蕈样肉芽肿皮损中树突细胞和凋亡的研究

目的探讨细胞凋亡与蕈样肉芽肿(MF)疾病进程和树突细胞(DC)的关系。方法皮肤肿瘤组织冰冻切片,单克隆抗体免疫组化多染及TdT介导的dUTP缺口末端标记技术(TUNEL)。结果MF斑片斑块期肿瘤组织Pautrier微脓肿内及真皮T细胞浸润区内可见少量凋亡细胞或凋亡小体,肿瘤期真皮T细胞浸润区内可见大量凋亡瘤细胞或凋亡小体(P<0.01)。部分肿瘤组织真皮深层凋亡细胞成片分布,这些区域常见许多未成熟的DC及包绕吞噬有凋亡瘤细胞或凋亡小体的未成熟的(Lag ,CD1a )DC存在,斑片及斑块期不易见到这种现象。结论凋亡瘤细胞或凋亡小体的出现和增多可能是MF疾病进展的一个病理征象,肿瘤期中包绕吞噬凋亡瘤细胞或凋亡小体的未成熟的CD1a DC出现提示在诱导抗肿瘤免疫方面存在交叉呈递,可能诱导抗肿瘤免疫耐受。     

蕈样肉芽肿浸润性皮损中树突细胞表型特征的研究

目的 探讨蕈样肉芽肿(MF)皮损中树突细胞(DC)表型及其临床意义。方法 检测DC表面分子的单克隆抗体和免疫组化技术。结果 MF斑片／斑块期的表皮及真皮浅层内存在大量的未成熟DC和成熟DC，主要是CD1a^ 、CD1c^ 、Lag^ ／Langerin^ 未成熟DC和CD83^ DC-Lamp^ 成熟DC。肿瘤期的真皮内也见大量的CD1a^ 、CD1c^ 未成熟DC和CD83^ DC-Lamp^ 成熟DC，但Lag^ ／Langerin^ DC更多见于表皮和真皮浅层．真皮深层少见，而此处CD1a^ 、CD1c^ 未成熟DC明显增多。结论 在MF斑片／斑块期，表皮朗格汉斯细胞发生了迁移，可能参与了抗肿瘤免疫反应，而肿瘤期真皮内大量CD1a^ DC可能对相应的免疫耐受产生作用。     

蕈样肉芽肿皮损中增生性T细胞与树突细胞的研究

目的 探讨蕈样肉芽肿(MF)不同时期浸润性皮损中增生性T细胞与树突细胞的分布特征。方法 皮肤肿瘤组织石蜡切片,单克隆抗体免疫组化染色。结果 MF皮损中Ki-67⁺细胞和皮肤淋巴细胞相关抗原阳性(CLA⁺)细胞均明显增多,多数Ki-67⁺细胞CD4⁺和cLA⁺,MF肿瘤期真皮浸润组织中的Ki-67⁺细胞明显多于斑片期/斑块期(P<0.01),且形态多不规则,异型性明显;未见CD83⁺/Ki-67⁺树突细胞,但树突细胞周围可见大量的Ki-67⁺增生性T细胞,CD83⁺树突细胞与Ki-67⁺肿瘤T细胞紧密接触。结论 CLA⁺/Ki-67⁺T细胞在MF皮损中表达的程度和免疫病理特征对于MF的早期发现和转化进展可能具有免疫病理学诊断意义。     

