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。     

Expression of T-cell receptor antigens in mycosis fungoides and inflammatory skin lesions

Using immunohistologic methods, we studied the expression of the T-cell receptor (TCR)-associated antigens CD3, TCR-beta, and TCR-delta by cutaneous T cells in mycosis fungoides (MF) (36 patients) and a variety of inflammatory diseases (16 patients). Most T cells in the inflammatory diseases and patch/plaque mycosis fungoides expressed the immunophenotype characteristic of the vast majority of mature peripheral T cells: CD3+ TCR-beta+ TCR-delta-. In contrast, abnormal CD3/TCR-beta antigen expression was seen in 3 of 6 cases (50%) of tumor stage mycosis fungoides. Furthermore, we were able to document its evolution from the normal pattern present in earlier patch/plaque lesions of the two cases in which serial biopsies were available for study. Divergence of epidermal versus dermal CD3/TCR-beta antigen expression was seen in 2 of 34 (6%) of biopsies of patch/plaque mycosis fungoides but not in inflammatory controls. The TCR-delta+ cells were generally rare regardless of diagnosis. We conclude that inflammatory skin diseases and most patch/plaque mycosis fungoides are typically composed of T lymphocytes that resemble mature peripheral T cells in regard to their expression of TCR-associated antigens. In contrast, aberrant patterns of TCR-associated antigen expression can be seen in tumor stage MF, and, more rarely in patch/plaque MF.     

Suppressor T cells in mycosis fungoides and so-called premycotic eruptions

The proportions of suppressor T cells (TG) and helper T cells (TM) were determined in 5 patients with Mycosis fungoides (MF), 4 patients with parapsoriasis en plaques/poikiloderma atrophicans vasculare and 3 patients with generalized chronic dermatitis. All the MF patients showed increased proportions of suppressor T cells, whereas the others did not differ from age- and sex-matched healthy controls.     

Immunological markers of Langerhans' cells in mycosis fungoides skin lesions

The patterns of two immunological markers of epidermal Langerhans' cells were compared in normal skin and mycosis fungoides (MF) skin lesions. A double-staining indirect immunofluorescence method was used. Cells suggestive of keratinocytes reacted with the anti-Ia antibodies in some cases of MF lesions, while no such pattern was seen with the thymocyte-antigen antibodies (OK T6). In other cases of MF, as in normal skin, the epidermal picture was mainly the same with the two markers. A great many of the dermal mononuclear cells in MF were reactive with the anti-Ia antibodies but only occasional dermal cells in MF and no dermal cells in normal skin stained with OK T6.     

蕈样肉芽肿中恶性T细胞亲表皮性机制研究进展

皮肤T细胞淋巴瘤中最常见的蕈样肉芽肿,早期可见不同程度的亲表皮现象,即恶性T细胞不同程度侵入表皮,对诊断有较大提示意义,当病情进展到肿瘤期时可逐渐消失.目前对此现象产生和维持的机制研究主要集中在以下两个方面,一为角质形成细胞表达的趋化因子及淋巴细胞表面的趋化因子受体相互作用,如IP-10/CXCR3、SDF-1/CXCR4、TARC/CCR4等,二为表皮内的朗格汉斯细胞及调节性T细胞的相互作用.

Abstract：

Mycosis fungoides is the most common cutaneous T cell lymphoma.Epidermotropism,manifested as the infiltration of epidermis with atypical CD4+ T lymphocytes, may be observed at the early stage of mycosis fungoides, and disappear at the tumor stage.Current studies concerning epidermotropism in mycosis fungoides are mainly focused on the following two aspects, i.e., the interaction of chemokines expressed by keratinocytes with their receptors on the surface of lymphocytes, such as IP-10/CXCR3, SDF-1/CXCR4, TARC/CCR4, as well as the interaction between intraepithelial Langerhans cells and regulatory T cells.     

The cellular microenvironment and neoplastic population in mycosis fungoides skin lesions: a clinicopathological correlation

Background

The cellular microenvironment has been proven to play a crucial role in solid tumours and seems to be important in haematologic malignancies, however, it has not been adequately investigated in primary cutaneous T cell lymphomas.

Objectives

The aim of this study was to register the composition of the cellular microenvironment in mycosis fungoides skin lesions and correlate the composing parameters with the clinical data and follow-up results.

