Proliferative Response of Cloned CTL line 10B-5 upon Stimulation with Soluble Clonotypic Monoclonal Antibody and its Blocking by Anti-Lyt-2 Antibody

Proliferation of the cloned cytotoxic T lymphocyte (CTL) line 10B-5 induced by anti-clonotypic antibody and its blocking by anti-Lyt-2 mAb were studied. Clone 10B-5 was derived from spleen cells of a (BALB/c X CS7BL/6)F₁ (CB6F₁)-nu/+ mouse immunized with UV♀1 sarcoma. 10B-5 cells lysed UV♀1 specifically and proliferated upon stimulation with UV♀1 and feeder cells in the presence of IL2. Anti-clonotypic monoclonal antibody (mAb) N1-56 was produced by a hybridoma established by fusion of spleen cells from a CB6F₁-nu/+ mouse that had been immunized by five injections of 10B-5 cells prefixed with 0.1% formaldehyde. N1-56 mAb immunoprecipitated 90 kd molecules cleavable to 45 kd molecules under reducing conditions, indicating its reaction with T cell antigen receptor (TCR). N1-56 mAb in its soluble form induced a proliferative response of 10B-5 cells. Thus, the antigen binding site of N1-56 mAb appeared to substitute for the specific antigen determinant on UV♀1 sarcoma. The F(ab')₂, but not the Fab fragment of purified N1-56 mAb, stimulated proliferation, indicating that cross-linking of TCR molecules was necessary for stimulation. The proliferative response of 10B-5 cells induced by soluble N1-56 mAb was blocked by addition of anti-Lyt-2.2 mAb to the cultures. The specificity of Lyt-2 blocking was confirmed by an absorption test. The proliferative response of 10B-5 cells induced by Con A, but not that induced by IL2, was blocked by anti-Lyt-2.2 mAb. These results indicated that blocking by anti-Lyt-2.2 mAb was at an early stage and that it could be bypassed by stimulation with IL2.