小鼠骨髓lin~-CD117~+造血干细胞定向分化成浆细胞样树突状细胞的研究

目的观察小鼠骨髓lin-CD117+造血干细胞大量增殖分化成浆细胞样树突状细胞(pDC)的方法。方法采用免疫磁珠法分离正常C57小鼠骨髓的lin-CD117+干细胞,加入干细胞因子(SCF)及白介素(IL)-3促进增殖。9天后,停用SCF和IL-3,加入粒细胞-巨噬细胞集落刺激因子(GM-CSF)、IL-4和IL-10诱导发育成pDC,或加入肿瘤坏死因子(TNF)-α促进细胞成熟。光学显微镜和扫描电镜观察pDC形态,经流式细胞法检测免疫表型和吞噬功能。结果SCF和IL-3促进造血干细胞大量增殖;与成熟DC比较,pDC具有明显的未成熟DC形态特征,表型呈CD117和CD11c阳性,I-A/I-E低表达及CD40、CD80和CD86阴性,且吞噬功能较弱。结论小鼠骨髓lin-CD117+造血干细胞可经细胞因子诱导大量增殖,并向pDC转化。     

白细胞介素2基因转染细胞因子诱导的杀伤细胞对恶性黑素瘤细胞的杀伤作用

目的 探讨白细胞介素2(IL-2)基因转染细胞因子诱导的杀伤细胞(CIK)对恶性黑素瘤的杀伤能力.方法 提取小鼠脾细胞,分离淋巴细胞,培养CIK细胞,用携带IL-2的质粒PEGF-N1-IL-2转染CIK细胞,荧光显微镜观察质粒转染情况,反转录-聚合酶链反应(RT-PCR)鉴定IL-2基因的表达.将效应细胞(CIK细胞或IL-2转染CIK细胞)和靶细胞(B16黑素瘤细胞)分别按照效靶比10∶1、20∶1和40∶1混合培养,采用4h乳酸脱氢酶释放测定法,检测两种CIK细胞对B16细胞的细胞毒活性.按照效靶比40∶1混合,采用酶联免疫吸附测定法(ELISA)检测两种CIK细胞IL-2、干扰素γ(IFN-γ)和肿瘤坏死因子α(TNF-α)水平.建立小鼠黑素瘤模型,将28只模型小鼠平均分为4组:对照组(瘤旁注射0.2 ml生理氯化钠溶液)、IL-2组(瘤旁注射100 IU IL-2)、CIK组(瘤旁注射细胞数约1×106 CIK细胞悬液)、IL-2转染CIK组(瘤旁注射细胞数约1×106 IL-2转染CIK细胞悬液),通过肿瘤形态学和抑瘤率、细胞凋亡率来评价荷瘤小鼠肿瘤生长情况.两组正态分布计量资料的比较行t检验,多组计量资料的比较采用方差分析,两两间多重比较采用LSD-t检验.结果 荧光显微镜及RT-PCR均显示IL-2转染CIK细胞成功.效靶比实验显示,40∶1时IL-2转染CIK细胞对B16细胞的毒性最强,IL-2转染CIK细胞组分泌IL-2 (1107.26±6.49 pg/ml)、IFN-γ(50.01±3.35 pg/ml)和TNF-α(39.86±3.25 pg/ml)的能力明显高于CIK细胞组(分别为51.09±3.85、32.71±2.43、30.11±3.08 pg/ml),两组比较,t值分别为442.60、14.93和6.89,差异均有统计学意义(P＜0.01).动物实验显示,与干预前相比,干预后对照组小鼠肿瘤体积明显增大(P＜0.05),而IL-2组、CIK组和IL-2转染CIK组小鼠肿瘤体积明显减小(P＜ 0.001),且IL-2转染CIK组肿瘤体积显著小于其他3组(均P＜0.01),但IL-2组和CIK组差异无统计学意义(P＞0.05).CIK组、IL-2组和IL-2转染CIK组的细胞凋亡率均显著大于对照组(P＜0.01),IL-2转染CIK组的细胞凋亡率及抑瘤率均显著大于IL-2组和CIK组(P＜ 0.01),而IL-2组和CIK组细胞凋亡率及抑瘤率差异无统计学意义(P＞0.05).结论 IL-2转染CIK细胞对恶性黑素瘤有更强的杀伤作用.     

