Absence of modulation of CD4+CD25high regulatory T cells in CTCL patients treated with bexarotene

Please cite this paper as: Absence of modulation of CD4+CD25^high regulatory T cells in CTCL patients treated with bexarotene. Experimental Dermatology 2010; 19 : e95–e102. Abstract: Cutaneous T‐cell lymphoma (CTCL) are a heterogeneous group of lymphoproliferative disorders, characterized by the infiltration of the epidermis by mature and activated malignant CD4+ T‐lymphocytes. Retinoids such as retinoic acid and synthetic analogues have long been used alone or in combination with other therapies for CTCL. Bexarotene, the first synthetic highly selective RXR retinoid, was approved for the treatment of all stages of CTCL in patients refractory to at least one systemic therapy. Recently, six cases in which the initiation of bexarotene therapy for CTCL was associated with the progression of internal disease despite improvement of cutaneous signs and symptoms were reported. Moreover, it has been established that retinoids promote the generation of CD4+ Foxp3+ regulatory T cells, raising the question of an induction of regulatory T‐cells by bexarotene. The aim of this work was to determine if bexarotene induces an increase of functional regulatory T cells which could play a role in the development of secondary extra‐cutaneous lymphomas. Regulatory T cells were studied both in cutaneous biopsy specimens using an immunohistochemical analysis of CD4, CD25 and Foxp3 and in blood where proportion and functionality of circulating CD4+CD25^high T‐cells were determined. The study was performed in 10 patients [five patients with Sézary syndrome (SS) and five mycosis fungoïdes (MF)], treated for 6 months with bexarotene. Four healthy donors were used as controls for phenotypic and functional analysis on PBL. We found that the frequency of CD4+CD25^high Treg cells was not significantly different before starting bexarotene and after 6 months of treatment in CTCL patients. However, we observed that the frequency of CD4+CD25^high Treg cells before the beginning of the treatment was significantly increased compared to healthy donors. In addition, functional assays demonstrated that Foxp3 expressing CD4+CD25^high T‐cells were capable of suppressing autologous CD4 + CD25? T‐cell proliferation. In the present work, we detected the presence of functional circulating CD4+CD25^high Foxp3+ regulatory T‐cells in CTCL patients, with an increased frequency compared to healthy donors. The treatment with bexarotene does not seem to affect the regulatory T‐cell compartment.     

A novel xenograft model of cutaneous T‐cell lymphoma

Please cite this paper as: A novel xenograft model of cutaneous T‐cell lymphoma. Experimental Dermatology 2010; 19 : 1096–1102. Abstract: Cutaneous T‐cell lymphomas (CTCLs) are characterized by accumulation of malignant T cells in the skin. Early disease resembles benign skin disorders but during disease progression cutaneous tumors develop, and eventually the malignant T cells can spread to lymph nodes and internal organs. However, because of the lack of suitable animal models, little is known about the mechanisms driving CTCL development and progression in vivo. Here, we describe a novel xenograft model of tumor stage CTCL, where malignant T cells (MyLa2059) are transplanted to NOD/SCID‐B2m^?/? (NOD.Cg‐Prkdc^scid B2m^tm1Unc/J) mice. Subcutaneous transplantation of the malignant T cells led to rapid tumor formation in 43 of 48 transplantations, whereas transplantation of non‐malignant T cells isolated from the same donor did not result in tumor development. Importantly, the tumor growth was significantly suppressed in mice treated with vorinostat when compared to mice treated with vehicle. Furthermore, in most mice the tumors displayed subcutaneous and/or lymphatic dissemination. Histological, immunohistochemical and flow cytometric analyses confirmed that both tumors at the inoculation site, as well as distant subcutaneous and lymphatic tumors, originated from the transplanted malignant T cells. In conclusion, we describe a novel mouse model of tumor stage CTCL for future studies of disease dissemination and preclinical evaluations of new therapeutic strategies.     

