CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> regulatory T cells in human lupus erythematosus

Natural CD4⁺CD25⁺ regulatory T cells (T_reg) show a potent immunosuppressive function and contribute to immunologic self-tolerance by suppressing potentially auto-reactive T cells. Depletion of these cells leads to the induction of severe autoimmune diseases in animal models; more recently, several studies have also reported an impairment of T_reg number and/or function in various human autoimmune diseases. For example, aberrant numbers of circulating CD4⁺CD25⁺ T_reg have been seen in patients with type I diabetes, mycosis fungoides, graft-versus-host-reaction, and rheumatoid arthritis. Moreover, increased numbers of functionally active CD4⁺CD25⁺ T_reg have been detected in the synovial fluid of patients with rheumatoid arthritis. In systemic lupus erythematosus (SLE), conflicting data on the role of CD4⁺CD25⁺ T_reg in human autoimmune diseases have been presented in the literature. Decreased numbers of peripheral blood T_reg have been reported by most studies on SLE patients with active disease, but non-impaired or even increased CD4⁺CD25⁺ T_reg numbers have also been described. In addition, both deficient and normal suppressive capacity of isolated T_reg have been observed in SLE. Analysis of CD4⁺FoxP3⁺ T_reg in skin lesions of patients with a primarily cutaneous manifestation of the disease showed a significant reduction in cell numbers as compared to other inflammatory skin diseases, suggesting the importance of analyzing T_reg numbers in the affected tissue. In this review, we discuss the role of CD4⁺CD25⁺ T_reg in autoimmunity and recent published data on SLE. Furthermore, we highlight the need for additional studies that address specific gaps of knowledge regarding the pathophysiological mechanisms as well as the identification of future therapeutic strategies for autoimmune diseases.     

In situ depletion of CD4<Superscript>+</Superscript> T cells in human skin by Zanolimumab

CD4⁺ T cells, in activated or malignant form, are involved in a number of diseases including inflammatory skin diseases such as psoriasis, and T cell lymphomas such as the majority of cutaneous T cell lymphomas (CTCL). Targeting CD4 with an antibody that inhibits and/or eliminates disease-driving T cells in situ may therefore be a useful approach in the treatment of inflammatory and malignant skin diseases. Depletion of CD4⁺ T cells in intact inflamed human skin tissue by Zanolimumab, a fully human therapeutic monoclonal antibody (IgG1, κ) against CD4, was studied in a human psoriasis xenograft mouse model. Zanolimumab treatment was shown to induce a significant reduction in the numbers of inflammatory mononuclear cells in upper dermis. This reduction in inflammatory mononuclear cells in situ was primarily due to a significant reduction in the numbers of skin-infiltrating CD4⁺, but not CD8⁺ CD3⁺ T cells. The capacity of Zanolimumab to deplete the CD4⁺ T cells in the skin may be of importance in diseases where CD4⁺ T cells play a central role. Indeed, in a phase II clinical trial Zanolimumab has shown a dose-dependent clinical response in patients with CTCL and the antibody is currently in a phase III clinical trial for CTCL, a disease for which there is no safe and effective treatment available today.     

Administration of substance P during a primary immune response amplifies the secondary immune response via a long-lasting effect on CD8<Superscript>+</Superscript> T lymphocytes

Atopic dermatitis can be exacerbated or induced by scratching or psychological stress; both cause the release of substance P (SP) from sensory nerves. Therefore, SP may have an etiological role in mechanisms underlying AD. Here, we show that administration of SP during the primary immune response (PIR) imprinted long-lasting pro-inflammatory immunity, resulting in exacerbation of the secondary immune response (SIR) in the absence of further SP. Five days after sensitization with dinitrofluorobenzene (DNFB), challenge with DNFB together with SP (“SP-Group”) resulted in an increased PIR (as evaluated by ear swelling and granulocyte infiltration) compared to DNFB only (“Control-Group”). On day 26, after inflammation completely subsided, a second challenge with DNFB only (without SP) caused an increased SIR in the “SP-Group” compared to controls. Pretreatment on day 5 with spantide, an SP receptor antagonist, prevented increased ear swelling in the “SP-Group” not only on day 5 (PIR) but also on day 26 (SIR). In contrast, spantide treatment on day 26 did not affect the SIR. Adoptive transfer experiments suggested that CD8⁺ T cells were involved in mediating enhanced SIR in animals pretreated on day 5 with SP. The present study offers a novel experimental approach to an uninvestigated facet of the pro-inflammatory effect of SP, i.e., exacerbation of inflammation via a long-term and indirect influence on CD8⁺ T lymphocytes.     