蕈样肉芽肿患者皮损中朗格汉斯细胞的迁移研究

目的：探讨蕈样肉芽肿（MF）患者皮损中皮肤淋巴细胞相关抗原（CLA）、CD62L、共刺激分子（CD40、CD80、CD86）分布及其在树突状细胞（DC）上的表达。方法：将MF皮损作冰冻切片,行单克隆抗体免疫组化染色。结果：肿瘤组织真皮及表皮内可见大量CLA、CD62L、主要组织相容性复合体（MHC）Ⅱ、共刺激分子表达,CLA的表达细胞明显多于CD62L,由斑块期到肿瘤期,表皮内CLA表达数目明显减少,真皮中CLA、CD62L表达增强。CD83^＋DC能表达强的共刺激分子,在基底层、真皮和血管内可见CD83^＋DC表达CD62L,但未成熟DC常表达CLA。结论：MF皮损中朗格汉斯细胞发生迁移,参与了抗肿瘤免疫反应。     

IL-10在蕈样肉芽肿中的免疫组化表达

IL-10在肿瘤性疾病,包括非何杰金淋巴瘤中的表达增加。且有报告,IL-10的这种过度表达与中、高度淋巴瘤的病情恶化有关,与蕈样肉芽肿(MF)的病程进展也有关。为了明确IL-10在MF中细胞来源与定位,该文作者采用免疫组化方法原位检测了MF皮损及来源于MF的T细胞株中IL-10的表达。     

蕈样肉芽肿白细胞介素13及其受体表达

目的 探讨白细胞介素13(IL-13)及其受体在蕈样肉芽肿(MF)皮损中的表达及临床意义.方法 2010年1月至2016年3月收集杭州市第三人民医院皮肤科经临床、病理、免疫表型和(或)T细胞受体基因重排检测确诊的MF石蜡组织标本34份,IA期5例,IB期9例,ⅡA期17例和ⅡB期3例.选择正常皮肤组织10份作为对照.应用免疫组化方法分别检测IL-13、IL-13Rαl及IL-13Rα2的表达.结果 IL-13、IL-13Rα1和IL-13Rα2表达于各期MF皮损组织的异形淋巴样细胞和亲表皮性淋巴样细胞,IL-13Rα2几乎高表达于所有的MF皮损组织.正常皮肤组织及淋巴细胞均不表达IL-13及其受体.随着MF病程的进展,皮损组织IL-13及其受体表达率升高.IL-13、IL-13Rαl及IL-13Rα2的表达率在Ⅰ期MF皮损(分别为10.00％±3.14％、21.43％±6.88％、31.14％±6.38％)均显著低于Ⅱ期MF皮损(分别为27.50％±11.00％、39.45％ ± 9.43％、44.40％±11.15％),差异均有统计学意义(P＜0.05),但在ⅠA期与ⅠB期之间以及ⅡA期与ⅡB期之间差异均无统计学意义(P＞0.05).结论 IL-13及其受体尤其是IL-13Rα2有望成为MF早期诊断和生物学行为预判的标志物.     

复发性生殖器疱疹患者外周血IL-12与Th1/Th2细胞因子的检测

目的检测复发性生殖器疱疹(RGH)患者不同病期外周血CD4+T细胞内IL-12,IFN-,γIL-4的水平,探讨IL-12,Th1与Th2亚群在疾病中的可能作用。方法应用流式细胞仪对20例发作期、15例恢复期RGH患者和15名健康人外周血CD4+T细胞IL-12,IFN-γ和IL-4进行检测。结果发作期患者外周血IFN-γ+-CD4+T细胞百分率显著低于正常对照组(P<0.05),IL-4+-CD4+T细胞百分率明显高于正常对照组(P<0.01),Th1/Th2比值显著低于正常对照组(P<0.01),同时IL-12+-CD4+T细胞百分率显著降低(P<0.01)。恢复期患者外周血IL-12+-CD4+T细胞百分率仍显著低于正常(P<0.05)。结论RGH患者存在Th1/Th2比例失衡和IL-12水平低下,而后者可能是导致Th1/Th2比例失衡和病情反复发作的重要原因。     