Malignant T cells in cutaneous T‐cell lymphoma lesions contain decreased levels of the antiapoptotic protein Ku70

Background Malignant T cells in primary cutaneous T‐cell lymphoma (CTCL) are genetically unstable and exhibit prolonged lifespans potentially explained by dysregulation of apoptosis, yet are responsive to apoptosis‐inducing therapies. The heterodimeric protein Ku70/80 is known to play a role in DNA repair (Ku70 and Ku80) and inhibition of apoptosis (Ku70 only). Objectives To investigate the expression of Ku70/80 in CD3+ T cells derived from skin and blood in patients with CTCL and normal samples, as well as benign dermatoses. Methods Normal (n = 10), CTCL (n = 9) and benign dermatoses (n = 13) skin samples were stained for confocal imaging of Ku70/80 and CD3 and analysed using imaging software. Circulating CD4+ T cells in normal and CTCL peripheral blood were analysed by flow cytometry and Western blot for Ku70/80 expression (n = 6). Results Ku70 and Ku80 were significantly diminished in T cells of CTCL lesions relative to T cells of control skin. Decreased T‐cell Ku70 expression was not a feature of the benign dermatoses psoriasis and contact dermatitis, suggesting that loss of Ku70/80 in CTCL is not simply the result of cutaneous inflammation. Reduced Ku70 was also noted in circulating CD4+ T cells in patients with CTCL with peripheral blood involvement. Conclusions Deficient expression or lack of Ku70/80 may result in genomic instability and play a role in tumorigenesis, as well as account for the increased susceptibility of malignant T cells to apoptosis‐inducing treatment modalities in the setting of intrinsic resistance to apoptosis.     

Cutaneous T‐Cell Lymphoma. A hypothesis on disease pathophysiology involving deficiency in DNA repair

Cutaneous T‐cell lymphoma (CTCL) is a rare disease occurring in Europe among two persons per million per year. It affects men more often than women (2:1). It is primarily a skin disease. In about 20% of patients, it becomes fatal with tumours in the skin and spreading to lymph glands. Approximately 3% of patients show a leukemic form called Sezary's syndrome, where malignant cells are present in blood with accompanying erythrodermia. CTCL is a T‐lymphocyte disease occurring late in life as the average age of patients is around 66 years in Europe, Japan and the US. This article focuses on cell lines and immune surveillance in CTCL, and especially the pronounced chromosomal instability. It leads to the hypothesis that chromosomal changes is the key event linked to DNA repair deficiencies, which in a subpopulation of T cells leads to CTCL development over years.     

About the cutaneous targets of bexarotene in CTCL patients

Please cite this paper as: About the cutaneous targets of bexarotene in CTCL patients. Experimental Dermatology 2010; 19 : e299–e301. Abstract: There are several approved therapies for cutaneous T‐cell lymphoma (CTCL). The retinoids are one of the major biologic response modifiers used in CTCL, producing good response rates but few complete responses. Bexarotene has been demonstrated to act on malignant T‐cells by inducing their apoptosis, but nothing is known about its role on keratinocytes and Langerhans cells. Immunohistochemical analysis using CD1a, HLA‐DR, ICAM‐1 (activation markers), CD95 and CD40 (apoptosis markers) was conducted on frozen sections of bexarotene‐exposed cutaneous explants and skin biopsy specimens from patients treated with bexarotene. None of the studied markers was significantly modulated both on cutaneous explants and on skin biopsy specimens after treatment with bexarotene, compared to controls. Langerhans cells and keratinocytes do not appear to play a central role in the therapeutic control of CTCL by bexarotene therapy. The main bexarotene’s target thus remains T‐cells by inducing their apoptosis, a mechanism that is different from the other retinoids used in CTCL.     

Mammalian target of rapamycin complex 1 (mTORC1) may modulate the timing of anagen entry in mouse hair follicles

Mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and survival. There is limited evidence that mTOR influences hair follicles (HFs), which undergo cycles of quiescence (telogen), growth (anagen) and regression (catagen). We sought to investigate whether mTOR, in particular mTOR complex 1 (mTORC1), regulates the hair growth cycle by employing biochemical, immunohistochemical and functional approaches in vivo. Here, we demonstrate that quantitative analysis of mTORC1 kinase activity shows phase‐dependent changes, and phosphorylated mTOR at S2448 (p‐mTOR) was localized in certain sites of HFs in a phase‐dependent manner. These results were indicative of mTOR's role in hair growth initiation. Finally, in a pharmacological challenge in vivo using the specific mTORC1 inhibitor, rapamycin, hair cycle initiation was delayed, suggesting a functional relevance of mTORC1 in anagen entry. Based on our findings, we propose that mTORC1 may participate in hair cycle regulation, namely the timing of anagen initiation.     

