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Year : 2016  |  Volume : 33  |  Issue : 1  |  Page : 44-48

A study of Foxp3+ regulatory T-cell in lesional skin of mycosis fungoides in comparison with some histological mimickers

1 Department of Dermatology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
2 Department of Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt

Date of Submission12-May-2015
Date of Acceptance12-May-2015
Date of Web Publication28-Nov-2016

Correspondence Address:
Marwa Zohdy A Moubarak
Department of Dermatology, Mansoura University, Mansoura
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1110-208X.194387

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Introduction Regulatory T-cells (Tregs) are essential to balance between proinflammatory and anti-inflammatory responses by protecting against autoimmune reactions. They have different roles in skin tumors, including cutaneous T-cell lymphomas. Foxp3 (forkhead box P3) is thought to be a reliable marker for Tregs. Contradictory results were reported in the literature when the density of Tregs in the skin of mycosis fungoides (MF, a type of cutaneous T-cell lymphoma) was compared with some pathologically mimicking inflammatory dermatoses.
Patients and methods This is a comparative cross-sectional study of lesional skin biopsies from 36 cases of MF versus those from 36 cases of histopathological mimickers, including 10 cases of psoriasis, 10 cases of spongiotic dermatitis, and 16 cases of lichenoid dermatoses (10 with pityriasis lichenoides chronica and six with discoid lupus erythematosus). We compared the densities of Foxp3+ Tregs in relation to CD4+ cells in the epidermis and dermis of these groups using Foxp3 and CD4 monoclonal antibodies.
Results The MF group showed a lower statistically significant difference as regards epidermal and dermal Foxp3/CD4+ ratios compared with the histologic mimicker group.
Conclusion MF lesions showed a significantly lower density of CD4+ Foxp3+ Tregs compared with its histological mimickers, which might be attributed to their limited proliferation at the expense of malignant T-lymphocytes.

Keywords: foxp3, T- cells, dermatoses, skin, mimickers

How to cite this article:
Moubarak MA, Sharaf L, Abdelnaby S, Zalata K, Fathy H. A study of Foxp3+ regulatory T-cell in lesional skin of mycosis fungoides in comparison with some histological mimickers. Benha Med J 2016;33:44-8

How to cite this URL:
Moubarak MA, Sharaf L, Abdelnaby S, Zalata K, Fathy H. A study of Foxp3+ regulatory T-cell in lesional skin of mycosis fungoides in comparison with some histological mimickers. Benha Med J [serial online] 2016 [cited 2021 Dec 5];33:44-8. Available from: http://www.bmfj.eg.net/text.asp?2016/33/1/44/194387

  Introduction Top

In the past decade, much was discovered about regulatory T-cells (Tregs) that develop from uncommitted (naive) CD4+ T-helper cells [1]. Disturbed effector functions of Tregs can lead to (sometimes fatal) autoimmune and allergic inflammation [2]. The FOXP3 gene (forkhead box P3) encodes a transcription factor thought to be important for the development and function of Tregs and represents a reliable marker [3]. In psoriasis, Tregs can differentiate toward interleukin-17-expressing Tregs. The combination of psoriatic Treg dysfunction and a propensity for differentiating into interleukin-17-producing regulatory cells contributes to the perpetuation of chronic autoimmunity [4]. Natural Tregs are responsible for the downregulation of eczema in allergic patients [5]. They block the migration of effector T-cells into inflamed tissue, suppress the production of immunoglobulin E, induce immunoglobulin G4 in B cells, and limit Th17-mediated inflammation [6]. In some lichenoid tissue reactions, such as those in pityriasis lichenoides chronica (PLC), Tregs may play an important role in controlling clonally restricted CD4+ T-cell proliferations [7]. Among several factors involved in the pathogenesis of the skin, discoid lupus erythematosus (DLE) has the decreased number of Tregs at the site of inflammation [8]. Although Tregs are essential in mediating immune homeostasis, their role is more complex in the context of cancer. As cancer cells express both self-associated and tumor-associated antigens, Tregs are thought to contribute to the progress of many tumors [9]. In lymphoma, being a malignant proliferation of T-cells, Tregs might play a fundamentally different role in comparison with solid tumors [10]. Mycosis fungoides (MF) is a type of cutaneous T-cell lymphoma (CTCL), characterized by a wide spectrum of clinical presentations: a classic form (patch, plaque, and tumor stages) and many clinicopathological variants [folliculotropic, hypopigmented, erythrodermic/Sezary syndrome (SS), etc.]. MF can pathologically mimic different psoriasiform, spongiotic and lichenoid dermatoses [11]. Tregs were found in MF lesions and have been correlated with improved survival, mostly through suppression of malignant cell proliferation [12]. Increased numbers of Tregs, in contrast, have been responsible for the immune deficiency accompanying SS [13]. This study was conducted to compare the densities of Tregs between MF and a group of its histologic mimickers, including psoriasis, spongiotic, and some lichenoid dermatoses (namely, PLC and DLE), aiming to clarify Tregs role in their pathogenesis.

