapy (Fig. 5C) having the same median PASI score (5.95 [1.2–32.2] versus 5.35 [0.6–22.4]). However, when we followed three patients before and 4 wk after successful therapy (PASI score reduced by >40% from baseline), we found that alleviation of psoriasis was accompanied by an increase of absolute T cell numbers (Fig. 5D) and percentage (data not shown) in peripheral blood. Possibly as a result of residual disease, Vγ9Vδ2 T cell levels did not consistently reach levels seen in healthy controls (data not shown).
Taken together, psoriasis severity correlated with decreased numbers of circulating Vγ9Vδ2 T cells and was reversed after successful treatment with psoriasis-targeted therapy, suggesting a clinically significant role of Vγ9Vδ2 T cells in psoriasis.
CLA+Vγ9Vδ2 T cells produce psoriasis-relevant proinflammatory cytokines, chemokines and growth factors
To further define the role of Vγ9Vδ2 T cells in psoriasis, we investigated a range of proinflammatory mediators with potential relevance to psoriasis pathogenesis. CLA+Vγ9Vδ2 T cells produced high levels of TNF-α and IFN-γ upon TCR-specific (HMB-PP) and unspecific stimulation, whereas constitutive production in resting cells was low to undetectable (representative experiment, n = 8) (Fig. 6A). Vγ9Vδ2 T cells produced IL-17A and IL-17A–producing Vγ9Vδ2 T cells were enriched in the CLA+ population (CLA+, 0.4% [± 0.07] versus CLA−, 0.15% [± 0.05%]; p = 0.0313) (Fig. 6B). CLA+Vγ9Vδ2 T cells did not produce IL-22 (data not shown).
Activated CLA+Vγ9Vδ2 T cells produced the psoriasis-relevant chemokine IL-8 (Fig. 6C). In addition, they produced the proinflammatory chemokines CCL3 (MIP-1α) and CCL4 (MIP-1β), which were markedly upregulated upon activation (Fig. 6C). CCL5 (RANTES) was constitutively produced at high levels (Fig. 6C).
Psoriasis is an immune-mediated disease associated with hyperproliferation of keratinocytes and angiogenesis; we therefore assessed whether CLA+Vγ9Vδ2 T cells produced growth factors such as VEGF, KGF, FGF-2, EGF, and IGF-1. CLA+Vγ9Vδ2 T cells upregulated IGF-1 production when stimulated with HMB-PP or PMA/ionomycin (Fig. 6D). This was confirmed at the RNA level where the psoriasis-relevant cytokine IFN-α was identified as a strong inducer of IGF-1 mRNA (n = 3) (Fig. 6D). CLA+Vγ9Vδ2 T cells produced low amounts of VEGF (<100 pg/ml) and FGF-2 (<15 pg/ml) upon activation, whereas KGF and EGF production was absent (data not shown). These data establish CLA+Vγ9Vδ2 T cells as potent proinflammatory cells and potential contributors to psoriasis immunopathogenesis.
CLA+Vγ9Vδ2 T cells activate keratinocytes
We next investigated the effects of CLA+Vγ9Vδ2 T cells on skin epithelial cells. Incubation of primary human keratinocytes with CLA+Vγ9Vδ2 T cell supernatant upregulated the activation markers ICAM-1, HLA-DR, and HLA-ABC on keratinocytes (Fig. 7A). Keratinocyte activation with CLA+Vγ9Vδ2 T cell supernatant was partly blocked by adding anti–IFN-γ and TNF-α Abs to the culture (Fig. 7B). CLA+Vγ9Vδ2 T cell supernatant induced keratinocytes to produce the psoriasis-relevant mediators TNF-α, IL-6, CXCL9, and CXCL10 in an IFN-γ– and TNF-α–dependent manner (Fig. 7C). A major defense mechanism of keratinocytes against invading pathogens is their production of antimicrobial peptides (28), which are also upregulated in psoriasis (29). CLA+Vγ9Vδ2 T cell supernatant induced production of β-defensin 2, S100A7, and S100A8 but not LL37 in keratinocytes (n = 6) (Fig. 7D and data not shown).
These data demonstrate a proinflammatory cross-talk between CLA+Vγ9Vδ2 T cells and keratinocytes.
Murine skin has an abundant population of epidermal γδ T cells, which serve an important role as early immune sentinels. γδ T cells are exceedingly rare in human skin, and it has remained controversial whether and to what extent γδ T cells play a role in human skin homeostasis or pathology. This study revisits the role of γδ T cells in skin of healthy individuals and in the chronic inflammatory skin disease psoriasis.