Bexarotene activates the p53/p73 pathway in human cutaneous T-cell lymphoma

Background Bexarotene is the first synthetic retinoid X receptor‐selective retinoid (rexinoid) approved for the treatment of cutaneous T‐cell lymphoma (CTCL). However, little is known about the signalling pathways by which it exerts its anticarcinogenic effect. Objectives To characterize the effects of bexarotene in CTCL cell lines and elucidate the underlying molecular pathways of its antineoplastic effect. Methods The cell lines Hut‐78, HH and MJ were used. Cell viability was assessed with the XTT assay. The self‐renewal potential of cells after bexarotene treatment was studied with the methylcellulose clonogenic assay. Flow cytometry was used to analyse the effects on cell cycle, Ki‐67 expression and apoptosis induction. Cell cycle and apoptosis‐related protein expression were determined by Western blot and immunofluorescence. Results Bexarotene induced a loss of viability and more pronounced inhibition of clonogenic proliferation in HH and Hut‐78 cells, whereas the MJ line exhibited resistance. Bexarotene upregulated and activated Bax in sensitive lines, although not enough to signal significant apoptosis. Instead, all data point to the inhibition of proliferation, rather than apoptosis, as the main mechanistic action of the rexinoid. Bexarotene signals both G₁ and G₂/M arrest by the modulation of critical checkpoint proteins. We further found that bexarotene activates p53 by phosphorylation at Ser15, which influences the binding of p53 to promoters for cell cycle arrest, induces p73 upregulation, and, in concordance, also modulates some p53/p73 downstream target genes, such as p21, Bax, survivin and cdc2. Bexarotene‐mediated ataxia telangiectasia mutated protein (ATM) activation in all studied lines suggests that ATM is likely to be the p53/p73 upstream activator. Conclusions Our data indicate for the first time that bexarotene exerts its effect in CTCL mainly by triggering the p53/p73‐dependent cell cycle inhibition pathway, probably by upstream ATM activation. Therefore, bexarotene‐modulated genes represent potential biomarkers to assess the response to treatment of patients with CTCL.     

Characterization of the novel Sezary lymphoma cell line BKP1

Cutaneous T‐cell lymphomas (CTCL) are a heterogeneous group of lymphomas primarily involving the skin. The most common types are mycosis fungoides (MF) and Sezary Syndrome (SS). We report a novel long‐term fast‐growing SS line termed BKP1 that was characterized by flow cytometry (FC), conventional and molecular cytogenetic [FISH/multi‐FISH together with array comparative genomic hybridization (aCGH)]. FC immunophenotype of the BKP1 is CD2+CD5+CD3+CD4+CD8?CD7?CD25?CD26?CD30?CD158k+. The TCRγ characterization of BKP1 by PCR identified a clonal rearrangement. The conventional cytogenetic and Multi‐FISH analysis showed complex chromosomal rearrangements. aCGH analysis highlighted the loss of genes involved in cell cycle control, in immune response (HLA, complement complex) and DNA damage repair mechanisms. The BKP1 is another lymphoma cell line thoroughly characterized that can be a valuable tool for both basic and applied research such as identification of deregulated genes and/or pathways and screening for new antilymphoma drugs.     

Paucity of intraepidermal FoxP3‐positive T cells in cutaneous T‐cell lymphoma in contrast with spongiotic and lichenoid dermatitis

Background: FoxP3 is the most specific available marker for regulatory T cells (Tregs). Tumor‐associated FoxP3‐positive Tregs have been identified in various neoplasms, including cutaneous T‐cell lymphoma (CTCL). FoxP3 expression in CTCL varies across groups; few studies have compared CTCL with inflammatory conditions. Methods: Lesional skin biopsies from 20 patients with CTCL [13 mycosis fungoides (MF); 7 Sézary syndrome (SS)] and 22 with inflammatory dermatoses (11 spongiotic; 11 lichenoid or interface) were examined for FoxP3 expression by immunohistochemistry. Epidermal FoxP3‐positive lymphocytes were counted as a percentage of the total epidermal CD3‐positive T‐cell population. Results: FoxP3‐positive T cells composed the minority of infiltrate in all major categories. Lower numbers of epidermal FoxP3‐positive T cells were observed in CTCL, particularly MF, than in inflammatory dermatoses (P < .001). CTCL neoplastic T cells did not express FoxP3. Conclusion: FoxP3‐positive T cells are less frequently encountered in MF than in inflammatory dermatoses. FoxP3‐positive T cells occur in higher proportions in the dermis than in the epidermis and probably correlate with coexisting inflammatory components. CTCL neoplastic cells do not typically express a Treg phenotype and are associated with low numbers of FoxP3‐positive Tregs in the infiltrate. FoxP3 expression by immunohistochemistry may aid histologic evaluation of these conditions. Wada DA, Wilcox RA, Weenig RH, Gibson LE. Paucity of intraepidermal FoxP3‐positive T cells in cutaneous T‐cell lymphoma in contrast with spongiotic and lichenoid dermatitis.     