Materials and methods

The presence of eosinophilic polymorphonuclear leukocytes, B lymphocytes, CD68⁺ macrophages, and CD1a⁺ epidermal Langerhans and antigen-presenting dermal dendritic cells, as well as their relation to clinicopathological parameters, were studied in 16 mycosis fungoides cases of different disease stages. The presence and nature of the participating T cell populations was also investigated.

Results

CD8⁺ tumour infiltrating T cells and CD56⁺ cells were found among neoplastic CD4⁺ T cells in the lesions. Generally, eosinophils and B lymphocytes were absent or in low numbers, regardless of clinical presentation, contrary to tumourous lesions. Macrophages and CD1a⁺ cells were constantly present, even in early-stage mycosis fungoides. The reduced presence of the CD1a⁺ population was associated with resistance to therapy (x²; p = 0.012).

Conclusion

There is a striking difference in cellular microenvironment composition between early and advanced mycosis fungoides lesions.

     

Chemokine receptor expression in non-melanoma skin cancer

Background:  Previous studies suggest that chemokines and chemokine receptors have a role in the metastatic process. A correlation exists between the specific expression of these chemoattractive, pro-inflammatory cytokines and the ability of cancer to disseminate. Prior studies have shown that in metastatic melanoma and squamous cell carcinoma of the head and neck upregulation of CXC (α) chemokine receptor (CXCR)4 and CC (β) chemokine receptor (CCR)7 expression is accompanied by downregulation of the chemokine receptor CCR6. However, the expression patterns of CCR6, CCR7 and CXCR4 in non-melanoma skin cancer have yet to be elucidated.
Methods:  The expression patterns of CCR6, CCR7 and CXCR4 were determined using an immunohistochemical approach on formalin-fixed, paraffin-embedded normal, pre-cancerous actinic (solar) keratosis, squamous cell carcinoma and basal cell carcinoma tissues.
Results:  Analysis of chemokine receptor expression showed downregulation of CCR6 and upregulation of CCR7 and CXCR4 in potentially metastatic non-melanoma skin cancer, invasive squamous cell carcinoma, but this pattern did not exist in non-melanoma skin cancer with no metastatic potential, basal cell carcinoma; or actinic keratosis, when compared with normal skin.
Conclusions:  Chemokine receptor expression may influence the biological behavior of non-melanoma skin cancer. The exact mechanism by which this occurs requires further study.     

Microanatomical compartments of clonal and reactive T cells in mycosis fungoides: molecular demonstration by single cell polymerase chain reaction of T cell receptor gene rearrangements

Mycosis fungoides (MF) is a cutaneous T cell lymphoma, clinically characterized by patches, plaques and tumors occurring in successive stages of the disease. In early MF, an infiltrate consisting of mainly reactive T cells is seen in the papillary dermis while tumor cells are mostly confined to the epidermis. By contrast, later stages show nodular infiltrates formed mostly of tumor cells in the dermis while the epidermis is relatively devoid of tumor cells; however, knowledge of the localization of clonal T cells has been based on histomorphologic features and immunohistochemical stainings visualizing certain V-beta subfamilies of the T cell receptor (TCR). As these techniques do not allow for an unequivocal identification of clonal tumor cells, we used micromanipulation and single cell PCR amplifying the TCR chain gene rearrangement. A total number of 387 single T cells was isolated from six skin biopsies in five patients in patch, plaque, and tumor stages. Of these, 180 T cells were picked from the epidermis and 207 from the dermal infiltrate. The rearranged TCR-gamma DNA could be sequenced from 181 of 387 T cells. In three of six patients representing all three stages, epidermal T cells with a clonal rearrangement could be amplified. In early plaque stage a higher degree of epidermal T lymphocytes was found than in initial patch, later plaque, and tumor stages with an inverse distribution found for reactive T lymphocytes. In two patients a biallelic rearrangement was demonstrated that had not been detected in prior PCR analysis from blood and skin samples. These data show that clonal (neoplastic) and non-clonal (reactive) T lymphocytes in MF preferentially infiltrate different microanatomical compartments of the skin, depending on the stage of disease. The microanatomically distinct localization of reactive and clonal T cells suggests that the absence of direct contact between tumor and host-defense lymphocytes may contribute to tumor persistence and progression in epidermis, peripheral blood, and deep dermal tumor cell nests, respectively.     

