By using polyethylene glycol 1540, BW5147 AKR T lymphoma cells were fused with splenocytes from A/J mice treated so as to induce suppressor T cells specific for azobenzenearsonate (ABA). Of 576 microwells originally seeded, 132 demonstrated growing cell clones, 4 of which produced an ABA-binding supernatant factor. When tested in vivo for suppression of delayed-type hypersensitivity to ABA, two of these cell lines, A4 and F12, were polypeptide (4-6), and tumor (7-9) models of murine immune response. One ofthe most extensively described pathways concerns the suppression of delayed-type hypersensitivity (DTH) to the hapten azobenzenearsonate (ABA) in A/J mice. This suppressor circuit relies on an idiotype-anti-idiotype system ofcellular interactions (2). The induction phase of this pathway involves the interaction of antigen with an antigen-binding idiotype-positive T suppressor cell (Ts,) and relies on an idiotype-anti-idiotype system of recognition. Although the participating T cell subsets and factors are well characterized (1, 2, 10, 11), their complexity has hindered attempts to analyze this suppressor circuit at a molecular level.Precise analysis of this important regulatory phenomenon requires the isolation of homogeneous suppressor cell populations and factors for detailed study. One approach has been to adapt the hybridoma fusion technique of Kohler and Milstein for use with T cells. Several such fusion products have been generated in various systems and have been reported to: (i) have suppressor activity when tested in vitro on antigen-specific responses (12-16), (ii) bind antigen directly (17,18), or (iii) produce antigen-binding materials (19,20). This report details the production and characterization ofan ABA-specific, suppressor factor-producing, Ts,-like T cell hybrid line. The hybrid-derived monoclonal factor abrogates the DTH response of A/J mice to ABA and therefore provides a useful reagent for analysis of the molecular basis of suppressor cell function. x 107 normal A/J spleen cells were derivatized with the diazonium salt ofABA and injected intravenously into nonimmune A/J mice. Recipient mice were splenectomized 7 days later and a single-cell suspension was prepared from their spleens by teasing with forceps and allowing larger fragments to settle out. The splenic lymphocytes were washed twice in 50 ml of phosphate-buffered saline (PJNaCI) (pH 7.4) with centrifugation at 250 X g for 10 min at 4°C. This cell population was demonstrated to contain ABA-specific suppressor cells by its ability to prevent subsequent DTH response to ABA when transferred to normal A/J mice at the time of initial priming, as described (10). BW5147 cells maintained in CDH (DH with 10% fetal calf serum) were harvested at the mid-logarithmic phase of growth and washed three times in PJNaCl. For fusion, Ts, containing A/J splenic lymphocytes were combined at a ratio of 5:1 with BW5147 T lymphoma cells and washed once in PJNaCl. The cell mixture was centrifuged at 200 x g for 8 min at 4°C, the supernatant...
We have found that an I-J+ I-A- antigen-presenting cell (APC) is required for Ts3 activation in vivo. Together with the I-J restriction previously reported for Ts3 induction (Takaoki, M., M.-S. Sy, A. Tominaga, A. Lowy, M. Tsurifiji, B. Benacerraf, R. Finberg, and M. I. Greene, 1982, J. Exp. Med., 156:1325), it appears that this I-J+ APC is responsible for I-J restriction in the triggering of Ts3. This restriction may be exerted via a pre-Ts3 associative recognition of antigen and I-J encoded determinants, analogous to the T helper recognition of antigen in the context of I-A and I-E determinants.
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