Studies of T cell-mediated immune response against spontaneously arising autologous human tumors have been markedly facilitated by functional analyses of T cells at the clonal level (1-7). This particular approach has now provided a strong support for the long-held belief by some, although viewed skeptically by others, that T cell-mediated host responses do indeed exist in different types ofhuman cancers. The evidence for such T cell antitumor response has been particularly impressive in malignant melanoma (1-4, 6, 7). In this system, the existence of CTL response (1-4, 6, 7), proliferative T cell response (8, 9), suggestion for regulatory T cell response (10, 11), and delayed-type hypersensitivity response (12) have all been demonstrated. While this impressive body of evidence certainly represents a serious beginning in investigation on T cell responses against autologous cancer, a critical analysis ofT cell-immune responses against a large number ofautologous melanoma at clonal levels is necessary for a more comprehensive understanding of T cell-immune response in host defense against human cancers or, for that matter, against malignant melanoma .We have undertaken clonal analyses of T cell-immune response in a larger group of patients with melanoma . In this work, we present our observation of clonal analyses of CTL and regulatory responses in 31 autologous case studies. Here we show that T cell responses (CTL as well as regulatory T cell responses), taken together, are demonstrable in approximately half of this cohort of subjects. Results of our studies clearly document the involvement of the entire T cell repertoire in response to autologous melanoma . The melanoma-specific (CTL) responses show appropriate MHC class restriction, and the cytotoxic response in the PBL is subject to regulation by the helper and suppressor arms of the T cell network. The amplification of cytotoxic response by Th cell clones is mediated by the elaboration of IL-2 and IFN-y, and the T cell-mediated downregulatory responses can be specific as well as nonspecific.
Surface membrane-associated growth factors are being recognized as important for developmental processes, including cell assembly, differentiation, and growth. To investigate the role of membrane-bound macrophage colony-stimulating factor (M-CSF) in myelopoiesis, and whether this factor is released from the cell surface in association with shed membrane-derived vesicles, COS-1 cells were transfected with cDNAs for M-CSF-tau (containing the transmembrane domain) or a soluble mutant form of the molecule lacking the transmembrane domain ([s]M-CSF- alpha). COS-1 cells transfected with either cDNA released activity into the spent culture medium. Conditioned medium was separated by centrifugation into supernatants and pellets were found to contain plasma membrane-derived vesicles by transmission electron microscopy. When medium fractions were assayed in marrow cultures, activity was localized to shed plasma membrane-derived vesicles in medium conditioned by cells transfected with cDNA for M-CSF-tau and in the vesicle-free supernatants of medium conditioned by cells transfected with cDNA for [s]M-CSF-alpha. In addition, nuclear, mitochondrial, and plasma membrane subfractions of stably transfected cells were prepared and assayed for activity. Concentration-dependent stimulation of macrophage colony formation was observed with purified plasma membranes (but not nuclear or cytosolic proteins) from cells transfected with cDNA for M-CSF-tau. By contrast, membranes from untransfected cells and cells transfected with cDNA for [s]M-CSF-alpha or control DNA expressed no activity. Together, the data indicate that human M-CSF is expressed at the cell surface and exfoliated in association with surface membrane- derived vesicles.
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