Thymus and activation-regulated chemokine in different stages of mycosis fungoides: tissue and serum levels

Background/Objectives: Thymus and activation‐regulated chemokine (CCL17) is a member of the CC chemokines known to attract T‐helper 2 type memory T cells and to participate in different T‐helper 2 diseases. The aim of this study is to determine both serum and tissue levels of thymus and activation‐regulated chemokine in patients with different stages of mycosis fungoides. Methods: Thirty‐two patients with different stages of mycosis fungoides and 10 controls were included in the study. Skin biopsies and blood samples were taken to evaluate both tissue and serum levels of thymus and activation‐regulated chemokine using the enzyme‐linked immunosorbent assay method. Results: The mean tissue level of thymus and activation‐regulated chemokine in 10 tumour‐stage patients was significantly higher (P = 0.002) than in the controls. The mean serum level of thymus and activation‐regulated chemokine in all stages of mycosis fungoides patients was not significantly elevated (P = 0.131, 0.725 and 0.622) compared with controls. Both tissue and serum levels of thymus and activation‐regulated chemokine correlated significantly with both the disease extent and duration in the three different stages of mycosis fungoides. Conclusion: Thymus and activation‐regulated chemokine may be a marker for disease activity of mycosis fungoides, and may have a role in monitoring disease progression.     

T cell receptor gene rearrangement analysis in mycosis fungoides and disseminated lymphocytoma cutis

A cDNA from the CT beta 2 region of the T cell receptor gene was used as a probe to investigate the clonal composition of T cells in the skin lesions of 3 patients with mycosis fungoides (MF) and 1 patient with disseminated lymphocytoma cutis (DLC). All of these skin lesions showed helper/inducer T cell dominant infiltration. Clonal T cell populations, as evidenced by rearranged DNA bands, were demonstrated in all the skin lesions of the 3 patients with MF, but not in the lesion of the patient with DLC. The patients with MF appeared to have the same gene rearrangement pattern in DNA obtained from separate lesions, providing evidence that the T cells in both sites were derived from the same clone. Our results show that this gene rearrangement analysis helps to diagnose difficult cases of lymphoproliferative disorders.     

Enzyme cytochemical and immunocytological differentiation of infiltrative cells in the skin in mycosis fungoides]

In 4 patients with mycosis fungoides in the praemycotic, infiltrative and tumour stage of the disease, a differentiation and functional characterization of the infiltrate cells, obtained from cell suspensions and cutaneous smears, was carried out by means of enzyme cytochemical and immunological methods. The lymphocyte-associated acid esterase is considered to be a marker for mature T-cell populations. Apart from monocytes and histiocytes, acid esterase-positive small lymphocytoid cells with T-cell properties are found in the praenycotic stage. In the tumour stage, large lymphocytoid cells become increasingly prevalent, they show no acid esterase activity, but an intracytoplasmatic-localized reaction in the acid phosphatase activity. On the basis of the cytochemical pattern, it is assumed that these cells represent proliferating lymphoblasts.     

Large cell mycosis fungoides at the tumor stage. Unusual T8, T4, T6 phenotypic expression

We report a case of mycosis fungoides in a 52-year-old woman with a huge skin tumor on her thigh in which the predominant cells were lymphoblastic and histiocytic large cells. The neoplastic cells showed strong suppressor and helper T-cell subset markers, and were partially positive for the cortical thymocyte (OKT6) subset marker.     

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.     

