Functional characterization of an IL-7-dependent CD4(+)CD8alphaalpha(+) Th3-type malignant cell line derived from a patient with a cutaneous T-cell lymphoma.

CDR3 of the functional rearranged T-cell receptor variable beta region (TCR-Vbeta) transcript was sequenced in order to demonstrate for the first time the identity between a long-term cultured T-cell line derived from a cutaneous T-cell lymphoma (CTCL) patient and the malignant T-cell clone present in the blood. The patient's peripheral blood lymphocyte-derived cultured T-cell line had a CD3(+)Vbeta22(+)CD4(+)CD8alphaalpha(+)CD25(-) phenotype. It was named Pno and had been cultured for more than 1 year. Both fresh and long-term-cultured tumor cells proliferated highly in response to interleukin-7 (IL-7), and exogeneous IL-7 prevented Pno lymphocytes from apoptosis and maintained high levels of Bcl-2 expression. This unique malignant cloned lymphocyte line was further used to carry out functional studies. The results indicated that the CD3/TCR structures expressed by the Pno lymphocytes were functional because an immobilized anti-CD3 monoclonal antibody (mAb) or the combination of a soluble anti-CD3 mAb with submitogenic doses of phorbol 12 beta-myristate 13 alpha-acetate induced a proliferative response. Further, the CD2 and CD28 coreceptors were functional because they were able to induce a strong proliferative response upon their specific stimulation. Finally, the Pno T cell line had a Th3-type cytokine profile because it produced high amounts of the immunosuppressor cytokine tumor growth factor-beta1 (TGF-beta1). This high production of TGF-beta1 may inhibit antitumor specific responses in CTCL.

Covalent binding of carbamazepine reactive metabolites to P450 isoforms present in the skin.

Carbamazepine is an anticonvulsant associated with a high risk for severe cutaneous reactions. Upon metabolism by cytochrome P450, carbamazepine may produce reactive metabolites. We evaluated in vitro the covalent binding of carbamazepine reactive metabolites on human P450s and then the presence of these P450s in human epidermis. Carbamazepine reactive metabolites covalent binding to human liver microsomes involved P450 subfamilies 1A, 2C and 3A. Specific covalent binding to yeasts expressing different P450s showed that carbamazepine reactive metabolites bound specifically to P450 1A2 and 3A4. We confirmed the constitutive presence of P450 3A in human epidermis and after induction with coaltar of P450 1A. Consequently, the production in epidermis of carbamazepine reactive metabolites is theoretically possible with formation of P450 adduct metabolites.

Isolation of tumor-specific cytotoxic CD4+ and CD4+CD8dim+ T-cell clones infiltrating a cutaneous T-cell lymphoma.

We have isolated several T-cell clones from lymphocytes infiltrating a human major histocompatibility class (MHC) II negative cutaneous T-cell lymphoma (CTCL). We describe here two of these clones, TC5 and TC7, with, respectively, a CD4(+)CD8dim+ and CD4(+)CD8(-) phenotype. Both clones mediated a specific MHC class I-restricted cytotoxic activity toward the fresh autologous tumor cells, and autologous tumor cell lines previously established with interleukin-2 (IL-2) and IL-7 from the skin and from the blood. Analysis of the T-cell receptor (TCR) Vbeta gene expression showed that the tumor cells, which were shown to have a trisomy 7 by fluorescent in situ hybridization, expressed Vbeta7/Jbeta2.3, Vbeta13/Jbeta2.5, and Vbeta22/Jbeta2.5 rearrangements. Phenotypic analysis using specific anti-Vbeta monoclonal antibodies indicated that only Vbeta13 could be detected on the cell membrane of the tumor cells. Analysis of the TCR Vbeta gene expression of the clones showed that TC5 and TC7 expressed a unique TCR-Vbeta transcript, corresponding, respectively, to Vbeta5/Jbeta2.3 and Vbeta17/Jbeta2.7 gene segments. To determine whether these reactive T lymphocytes were present in vivo, we used specific primers corresponding to TC5- and TC7-Vbeta TCR transcripts. The results showed that both cytotoxic T-cell clones were present at the lesional skin site and amplified in vitro. TC7 was found in the patient peripheral blood invaded by tumoral cells, whereas TC5 was not, indicating that the repertoire of the reactional lymphocytes differs in the blood and at the tumor site. These results show for the first time the presence of reactive T lymphocytes with CD4 or double-positive phenotype infiltrating a CTCL. These findings raise the question of the role of these antitumoral effector T cells in the tumor growth.

CD101 is expressed by skin dendritic cells. Role in T-lymphocyte activation.