Changes in Na<Superscript>+</Superscript>, K<Superscript>+</Superscript> concentrations in perspiration and perspiration volume with alternating current iontophoresis in palmoplantar hyperhidrosis patients

Various treatments are currently available for palmoplantar hyperhidrosis. We have treated palmoplantar hyperhidrosis patients effectively with the use of alternating current (AC) iontophoresis. However, much remains unknown about the physiological changes that occur with AC iontophoresis, and its mechanism of action. We measured the changes in Na⁺, K⁺ concentration in perspiration and perspiration volume with AC iontophoresis in palmoplantar hyperhidrosis patients. We found that hyperhidrosis patients have significantly higher perspiration volume and Na⁺ concentration in perspiration than healthy controls. Looking at the temporal changes with AC iontophoresis, we found a significant decrease in perspiration volume and Na⁺ concentration in perspiration after six iontophoresis treatments. This result is further evidence that Na⁺ concentration in perspiration is closely involved with perspiration volume. However, looking at the changes in perspiration volume and Na⁺ concentration in perspiration before and after a single AC iontophoresis treatment, we found that while perspiration volume did not decrease in hyperhidrosis patients after a single treatment, there was a significant decrease in Na⁺ concentration. In healthy controls as well, Na⁺ concentration in perspiration decreased significantly after a single treatment. These findings suggest that the effect of AC iontophoresis may be due to a complex mechanism involving changes in reabsorption of ductal Na⁺.     

Fractional CO<Subscript>2</Subscript> lasers contribute to the treatment of stable non-segmental vitiligo

Background

Stable non-segmental vitiligo is often resistant to conventional therapies.

Objectives

The purpose of this study was to investigate the effect of three types of fractional lasers in the treatment of stable nonsegmental vitiligo.

Materials & methods

Twenty patients were enrolled in the study. The vitiligo lesions of each patient were divided into four treatment parts, and all parts were treated with narrowband ultraviolet-B (NB-UVB). Three of the four parts were respectively treated with three types of fractional lasers (two ablative 10,600-nm CO₂ lasers and one non-ablative 1,565-nm laser), followed by topical betamethasone solution application. The treatment period lasted six months. Efficacy and satisfaction were respectively assessed by dermatologists and patients.

Results

The ablative CO₂ lasers, in combination with topical betamethasone solution and NB-UVB, achieved marked to excellent improvement on white patches assessed by dermatologists. Patients showed high satisfaction scores for the treatments. The non-ablative 1,565-nm fractional laser did not provide any further benefit in the treatment of vitiligo. No severe adverse events developed for any of the treatments.

Conclusion

The treatment protocol with ablative CO₂ lasers, in combination with topical betamethasone solution and NB-UVB, was suitable for stable non-segmental vitiligo. For vitiligo, the ablative fractional CO₂ laser is more effective than the non-ablative fractional laser.

     

A possible mechanism in the recruitment of eosinophils and Th2 cells through CD163<Superscript>+</Superscript> M2 macrophages in the lesional skin of eosinophilic cellulitis

Background

M2 macrophages play a critical role in the recruitment of T helper 2 (Th2) regulatory T cells (Treg).

Objectives

To study the role of M2 macrophages and Treg cells in eosinophilic celulitis.

Material and Methods

We employed immunohistochemical staining for CD163 and CD206 (macrophages) as well as FoxP3 (Treg), in lesional skin of four cases of eosinophilic cellulitis.

Results

CD163⁺ CD206⁺ M2 macrophages, which were previously reported to produce CCL17 to induce Th2 cells and Treg cells, were predominantly infiltrating the subcutaneous tissues and interstitial area of the dermis. M2 macrophages derived from PBMC showed significantly increased expression of CCL11, CCL17, CCL24 and CCL26 mRNA and production of CCL17 and CCL24, when stimulated by IL-4 or IL-13. In addition, CCL17-producing cells and CCL24-producing cells were prominent in the lesional skin of EC.

Conclusion

Our study sheds light on one of the possible immunological mechanisms of eosinophilic cellulitis.

