Eur J Dermatol 2014; 24(6): 676-82
Investigative report Mao LIN1,2,a Bao-Xiang ZHANG1,a Nan SHEN1 Xue-Jiao DONG1 Ci ZHANG1 Xiao-Yi QI Jie ZHU3 Yu-Zhong LI3 Mao-Qiang MAN4 Cai-Xia TU1,2 1
Department of Dermatology, College of Integrative Medicine, 3 Department of Clinical Laboratory, 2nd Affiliated Hospital of Dalian Medical University, No.467 Zhongshan Road, 116027 Dalian, Liaoning Province, P. R. China 4 Department of Dermatology, Northern California Institute for Research and Education, San Francisco CA 94121, USA a These authors contributed equally to this work. 2
Reprints: Cai-Xia Tu
Article accepted on 8/17/2014
V
Regulatory T cells from active non-segmental vitiligo exhibit lower suppressive ability on CD8+ CLA+ T cells Background: Recent studies have shown that vitiligo is a T-cell mediated autoimmune disease. Skin-homing cytotoxic T lymphocytes expressing cutaneous lymphocyte-associated antigen (CLA) have been suggested to be responsible for the destruction of melanocytes in vitiligo. An aberration in the suppressive function of regulatory T cells (Tregs) has been reported in vitiligo patients. However, whether the weakened suppressive ability of the Tregs contributes to hyper-activated skin homing CD8+ CLA+ T cells remains to be determined. Objectives: To investigate the inhibition of circulating Tregs on the proliferation of autologous CD8+ CLA+ T cells in non-segmental vitiligo patients. Methods: CD8+ CLA+ T cells and Tregs were obtained from the peripheral blood of 13 non-segmental vitiligo patients and 7 controls. The proliferative responses of CD8+ CLA+ T cells were assessed in the absence or presence of autologous Tregs, and the levels of Transforming Growth Factor 1(TGF-1) and IL-10 in culture supernatants were detected by enzyme-linked immunosorbent assay. Results: The proliferative responses of circulating CD8+ CLA+ T cells in the presence of Tregs were significantly higher in the active vitiligo than in the stable vitiligo and control groups. Tregs from active vitiligo patients exhibited a lower inhibitory effect on proliferation of CD8+ CLA+ T cells. The levels of TGF-1 produced by Tregs were significantly lower in active vitiligo than other groups and anti-TGF-1 antibodies could abrogate the suppressive function of Tregs. Conclusions: The functional activity of Tregs is compromised in active vitiligo patients. TGF-1 plays an important role in the autoimmune mechanism of the disease. Key words: vitiligo, regulatory T cells, cutaneous lymphocyteassociated antigen, CD8+ T cell
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antigen (CLA). These cytotoxic T cells are often found clustered in the vicinity of disappearing melanocytes [10, 11]. Moreover, these perilesional CD8+ T cells can infiltrate autologous normally-pigmented skin explants in vitro and efficiently kill melanocytes within this microenvironment [10]. Co-culturing of CD8+ T cells isolated from skin lesions with autologous melanocytes could induce apoptosis of melanocytes [12]. Recently, we showed that patients with active vitiligo had a higher frequency of skin homing CD8+ CLA+ T cells and an increased expression of cytotoxic molecules [13]. It is well-known that regulatory T cells (Tregs) play a crucial role in the maintenance of peripheral tolerance via suppression of auto-reactive T cells [14]. It has been demonstrated that circulating Tregs are impaired in their ability to suppress auto-reactive T cells in vitiligo patients [15, 16]. An expansion of CD8+ T cells with a concomitant decrease in Tregs is observed in generalized vitiligo patients and the decrease in the Treg cells is significantly higher in active patients as compared with stable patients [6]. Moreover, peripheral Tregs are not able to sufficiently suppress the proliferation and cytotoxic capacity of autologous CD8+ T cells in vitiligo. All these results EJD, vol. 24, n◦ 6, November-December 2014
To cite this article: Lin M, Zhang BX, Shen N, Dong XJ, Zhang C, Qi XY, Zhu J, Li YZ, Man MQ, Tu CX. Regulatory T cells from active non-segmental vitiligo exhibit lower suppressive ability on CD8+ CLA+ T cells. Eur J Dermatol 2014; 24(6): 676-82 doi:10.1684/ejd.2014.2436
doi:10.1684/ejd.2014.2436