Dermal infiltrates of cutaneous T‐cell lymphomas with epidermotropism but not other cutaneous lymphomas are abundant with langerin+ dendritic cells

Background The Langerhans cell (LC) hypothesis suggests that cutaneous T‐cell lymphomas (CTCL) are diseases of chronic T‐cell stimulation by LC‐mediated antigen presentation. Objective To investigate a broad panel of CTCL and cutaneous B‐cell lymphomas (CBCL) for the spatial association of langerin⁺ dendritic cells (DC) with T and B cells in the skin, respectively. Methods Fifty‐five specimens of CTCL and 10 of CBCL were double‐stained with monoclonal antibodies against langerin and CD3 or CD20, respectively, and evaluated by confocal laser scan microscopy. Results Dermal infiltrates in mycosis fungoides (n = 38), primary cutaneous CD4+ small/medium‐sized pleomorphic T‐cell lymphoma (n = 3) and primary cutaneous peripheral T‐cell lymphoma, unspecified (n = 3) were characterized by a high frequency of dermal langerin⁺ DCs. These cells were exclusively present in the malignant infiltrates. No direct co‐localization of CD3 and langerin could be resolved. Dermal langerin⁺ cells were detected only in one of six primary cutaneous anaplastic large cell lymphomas (C‐ALCL), characterized by epidermotropism. In other C‐ALCL cases (five of six), in lymphomatoid papulosis (n = 3), subcutaneous panniculitis‐like T‐cell lymphoma (n = 2), and all variants of CBCL no dermal langerin⁺ DCs could be found. Conclusions Langerin⁺ DCs are abundant in the dermal infiltrates of T‐cell lymphomas with specific involvement of the epidermis. This might indicate that immature LC and neoplastic T cells interact and gives rise to further studies to characterize the phenotype of the langerin⁺ cell population described here and its role in the pathology of CTCL.     

Combination therapy with extracorporeal photopheresis,interferon‐α, PUVA and topical corticosteroids in the management of Sézary syndrome

Background: Extracorporeal photopheresis (ECP) is recommended for the treatment of Sézary syndrome (SS), the leukemic variant of cutaneous T‐cell lymphoma (CTCL). Several combination therapies are used to increase response rates to ECP. Patients and Methods: We report our experience with the combination therapy of ECP, interferon‐α, PUVA and topical corticosteroids in SS. Results: The treatment outcome in 12 SS patients was retrospectively analyzed and showed an overall response rate to this combination treatment of 42 % with 4/12 patients achieving a partial remission and 1/12 patients a stable disease. The median overall survival time was 42 months. We investigated several clinical and laboratory parameters as an indicator for a response to treatment in our patient cohort. A combined analysis of the erythroderma assessment scale, WBC, LDH, CD4/CD8 ratio and the number of Sézary cells revealed that a reduction of several parameters significantly correlated with response to treatment. The parameters which correlated best with response were number of Sézary cells, CD4/CD8 ratio and WBC. Conclusions: The investigated combination therapy was effective and well‐tolerated in a subgroup of SS patients but needs to be evaluated in a larger patient population.     

Hematopoietic stem cell transplantation in advanced cutaneous T‐cell lymphoma

We retrospectively reviewed data pertaining to five patients with cutaneous T‐cell lymphoma (CTCL) who had received hematopoietic stem cell transplantation (HSCT) between 2004 and 2015 at Kurume University Hospital, along with their clinical data until March 2016. For patients with advanced CTCL eligible for HSCT, autologous HSCT was performed when they responded well to chemotherapy, and allogeneic HSCT was selected for patients with advanced mycosis fungoides (MF)/Sézary syndrome (SS) and CTCL other than MF/SS with poor chemosensitivity. Two patients (primary cutaneous anaplastic large cell lymphoma and primary cutaneous CD8⁺ aggressive epidermotropic cytotoxic T‐cell lymphoma) who responded well to chemotherapy received autologous HSCT: one patient was alive in partial remission and the other died due to therapy‐related acute myeloid leukemia without disease relapse. In the remaining three patients with MF or SS, allogeneic HSCT was performed. Although one patient with MF died due to disease progression, the remaining two patients were alive in complete remission. Although there were two deaths in this study, the outcomes were considered satisfactory.     