In a first instance, we had performed an extensive flow cytometry-based screen of unconventional T cell populations including NKT cells and γδ T cells in healthy subjects and psoriasis patients. Although we were unable to find significant differences in most of the cell populations investigated, there was a reproducible and significant reduction of Vγ9Vδ2 cells in the blood of psoriasis patents compared with healthy controls.
In this study, we identify and characterize a novel subset of skin homing, proinflammatory Vγ9Vδ2 T cells that migrates to perturbed human skin in vivo. We suggest that Vγ9Vδ2 T cells are clinically relevant in psoriasis because reduction of circulating Vγ9Vδ2 T cell numbers correlated with increased psoriasis severity and was restored by successful psoriasis-targeted therapy.
Few studies have investigated Vγ9Vδ2 T cell in skin homeostasis and pathology, and even though several reports show that Vγ9Vδ2 T cells are present in skin, their role and function has remained ill explored. Using spectrotyping techniques, Holtmeier et al. (12) described the presence ofVγ9Vδ2 T cells in healthy human skin. Other studies using immunohistology techniques detected Vγ9Vδ2 T cells in a variety of infectious, inflammatory, and malignant cutaneous diseases (8, 9, 30–32). Interestingly, the majority of primary cutaneous γδ T cell lymphomas display a Vδ2 gene usage (33) supporting the concept of a specialized subset of skin homing Vγ9Vδ2 T cells.
We show that a reduction of circulating Vγ9Vδ2 T cells in psoriasis patients compared with healthy controls is specific for Vγ9Vδ2 T cells as other circulating lymphocyte subsets, such as conventional T cells, Vδ1 T cells, NKT cells, or NK cells, did not show significant differences. More detailed analysis revealed the existence of a Vγ9Vδ2 T cell subset expressing the E-selectin ligand and skin homing marker CLA. This subset was selectively decreased in psoriasis compared with the CLA− subset, indicating the existence of a skin homing Vγ9Vδ2 T cell subset with the potential to be recruited into psoriatic skin. This was further supported by the reduction of peripheral Vγ9Vδ2 T cells expressing skin homing chemokine receptors in psoriasis.
Although the majority of CLA+ conventional αβ T cells coexpresses the homeostatic skin homing chemokine receptors CCR4 and CCR10 (20), only a minority of Vγ9Vδ2 T cells expressed these receptors indicating differences in skin homing behavior between conventional αβ T cells and Vγ9Vδ2 T cells. Predominant expression of the inflammatory skin chemokine receptor CCR6 on Vγ9Vδ2 T cells supported the possibility of a coordinated and selective recruitment of circulating Vγ9Vδ2 T cells into skin upon skin perturbation as opposed to homeostatic migration into the skin.
We studied the skin homing capability of Vγ9Vδ2 T cells in vivo using a skin suction blister model. This model has been widely used to study skin immune cells under homeostasis and in the context of skin perturbation (26, 27, 34). Immune cells present in blister fluid have been shown to represent cells, phenotypically and numerically, found in situ by histology (35). Vγ9Vδ2 T cells were significantly increased following skin perturbation against a background of Vγ9Vδ2 T cells present in unperturbed skin. Vγ9Vδ2 T cells in unperturbed skin could represent a rare skin resident population (14). Vγ9Vδ2 T cells in perturbed skin increased to a significantly higher extent than total T cells and expressed significantly more CLA than circulating Vγ9Vδ2 T cells. Taken together, these results support the existence of a specialized circulating skin homing Vγ9Vδ2 T cell subset in vivo.
To place Vγ9Vδ2 T cells in the context of pathology we investigated psoriasis, one of the most common inflammatory skin conditions. Psoriasis has served a model of inflammatory disease in which to study the involvement of immune cell subsets.
Although the presence of cells expressing the pan-γδ TCR in psoriasis has been previously described (30), our results show for the first time, to our knowledge, the infiltration of Vγ9Vδ2 T cells in psoriasis skin. Calculating absolute Vγ9Vδ2 T cell numbers in psoriatic skin, we found substantial amounts of Vγ9Vδ2 T cells in psoriatic lesions. The number of Vγ9Vδ2 T cells in the skin of patients with severe psoriasis was an order of magnitude higher than the number of circulating Vγ9Vδ2 T cells (e.g., patient A had 14-fold higher numbers of Vγ9Vδ2 T cells in inflamed skin compared with that in the peripheral circulation [770 million Vγ9Vδ2 T cells in skin versus 53 million circulating Vγ9Vδ2 T cells]).
