Our previous work demonstrated that cytotoxic T lymphocyte (CTL)-mediated tumor immunosurveillance of the 15-12RM tumor could be suppressed by a CD1d-restricted lymphocyte, most likely a natural killer (NK) T cell, which produces interleukin (IL)-13. Here we present evidence for the effector elements in this suppressive pathway. T cell–reconstituted recombination activating gene (RAG)2 knockout (KO) and RAG2/IL-4 receptor α double KO mice showed that inhibition of immunosurveillance requires IL-13 responsiveness by a non–T non–B cell. Such nonlymphoid splenocytes from tumor-bearing mice produced more transforming growth factor (TGF)-β, a potent inhibitor of CTL, ex vivo than such cells from naive mice, and this TGF-β production was dependent on the presence in vivo of both IL-13 and CD1d-restricted T cells. Ex vivo TGF-β production was also abrogated by depleting either CD11b+ or Gr-1+ cells from the nonlymphoid cells of tumor-bearing mice. Further, blocking TGF-β or depleting Gr-1+ cells in vivo prevented the tumor recurrence, implying that TGF-β made by a CD11b+ Gr-1+ myeloid cell, in an IL-13 and CD1d-restricted T cell–dependent mechanism, is necessary for down-regulation of tumor immunosurveillance. Identification of this stepwise regulation of immunosurveillance, involving CD1-restricted T cells, IL-13, myeloid cells, and TGF-β, explains previous observations on myeloid suppressor cells or TGF-β and provides insights for targeted approaches for cancer immunotherapy, including synergistic blockade of TGF-β and IL-13.

Abstract. Transforming growth factor beta (TGF-13) is angiogenic in vivo. In vitro, endothelial cell proliferation is inhibited by TGF-13. We have correlated this inhibitory effect with an increase in cellular fibronectin synthesis and deposition in a two-dimensional culture system using specific matrix coatings. The inhibitory effect was mimicked by addition of soluble fibronectin to cultures. In contrast, TGF-13 was found to elicit the formation of tube-like structures (mimicking angiogenesis) when microvascular endothelial cells were grown in three-dimensional collagen gels. In this culture system TGF-[3 elicited rapid extensive formation of complex, branching, tube-like structures, while cell proliferation was not inhibited. These data confirm and support the hypothesis that TGF-13 is angiogenic and may exert some of its effects through modulation of matrix synthesis and are consistent with the hypothesis that the organization of the extracellular environment influences cellular responses to this "panregulin" GIOGENESIS (the formation of new blood vessels from endothelial cells) is a complex process involving endothelial cell activation (including synthesis and secretion of proteases), migration, and proliferation, as well as matrix synthesis, differentiation, multicellular organization, stabilization, and capillary endothelial cell regression of local endothelial cell populations during healing and repair (Furcht, 1986;. Previously, we have demonstrated the importance of extracellular matrix (ECM) t composition and organization (laminin and type IV collagen) in the regulation of endothelial cell migration, proliferation, and multicellular organization during angiogenesis (Madri and Williams, 1983;Pratt et al., 1984Pratt et al., , 1985Form et al., 1986). Although ECM appears to be an important modulator of angiogenesis, it is but one element in a complex control mechanism which also makes use of various soluble factors including platelet-derived growth factor and transforming growth factor beta (TGF-[3). Recently, TGF-[3 (a 25-kD homodimer that has been shown to control cell growth and differentiation) has been noted to elicit a striking angiogenic response in mice as well as to dramatically affect synthesis in vivo and in vitro . Ignotz and Massague (1986) have demonstrated a functional involvement of fibronectin in mediating some cellular responses to TGF-13 in fibroblasts and have suggested a model for TGF-13 action based on the modulation of ECM in the target cell. Since TGF-13 is present in platelets and inflammatory cells it is reasonable to postulate that this protein may function as 1. Abbreviations used in this paper: ECM, extracellular matrix; Fn, fibronectin; Ln, laminin; TGF-[3, transforming growth factor beta; IV, type IV collagen; V, type V collagen. a paracrine mediator of the repair process, affecting local endothelial cell behavior by modulating matrix synthesis and degradation in the local cell populations in the injured area Saksela et al., 1987). In this report we show that in two-dimensi...