  Patients and methods Top

We carried out a comparative cross-sectional study on lesional biopsies from 36 patients with MF versus those from 36 patients with benign histological mimickers, including 10 patients with psoriasis, 10 patients with eczema, and 16 with lichenoid dermatoses (10 with pityriasis lichenoides and six with DLE). Ten biopsies were taken from normal skin as control. Patients were selected from the Dermatology outpatient clinic and biopsied in the Dermatopathology Laboratory at Dermatology, Andrology, and Sexually Transmitted Diseases Department, Mansoura University Hospital, Mansoura, Egypt, during the period from February 2012 to November 2014. Oral consents were taken from all patients. MF cases included in the study were newly diagnosed patients who had not received any topical or systemic treatment and patients who had not undergone phototherapy or systemic therapy for at least 6 months. Patients with psoriasis were either new cases or those who had stopped topical treatment for at least 4 weeks, or stopped systemic therapy (psoralen with ultraviolet A, methotrexate, cyclosporine, and retinoides) for at least 12 months. Patients with eczema, pityriasis lichenoides, and DLE were selected as newly diagnosed cases. Patients with concurrent autoimmune disease or any other systemic disease with immune dysregulation that may alter Tregs numbers were excluded from the study. All patients were subjected to thorough history taking, general examination, and skin examination. MF cases were admitted to the Dermatology Inpatient Department, Mansoura University for tumor–node–metastasis–blood staging. Skin punch biopsies from MF and skin lesions of benign histological mimickers (psoriasis, eczematous, and lichenoid) were taken and fixed with 10% formaldehyde, and paraffin-embedded sections were stained with hematoxylin and eosin and examined. For established MF cases, assessment of superficial lymphoid infiltrate for the presence of epidermotropism and cellular atypia was carried out. Before immunohistochemistry, antigen retrieval process was carried out by placing skin specimens of 3 mm on positively charged slides. Specimens were then deparaffinized, and rehydrated skin sections were rated through a series of xylenes and graded alcohols. Specimens were then quenched for endogenous peroxidase in 3% H2O2 for 5 min. Thereafter, the slides were placed in a vegetable steamer containing Dako’s TRS solution. All skin specimens of MF and histologic mimickers were stained with anti-CD4 antibody (mouse monoclonal anti-CD4, clone 4B12, dilution 1 : 50; Genemed, San Francisco, California, USA) and antiforkhead box protein 3 (FoxP3) mAb (FoxP3 mAb, clone NB100–39002, dilution 1 : 400; Novus, St Charles, Missouri, USA) to define Tregs. MF specimens were additionally stained with anti-CD8 (mouse monoclonal anti-CD4, clone C8/144B, dilution 1 : 50; Dako, Glostrup, Denmark) to define CD8+ predominant cases. For FoxP3 staining, Dako LSAB+/AP was used; for other immunohistochemical staining, ‘DakoChem Mate Envision Kit/horseradish’ peroxidase was used. Quantification of the frequency of immunostained cells in the epidermis and upper dermis was performed in single-stained sections. In each run, negative and positive controls were applied. The negative control was a section from the tested case but lacking the antibody. The positive control was taken from tonsil and lymph node tissues. CD4 expressed a membranous reaction, whereas Foxp3 expressed nuclear and/or cytoplasmic reactions. The slides stained with CD4 and Foxp3 were examined for each case in one session and compared with the hematoxylin and eosin-stained slides. The specifically stained lymphocytes were calculated in three different high power fields (×40) and then the number was recorded for both the epidermis and the upper dermis separately. The mean number of each specifically immunostained lymphocytes (CD4+ and Foxp3+) was calculated by dividing the sum in the three fields by 3. CD4 was considered as the reference standard for which a ratio for Foxp3+ was calculated. Divided numbers were approximated for the highest value. Data were entered and statistically analyzed using the statistical package for social sciences, version 17 (SPSS Inc, Chicago, IL, USA). Qualitative data were described as numbers and percentages. The χ2-test was used for comparison between groups. Quantitative data were described as median after testing normality using the Kolmogorov–Smirnov test. The Kruskal–Wallis test was used for comparison between several groups. Nonparametric correlations were carried out using Spearman’s rank correlation. A P value of 0.05 or less was considered to be statistically significant.