Topical application of rapamycin ointment ameliorates Dermatophagoides farina body extract‐induced atopic dermatitis in NC/Nga mice

Atopic dermatitis (AD), a chronic inflammatory skin disease characterized by relapsing eczema and intense prurigo, requires effective and safe pharmacological therapy. Recently, rapamycin, an mTOR (mammalian target of rapamycin) inhibitor, has been reported to play a critical role in immune responses and has emerged as an effective immunosuppressive drug. In this study, we assessed whether inhibition of mTOR signalling could suppress dermatitis in mice. Rapamycin was topically applied to inflamed skin in a murine AD model that was developed by repeated topical application of Dermatophagoides farina body (Dfb) extract antigen twice weekly for 7 weeks in NC/Nga mice. The efficacy of topical rapamycin treatment was evaluated immunologically and serologically. Topical application of rapamycin reduced inflammatory cell infiltration in the dermis, alleviated the increase of serum IgE levels and resulted in a significant reduction in clinical skin condition score and marked improvement of histological findings. In addition, increased mTOR phosphorylation in the lesional skin was observed in our murine AD model. Topical application of rapamycin ointment inhibited Dfb antigen‐induced dermatitis in NC/Nga mice, promising a new therapy for atopic dermatitis.     

Proteasome inhibition as a novel mechanism of the proapoptotic activity of γ‐secretase inhibitor I in cutaneous T‐cell lymphoma

Background We have previously discovered that Notch1 is expressed on malignant T cells in cutaneous T‐cell lymphoma (CTCL), and is required for survival of CTCL cell lines. Notch can be inhibited by γ‐secretase inhibitors (GSIs), which differ widely in their ability to induce apoptosis in CTCL. Objectives To investigate whether GSI‐I, in addition to inhibiting Notch, induces apoptosis in CTCL by proteasome inhibition, as GSI‐I is very potent and has structural similarity to the proteasome inhibitor MG‐132. Methods Cell lines derived from CTCL (MyLa, SeAx, JK, Mac1 and Mac2a) were treated with GSI‐I and two other proteasome inhibitors (MG‐132 and bortezomib). The effects on cell viability, apoptosis and proteasome activity were measured, as was the impact on the prosurvival, nuclear factor κB (NF‐κB) pathway. Results In CTCL, GSI‐I had proteasome‐blocking activity with a potency comparable to the proteasome inhibitors MG‐132 and bortezomib. Proteasome inhibition was the main mechanism responsible for GSI‐I‐induced cell death, as tiron, a compound known to reverse the effect of MG‐132, restored proteasome activity and largely abrogated the cytotoxic effect of GSI‐I. Although inactivation of NF‐κB is an important mechanism of action for proteasome inhibitors, we demonstrated an apparent activation of NF‐κB. Furthermore, we showed that while the tumour suppressor protein p53 was induced during proteasome inhibition, it was dispensable for CTCL apoptosis, as both SeAx cells, which harbour p53 mutations that attenuate the apoptotic capacity, and HuT‐78 cells, which have a deleted p53 gene, demonstrated potent apoptotic response. Conclusions GSI‐I represents an interesting drug with a dual mechanism of action comprising inhibition of both Notch and the proteasome.     

Inability to culture the dominant T-cell clone from the skin of primary cutaneous T-cell lymphoma as proven by TCR γ-chain gene sequencing

Abstract Molecular analysis of T-cell receptor (TCR) chain rearrangement has recently become an attractive tool for demonstrating the clonal origin of cutaneous T-cell lymphoma (CTCL) and for identifying the malignant clone at the molecular level. Over the past decade a number of attempts have been made to culture malignant CTCL cells using standard procedures and these attempts have resulted in several cell lines from the peripheral blood of Sézary syndrome, mycosis fungoides and CD30⁺ lymphoma patients. However, so far it has not been proven by sequence analysis that the cultured T cells truly represent the malignant cells. Aiming to functionally analyze the malignant T cells at a clonal level, we generated a total of 150 T-cell clones (TCC) from lesional skin and peripheral blood of three patients with mycosis fungoides and one patient with a CD30⁺ lymphoma. Cells were grown either in the presence of autologous irradiated peripheral blood feeder cells using various conditions for T-cell stimulation by direct outgrowth or from skin specimens with various cytokine combinations. In order to identify the malignant TCC we used N-region-specific PCR and compared TCR á-chain sequences from clones of lesional skin with in vitro-generated TCC. With the methods employed, none of the 150 established cell lines was found to be identical to the malignant TCC which was readily detected in lesional skin. Our results indicate that standard cell culture methods are not suitable for growing low-grade CTCL cells from the skin but give rise only to benign infiltrating T cells. Received: 8 September 2000 / Revised: 11 November 2000 / Accepted: 10 December 2000