itiligo is an acquired skin disorder characterized by achromic or hypochromic patches on the skin and mucous membranes, due to the loss of melanocytes. Non-segmental vitiligo is the most common type of vitiligo and is generally described as having an autoimmune etiology [1]. Altered CD4+ T cell function and auto-reactive melanocyte-specific cytotoxic T cells have been reported in the pathogenesis of vitiligo, supporting an autoimmune hypothesis of the disease [2-4]. Several studies suggest that sensitized CD8+ T-cells target melanocyte differentiation antigens and destroy the melanocytes, leading to white patches [3]. In vitiligo patients, CD8+ T-cell counts are significantly higher and melanocyte-specific cytotoxic CD8+ T cells are detected in the peripheral blood and skin lesions [4, 5]. The frequency of melanocyte-specific cytotoxic CD8+ T cells correlates with the severity of disease and active vitiligo patients showed higher CD8+ T-cell counts compared with stable vitiligo patients [6-9]. Recent reports have shown that the auto-reactive CD8+ T cells causing the destruction of melanocytes in vitiligo are the skin homing cytotoxic T lymphocytes that express cutaneous lymphocyte-associated
suggest that dysregulation of Tregs may break the peripheral tolerance to melanocyte self-antigens and contribute to the pathogenesis of vitiligo [17]. Tregs can mediate their suppressive activity by a cellular contact dependent mechanism or by suppressor cytokines, such as Transforming Growth Factor 1 (TGF-1 ) or IL-10 [18, 19]. Recently we demonstrated that the levels of TGF-1 in serum and culture supernatants of Tregs are significantly reduced in active vitiligo patients, suggesting a defective suppressive function of Tregs [20]. Based on these results, we hypothesized that an imbalance between activated CD8+ CLA+ T cells and Tregs in the peripheral blood of patients may play a role in the development of vitiligo. Therefore, in this study we analyzed the circulating Tregs and their functional activity in inhibiting the cytotoxic activity of autologous CD8+ CLA+ T cells in vitiligo patients.
cedure using the CD4+ CD25+ Regulatory T Cell Isolation Kit according to the manufacturer’s instructions, and the purities were confirmed >95%.
CFSE labeling For the proliferation assay, freshly isolated CD8+ CLA+ T cells or Tregs were immediately labeled with CFSE. Briefly, 2.5 M of CFSE was added to 1 × 106 cells/mL in PBS/0.1%BSA at 37◦ C for 10 min. The staining was quenched by adding 5 volumes of ice-cold culture media (RPMI 1640 supplemented with 10%FBS, 100 U/mL penicillin G and 100 g/mL streptomycin) and the cells were incubated on ice for 5 min. Cells were then washed 3 times and suspended in the culture media.
T cell culture and proliferation assay
Materials and methods Study participants Patients with history of non-segmental vitiligo (N = 13) from the department of dermatology of the 2nd affiliated hospital of Dalian Medical University and healthy controls (N = 7) were included in the study. Six patients had stable, while 7 had active vitiligo (with enlarged lesions or the development of new lesions in the previous 6 months). The disease duration ranged from 4 weeks to 28 years and the two patient groups were matched for age and gender. The healthy volunteers had no other autoimmune disorders and had not used any medications in the last four weeks before the study. All participants signed an informed consent and the study was approved by the hospital Ethics Committee.
Reagents The following monoclonal antibodies were used for flow cytometry experiments: anti-CD8-PerCP, anti-CLA-PE, anti-CD4-PerCP, anti-CD25-PE, purified anti-CD3 NA/LE and anti-CD28 NA/LE, all of which were obtained from BD Biosciences (Franklin Lakes, USA). CD8+ T Cell Isolation Kit, anti-CLA MicroBead Kit, CD4+ CD25+ Regulatory T Cell Isolation Kit, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), MS/LD Columns, MiniMACS separator were purchased from Miltenyi Biotec (Cologne, Germany). Biotin-conjugated anti-TGF-1 or biotin-conjugated chicken IgG for a negative control were from calbiochem (Darmstadt, Germany). RPMI 1640 medium, 100 U/mL penicillin G and 100 g/mL streptomycin were purchased from Invitrogen (Carlsbad, USA).