Th17和Treg细胞与蕈样肉芽肿相关性研究

目的 探讨调节性T（Treg）细胞和Th17细胞在蕈样肉芽肿不同分期中的变化。 方法 流式细胞仪检测28例蕈样肉芽肿、13例大斑块型副银屑病、17例扁平苔藓患者及10例健康对照外周血Treg细胞和Th17细胞百分率,同时应用免疫组化法检测40例蕈样肉芽肿、13例副银屑病、17例扁平苔藓及10例健康对照蜡块组织中叉头状转录因子P3（FOXP3）和白细胞介素17（IL-17）的表达。 结果 蕈样肉芽肿、副银屑病、扁平苔藓患者外周血Treg细胞百分率分别为（8.09 ± 1.68）%,（6.53 ± 1.67）%,（2.84 ± 1.16）%较健康对照［（1.01 ± 0.35）%］升高,差异均有统计学意义。蕈样肉芽肿、副银屑病患者外周血Treg细胞百分率亦高于扁平苔藓（均P < 0.05）;蕈样肉芽肿与副银屑病患者差异无统计学意义（P > 0.05）。外周血Th17细胞百分率在蕈样肉芽肿较副银屑病、扁平苔藓患者比健康人升高［（3.22 ± 0.82）%比（2.46 ± 0.79）%,（1.38 ± 0.47）%和（0.59 ± 0.30）%,均P < 0.05］。FOXP3阳性率在蕈样肉芽肿、副银屑病及扁平苔藓均高于正常皮肤组织［（14.94 ± 4.46）%,（11.95 ± 4.72）%,（6.32 ± 2.81）%比（3.43 ± 1.79）%,均P < 0.05］,蕈样肉芽肿及副银屑病比扁平苔藓高（均P < 0.05）,蕈样肉芽肿比副银屑病差异无统计学意义（P > 0.05）。IL-17阳性率在蕈样肉芽肿、副银屑病、扁平苔藓和正常组织中分别为（15.89 ± 4.27）%,（12.02 ± 3.34）%,（4.84 ± 1.93）%和（2.62 ± 0.89）%,其中,蕈样肉芽肿均高于副银屑病、扁平苔藓组织及正常组织（均P < 0.05）。蕈样肉芽肿、副银屑病外周血Th17/Treg细胞比率比扁平苔藓、健康对照低（0.41 ± 0.11,0.39 ± 0.12比0.50 ± 0.06,0.57 ± 0.19（均P < 0.05）。早期蕈样肉芽肿Th17细胞与Treg细胞呈正相关（r = 0.423,P < 0.05）,肿瘤期蕈样肉芽肿Th17细胞有所下降,而Treg细胞继续升高,但蕈样肉芽肿各期差异无统计学意义,且二者在肿瘤期无相关性。 结论 Treg和Th17细胞失衡可能参与了蕈样肉芽肿的发生与发展。     

Decreased interleukin‐21 expression in skin and blood in advanced mycosis fungoides

Interleukin (IL)‐21 is regarded as a potent antitumor agent, which increases the cytotoxicity of both natural killer (NK) and CD8⁺ T cells. In this study, we investigated the role of IL‐21 in mycosis fungoides (MF). IL‐21 mRNA expression levels in patch and plaque MF were significantly higher than those in normal skin. IL‐21 mRNA expression levels in tumor MF were significantly decreased compared with those in patch and plaque MF. Interestingly, mRNA expression levels of IL‐21 in MF lesional skin significantly correlated with those of T‐helper type‐1 cytokines/chemokines such as CXCL10, CXCL11 and γ‐interferon. Immunohistochemistry showed that IL‐21 was expressed by keratinocytes in patch and plaque MF. Furthermore, serum IL‐21 levels in patients with tumor MF were significantly lower than those of healthy controls and plaque MF. Thus, IL‐21 expression was significantly downregulated in skin and blood of patients with tumor MF, which may contribute to progression of MF. Our study suggests that recombinant IL‐21 would be a promising therapy for MF.     

Immunohistopathological observations in the skin of patients with mycosis fungoides

Summary With the direct and indirect immunofluorescence technique, deposits of immunoglobulins (predominantly IgG, less frequently IgA, IgM and IgD) and complement were demonstrated in lesional skin of patients with mycosis fungoides. The occurrence of these deposits proved to be related to the infiltrative stage of the disease. Immunoglobulin and complement deposits were not found in the premycotic stage of mycosis fungoides, but in the tumour stage a small number were demonstrable. In 2 patients with other forms of malignant reticulosis, i.e. Brill-Symmers' disease and lymphosarcoma such deposits could not be detected in the lesional skin.The immunoglobulin and complement deposits were located in the walls of bloodvessels and scattered in the dermis.The possible significance of the deposition of immunoglobulin and complement in the skin of patients suffering from mycosis fungoides is discussed.

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Zusammenfassung Immunglobuline (vorwiegend IgG, seltener IgA, IgM und IgD) sowie Complement wurden in der erkrankten Haut von Patienten mit Mycosis fungoides mit direktem und indirektem Immunfluorescenzverfahren nachgewiesen. Das Auftreten der Immunkomplexe zeigte sich im infiltrativen Stadium der Erkrankung. Immunglobulin- und Complementniederschläge fehlten im prämykotischen Stadium, während sie im Tumorstadium in geringer Menge nachweisbar wurden. Bei 2 Patienten, die an anderen Formen der malignen Reticulose (Brill-Symmers, Lymphosarkom) erkrankt waren, konnten keine Immunkomplexe nachgewiesen werden.Immunglobulin und Complement befanden sich in den Gefäßwänden und in der Dermis verstreut.Die Bedeutung von Immunglobulin- und Complementniederschlägen bei Mycosis fungoides wird besprochen.