     

寻常型进展期白癜风患者外周血 CD4+CD25+调节性T细胞检测

目的：探讨寻常型进展期白癜风患者外周血T细胞亚群及CD4^＋CD25^＋调节性T细胞（Treg）的水平变化及其意义。方法：采用流式细胞分析技术对45例寻常型进展期白癜风患者和24例正常人外周血CD4^＋、CD25^＋、Foxp^3＋,CD^4＋CD25^＋,CD4^＋CD25^＋Foxp3^＋T细胞水平。结果：寻常型进展期白癜风患者外周血CD4^＋CD25^＋Treg和CD4^＋CD25^＋Foxp3^＋nTreg细胞数量显著高于正常对照组（P〈0．05）。结论：寻常型进展期白癜风患者外周血Treg水平发生变化,使体内免疫功能处于失衡状态,细胞免疫尤其是Treg可能参与了白癜风的发病。     

CD8~+T细胞对白癜风患者黑素细胞的杀伤机制

目的:明确CD8~+T细胞对白癜风患者黑素细胞的杀伤机制。方法:分选白癜风患者及正常人外周血CD8~+T细胞,分别与原代黑素细胞及永生化正常黑素细胞系PIG1及永生化白癜风黑素细胞系PIG3V建立共孵育杀伤模型,采用流式细胞术检测靶细胞被杀伤情况,酶联免疫吸附法(ELISA)检测杀伤环境中IFN-γ、TNF-α、颗粒酶B及穿孔素的分泌水平。结果:白癜风患者外周血CD8~+T细胞杀伤原代黑素细胞及PIG3V能力,分泌IFN-γ、颗粒酶B及穿孔素水平均高于健康对照,而杀伤PIG1能力和TNF-α水平并无显著差异;白癜风患者CD8~+T细胞IFN-γ分泌水平与细胞杀伤率呈明显正相关。结论:白癜风患者CD8~+T细胞杀伤黑素细胞是白癜风发病的重要机制,且该杀伤作用可能依赖IFN-γ、颗粒酶B及穿孔素分泌水平的上调。     

银屑病患者皮损和外周血中CD8α+α+ T细胞的研究

目的 观察银屑病患者皮损和外周血中CD8α+α+ T细胞的分布和比例,探讨其在银屑病发病中的作用。 方法 采用免疫荧光技术观察5例进展期寻常性银屑病患者和5例健康人皮肤中CD8α+α+ T细胞的分布,流式细胞仪分别检测10例进展期寻常性银屑病患者和8例健康人外周血中CD8α+α+ T细胞比例及干扰素γ（IFN-γ）和肿瘤坏死因子α（TNF-α）的表达。采用Graphpad Prism统计软件对实验组和对照组各数据进行t检验。 结果 在5例银屑病患者皮损真皮浅层均可见以CD8α+α+ T细胞为主的浸润,未见明显的CD8α+β+ T细胞浸润;而5例健康人皮肤中均未见CD8α+α+ T细胞和CD8α+β+ T细胞浸润。流式细胞仪检测,8例健康人外周血中CD8α+α+ T细胞比例为9.12% ± 4.80%,10例银屑病患者组为26.47% ± 12.99%,银屑病患者组明显高于对照组,两组比较,t = 3.96,P ＜ 0.001。在银屑病患者外周血CD8α+α+ T细胞中分泌IFN-γ和TNF-α的细胞比例分别为47.36% ± 19.38%和54.14% ± 21.14%,健康对照组分别为13.44% ± 9.21%和34.03% ± 17.22%,两组差异均有统计学意义（t值分别为4.54和2.17,P值分别 ＜ 0.001和 ＜ 0.05）。 结论 银屑病患者皮损和外周血中存在CD8α+α+ T细胞的分布和比例增多,CD8α+α+ T细胞可能是参与银屑病发病的主要CD8+ T细胞亚群。     

Direct evidence to support the role of antigen-specific CD8(+) T cells in melanoma-associated vitiligo

Vitiligo is a cutaneous pigmentary disorder characterized by the loss of melanocytes. An autoimmune mechanism is strongly suspected to be involved in this affection given that it is frequently associated with autoimmune hormonal disorders, and because antibodies directed against melanocytic antigens are found in the serum of patients with vitiligo. We examined the role of cellular immunity in melanoma-associated vitiligo by expanding infiltrating lymphocytes from fresh biopsy specimens of vitiligo patches in melanoma patients. The vitiligo-infiltrating lymphocytes were almost exclusively T lymphocytes, and most were CD8(+). Following in vitro expansion, vitiligo-infiltrating lymphocytes remained predominantly CD8(+) and expressed the cutaneous homing receptor CLA. Furthermore, vitiligo-infiltrating lymphocytes had a clonal or oligoclonal T cell receptor profile, possibly reflecting specific antigenic stimulation. Finally, vitiligo- infiltrating lymphocytes specifically recognized differentiation antigens shared by normal melanocytes and melanoma cells. This direct demonstration of CD8(+) T cell involvement in vitiligo suggests that, in melanoma patients, vitiligo may be a visible effect of a spontaneous antitumoral immune response.     