Cell purification Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation. CD8+ T cells were purified by negative selection (using CD8+ T Cell Isolation Kit, Milteny Biotech), followed by a second step purification using anti-CLA MicroBead Kit to get CD8+ CLA+ cells. Milteny MidiMACS magnetic separation system was used for cell purification according to the manufacturer’s instructions, with final purities >92%. The isolation of Tregs was performed in a two-step proEJD, vol. 24, n◦ 6, November-December 2014
CFSE-labeled CD8+ CLA+ T cells (5 × 104 cells/well) were cultured in the absence or in the presence of unlabeled autologous Tregs (5 × 104 cells/well) in 96-well U-bottom plates which were pre-coated overnight with 5 g/mL anti-CD3 NA/LE and 5 g/mL anti-CD28 NA/LE (antiCD3/CD28). After incubation for 4 days, the cells were harvested and washed in PBS containing 0.5 mM EDTA, CFSE labeled cells were acquired on the Flow Cytometer (FACS caliber, BD Biosystems). According to the fluorescence intensity of CFSE, the proliferating (CFSEDIM ) and non-proliferating (CFSEBRIGHT ) populations of T cells were gated and analyzed.
Detection of the levels of TGF-1 and IL-10 The levels of TGF-1 and IL-10 in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits (Endogen, USA) according to the manufacturer’s instructions.
Transwell experiment Freshly isolated CD8+ CLA+ T cells were labeled with CFSE and cultured in 96-well plates (5 × 104 cells/well), pre-coated with 5 g/mL anti-CD3 NA/LE and 5 g/mL anti-CD28 NA/LE (anti-CD3/CD28). Autologous Tregs were either directly added in wells containing CD8+ CLA+ T cells (5 × 104 cells/well) or were added in transwell inserts (Corning, Acton, MA) (5 × 104 cells/well), which were inserted in the wells of CD8+ CLA+ T cells. After incubating for 4 days, cells were harvested and washed in PBS containing 0.5 mM EDTA. CFSE labeled cells were analyzed by Flow Cytometer. According to the fluorescence intensity of CFSE, the proliferating (CFSEDIM ) and non-proliferating (CFSEBRIGHT ) populations of T cells were gated and analyzed.
Statistical analysis Data were expressed as mean ± SD. Statistical differences between the groups were analyzed by one-way ANOVA. A value of P analyzed
Investigative report Mao LIN1,2,a Bao-Xiang ZHANG1,a Nan SHEN1 Xue-Jiao DONG1 Ci ZHANG1 Xiao-Yi QI Jie ZHU3 Yu-Zhong LI3 Mao-Qiang MAN4 Cai-Xia TU1,2 1
Department of Dermatology, College of Integrative Medicine, 3 Department of Clinical Laboratory, 2nd Affiliated Hospital of Dalian Medical University, No.467 Zhongshan Road, 116027 Dalian, Liaoning Province, P. R. China 4 Department of Dermatology, Northern California Institute for Research and Education, San Francisco CA 94121, USA a These authors contributed equally to this work. 2
Reprints: Cai-Xia Tu
Article accepted on 8/17/2014
V
Regulatory T cells from active non-segmental vitiligo exhibit lower suppressive ability on CD8+ CLA+ T cells Background: Recent studies have shown that vitiligo is a T-cell mediated autoimmune disease. Skin-homing cytotoxic T lymphocytes expressing cutaneous lymphocyte-associated antigen (CLA) have been suggested to be responsible for the destruction of melanocytes in vitiligo. An aberration in the suppressive function of regulatory T cells (Tregs) has been reported in vitiligo patients. However, whether the weakened suppressive ability of the Tregs contributes to hyper-activated skin homing CD8+ CLA+ T cells remains to be determined. Objectives: To investigate the inhibition of circulating Tregs on the proliferation of autologous CD8+ CLA+ T cells in non-segmental vitiligo patients. Methods: CD8+ CLA+ T cells and Tregs were obtained from the peripheral blood of 13 non-segmental vitiligo patients and 7 controls. The proliferative responses of CD8+ CLA+ T cells were assessed in the absence or presence of autologous Tregs, and the levels of Transforming Growth Factor 1(TGF-1) and IL-10 in culture supernatants were detected by enzyme-linked immunosorbent assay. Results: The proliferative responses of circulating CD8+ CLA+ T cells in the presence of Tregs were significantly higher in the active vitiligo than in the stable vitiligo and control groups. Tregs from active vitiligo patients exhibited a lower inhibitory effect on proliferation of CD8+ CLA+ T cells. The levels of TGF-1 produced by Tregs were significantly lower in active vitiligo than other groups and anti-TGF-1 antibodies could abrogate the suppressive function of Tregs. Conclusions: The functional activity of Tregs is compromised in active vitiligo patients. TGF-1 plays an important role in the autoimmune mechanism of the disease. Key words: vitiligo, regulatory T cells, cutaneous lymphocyteassociated antigen, CD8+ T cell
