Figure 1. Four different methods for inducing the nonresponsive state in IL-2-producing T-cell clones.(A) The T-cell clone A. E7, specific for the antigen pigeon cytochrome c, was stimulated with a synthetic peptide analog of the T-cell determinant" DASP"= NH2-KKANELIAYLKQATK-COOH and B10. A antigen-presenting cells (normal APC= 3000 R irradiated spleen cells)(@) or APC pretreated with 0.5% paraformaldehyde for 15 min at room temperature (9 After 1 day, the T cells were recovered and rested. After 4 more days, they were stimulated with various concentrations of DASP and irradiated B10. A spleen cells (normal APC) or with IL-2 (5% supernatant from the Gibbon cell line, MLA) as a control of viability. The stimulation of T cells not pretreated in any way is shown as the" rested" control (11). The proliferative response (CPM) was measured 3 days later by assessing the incorporation of [3H] thymidine into DNA. Note that pretreatment with chemically fixed APC makes the T cells hyporesponsive to restimulation, compared to rested cells. Pretreatment with normal APC and antigen gives a" preactivated" pattern on restimulation, ie, an increased maximal response and a shift in the ECs0 to a larger antigen concentration. See Jenkins et al.(1988) for more details.(B) The T-cell F1. A2 was cultured for 24 hr with planar lipid membranes containing the murine MHC class II molecule Ek~: Ek~ either alone (&) or in the presence of 10/. LM DASP (&). The cells were then recovered and rested. After 8 days, the T cells were restimulated with irradiated B10. A spleen cells (normal APC) and various concentrations of DASP. The proliferative response (CPM) was measured 3 days later. Note that pretreatment with peptide-MHC molecule complexes alone, a pure T-cell antigen-receptor occupancy event, induces a hyporesponsive state lasting at least 8 days. See Quill and Schwartz (1987) for more details.(C) The T-cell clone A. E7 (5 x 105 cells/well) was precultured for 24 hr in tissue culture plates precoated with 10/zg/ml anti-CD3 monoclonal antibody 145-2Cll (9 or medium (@). The cells were then recovered and rested in fresh plates without any antibody. After 7 days, surviving cells (20%) were stimulated with either IL-2 or irradiated B10. A spleen ceils and various concentrations of the carboxy-terminal cyanogen bromide (CNBr) fragment of pigeon cytochrome c (residues 81-104). The proliferative response (CPM) was measured 3 days later. Note that pretreatment with anti-CD3, which binds to the 9 chain of the T-cell antigen receptor, induces a hyporesponsive state for antigen restimulation, even though the response to IL-2 is augmented. See MK Jenkins et al.(in prep.) for more details.(D) The T-cell clone A. E7 was stimulated for 48 hr with 5/zg/ml concanavalin A ([]) or medium (@) in the absence of any APC. The cells were treated with amethylmannoside, recovered on a Ficoll-Hypaque gradient, washed with medium, and rested. After 2 days, the T cells were stimulated with either IL-2 or irradiated B10. A spleen cells and various concentrations of DASP. The proliferative response (CPM) was measured 3 days later. Note that, in the absence of APC, pretreatment with concanavalin A, which binds to many cell-surface glycoproteins including the T-cell antigen receptor, induces the hyporesponsive state. See Mueller et al.(1989) for more details.