Yonsei Med J.  2015 Jan;56(1):196-203. 10.3349/ymj.2015.56.1.196.

Attenuation of Peripheral Regulatory T-Cell Suppression of Skin-Homing CD8+T Cells in Atopic Dermatitis

• Zhang BX
• Lyu JC
• Liu HB
• Feng DQ
• Zhang DC
• Bi XJ
• Duan ZW
• Ding G

• KMID: 2352807
• DOI: http://doi.org/10.3349/ymj.2015.56.1.196

Abstract

PURPOSE
Cutaneous lymphocyte-associated antigen (CLA)-expressing CD8+T cells have been known to play an important role in the pathogenesis of atopic dermatitis (AD). However, the mechanisms underlying the loss of self-tolerance remain unclear. Regulatory T cells (Tregs) play a key role in the development of homeostasis in the immune system. We, therefore, hypothesized that a reduced ability of Tregs to inhibit autologous CD8+CLA+T cells might be underlying mechanism in AD.
MATERIALS AND METHODS
CD8+CLA+T cells and Tregs were obtained from the peripheral blood of AD patients and control volunteers. The frequencies of CD8+CLA+T cells were evaluated. The proliferative responses of CD8+CLA+T cells were assessed by flow cytometry, and the levels of transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) in culture supernatants were detected by enzyme-linked immunosorbent assay.
RESULTS
Our results revealed higher frequency and increased expression of perforin and granzyme-B in peripheral CD8+CLA+T cells in AD, and lower inhibitory ability of Tregs on proliferation of CD8+CLA+T cells in AD. Meanwhile, the levels of TGF-beta1 produced by Tregs were significantly lower in AD, and anti-TGF-beta1 abolished such suppression.
CONCLUSION
The attenuated inhibitory ability of Tregs on hyper-activated autologous CD8+CLA+T cells, mediated by TGF-beta1, plays an important role in the pathogenesis of AD.

Keyword

Cutaneous lymphocyte-associated antigen; CD8+T cell; regulatory; T cells; atopic dermatitis

MeSH Terms

Adult
Aged
CD8-Positive T-Lymphocytes/drug effects/*immunology
Case-Control Studies
Cell Proliferation
Cell Separation
Dermatitis, Atopic/*immunology/pathology
Female
Granzymes/metabolism
Humans
Interleukin-10/metabolism
Lymphocyte Count
Male
Perforin/metabolism
Skin/*immunology/pathology
T-Lymphocytes, Cytotoxic/drug effects/immunology
T-Lymphocytes, Regulatory/drug effects/*immunology
Transforming Growth Factor beta1/pharmacology
Granzymes
Interleukin-10
Perforin
Transforming Growth Factor beta1

Figure

• 

  Fig. 1 The purities of isolated Tregs (CD4+CD25+T cells). PBMCs were isolated on Ficoll-Hypaque gradient. According to the manufacturer's instructions of CD4+CD25+ Regulatory T Cell Isolation Kit, CD4+T cells were purified by negtive selection, and then CD25+T cells were isolated by positive selection. The purities were confirmed >95%. A representative profile of purified Tregs was showed. PBMC, peripheral blood mononuclear cell; FITC, fluorescein isothiocyanate; PE, phycoerythrin.

• 

  Fig. 2 Frequencies of peripheral CD8+CLA+T cells. The frequencies of CD8+CLA+T cells in peripheral blood were measured by flow cytometry, and the representative results (A) from AD patients and healthy volunteers were shown by dot plot graphs. (B) The data showed the frequencies of CD8+CLA+T cells were significantly higher in the AD group (n=30) compared to the control group (n=25, *p<0.05). CLA, cutaneous lymphocyte-associated antigen; AD, atopic dermatitis; PE, phycoerythrin.