676
antigen (CLA). These cytotoxic T cells are often found clustered in the vicinity of disappearing melanocytes [10, 11]. Moreover, these perilesional CD8+ T cells can infiltrate autologous normally-pigmented skin explants in vitro and efficiently kill melanocytes within this microenvironment [10]. Co-culturing of CD8+ T cells isolated from skin lesions with autologous melanocytes could induce apoptosis of melanocytes [12]. Recently, we showed that patients with active vitiligo had a higher frequency of skin homing CD8+ CLA+ T cells and an increased expression of cytotoxic molecules [13]. It is well-known that regulatory T cells (Tregs) play a crucial role in the maintenance of peripheral tolerance via suppression of auto-reactive T cells [14]. It has been demonstrated that circulating Tregs are impaired in their ability to suppress auto-reactive T cells in vitiligo patients [15, 16]. An expansion of CD8+ T cells with a concomitant decrease in Tregs is observed in generalized vitiligo patients and the decrease in the Treg cells is significantly higher in active patients as compared with stable patients [6]. Moreover, peripheral Tregs are not able to sufficiently suppress the proliferation and cytotoxic capacity of autologous CD8+ T cells in vitiligo. All these results EJD, vol. 24, n◦ 6, November-December 2014
To cite this article: Lin M, Zhang BX, Shen N, Dong XJ, Zhang C, Qi XY, Zhu J, Li YZ, Man MQ, Tu CX. Regulatory T cells from active non-segmental vitiligo exhibit lower suppressive ability on CD8+ CLA+ T cells. Eur J Dermatol 2014; 24(6): 676-82 doi:10.1684/ejd.2014.2436
doi:10.1684/ejd.2014.2436
itiligo is an acquired skin disorder characterized by achromic or hypochromic patches on the skin and mucous membranes, due to the loss of melanocytes. Non-segmental vitiligo is the most common type of vitiligo and is generally described as having an autoimmune etiology [1]. Altered CD4+ T cell function and auto-reactive melanocyte-specific cytotoxic T cells have been reported in the pathogenesis of vitiligo, supporting an autoimmune hypothesis of the disease [2-4]. Several studies suggest that sensitized CD8+ T-cells target melanocyte differentiation antigens and destroy the melanocytes, leading to white patches [3]. In vitiligo patients, CD8+ T-cell counts are significantly higher and melanocyte-specific cytotoxic CD8+ T cells are detected in the peripheral blood and skin lesions [4, 5]. The frequency of melanocyte-specific cytotoxic CD8+ T cells correlates with the severity of disease and active vitiligo patients showed higher CD8+ T-cell counts compared with stable vitiligo patients [6-9]. Recent reports have shown that the auto-reactive CD8+ T cells causing the destruction of melanocytes in vitiligo are the skin homing cytotoxic T lymphocytes that express cutaneous lymphocyte-associated
suggest that dysregulation of Tregs may break the peripheral tolerance to melanocyte self-antigens and contribute to the pathogenesis of vitiligo [17]. Tregs can mediate their suppressive activity by a cellular contact dependent mechanism or by suppressor cytokines, such as Transforming Growth Factor 1 (TGF-1 ) or IL-10 [18, 19]. Recently we demonstrated that the levels of TGF-1 in serum and culture supernatants of Tregs are significantly reduced in active vitiligo patients, suggesting a defective suppressive function of Tregs [20]. Based on these results, we hypothesized that an imbalance between activated CD8+ CLA+ T cells and Tregs in the peripheral blood of patients may play a role in the development of vitiligo. Therefore, in this study we analyzed the circulating Tregs and their functional activity in inhibiting the cytotoxic activity of autologous CD8+ CLA+ T cells in vitiligo patients.
cedure using the CD4+ CD25+ Regulatory T Cell Isolation Kit according to the manufacturer’s instructions, and the purities were confirmed >95%.