蕈样肉芽肿的端粒酶研究

目的 检测各期蕈样肉芽肿(MF)的端粒酶活性,探讨端粒酶在MF肿瘤发生机制中的作用.方法 采用端粒酶聚合酶链反应-酶联免疫吸附测定法(PCR-ELISA),对35例MF患者进行端粒酶的定性和定量分析.结果 92.3%的肿瘤期MF、78.6%的斑块期MF和75.0%的斑片期MF显示端粒酶阳性;肿瘤期MF的端粒酶活性水平高于斑块期和斑片期,且差异有显着性;而斑块期与斑片期之间差异无显着性.对照组的端粒酶均为阴性.讨论 MF患者存在高水平的端粒酶活性,提示端粒酶可能在MF的肿瘤发生中起重要作用,对MF的诊断有一定的临床意义.     

Dendritic cells and apoptosis in mycosis fungoides

BACKGROUND: The existence of an effective antitumour immune response in mycosis fungoides (MF) has been shown by the isolation of tumour-specific T-cell clones from such patients. Dendritic cells (DCs) are considered crucial for the induction of immunity, including resistance to tumours. Apoptotic tumour cells are a major source for tumour antigens processed and presented by DCs via cross-presentation. The production of interleukin (IL)-10 by MF tumour cells is acknowledged and may block DCs maturation leading to tumour tolerance. OBJECTIVES: Cross-presentation of apoptotic tumour cells by DCs will induce immunity if the DCs mature, but tolerance if maturation does not occur. We now further characterize the DCs in skin infiltrates of patch/plaque-stage MF (PS) and tumour-stage MF (TS) in situ. Secondly, we demonstrate apoptosis in MF infiltrates in situ and analyse the association of apoptotic cells to immature DCs, mature DCs and IL-10-positive cells. METHODS: Immunohistochemical staining (single, double, triple) employing novel markers specific for immature and mature DCs, IL-10 and a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) test were done on representative skin biopsies from PS and TS. RESULTS: In PS, the immature DCs are mostly lag/langerin + Langerhans cells (LCs). In the epidermis of PS, LCs predominate over fully mature DCs (non-LC type, CD83+, DC-lamp+). In the dermis of PS and TS, equal numbers of mature and immature (CD1a+, CD1c+) DCs are densely interspersed between the lymphocytic infiltrate. In TS, immature DCs mostly lack lag or langerin expression. Immature DCs with incorporated apoptotic cells were found rarely in PS but increasingly in TS. By triple staining in situ we could now show that strongly IL-10+ cells frequently surround immature DCs, some of them with incorporated apoptotic cells. CONCLUSIONS; DCs in MF perform a dual role, namely induction and maintenance of antitumour immunity, or, under less favourable circumstances such as production of IL-10 downregulation of antitumour immunity. The latter condition was mainly seen in TS, possibly explaining disease progression. Further in vitro studies are now required illuminating the role of DCs for the antitumour immune response in MF.     

Antitumour necrosis factor-alpha chimeric antibody (infliximab) inhibits activation of skin-homing CD4+ and CD8+ T lymphocytes and impairs dendritic cell function

