The ability of shed Thy-1 antigenic moiety from S.49.1 (Thy-1.2, H-2d) and BW5147 (Thy-1.1, H-2k) lymphoblastoid cells to induce primary antibody responses to Thy-1.1 and Thy-1.2 was investigated by using thymocytes as target cells for a plaque-forming cell (PFC) assay. Addition of S.49.1 culture medium to AKR/J (Thy-1.1, H-2k) spleen cells induced a significant anti-Thy-1.2 PFC response against target CBA/J (Thy-1.2, H-2k) thymocytes. In the reciprocal protocol anti-Thy-1.1 PFC responses against target AKR/J thymocytes were elicited by CBA/J spleen cells cultured with BW5147 cell culture medium. Congenic anti-Thy-1.1 sera added to immunizing culture medium provided still another test of specificity because anti-Thy-1.1 PFC responses were abrogated whereas anti-Thy-1.2 PFC responses remained unaffected. In the reverse experiment, addition of congenic anti-Thy-1.2 sera blocked the induction of anti-Thy-1.2 PFC responses.
Kinetics of Thy-1.2 release from S.49.1 cells was studied by radiolabeling the lymphoblastoid cells with 14C-galactose or 14C-glucosamine followed by specific immunoprecipitation of solubilized cell-associated Thy-1.2 and of shed Thy-1.2 with anti-Thy-1.2 sera. Rapid disappearance of radiolabeled Thy-1.2 from S.49.1 cells occurred during the first 2 hr of incubation followed by a gradual synthesis of Thy-1.2 over the next 10 hr. A second phase of release took place between 11.5 and 27.5 hr of incubation. Shed radiolabeled Thy-1.2 appeared rapidly in the culture medium during the first 11.5 hr phase of incubation when more than 60% of the labeled Thy-1.2 material was found to be released. Accumulation of Thy-1.2 in culture medium continued during the prolonged periods of incubation and provided increased anti-Thy-1.2 PFC responses.
The molecular properties of shed Thy-1.2 were studied by chromatography of supernatants from a 45 hr culture of 14C-glucosamine labeled S.49.1 cells on a Sepharose-6B column. Thy-1.2 antigenic activity was primarily detected in fractions from a 14C-radioactive peak of greater than 2 × 106 daltons. In addition, Thy-1 antigenic activity was found in fractions with a m.w. estimated at 3 × 105 daltons. These results indicate that Thy-1 is synthesized and released as a large complex from lymphoblastoid cells.