The cytoplasmic, dense granules of cloned T cell lines were isolated and analyzed for their functional and biochemical properties. Isolated granules of approximately 90% homogeneity, in the presence of Ca, effect strong tumoricidal and hemolytic activity. Tumor cell lysis is complete in less than 30 min, with less than 2 micrograms granule protein corresponding to a killer/target ratio of 3-6:1 by assuming 50% yield for granule isolation. The granules contain a set of unique proteins, responsible for cytolytic activity and designated K1 to K6, in the molecular weight range of 14,000 to 75,000, as defined by sodium dodecyl sulfate (SDS) polyacrylamide slab gel analysis under reducing and nonreducing conditions. Cytolysis mediated by isolated granules is accompanied by the assembly of tubular complexes of 160 A (poly P1) and of approximately 70 A width (poly P2) that are inserted into membranes and form ultrastructural membrane lesions. As shown by immunofluorescence and by Percoll gradient fractionation, cytolytic granules are detected in cells of cytolytic T cell lineage and not in the T cell lymphomas E14 and S194. Poly perforin 1 assembled by CTLL-2 upon stimulation with concanavalin A (Con A) and phorbol myristate acetate (PMA) was isolated by detergent extraction and gel filtration. Poly P1 is composed of disulfide-linked subunits that, after reduction, co-migrate with certain granule proteins. The results are compatible with the hypothesis that the dense granules of cytolytic T cells contain cytolytic proteins that polymerize to disulfide-linked tubular poly perforins in a Ca-dependent reaction and may cause cytolysis by membrane insertion and transmembrane channel formation.
A unique immunoliposome has been developed as a drug delivery vehicle for immunotherapy. Human recombinant interleukin-2 (IL-2) has been chemically coupled to the external surface of small unilamellar vesicles (SUVs) containing methotrexate as a candidate immunosuppresive agent in order to specifically direct the drug-bearing liposome to activated T-cells expressing the high affinity IL-2 receptor. This drug delivery system is designed to deliver an immunosuppressive agent to those cells that actively participate in disorders such as graft rejection without delivering an effective but potentially toxic drug to all cells of the immune system as well as other healthy tissues. IL-2 was chemically modified with succinimidyl 4-[p-maleidophenyl butyrate](SMPB) while the receptor binding domain on IL-2 was protected by monoclonal anti-IL-2 bound to Protein A-Silica Gel. The antibody recognizes the receptor binding domain of the IL-2 molecule. The IL-2 was derivatized with S-succinimidyl-S-thioacetate (SATA) in order to add an acetyl thioester group to the lipid and create the complex. The derivatized lipid (SATA-PE) was then part of the liposome formulation containing DSPC:cholesterol: SATA-PE at a mole ratio of 1.5:1.0:0.26. SMPB-IL-2 was covalently coupled to the external surface of the SUV after deacetylation of the thioester moiety at pH 7.4 in PBS. Liposomes prepared by sonication or extrusion had an average diameter of 46-50 nm. SUV-IL-2 bound to the high affinity IL-2 receptor as measured by competitive binding assays and Scatchard analysis using 111InCl2-loaded liposomes The preparation exhibited a binding constant of 30 pM, consistent with values for free IL-2 cited in the literature. SUV IL-2 could be used as the sole source of IL-2 for the murine CTLL-2 T-cell line or for human mitogen-activated PBLs. The presence of IL-2 coupled to the surface was absolutely required for delivery of the drug to the cell. When methotrexate was encapsulated within the internal aqueous space, receptor-mediated endocytosis led to the inhibition of proliferation due to delivery of MTX to the cytoplasm of the cell. More than 90% of the methotrexate was retained within the liposome during storage over a 24-h period at 4 degrees C. This immunoliposome represents a new class of cell specific immunoliposomes whose entry into the cell is controlled by a cell surface receptor.
Collagen-induced arthritis (CIA) generated in rats or mice has long been a model system for the study of rheumatoid arthritis in humans. In particular, this system has been used to study the mechanisms and effects of anti-arthritic drugs in the treatment of the disease. Sodium aurothiomalate (ATM) is an agent often used to treat rheumatoid arthritis in humans; however, it possesses inherent toxicities which limits its usefulness. Liposome-encapsulated drugs are currently being developed to minimize the toxicities associated with a variety of potentially beneficial drugs. We have chosen to encapsulate ATM into small unilamellar vesicles (SUVs) to determine whether greater efficacy would be achieved in treating CIA with SUV ATM as compared to using the free drug. SUVs were prepared from hydrogenated egg phosphatidylcholine and cholesterol. These SUVs were very stable. Vesicles stored at 4 degrees C lost only 0.09% of encapsulated ATM (SUV ATM) after 14 days and were able to reduce collagen-induced arthritis in these mice. Animals treated by i.m. injections of SUV ATM exhibited a 50% reduction in symptoms. More importantly, histological examination of knee joints of the affected animals verified that SUV ATM treatment prevented cellular infiltration of lymphocytes into the synovia of the collagen-sensitized mice. Conditioned media from spleen cell cultures was assayed for the presence of inflammatory lymphokines that might be affected by SUV ATM to account for the success in suppressing collagen-induced arthritis.
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