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and altered differentiation of keratinocytes in reply to cytokines such as interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha, provided by infiltrating CD4+ and CD8+ T cells and natural killer cells. Infliximab is a chimeric monoclonal antibody that neutralizes both soluble and membrane-bound TNF-alpha, and that may give a long-term disease remission. OBJECTIVES: To determine the in vitro effects of infliximab on CD4+ and CD8+ T cells derived from lesional skin, and on dendritic cells (DCs). METHODS: Psoriatic T-cell lines were isolated from lesional skin of four patients with psoriasis and assayed for their proliferation, cytokine release and susceptibility to apoptotic stimuli in the presence of graded (1-100 microg mL(-1)) concentrations of infliximab. DCs were differentiated in the presence of infliximab from peripheral blood monocytes. Phenotype was assessed by fluorescence-activated cell sorting and antigen-presenting capacity in functional assays. RESULTS: In vitro activation of psoriatic as well as antigen (nickel)-specific skin-homing T cells was strongly and dose-dependently impaired by infliximab, in terms both of proliferation and of IFN-gamma release. Despite the significant reduction of IFN-gamma secretion, infliximab only marginally affected the release of interleukin (IL)-10 by skin T cells, thus determining a reduction of the IFN-gamma/IL-10 ratio at the site of inflammation. The effects were maximal when T-cell activation occurred in the absence of costimulation, or when T cells were activated by immature compared with mature DCs. In addition, skin-homing CD8+ T cells were more prominently affected by infliximab compared with CD4+ T lymphocytes, both in terms of inhibition of activation and in their susceptibility to apoptosis. Finally, infliximab directly affected the differentiation of monocyte-derived DCs, by inhibiting the expression of CD1a and CD86, and strongly impaired the antigen-presenting capacity of immature and, to a lesser extent, mature DCs. CONCLUSIONS: Infliximab directly affects psoriatic T cells and impairs the antigen-presenting capacity of DCs. These effects may help to explain the long-term disease remission obtained with the drug.     

Human basal cell carcinoma is associated with Foxp3+ T cells in a Th2 dominant microenvironment

Basal cell carcinoma (BCC), the most common human cancer, undergoes spontaneous regression in certain circumstances, which is potentially immune-mediated. To understand the immune response surrounding BCCs, we characterized the genomic, protein, and cellular microenvironment associated with BCC in comparison to normal skin. Our results demonstrated the following: (1) CD4+ CD25+ Foxp3+ surround epithelial tumor aggregates; (2) Immature dendritic cells (DCs) were abundant in the tumor microenvironment; (3) BCC showed increased expression of IL-4, IL-10, and CCL22 and increased expression of interferon-associated genes (IFI27, IRF1, IRF7, and G1P2) and IL-12/23, gene indicating a Th2 dominant microenvironment. Our findings suggest a dynamic state within the immune microenvironment associated with BCC. The finding of phenotypic T regs, in conjunction with immature DCs and Th2 cytokines, suggests an attenuated state of immunity to human BCC. In contrast, abundant CD8+ T cells, an interferon signal, and IL-12/23 suggest partial host antitumor response. A better understanding of these opposing forces within the immune microenvironment may facilitate development of more potent immune-based treatment for BCC and other human carcinomas.     

IL-15 and IL-16 overexpression in cutaneous T-cell lymphomas: stage-dependent increase in mycosis fungoides progression

Cytokines are of major importance for the pathogenesis of cutaneous T-cell lymphomas (CTCL). Recent data suggested that IL-15 and IL-16 are survival/growth factors for the malignant T cells in these entities. To investigate the expression of IL-15 and IL-16 in mycosis fungoides (MF) and CD30+ pleomorphic T-cell lymphoma in vivo, we established a competitive RT-PCR technique. Analyzing skin biopsies from CTCL patients at different stages in comparison to psoriatic and healthy skin, we found IL-15 and IL-16 mRNA overexpression in both CTCL entities. Remarkably, there was some evidence for a stage-dependent increase during MF progression. We found only slight overexpression in early stage MF, when only few tumor cells are detectable within the infiltrates, whereas marked overexpression was found in more advanced lesions, which are characterized by a higher density of malignant cells. These results suggested that CTCL cells themselves might produce the cytokines. To further elucidate this hypothesis, two CTCL cell lines were analyzed but gave conflicting results. Therefore, the cellular origin of the IL-15 and IL-16 overexpression in CTCL remains unclear. Considering the significant overexpression of IL-15 and IL-16 and their biological capacities it is likely that these cytokines contribute to the tumor development. So, they might be involved in growth and skin homing of CTCL cells.     