CFSE labeling For the proliferation assay, freshly isolated CD8+ CLA+ T cells or Tregs were immediately labeled with CFSE. Briefly, 2.5 M of CFSE was added to 1 × 106 cells/mL in PBS/0.1%BSA at 37◦ C for 10 min. The staining was quenched by adding 5 volumes of ice-cold culture media (RPMI 1640 supplemented with 10%FBS, 100 U/mL penicillin G and 100 g/mL streptomycin) and the cells were incubated on ice for 5 min. Cells were then washed 3 times and suspended in the culture media.
T cell culture and proliferation assay
Materials and methods Study participants Patients with history of non-segmental vitiligo (N = 13) from the department of dermatology of the 2nd affiliated hospital of Dalian Medical University and healthy controls (N = 7) were included in the study. Six patients had stable, while 7 had active vitiligo (with enlarged lesions or the development of new lesions in the previous 6 months). The disease duration ranged from 4 weeks to 28 years and the two patient groups were matched for age and gender. The healthy volunteers had no other autoimmune disorders and had not used any medications in the last four weeks before the study. All participants signed an informed consent and the study was approved by the hospital Ethics Committee.
Reagents The following monoclonal antibodies were used for flow cytometry experiments: anti-CD8-PerCP, anti-CLA-PE, anti-CD4-PerCP, anti-CD25-PE, purified anti-CD3 NA/LE and anti-CD28 NA/LE, all of which were obtained from BD Biosciences (Franklin Lakes, USA). CD8+ T Cell Isolation Kit, anti-CLA MicroBead Kit, CD4+ CD25+ Regulatory T Cell Isolation Kit, 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), MS/LD Columns, MiniMACS separator were purchased from Miltenyi Biotec (Cologne, Germany). Biotin-conjugated anti-TGF-1 or biotin-conjugated chicken IgG for a negative control were from calbiochem (Darmstadt, Germany). RPMI 1640 medium, 100 U/mL penicillin G and 100 g/mL streptomycin were purchased from Invitrogen (Carlsbad, USA).
Cell purification Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation. CD8+ T cells were purified by negative selection (using CD8+ T Cell Isolation Kit, Milteny Biotech), followed by a second step purification using anti-CLA MicroBead Kit to get CD8+ CLA+ cells. Milteny MidiMACS magnetic separation system was used for cell purification according to the manufacturer’s instructions, with final purities >92%. The isolation of Tregs was performed in a two-step proEJD, vol. 24, n◦ 6, November-December 2014
CFSE-labeled CD8+ CLA+ T cells (5 × 104 cells/well) were cultured in the absence or in the presence of unlabeled autologous Tregs (5 × 104 cells/well) in 96-well U-bottom plates which were pre-coated overnight with 5 g/mL anti-CD3 NA/LE and 5 g/mL anti-CD28 NA/LE (antiCD3/CD28). After incubation for 4 days, the cells were harvested and washed in PBS containing 0.5 mM EDTA, CFSE labeled cells were acquired on the Flow Cytometer (FACS caliber, BD Biosystems). According to the fluorescence intensity of CFSE, the proliferating (CFSEDIM ) and non-proliferating (CFSEBRIGHT ) populations of T cells were gated and analyzed.
Detection of the levels of TGF-1 and IL-10 The levels of TGF-1 and IL-10 in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits (Endogen, USA) according to the manufacturer’s instructions.
Transwell experiment Freshly isolated CD8+ CLA+ T cells were labeled with CFSE and cultured in 96-well plates (5 × 104 cells/well), pre-coated with 5 g/mL anti-CD3 NA/LE and 5 g/mL anti-CD28 NA/LE (anti-CD3/CD28). Autologous Tregs were either directly added in wells containing CD8+ CLA+ T cells (5 × 104 cells/well) or were added in transwell inserts (Corning, Acton, MA) (5 × 104 cells/well), which were inserted in the wells of CD8+ CLA+ T cells. After incubating for 4 days, cells were harvested and washed in PBS containing 0.5 mM EDTA. CFSE labeled cells were analyzed by Flow Cytometer. According to the fluorescence intensity of CFSE, the proliferating (CFSEDIM ) and non-proliferating (CFSEBRIGHT ) populations of T cells were gated and analyzed.
Statistical analysis Data were expressed as mean ± SD. Statistical differences between the groups were analyzed by one-way ANOVA. A value of P analyzed
