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In order to determine whether the neoplastic T cells from patients with cutaneous T-cell lymphoma express tumor-specific antigens that can serve as the targets of an immune response, we took advantage of family-specific monoclonal antibodies, magnetic bead technology, and recombinant cytokines, which provided the previously precluded ability to isolate and expand populations of purified tumor and autologous CD8 cytotoxic T cells. Four patients with advanced cutaneous T-cell lymphoma had CD8 cells that specifically killed autologous tumor in a class I limited fashion. Tumor cell cytolysis could be specifically enhanced by pre-culture with autologous gamma-irradiated tumor. The cytolytic T cells produced tumor necrosis factor-alpha in response to stimulation with autologous tumor. The presence of tumor-specific cytotoxic T cells recognizing distinctive class I associated molecules on cutaneous T-cell lymphoma tumor cells suggests that infiltration of early lesions by CD8 cells reflects host immunity to the neoplasm. These studies provide the foundation for the development of tumor vaccines through the use of cytotoxic T cells to isolate and characterize tumor-associated cutaneous T-cell lymphoma peptides.

IntroductionKIT, the stem cell factor (SCF) receptor, is a receptor protein tyrosine kinase (RTK) that is primarily expressed on mast cells, hematopoietic stem cells, germ cells, melanocytes, and the interstitial cells of Cajal. 1,2 Studies in white-spotting and steel mice have shown that functional SCF and KIT are essential for the development of stem cells involved in hematopoiesis, pigmentation, and reproduction. 3 Stimulation of KIT with its ligand, SCF, induces the activation of KIT including its dimerization and autophosphorylation and its interaction with other proteins to initiate a signaling cascade. 4 Control of the KIT kinase activity is essential for mast cell growth and development and immune response regulation. Constitutive KIT activation caused by mutation of the c-KIT gene has been associated with cellular transformation and the pathogenesis of human mastocytosis and gastrointestinal stromal tumors. [4][5][6] However, despite the central role that KIT plays in diverse cellular systems, the mechanisms that regulate KIT signaling have not been completely elucidated.Ubiquitination and subsequent degradation of proteins has been implicated as a key mechanism regulating duration and intensity of many intracellular signals, and it has been suggested that activated KIT is degraded through the polyubiquitination-dependent protein degradation pathway. 7 However, specific ubiquitin ligases responsible for KIT receptor ubiquitination have not been reported.Cbl family members are newly established as components of the ubiquitin ligation machinery involved in the degradation of phosphorylated proteins. 8 In vitro assays have shown that Cbl proteins can function as E3 ubiquitin (Ub) ligases, and Cbl proteins have recently been implicated in the negative regulation of various RTK and non-receptor tyrosine kinase activities with diverse effects including regulation of the T-cell activation threshold. [9][10][11][12][13] The Cbl family proteins consist of an N-terminal tyrosine kinase binding (TKB) domain, a RING finger (RF) domain, and a C-terminal proline-rich domain with potential tyrosine phosphorylation sites. 14 The Cbl RF domain recruits a Ub-conjugating enzyme (E2), whereas the Cbl N-terminal TKB domain mediates Ub conjugation of target proteins. Uncoupling c-Cbl from RTKs may lead to receptor deregulation and tumor formation. 15,16 We hypothesized that Cbl proteins may mediate the ubiquitination and degradation of activated KIT. In this study, we provide evidence that SCF stimulation induces KIT binding and phosphorylation of Cbl proteins. Furthermore, by using Cbl-b or c-Cbl and their ubiquitin ligase-deficient mutants in a reconstituted 293 cell system and in mast cell lines, we show that Cbl-b or c-Cbl acts as an E3 ligase and binds and leads to the ubiquitination and degradation of active KIT, at least partially through the proteasomal pathway upon SCF stimulation. Materials and methods MaterialsAntibodies against KIT (C-19), c-Cbl (C-20), and Cbl-b (G-1) were obtained from Santa Cruz Biotechnology ...