Perhaps our most striking finding was the fact that psoriasis disease severity significantly correlated with lower relative and absolute numbers of Vγ9Vδ2 T cells in the circulation and that successful antipsoriatic therapy leads to increase of peripheral Vγ9Vδ2 T cells. This establishes Vγ9Vδ2 T cells as potential biomarkers for psoriasis and supports their clinical significance. The mechanism by which Vγ9Vδ2 T cells increase with successful therapy remains to be determined. Possible scenarios involve re-entry of skin resident Vγ9Vδ2 T cells into the circulation, reduced recruitment of peripheral Vγ9Vδ2 T cells to resolving psoriatic lesions or decrease of CLA expression on peripheral Vγ9Vδ2 T cells.
We suggest that circulating CLA+Vγ9Vδ2 T cells are early immune sentinels recruited to perturbed skin in the context of trauma or infection. In this scenario, Vγ9Vδ2 T cells might be protective/beneficial in conditions of skin homeostasis and help in maintenance of skin integrity and in antimicrobial responses (31, 32). In contrast, Vγ9Vδ2 T cells might be pathogenic in an inflammatory environment contributing to psoriasis pathogenesis as potentially early players in the development of a psoriatic plaque (Supplemental Fig. 4).
Plasmacytoid DCs triggering the development of psoriatic lesions through their IFN-α production (36). Thus, it is conceivable that Vγ9Vδ2 T cells are activated by IFN-α in psoriatic skin as IFN-α potently induces IFN-γ production in Vγ9Vδ2 T cells (37). In addition we show that IFN-α induces production of the psoriasis relevant growth factor IGF-1 (38, 39) in Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells produce the psoriasis-relevant cytokines IFN-γ, TNF-α, and IL-17A, in line with previously reported IL-17A production of blood-derived Vγ9Vδ2 T cells (40). Vγ9Vδ2 T cells also release large amounts of proinflammatory chemokines such as IL-8, CCL3, CCL4, and CCL5 which have been shown to be of importance for recruitment of key immune effector cells to skin (41, 42) and efficiently activate keratinocytes. Thus, Vγ9Vδ2 T cells might play an important role in psoriasis disease pathogenesis.
There are questions remaining. Although it is the nature of translational human immunology studies that most of the data are observational, it will be interesting to study the role of Vγ9Vδ2 T cells in the context of targeted interventions using biologics. Also, more insights into the functional nature of these cells might be obtained in clinically relevant model systems such as animal xenotransplantation models.
Taken together, we propose that CLA+Vγ9Vδ2 T cells represent an innate immediate response tissue surveillance cell, constituting a first wave of T cells that enter skin upon tissue perturbation, such as trauma, adding to the intricate immune surveillance network that operates in the skin (43). Our data point toward three major roles for skin homing Vγ9Vδ2 T cells: 1) the rapid release of proinflammatory cytokines influencing resident immune and epithelial cells; 2) the recruitment of immune cells from the circulation; and 3) the release of growth factors resulting in tissue remodeling.
Our data put the emphasis on a previously overlooked T cell subset in skin immunology. The discovery of a potential role of Vγ9Vδ2 T cells in psoriasis pathogenesis provides the basis for the discovery of novel biomarkers and potential therapeutic targets in chronic inflammatory skin diseases.
The authors have no financial conflicts of interest.
We thank Karen Robertson, Naomi Hare, Gemma Ash, and Darren Geoghegan from the skin therapy research unit, Jenny Geh and Ciaran Healy from plastic surgery, as well as Dr. Rose Mak, Angela Clifford, and Sharon Jones for help with collecting clinical samples. In addition, we thank Isabella Tosi for excellent management and coordination of the tissue bank as well as Jennifer Mollon for advice on statistical analysis. This work would not have been possible without the generous help of healthy volunteers and patients from Guy’s and St. Thomas’ Hospital.
This work was supported by National Institutes of Health Grant RO1AR040065, Wellcome Trust Programme Grant GR078173MA, the Department of Health via the National Institute for Health Research Comprehensive Biomedical Research Centre award to Guy’s and St Thomas’ National Health Service Foundation Trust in partnership with King’s College London and King’s College Hospital National Health Service Foundation Trust, Medical Research Council U.K. Programme Grant G0601387, and the Dunhill Medical Trust.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- avidin/biotin complex
- body surface area
- cutaneous lymphocyte Ag
- dendritic epidermal T cell
- epidermal growth factor
- fibroblast growth factor
- 4-hydroxy-3-methyl-but-2-enyl pyrophosphate
- insulin-like growth factor
- keratinocyte growth factor
- Psoriasis Area and Severity Index
- vascular endothelial growth factor.
- Received March 25, 2011.
- Accepted June 29, 2011.
- Copyright © 2011 by The American Association of Immunologists, Inc.