Reduction of skin-homing cytotoxic T cells (CD8+ -CLA+) in patients with vitiligo

Background: Vitiligo is a frequently acquired, hereditary disease, characterized by achromic macules due to the absence of melanocytes. In contrast with earlier studies, in which the main pathogenic role was attributed to anti‐melanocyte antibodies, recent papers have emphasized a role for CD8⁺ cytotoxic T lymphocytes in melanocyte destruction. Fifteen percent of peripheral T cell express cutaneous lymphocyte‐associated antigen (CLA), responsible for skin‐homing T cell. Phototherapy is used to treat patients with generalized vitiligo and it has been shown to interfere with CLA⁺ T cells in other skin diseases. Objective: To describe peripheral blood T cell subpopulations' frequency and ability to express the skin‐homing molecule (CLA) in patients with non‐segmental vitiligo, before and after photochemotherapy (PUVA). Patients and Methods: Twenty‐two patients with generalized and active spreading vitiligo were submitted to 30 PUVA‐8MOP sessions. Lymphocyte immunophenotyping was performed by flow cytometry using anti‐CD3, anti‐CD8 and anti‐CLA monoclonal antibodies. Fifteen healthy volunteers, sex‐ and age‐matched, were included as a control group. Results: CD8⁺–CLA⁺ T cells were significantly reduced in number in untreated vitiligo patients (P=0.008) when compared with control individuals, albeit with a more intense CLA expression (P=0.028). These findings were not altered after PUVA. No significant difference was noticed in CD4/CD8 ratios nor in CD4–CLA⁺ T cell numbers between vitiligo patients and controls, both before and after PUVA. Conclusions: CD8–CLA⁺ T cells are reduced in peripheral blood of patients with non‐segmental vitiligo. This finding may be related to the previously reported increase of CD8⁺ cells in both lesions and perilesional skin of these patients.     

Decreased suppression of CD8+ and CD4+ T cells by peripheral regulatory T cells in generalized vitiligo due to reduced NFATC1 and FOXP3 proteins

Regulatory T cells (Tregs) are involved in the suppression of activated T cells in generalized vitiligo (GV). The study was aimed to investigate Tregs functional defects in Treg:CD8⁺ and Treg:CD4⁺ T cells' co-culture systems of 55 GV patients and 45 controls. CD8⁺ and CD4⁺ T-cell proliferation was assessed by BrdU assay; production of IL-10, TGF-β and IFN-γ cytokines was assessed by ELISA; and FOXP3, CD25, NFATC1 and CD44 proteins were measured by flow cytometry. Generalized vitiligo patients showed reduced suppression of CD8⁺ and CD4⁺ T cells (P = .0384, P = .0084), increased IFN-γ (P < .0001, P = .0019), decreased IL-10 and TGF-β (P < .0001) and decreased FOXP3, CD25 and NFATC1 proteins (P < .0001). Active vitiligo (AV) patients showed reduced suppression of CD8⁺ & CD4⁺ T cells (P = .006, P = .015), increased IFN-γ (P = .036, P = .045), decreased IL-10 (P = .009, P = .021), FOXP3 (P = .0244) and NFATC1 (P = .019). Severe GV (50%-75% VASI) patients showed reduced suppression of CD8⁺ and CD4⁺ T cells (P = .0003, P = .001), increased IFN-γ (P = .0029, P < .0001), decreased IL-10 (P = .0057, P = .0017), FOXP3 (P = .002) and NFATC1 (P = .0347). VASI score was positively correlated with the suppression of CD8⁺ and CD4⁺ T cells (P = .0006, P < .0001), IL-10 (P = .0096, P = .029), FOXP3 (P = .0008) and NFATC1 (P = .043), whereas it was negatively correlated with IFN-γ (P = .0029, P = .0017). Early age of onset patients' Tregs demonstrated decreased suppression of CD8⁺ and CD4⁺ T cells (P = .0156, P = .0074), decreased TGF-β (P = .0212, P = .0083) and NFATC1 (P = .0103). NFATC1 was positively correlated with FOXP3 in Tregs (P < .0001). Our results suggest impaired Tregs suppressive function in GV patients due to decreased NFATC1, FOXP3, CD25, IL-10 and TGF-β resulting into increased CD8⁺ and CD4⁺ T-cell proliferation and IFN-γ production. For the first time, decreased NFATC1 levels were correlated with decreased FOXP3, thereby altering Treg cell function in GV patients. Additionally, decreased Treg cell function also affected onset, activity and severity of GV.     