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Abstract. TGF-␤ is believed to play a central role in the development of Cyclosporin A (CsA)-induced nephropathy. This study investigated the effects of 1D11, a murine panspecific TGF-␤-neutralizing monoclonal antibody, in an ICR mouse model of chronic CsA nephropathy. Mice were administered a low-salt diet (0.01% sodium) for 1 wk followed by CsA treatment (30 mg/kg, subcutaneously, daily) for 4 wk. 1D11 was administered (2.5 mg/kg, intraperitoneally, 3 times/ wk) beginning immediately after the termination of CsA dosing and continued through 8 wk. CsA caused extensive renal histopathologic alterations, including tubular damage, interstitial infiltrates and fibrosis, deposition of collagen III, and apoptosis of tubular epithelial cells. 1D11 ameliorated the CsA-induced histopathologic alterations, with significant reduction in collagen III expression and deposition. Additionally, elevated levels of mRNA encoding TGF-␤1 and TGF-␤2 were significantly reduced. 1D11 also protected tubular epithelial cells from apoptosis by 48% (P Ͻ 0.05). In contrast, 13C4 (a control antibody) had no significant effect on any of the endpoints described above. Importantly, the effects of 1D11 on the CsA-induced morphologic alterations were followed by a reduction in serum creatinine level when compared with CsA mice treated with 13C4 (13C4, 0.45 Ϯ 0.09; 1D11, 0.30 Ϯ 0.08; P Ͻ 0.05) after 8 wk of treatment. Endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), nitrotyrosine, and tissue hypoxia were examined by immunostaining using specific antibodies. eNOS was significantly reduced in the endothelium of arterioles in the kidneys of mice treated with CsA, whereas iNOS was induced in the cortical tubules. Tissue hypoxia was found in both the arterioles and tubules, whereas nitrotyrosine was localized in the tubules. Administration of 1D11 improved tissue hypoxia and reduced nitrotyrosine formation. Moreover, the reciprocal changes in iNOS and eNOS expression were normalized by 1D11. This study demonstrates that 1D11 administration ameliorated morphologic alterations and preserved renal function in the context of existing chronic CsA nephropathy.

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The presence and specific structures of the oligosaccharides on TSH have been shown to be important for its production and bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the biological activity and metabolic clearance of a preparation of purified recombinant human (rh) TSH derived from a stable transfectant of Chinese hamster ovary cells. Carbohydrate compositional analysis of this rTSH showed it to be more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. In addition, no N-acetyl galactosamine was detectable in rhTSH, which implies the absence of terminal sulfate moieties, both of which are present in pituitary-derived TSH. The immunologic activity and porcine TSH receptor-binding activity of the preparation of rhTSH were 3- to 4-fold lower than those of a standard pituitary hTSH. The rhTSH showed a maximum stimulatory activity similar to that of pituitary hTSH in two different in vitro bioassays. However, rhTSH elicited about 3-fold and 5-fold less cAMP than pituitary TSH after stimulation of adenylyl cyclase in bovine thyroid membranes and the rat FRTL-5 cell line, respectively. Removal of sialic acid did not alter the immunologic activity of rhTSH. However, the potencies of rhTSH in receptor-binding, adenylyl cyclase, and FRTL-5 assays were increased 2.4-, 2.6- and 26.7-fold, respectively after sialic acid removal. These data suggest that the in vitro biological activity of rhTSH is influenced by its highly sialylated oligosaccharide chains. The rhTSH had a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH at 3 h as compared to pituitary hTSH. After sialic acid removal, the rhTSH was cleared faster (7.5-fold) than pituitary hTSH, showing that its longer plasma half-life was due to its higher sialylation. Biologically active rhTSH should be of clinical value in the diagnosis and treatment of patients with thyroid cancer and as a pure hTSH reference preparation.

We have genetically engineered a cell line, and developed a reproducible process, for the expression and purification of biologically active recombinant human thyroid stimulating hormone (rhTSH).rhTSH was expressed by co-transfecting a human alpha-subunit cDNA with a human beta-subunit partial genomic clone into Chinese Hamster Ovary (CHO) cells. Stable transfectants which expressed high levels of rhTSH were selected, and subsequently cultured on microcarrier beads. The rhTSH-containing media, produced under serum-free conditions, was clarified and purified by a combination of ion exchange, dye and gel filtration chromatographies. Individual step recoveries were greater than 90% with the exception of a very conservative pooling of the final gel filtration step (78% recovery) that resulted in a cumulative yield of 54% for the purification process. Purity of the final bulk material was judged to be > 99% by SDS polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase HPLC, and size exclusion chromatography. Initial characterization of the oligosaccharide composition indicated the presence of partially sialylated bi- and triantenary complex oligosaccharides. Purified rhTSH was active in a thyroid membrane bioactivity assay with a specific activity of 8.2 IU/mg. The in vivo activity of rhTSH in cynomolgus monkeys appeared to be equal to or greater than that reported for bovine TSH (bTSH) in human subjects. The rapid clearance phase half-life of rhTSH was approximately 35 minutes while the post-distribution phase half life was approximately 9.8 hours. Furthermore, the monkeys showed cumulative increases in minimum plasma rhTSH levels when given three daily intramuscular (IM) rhTSH injections; a phenomenon not observed when bTSH had been administered to humans. The rhTSH showed no evidence of toxic or adverse effects when administered at doses up to 7.2 IU/kg and 0.52 IU/kg in rat and monkey, respectively. These are 50X and 4X multiples of the bTSH doses of 0.143 IU/kg (10 IU/70kg) previously administered to humans.

We conclude that there are significant histopathologic consequences, focused in the kidney, resulting from the daily administration of high doses of human recombinant TGF-beta2, and we propose that selective vascular constriction with consequent tissue hypoxia is a contributing factor.