CD101 was first described in our laboratory using two different monoclonal antibodies, BA27 and BB27, recognizing a 140-kDa disulfide-bonded homodimeric polypeptide on a small subset of circulating T lymphocytes and on most activated T cells in vitro. Further, it has been reported that most intestinal mucosal T lymphocytes expressed CD101. The gene coding for the CD101 antigen has been cloned and found to be identical to the gene coding for the recently described V7 antigen, corresponding to a type I trans-membrane protein with seven immunoglobulin-like loops in its extracellular domain. To define surface proteins that are involved in skin dendritic cell (DC) localization or function, we looked for the expression of CD1O1 on skin DC migrating from human skin explants. The majority of these DC had a phenotype of Langerhans cell (LC)-like mature DC, i.e., HLA-DR+ CD1a+ CD1c+ CD11a+ CD11c+ CD40+ CD50+ CD54+ CD58+ CD80+ CD83+ CD86+. We found that CD101 was expressed by a major subset of these HLA-DR+ CD1a+ CD1c+ LC-like skin DC. Next, we studied the effect of anti-CD101 monoclonal antibodies on primary allogeneic and on soluble antigen-specific mixed skin DC-lymphocyte reactions. We showed that two different monoclonal antibodies, BB27 and V7.1, inhibited the T-lymphocyte proliferative responses and that the inhibitory effect was overcome by high doses of exogenous IL-2. As both DC and T lymphocytes expressed CD101 molecules, we determined that the inhibitory effect was achieved both at the responder T-cell level and at the DC level. Thus, CD101 which is expressed on a subset of circulating T lymphocytes, has also been found on a subpopulation of LC-like DC. This molecule plays a major role in the activation of T cells by skin DC.

Interleukin-7 receptor expression in cutaneous T-cell lymphomas.

Keratinocyte-derived interleukin-7 (IL-) is a potent growth factor for some cutaneous T-cell lymphomas (CTCL). We investigated the expression of IL-7 receptor (IL-7R) in several types of cutaneous and nodal lymphomas. We studied 44 CTCL (13 mycosis fungoides, six Sézary syndromes, eight pleomorphic small cell, and 17 pleomorphic medium and large cell), 10 lymphomatoid papulosis (LP), five cutaneous B-cell lymphomas, and five reactive lymphocytic infiltrates. Twenty nodal T-cell lymphomas, and three reactive lymph nodes were also analysed. Frozen sections were stained with monoclonal antibodies directed against IL-7R, CD25, CD30 and T antigens (CD3, CD2, CD5, CD7, CD4, CD8), using the alkaline phosphatase-antialkaline phosphatase technique. No expression of IL-7R was observed in cutaneous B-cell lymphomas, benign cutaneous lymphoid infiltrates, and reactive lymph nodes. IL-7R was expressed by more than 20% of lymphoid cells in 50-75% of all histological subtypes of CTCL, and by more than 50% of cells in 15-50%. IL-7R was expressed by more than 20% and 50% of cells in 40% and 10% of nodal large T-cell lymphomas, respectively. Eighty-nine per cent of CTCL and LP expressing IL7-R also expressed CD25+, compared with 58% of IL-7R--CTCL and LP (P < 0.05). No association of IL7-R and CD30 expression was found. In conclusion, CTCL frequently express IL-7R. This expression is not related to the epidermotropic characteristic of the infiltrate. In CTCL and LP, IL-7R expression is associated to CD25 expression, but not to CD30 expression.

Induction of rat CD4+ proliferative and mouse CD8+ cytotoxic T-cell lines specific for human papillomavirus type 16 antigens.

Infection by human papillomavirus type 16 (HPV16) has been associated with cervical dysplasia and carcinoma. To detect a possible cellular immune response against HPV16, we investigated the induction of T cells specific for antigens encoded by the virus. Two CD4+ T-cell lines with a specific proliferative response were isolated from the spleens of rats vaccinated using vaccinia virus vectors expressing E6 or E7 and challenged with syngeneic HPV-transformed cells. Two CD8+ T-cell lines with a specific cytotoxic activity were obtained from mice immunized using a syngeneic squamous cell tumour cell line transfected with the full-length HPV16 DNA. These results demonstrate that both CD8-mediated cytotoxic responses and CD4-mediated delayed-type hypersensitivity responses are involved in immunologic reactions to HPV antigens.

Metabolic predisposition to cutaneous adverse drug reactions. Role in toxic epidermal necrolysis caused by sulfonamides and anticonvulsants.

BACKGROUND AND DESIGN: Cutaneous adverse drug reactions (ADRs) have been hypothesized to have a metabolic basis. Our aim was to identify detoxification defects involved in toxic epidermal necrolysis and other severe cutaneous ADRs. Lymphoid cells of 33 patients with cutaneous ADRs were challenged with reactive metabolites generated from drugs by a microsomal oxidation system. To be precise in the detoxification defect involved in sulfonamide and anticonvulsant reactions, we challenged lymphoid cells from 11 patients (seven patients with sulfonamide ADRs and four patients with anticonvulsants ADRs) to menadione and formaldehyde. Menadione induces toxic effects by oxygen species; formaldehyde is detoxified by aldehyde dehydrogenase, oxidase, and reductase. RESULTS: When the culprit drug was a sulfonamide or an anticonvulsant (used in 13 and 13 patients, respectively), the toxic effects of culprit drug-reactive metabolites toward patients' lymphoid cells were higher than toward controls'. First-degree relatives of four patients with sulfonamide- and phenobarbital-induced toxic epidermal necrolysis were also tested. In each family, a relative was more susceptible to culprit drug-reactive metabolites than were controls. After incubation with menadione, or formaldehyde, no difference in toxicity was found between patients' and controls' lymphoid cells. CONCLUSIONS: Toxic epidermal necrolysis and other severe cutaneous ADRs to sulfonamides and anticonvulsant drugs may be linked to a highly specific defect in the detoxification of culprit drug-reactive metabolites. Our results suggest that this defect is constitutional and inherited and does not involve oxygen free radicals and/or aldehyde detoxification pathways.