人真皮间充质干细胞对白癜风患者皮损周围CD8+ T淋巴细胞表达和分泌白介素13的影响

【摘要】 目的 探讨人真皮间充质干细胞（DMSC）对白癜风患者皮损周围CD8+ T淋巴细胞分泌表达白介素（IL）13的影响。 方法 细胞增殖检测法（MTS）检测重组IL-13（rIL-13）对黑素细胞增殖的影响。逆转录聚合酶链反应（RT-PCR）和Western印迹法分别检测6例进展期寻常型白癜风患者皮损周围及外周血中CD8+ T淋巴细胞中IL-13基因和蛋白表达。实时荧光定量反转录聚合酶链反应（qRT-PCR）技术和酶联免疫吸附试验（ELISA）分别检测DMSC与白癜风患者皮损周围CD8+ T淋巴细胞共培养前后细胞IL-13 mRNA水平和上清蛋白水平。 结果 不同浓度（10、50、100、250、500 μg/L）的rIL-13作用黑素细胞24、48、72、96 h后黑素细胞的增殖与对照组比较均未见明显变化（均P > 0.05）。白癜风患者皮损周围及外周血中CD8+ T淋巴细胞均可表达IL-13,但皮损周围CD8+ T淋巴细胞表达IL-13更显著。将皮损周围CD8+ T淋巴细胞和DMSC共培养,CD8+ T淋巴细胞IL-13 mRNA（0.100 0 ± 0.002 4）和蛋白［（1 509.62 ± 48.44） ng/L］的表达水平均显著低于单独培养的CD8+ T淋巴细胞［mRNA：0.383 2 ± 0.018 7,蛋白：（5 507.98 ± 34.11） ng/L,均P < 0.05］。 结论 白癜风患者皮损周围CD8+ T淋巴细胞高表达IL-13,DMSC能够有效地抑制其表达IL-13,或许可作为白癜风的治疗靶点之一。     

Molecular and functional bases of self-antigen recognition in long-term persistent melanocyte-specific CD8+ T cells in one vitiligo patient

Vitiligo patients possess high frequencies of circulating CD8+ T lymphocytes specific for the melanocyte differentiation antigen Melan-A/MART-1. These self-specific T cells exhibit intact functional properties and their T cell receptors are selected for a narrow range of high affinities of antigen recognition, suggesting their important role in the pathogenesis of vitiligo. In order to understand the molecular base for this unexpected, optimal T cell receptor recognition of a self-antigen, a tetramer-guided ex vivo analysis of the T cell receptor repertoire specific for the Melan-A antigen in a patient affected by vitiligo is reported. All T cell receptors sequenced corresponded to different clonotypes, excluding extensive clonal expansions and revealing a large repertoire of circulating Melan-A-specific T lymphocytes. A certain degree of T cell receptor structural conservation was noticed, however, as a single AV segment contributed to the alpha chain rearrangement in 100% of clones and a conserved amino acid sequence was found in the beta chain complementarity determining region 3 of various high affinity cells. We suggest that the conserved alpha chain confers self-antigen recognition, necessary for intrathymic selection and peripheral homeostasis, to many synonymous T cell receptors, whereas the beta chain fine tunes the T cell receptor affinity of the specific cells. In addition, we demonstrate that many high avidity T cell clones from this patient were capable of specifically lysing normal, HLA-matched melanocytes. These autoreactive clones persisted for more than 3 y in the patient's peripheral blood. These data, together with the skin-homing potential of the clones, directly point to the in vivo pathogenic role of melanocyte-specific cytotoxic T lymphocytes in vitiligo.     

寻常型银屑病患者外周血CD4~+CD25~+及CD8~+CD25~+细胞的检测

目的研究进展期寻常型银屑病患者外周血CD4+CD25+和CD8+CD25+调节性T细胞的数量变化及其在银屑病免疫病理学发病机制中的作用。方法应用流式细胞术对进展期寻常型银屑病患者外周血CD4+CD25+和CD8+CD25+调节性T细胞进行检测。结果进展期寻常型银屑病外周血CD4+CD25+细胞及CD8+CD25+调节性T细胞数量与正常对照组相比,均显著降低(P<0.05,P<0.005),而CD4+CD25+/CD8+CD25+比值无显著性差异(P>0.05)。结论寻常型银屑病的发病与CD4+CD25+和CD8+CD25+调节性T细胞的同步降低有关,与二者的比值无关。