  Results Top

In total, this comparative study included 36 cases of MF versus 36 cases of histological mimickers. The age range of the MF group at presentation was 6–78 years (median = 50.0 years) and that for the mimicker group was 2–75 years (median = 28 years), with significant age difference between the two groups (P = 0.004). Among the MF patients, there were 18 male and 18 female patients, whereas in the mimicker group there were 16 male and 20 female patients, with no significant difference as regards sex between the two groups (P = 0.0637). The MF stages in the 36 cases ranged from stage I to III. In another spectrum, MF cases included the different clinicopathological variants (26 classic, five hypopigmented, two poikilodermic, two erythrodermic, and one folliculotropic). The 36 cases of MF mimickers were clinically diagnosed as psoriasis (four cases of psoriasis vulgaris, four cases of guttate, and two cases of erythrodermic psoriasis), eczema (five atopic, four contact, and one seborrhic dermatitis), 10 patients with PLC, and six with DLE. In all 36 MF cases, there was epidermotropism with varying degrees of cellular atypia ([Figure 1]). Immunophenotyping revealed that 30 cases (83.3%) were predominantly CD4 positive, whereas six cases (16.7%) were predominantly CD8+, including the five cases of hypopigmented MF and one folliculotropic variant. In the MF mimicker group, there were 10 cases with typical pathologic features of psoriasis (hyperkeratosis, acanthosis, regular elongation of rete ridges, and diminished granular cell layer), 10 cases with variable features of spongiotic dermatitis (intraepidermal vesiculation, serum crusts, lymphocytic exocytosis, and collections of Langerhans cell microvesicles within the epidermis), and 16 cases of lichenoid interface changes, with occasional extravasated erythrocytes in 10 cases of PLC and variable epidermal atrophy with follicular plugging in six cases of DLE. Foxp3 immunohistochemistry was carried out in both groups and positivity was expressed as a ratio to the CD4+ T-cells in the epidermis and dermis. All 10 biopsies of normal skin showed negative staining for Foxp3+ CD4+ Tregs with a median that equals 0. On comparing the MF group and the mimicker group, the MF group showed a lower statistically significant difference as regards epidermal (P = 0.012) and dermal Foxp3/CD4+ ratios (P < 0.001) ([Table 1]).
Figure 1 A section of mycosis fungoides skin stained with Foxp3 monoclonal antibodies. The epidermis showed epidermotropism with Foxp3 positively stained atypical intraepidermal lymphocytes (×40).

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Table 1 Foxp3+/CD4+ Treg ratio in the study groups