Immunosuppressive effects of 1,25-dihydroxyvitamin D3 and its analogue calcipotriol on epidermal cells.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3; calcitriol) is the biologically active form of vitamin D. This hormone is a potent immunoregulatory agent. Calcipotriol is a synthetic analogue of 1,25(OH)2D3, with similar receptor binding, and comparable effects on cell proliferation and differentiation, but less potent effects on calcium metabolism. As a step towards understanding the mechanisms by which vitamin D compounds affect T-cell activation by epidermal cells (EC), we assessed the effects of 1,25(OH)2D3 and calcipotriol on the human allogeneic mixed epidermal cell-lymphocyte reaction. All experiments were performed both with 1,25(OH)2D3, and calcipotriol, with similar results. Both compounds had potent immunoinhibitory properties on this model, and enhanced the immunosuppressive effects of cyclosporin A. Using preincubation experiments, we found that pretreatment of EC with 1,25(OH)2D3 resulted in a more pronounced inhibition than preincubation of lymphoid cells. The epidermal targets of this inhibitory effect have been further investigated, using cultures with freshly isolated Langerhans cells (LC) or LC-depleted keratinocytes, separated by an immunomagnetic particle technique. Pretreatment of LC induced a 30% decrease of proliferation, compared with vehicle-treated LC. These calcitriol-pulsed LC did not decrease the proliferation induced by unmodified autologous EC. As expected, LC-depleted keratinocytes failed to stimulate allogenic lymphocytes. When added to autologous unmodified EC, however, calcitriol-pulsed keratinocytes induced an 85% decrease of proliferation, compared with vehicle-treated keratinocytes. The phenotypic expression of HLA-DR, -DQ, and -DP antigens on EC, assessed by immunoalkaline phosphatase staining, was not modified after a 2-h or 24-h pulse with 1,25(OH)2D3 or calcipotriol.(ABSTRACT TRUNCATED AT 250 WORDS)

Additive effects of calcipotriol and cyclosporine A: from in vitro experiments to in vivo applications in the treatment of severe psoriasis.

We report that calcipotriol (CPT) is a potent inhibitor of lymphocyte proliferation in mixed epidermal cell lymphocyte reactions. In this model, CPT and cyclosporine A (CsA) have synergistic effects. These results have led to a randomised double-blind therapeutic study protocol in patients with severe psoriasis. In these patients, topical CPT and sub-therapeutic doses of CsA (2mg/kg/d) induce disappearance of cutaneous lesions. This therapeutic association may minimize secondary side effects of both drugs.

Impaired antigen presentation in toxic epidermal necrolysis.

BACKGROUND AND DESIGN--The pathophysiology of toxic epidermal necrolysis (TEN) remains largely unknown. Toxic epidermal necrolysis is considered a hypersensitivity reaction to drugs, but direct evidence of an immunologic mechanism is still lacking. Lymphopenia and a decrease in the numbers of circulating CD3- and CD4-positive lymphocytes have been reported in acute phase, but, to our knowledge, no study of cellular immune functions of patients with TEN has been reported so far. Herein, we investigated several T-cell functions in a series of 11 patients with TEN. Peripheral blood mononuclear cells (PBMC) obtained in the acute phase were tested together with PBMC obtained after the patient's recovery and compared with those of age- and sex-matched healthy control subjects. RESULTS--Phytohemagglutinin-induced proliferations and lymphocyte responses in allogeneic mixed lymphocyte reactions were not impaired in the acute phase compared with those after recovery in the same patients and with those in control subjects. In contrast, natural killer cytotoxicity and allogeneic cytotoxic responses were significantly decreased in early TEN. The most striking feature was the significantly impaired ability of acute-phase lymphoid cells to activate allogeneic T cells. Patient PBMC in acute-phase TEN did not inhibit the proliferation induced by patient PBMC after recovery, suggesting that their defect was not related to the presence of radioresistant suppressor cells. The phenotypic expression of HLA-DR, -DQ, and -DP antigens on circulating peripheral blood lymphocytes was then assessed by immunoalkaline phosphatase staining and flow cytometry. Results showed decreased percentages of HLA-DR-positive mononuclear cells and a decreased density of HLA-DR antigens, mainly on monocytes, in acute-phase TEN. CONCLUSIONS--These results demonstrate that peripheral blood lymphocytes of patients with TEN have an impaired ability to activate allogeneic T cells. This defective antigen presentation is not secondary to the presence of suppressor lymphocytes, but it is probably related to a decreased expression of HLA-DR antigens on circulating mononuclear cells in acute-phase TEN.