FOXP3 in sequential biopsies of progressive mycosis fungoides

A recent report has suggested that the numbers of regulatory T cells correlate with stage of disease and prognosis in mycosis fungoides (MF). To evaluate the role of FOXP3+ Tregs in different stages of MF, we investigated sequential biopsies in 14 patients with patch/plaque and subsequent tumor stage using FOXP3 antibody. Our data neither show a significant difference in the percentage of FOXP3+ cells between patch/plaque and tumor stage biopsies of MF nor demonstrate a predictable shift of Tregs in the course of disease progression. Additionally, we could observe FOXP3-expressing neoplastic cells in 4 patch/plaque stage biopsies, where they represented almost 100% of the epidermotropic infiltrate. Only in one of these patients, FOXP3+ cells could also be detected in the tumor stage biopsy, indicating that FOXP3 expression can be acquired or lost during the course of the disease, comparable to other phenotypic markers.     

Distribution and maturation of skin dendritic cell subsets in two forms of cutaneous T-cell lymphoma: mycosis fungoides and Sézary syndrome

Dendritic cells (DCs) critically regulate immune responses and the "immune-surveillance" of tumours. This study retrospectively analysed the distribution and maturation status of DC-subsets in T-cell lymphoma of the skin. Mycosis fungoides and Sézary syndrome (n = 25) were investigated immunohistochemically for DC subsets, based on C-type lectin receptor expression: Langerhans' cells (langerin/CD207+, DEC-205/CD205+), dermal DCs (DC-SIGN/CD209+, CD205+) and plasmacytoid DC (BDCA-2/CD303+). Maturation status was assessed by double-labelling for CD83 and CD208/DC-LAMP. DCs were interspersed between the neoplastic infiltrate, and a marked increase in numbers of all three subsets was noted, DC-SIGN+ dermal DCs constituting the majority. Substantial numbers of plasmacytoid DCs were consistently observed. Most DCs in epidermis and dermis were phenotypically immature. Amongst the relatively few mature DCs in the dermis, langerin+ cells predominated. There was a positive correlation between the histological intensity of the tumour infiltrate and DC numbers. It is possible that mature DCs reflect ongoing anti-tumour immune responses, and immature DCs the induction of tumour tolerance.     

Artificial phosphatidylserine liposome mimics apoptotic cells in inhibiting maturation and immunostimulatory function of murine myeloid dendritic cells in response to 1-chloro-2,4-dinitrobenze in vitro

Phosphatidylserine (PS) exposed on the apoptotic cell surface inhibits inflammatory responses, implying that PS may regulate the function of dendritic cells (DCs) after being phagocytosed by the latter. Here we use PS liposomes to investigate the effects of PS on the maturation and immunostimulatory functions of DCs in response to the challenge of 1-chloro-2,4-dinitrobenze (DNCB) in vitro. We demonstrate that after treatment with PS, murine DCs display reduced expression of MHC II, CD80, CD86 and CD40, but increased programmed death ligand-1 (PD-L1 and PD-L2); and increased IL-10 and inhibited IL-12 cytokine production. PS-treated DCs exhibit normal endocytic function, but ability to stimulate allogeneic T cells is reduced, similar to immature dendritic cell (iDCs). Treatment of DCs with PS liposomes also suppressed DNCB induced CD4 + T cell proliferation and IFN-γ production. Addition of exogenous IL-12p70 during the DC-T cell co-culture restored their IFN-γ production. Furthermore, PS-treated DCs enhance the ratio of CD4 ⁺ CD25^highFoxp3⁺ T cells to CD4⁺ T cells and PD-1 expression on CD4⁺ T cells. These data demonstrate that PS liposomes have therapeutic potential in allergic contact dermatitis (ACD). Dongmei Shi, Meng Fu and Pingshen Fan contributed equally to this article.