[CANCER RESEARCH 61, 452– 454, January 15, 2001] Advances in Brief Coexpression of CD40 and CD40 Ligand in Cutaneous T-Cell Lymphoma (Mycosis Fungoides) 1 Monique Storz, Karoline Zepter, Jivko Kamarashev, Reinhard Dummer, Gu ¨ nter Burg, and Andreas C. Ha ¨ffner 2 Department of Dermatology, University Hospital of Zurich, CH-8091 Zurich, Switzerland Abstract Microarray analysis is a promising new approach for creating specific expression profiles of multiple genes simultaneously. We quantitatively analyzed differential gene expression patterns in mycosis fungoides- derived clonal T cells and autologous, identically cultured CD4 lympho- cytes using microarrays containing 588 cDNA segments from genes rele- vant to cell signaling, carcinogenesis, and apoptosis. Among other dissimilarities, neoplastic T cells showed coexpression of CD40 (Bp50) and CD40 ligand (gp39, CD154). These results could be corroborated by reverse transcription-PCR, immunohistochemistry, and two-color immu- nofluorescence staining. Our data suggest that in cutaneous T-cell lym- phoma, CD40/CD40 ligand interactions might represent a paracrine loop that is crucial not only in preventing apoptosis or positively regulating growth but also in homing of neoplastic cells to the skin. Introduction CD40 receptor is a transmembrane protein and has been clustered as a member of the tumor necrosis factor/nerve growth factor receptor superfamily (1). It has been extensively studied and demonstrated to be present on a variety of cell types ranging from benign B cells, monocytes, dendritic cells, endothelial cells, keratinocytes, fibro- blasts, and thymocytes (2–5) to neoplastic B-cell non-Hodgkin’s lymphomas and leukemias, Reed-Sternberg cells in Hodgkin’s dis- ease, myeloma plasma cells, CTCLs, 3 a number of carcinomas (in- cluding bladder, breast, and ovarian cancer), epidermal tumors, and melanoma cells (6 –11). Low-level CD40 expression is also found on some human T-cell lines and on activated peripheral blood T cells (12). Its counterpart, CD40L, which shares significant sequence ho- mology to tumor necrosis factor, is transiently induced and tightly regulated on the surface of CD4+ T cells after their activation (13) but is also weakly expressed on some activated CD8+ T cells, basophils, mast cells, eosinophils, natural killer cells, and monocytes (14). A consistent fraction (40%) of peripheral T-cell non- Hodgkin’s lymphomas and CTCLs displaying a CD4+/CD8- phe- notype, along with a subset of T-lineage acute lymphoblastic leuke- mias with stem cell-like phenotype, constitutively display surface CD40L (15). The CD40/CD40 L interaction is known to be an important feature of B-cell/T-cell collaboration, leading to T-cell-dependent activation, proliferation, and differentiation of B lymphocytes; immunoglobulin isotype switching; and memory B-cell formation (16). Mutations of the CD40L gene have been associated with X-linked hyper-IgM immunodeficiency syndrome, pointing to the critical role of the CD40/CD40L interaction in the T-cell/B-cell interplay (17). In addi- tion, several groups have implicated CD40 in the regulation of B-cell survival via molecules of the Bcl family (18) and of B-cell apoptosis via Fas (19). It has been reported that binding of CD40L to its CD40 receptor may activate not only B lymphocytes but T lymphocytes as well and that CD40L can act as a stimulatory molecule for T lymphocytes (20). The fact that neoplastic CD4+ T cells constitutively express detect- able amounts of surface and/or cytoplasmic CD40L molecules indi- cates a possible physiological role of this molecule in these neoplasms (21). Further data demonstrated coexpression of CD40 and CD40L in B-cell lymphoma cells, raising the possibility of an autocrine loop that may contribute to the growth regulation of malignant B cells in vivo (22) and may potentially exist at the T-cell branch as well. Comparative microarray analysis is a new and promising approach to establish gene expression profiles that may finally help to delineate the intricate interactions responsible for pathogenesis and clinical phenotype of this particular disease (23, 24). We performed comparative analyses of clonal lymphocytes derived from MF lesions and their autologous CD4+ counterparts originating from peripheral blood by microarray and obtained further confirma- tion by RT-PCR, immunohistochemistry, and two-color immunoflu- orescence staining. Materials and Methods Cell Culture. All human material was obtained with the patient’s informed consent. Lymphocytic cells from a diagnostic MF tumor sample were cultured for 8 weeks in 500 ml of RPMI 1640 supplemented with 100 ml of 10% FCS, 10 ml of fungizone/streptomycin, 10 ml of glutamin, 5 ml of sodium pyruvate (all from Life Technologies, Inc., Grand Island, NY), 100 l of human epidermal growth factor (1 g), 200 ml of interleukin 2 (10 6 units/ml), 2 ml of interleukin 4 (50,000 units/ml), 2 ml of granulocyte macrophage colony- stimulating factor (80,000 units/ml), and 400 l of basic fibroblast growth factor (250,000 units/ml; all from Becton Dickinson, San Jose, CA). Clonality of the morphologically homogenous tumor cell population was determined by PCR/denaturing gradient gel electrophoresis as described previously (25) and compared with original cryopreserved biopsy material. Autologous peripheral CD4+ T cells were cultured for 3 days under identical conditions to normalize for tissue culture artifacts. Separation and Activation of Autologous CD4 Cells from Peripheral Blood. CD4 cells were separated by an immunomagnetic procedure using paramagnetic polystyrene beads (Dynal, Lake Success, NY) and a magnetic separation device (Dynal). An antihuman CD4-specific murine IgM antibody (DAKO, Glostrup, Denmark) was conjugated to Dynabeads M-450 (Dynal) by overnight incubation at pH 9.5, washed twice in PBS containing 10% FCS, and incubated under constant slow agitation with peripheral blood mononuclear cells generated by Ficoll gradient enrichment from the patient’s blood at 4°C. Purified CD4+ T cells were stimulated by exposure to 10 g/ml phytohem- agglutinin (Life Technologies, Inc.) and harvested on day 3. RNA Isolation. Total mRNA was extracted from CTCL cell culture and peripheral blood mononuclear cell culture using RNAeasy Mini Kit (Qiagen AG, Basel, Switzerland) according to the manufacturer’s instructions. RT-PCR. RNA samples (500 ng) were reverse transcribed using avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) according Received 6/26/00; accepted 11/28/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by a grant from the Cancer League of the Canton Zurich (to A. C. H.). 2 To whom requests for reprints should be addressed, at Department of Dermatology, University Hospital of Zurich, Gloriastrasse 31, CH-8091 Zurich, Switzerland. Phone: 41-1-255-4049; Fax: 41-1-255-4403; E-mail: ach5@derm.unizh.ch. 3 The abbreviations used are: CTCL, cutaneous T-cell lymphoma; MF, mycosis fungoides; CD40L, CD40 ligand; RT-PCR, reverse transcription-PCR. 452 on July 6, 2015. © 2001 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from