• 

  Fig. 3 Expression levels of cytotoxic molecules (perforin and granzyme-B) on peripheral CD8+CLA+T cells. The percentages of cytotoxic molecules (perforin and granzyme-B) on CD8+CLA+T cells in peripheral blood were measured by flow cytometry, and the representative results (A and C) from AD patients and healthy volunteers were shown by dot plot graphs. (B and D) The data showed the expression levels of both perforin and granzyme-B on CD8+CLA+T cells were significantly higher in the AD group (n=30) compared to the control group (n=25, *p<0.05). CLA, cutaneous lymphocyte-associated antigen; AD, atopic dermatitis; PE, phycoerythrin; FITC, fluorescein isothiocyanate.

• 

  Fig. 4 Proliferation of peripheral CD8+CLA+T cells. CFSE-labeled peripheral CD8+CLA+T cells stimulated with anti-CD3/CD28 in the absence of Tregs for 4 days. According to the CFSE intensity by flow cytometry, the percentages of proliferating CD8+CLA+T cells were detected. (A) Representative CFSE profiles from an AD patient and a healthy volunteer were shown. (B) The data showed the percentages of proliferating CD8+CLA+T cells were of no significant difference between the AD group (n=8) and the control group (n=8). CFSE, carboxyfluorescein diacetate succinimidyl ester; CLA, cutaneous lymphocyte-associated antigen; AD, atopic dermatitis.

• 

  Fig. 5 Tregs suppress proliferation of autologous CD8+CLA+T cells. CFSE-labeled peripheral CD8+CLA+T cells stimulated with anti-CD3/CD28 in the presence of autologous Tregs for 4 days. According to the CFSE intensity by flow cytometry, the percentages of proliferating CD8+CLA+T cells were detected. (A) Representative CFSE profiles from an AD patient and a healthy volunteer were shown. (B) The data showed the percentages of proliferating CD8+CLA+T cells were significantly higher in the AD group (n=8) compared to the control group (n=8, *p<0.05). (C) The suppressive function of Tregs on the proliferation of autologous CD8+CLA+T cells was significantly lower in the AD group (n=8) compared to the control group (n=8, *p<0.05). CFSE, carboxyfluorescein diacetate succinimidyl ester; Tregs, regulatory T cells; CLA, cutaneous lymphocyte-associated antigen; AD, atopic dermatitis.

• 

  Fig. 6 Levels of TGF-β1 and IL-10 in culture supernatants of Tregs and/or CD8+CLA+T cells. 5×104 Tregs co-cultured with 5×104 autologous CD8+CLA+T cells stimulated with anti-CD3/CD28 in 96-well plates. Four days later, the amount of TGF-β1 (A) and IL-10 (B) in culture supernatant was measured by ELISA. The levels of TGF-β1 produced by Tregs were significantly lower in the AD group (n=8) compared to the control group (n=8, *p<0.05). However, the levels of IL-10 produced by Tregs showed no difference between the two groups. And 5×104 CD8+CLA+T cells stimulated with anti-CD3/CD28 in 96-well plates for 4 days, secreted undetectable amounts of TGF-β1 and minimal amounts of IL-10 in the two groups. CLA, cutaneous lymphocyte-associated antigen; AD, atopic dermatitis; Tregs, regulatory T cells; ELISA, enzyme-linked immunosorbent assay; TGF-β1, transforming growth factor-β1; IL-10, interleukin-10.

• 

  Fig. 7 Suppression of CD8+CLA+T cells proliferation by Tregs is mediated by TGF-β1. 5×104 Tregs co-cultured with 5×104 autologous CD8+CLA+T cells from AD patients (n=8, gray bars) or healthy volunteers (n=8, white bars) stimulated with anti-CD3/CD28 in 96-well plates in the presence of anti-TGF-β1 or control IgG. Four days later, the percentages of proliferating CD8+CLA+T cells were detected. The CD8+CLA+T cells exhibited increased proliferation in the presence of 50 µg/mL of anti-TGF-β1, and their proliferation were further increased and of no significant difference between the two groups in the presence of 100 µg/mL of anti-TGF-β1. Tregs, regulatory T cells; CLA, cutaneous lymphocyte-associated antigen; AD, atopic dermatitis; TGF-β1, transforming growth factor-β1.

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