Mycosis fungoides (MF) is a low-grade cutaneous T cell lymphoma of unknown etiology. In this report, the Jak͞Stat (Janus kinase͞signal transducer and activator of transcription) signaling pathway was investigated in tumor cell lines established from skin biopsy specimens from a patient with MF. Jaks link cytokine receptors to Stats, and abnormal Jak͞Stat signaling has been observed in some hemopoietic cancers. In MF tumor cells, a slowly migrating isoform of Stat3, Stat3 sm , was found to be constitutively activated, i.e., (i) Stat3 sm was constitutively phosphorylated on tyrosine residues, and tyrosine phosphorylation was not enhanced by growth factor stimulation; (ii) band shift assays and immunoprecipitations of DNA͞Stat complexes showed constitutive DNA-binding properties of Stat3 sm ; and (iii) Stat3 sm was constitutively associated with Jak3. The abnormal activation of Stat3 sm was highly specific. Thus, neither the fast migrating isoform of Stat3 (Stat3 fm ) nor other Stats (Stat1, Stat2, and Stat4 through Stat6) were constitutively activated. The Jak kinase inhibitor, tyrphostin AG490, blocked the constitutive activation of Stat3 sm and inhibited spontaneous as well as interleukin 2-induced growth of MF tumor cells. In conclusion, we have provided evidence for an abnormal Jak͞ Stat signaling and growth regulation in tumor cells obtained from affected skin of an MF patient.

A characteristic feature of neoplastic transformation is a perpetual activation of oncogenic proteins. Here, we studied signal transducers and activators of transcription (STAT) in patients with mycosis fungoides (MF)/cutaneous T-cell lymphoma (CTCL). Malignant lymphocytes in dermal infiltrates of CTCL tumors showed frequent and intense nuclear staining with anti-PY-STAT3 antibody, indicating a constitutive activation of STAT3 in vivo in tumor stages. In contrast, only sporadic and faint staining was observed in indolent lesions of patch and plaque stages of MF. Moreover, neoplastic lymphocytes in the epidermal Pautrier abscesses associated with early stages of MF did not express activated STAT3. To address the role of STAT3 in survival/apoptosis, CTCL tumor cells from an advanced skin tumor were transfected with either wild-type STAT3 (STAT3wt) or dominant-negative STAT3 (STAT3D). Forced inducible expression of STAT3D triggered a significant increase in tumor cells undergoing apoptosis, whereas forced expression of STAT3wt or empty vector had no effect. In conclusion, a profound in vivo activation of STAT3 is observed in MF tumors but not in the early stages of MF. Moreover, STAT3 protects tumor cells from apoptosis in vitro. Taken together, these findings suggest that STAT3 is a malignancy factor in CTCL.

The mechanism of positive regulation of cytokine signalling pathways has been well investigated, whereas our knowledge of negative regulation is relatively sparse. Here we review recent literature on important negative regulators: the family of suppressors of cytokine signalling, SOCS, consisting of eight members (SOCS-1 to SOCS-7 and CIS) all sharing a central SH2 domain and a C-terminal SOCS box. Expression of CIS, SOCS-1, SOCS-2 and SOCS-3 is induced by various cytokines, and overexpression studies in various cell lines have demonstrated their inhibitory roles. These family members have been implicated in the negative regulation of several pathways, particularly the JAK/STAT pathway, and since this signalling pathway is responsible for their induction, they form part of a classical negative feedback circuit. To date, at least three different modulating mechanisms have been demonstrated: through the SH2 domain they bind to phosphotyrosines on the target protein, leading to inhibition of signal transduction by N-terminal inactivation of JAK, by blocking access of STAT to the receptor sites, or by SOCS box-targeting bound proteins to proteasomal degradation. In gene modification studies in mice, it has been demonstrated that SOCS-1 plays an important role in IFNgamma-regulation and T-cell differentiation, while SOCS-2 seems necessary for normal growth regulation. SOCS-3(-/-) mice die during embryogenesis for a reason still not fully understood, but insufficient control of fetal erythropoiesis or defects in placental development may be involved. The physiological role for the other family members, as well as their molecular regulation mechanisms, remain to be revealed.

Interleukin-2 (IL-2

The Jak/Stat signaling pathway transmits signals from many cytokine and growth factor receptors to target genes in the nucleus. Constitutive activation of Stat3 has recently been observed in many tumor cells and dysregulation of the Stat signaling pathway has been proposed to be implicated in malignant transformation. In a previous study, we found constitutively tyrosine phosphorylated Stat3 in mycosis fungoides tumor cells. Here, we show that the Jak kinase inhibitor, Ag490, inhibits the constitutive binding of Stat3 to an oligonucleotide representing the Stat-binding sequence from the ICAM promotor. The decreased ability of Stat3 to bind DNA precedes dynamic alterations in the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (decreased Bcl-2 expression and increased Bax expression) and induction of apoptosis. Thus, our data suggest that the involvement of Stat3 in oncogenic transformation could be mediated through regulation of survival signals.

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Mycosis fungoides is a low-grade cutanous T-cell lymphoma (CTCL) of unknown etiology. In advanced stages of CTCL, a shift in cytokine profile from T H 1 to T H 2 is observed, which coincides with eosinophilia, high levels of immunoglobulin E, and increased susceptibility to bacterial infections. It is, however, unknown why T H 2 cytokines predominate in advanced CTCL, and the cellular source of these cytokines also remains to be identified. In several leukemias and lymphomas, constitutively activated signal transducer and activator of transcription (Stat) signaling pathways have been detected. In a previous study, constitutive activation of Stat3 was found in tumor cells isolated from affected skin and blood from CTCL patients. Here, it is shown that CTCL tumor cell lines, but not nonmalignant cell lines, spontaneously produce interleukin-5 (IL-5), IL-6, and IL-13. Transfection of tumor cells with dominantnegative Stat3 almost completely blocks IL-5 production and strongly inhibits IL-13 production, whereas IL-6 production is unaffected. Thus, the data show that malignant CTCL cells themselves might contribute to the change in cytokine pattern accompanying progression of CTCL. In conclusion, constitutively activated Stat3 is found to mediate a spontaneous IL-5 production and regulate IL-13 production in CTCL cell lines, pointing toward a new role of Stat3 in malignant transformation. (Blood. 2002;99:973-977)

In the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of such microenvironments and more particularly the nature of communicative signals in lympho-stromal interaction during T cell development, we have employed the phage antibody display technology, with the specific aim of isolating thymic stromal cellspecific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intact, mildly fixed thymic fragments as target tissue and lymphocytes as absorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4-4, TB4-20, and TB4-28. While TB4-4 and TB4-20 are both epithelium specific, TB4-28 detects an epitope expressed on both epithelial-and mesenchymal-derived stromal cells. TB4-4 reacts with all cortical epithelial cells and with other endoderm-derived epithelia, but this reagent leaves the majority of medullary epithelial cells unstained. In contrast, MoPhab TB4-20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm-and ectoderm-derived epithelial cells. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection of evolutionary conserved epitopes expressed on subsets of thymic stromal cells.