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  Discussion Top

As the importance of CD4+ CD25+ Foxp3+ Treg cells in the maintenance of peripheral immunologic tolerance has been well established, different studies over the past few years have addressed the number and/or function of Treg cells in the skin of different dermatoses. Furthermore, their role in tumor progression has attracted authors’ attention to study these cells in MF in comparison with some inflammatory mimickers, aiming to clarify their role in the pathogenesis of such long standing, great imitator [14–17]. As a whole, our comparative results showed markedly significant differences between MF (n = 36) and histologic mimickers (n = 36) in epidermal (P = 0.01) and dermal Foxp3+/CD4+ ratios (P < 0.001) as well as epidermal (P = 0.009) – that is, fewer number of Treg cells was found in MF in comparison with histologic mimickers ([Table 1] and Diagram 1a and b). This can be possibly explained by the fact that Tregs are increased in inflammatory skin lesions to suppress skin inflammation due to their anti-inflammatory and immunosuppressive properties [17], whereas they are decreased in MF possibly due their limited proliferation at the expense of malignant T-lymphocytes. This is supported by the hypothesis that Tregs might suppress the propagation of the neoplastic T-cells and implies that several mechanisms, which make the malignant T-cells resistant to this suppression, impair the function and numbers of infiltrating Tregs that can promote disease progression [12]. Our results were concordant with similar comparative studies published in the literature [15],[16] and was in disagreement with others [14]. Solomon and Magro found that the number of Foxp3+ Treg cells was highest in reactive lymphomatoid infiltrates (n = 33; including spongiotic and lichenoid subsets), intermediate in endogenous prelymphomatous T-cell dyscrasias (n = 40; including 19 PLC), and lowest in T-cell lymphomas (P = 0.0001). These results were in accordance with our results, but the authors had not specified epidermal and dermal subsets. Moreover, they used Foxp3 as sole markers of Tregs, and positivity of Foxp3-stained cells were estimated as a percentage regardless of CD4 staining. In addition, the cutaneous lymphoma group (n = 22) included, in addition to MF (n = 4), a wider spectrum compared with our study (subcutaneous panniculitis-like T-cell lymphoma, extranodal natural killer-like T-cell lymphoma, primary cutaneous anaplastic large cell lymphoma, secondary peripheral T-cell lymphoma). Reactive lymphoid dermatoses included only lichenoid (including collagen vascular disease) and eczematous, whereas lymphoid dyscrasias included PLC, pigmented purpuric dermatosis, and alopecia mucinosa and did not include psoriasis that was included in our study. Similar results were observed by Wada and colleagues, who found significantly lower percentages of intraepidermal Foxp3+ lymphocytes (P < 0.001) in patients with CTCL (n = 20), including MF (n = 13) and SS (n = 7), compared with those observed in patients with spongiotic (n = 11) and lichenoid or interface dermatoses (n = 11). They counted Foxp3+ Treg cells in the context of CD3+ cells in the epidermis and dermis separately and noted a higher proportion of Foxp3+ T-cells in the dermal infiltrate compared with those in the epidermis of CTCL cases and in the inflammatory group. Fujimura and colleagues evaluated the percentage of epidermal Foxp3+ cells among CD3+, CD4+, CD8+, and CD25+ cell populations in skin biopsies of psoriasis (n = 16), MF (n = 17), and eczematous dermatitis (n = 18), in addition to normal skin (n = 10), using double staining techniques. They concluded that there was a lower percentage of epidermal and dermal Foxp3+ cells in eczematous dermatitis and psoriasis vulgaris compared with MF, which was in disagreement with our results. They also reported lower percentage of epidermal Foxp3+ cells in eczematous dermatitis than in psoriasis vulgaris or MF, and significantly fewer dermal Foxp3+ cells in psoriasis vulgaris than in eczematous dermatitis or MF (P < 0.05). The difference between the previous data and our results could be attributed to an important factor, which is the method of calculation of Foxp3+ Treg among CD3+, CD8+, and CD25+ cells, in addition to CD4+ cells. Many other factors could explain the discrepancy, such as the difference in the pathological spectrum of the studied groups – as the previous study did not include lichenoid dermatoses among the mimickers of MF, instead it included normal skin as a control – different staining techniques (single staining vs. double staining), and the use of different antibodies for FoxP3 in the three studies. We used mouse monoclonal antibody as did Solomon and Magro and Wada and colleagues, whereas Fujimura and colleagues used rabbit anti-FoxP3 antibody (clone not specified). In our study, the dermal Foxp3+/CD4+ Tregs in MF (median = 41%) were fewer compared with the epidermal Treg cells (median = 50%), in contrast to the inflammatory group (median = 50 and 80% for epidermal and dermal, respectively) ([Table 1]). This can be explained by the presence of a higher dermal population of CD8+ T-cells in case of inflammatory MF mimickers, which may have acquired Foxp3, which could substantially increase the calculated number of Tregs. Our results were in agreement with Fujimura and colleagues, who observed significantly smaller percentage of dermal Foxp3+ cells, in MF cases, compared with epidermal Foxp3+ cells (P < 0.05).

  Conclusion Top

Contradictory results were reported in the literature as regards Treg numbers in lesional skin from different inflammatory and neoplastic dermatoses. MF is a type of CTCL that can pathologically mimic different psoriasiform, spongiotic and lichenoid dermatoses. The aim of this study was to assess the density of Foxp3+ Treg cells in lesional skin of MF in comparison with its pathological mimickers, which may help in clarifying their possible role in the pathogenesis of these diseases. This comparative study was performed on lesional skin biopsies from 36 patients with MF versus those from 36 patients with benign histological mimickers, including 10 patients with psoriasis, 10 patients with eczema, and 16 with lichenoid dermatoses (10 patients with PLC and six with DLE) (during the period February 2012–November 2014) using Foxp3 monoclonal antibodies. Treg cells were calculated as a ratio to the total CD4+ cells, both epidermal and upper dermal, and compared between the two groups. We found that MF lesions showed a significantly lower density of CD4+ Foxp3+ Tregs compared with its histological mimickers, which might be attributed to their limited proliferation at the expense of malignant T-lymphocytes.



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