It has been proposed that under the usual conditions, immunization with thymocytes that are H-2-compatible and incompatible with the recipient at non-H-2 loci results in the presentation of the Thy-1 antigen in the cell-bound form. This form is recognized in the context of donor class I molecules (Zaleski and Reichner 1982). By contrast, immunization with H-2-incompatible thymocytes under appropriate conditions leads to the presentation of the Thy-1 antigen in the cell-free form. In order to be recognized and to elicit an anti-Thy-1 response, this form must be presented in the context of class II molecules, presumably expressed by antigen processing cells of the responder. Earlier studies have shown that the hybrid A molecules of Ab/A d heterozygotes are capable of presenting the cell-free form of the Thy-1 antigen, and that the two genes encoding each of these molecules act as complementary Ir-Thy-1 genes (Zaleski and Reichner 1982). This concept was recently given further credence by showing that mutation of the A b allele (McKean et al. 1981) affects the anti-Thy-1 response (Quackenbush et al. 1985). However, direct proof of the physical involvement of the A molecules in the anti-Thy-1 response was still missing. This paper describes the results of preliminary, but definitive experiments in which such proof has been obtained.
Because so far it has not been possible to produce a monoclonal antibody specific for hybrid Ab: A d molecules (J. S. Reichner, unpublished data), it was decided to use antibodies specific for parental A molecules. Table 1 contains basic information concerning the two monoclonal antibodies used in the present experiments (Kappler et al. 1981, Ozato et al. 1981). Hybridoma cell lines producing these antibodies were grown in vitro using either RPMI 1640 or Iscove's modified Dulbecco's medium supplemented with fetal bovine serum (10%), glutamine (2 mM), penicillin (100 units/ml), and streptomycin (100 gg/ml). 2-mercaptoethanol (5x10 -s iV/), insulin, transferrin, and sodium selenate (ITS, Collaborative Research, Inc., Lexington, Massachusetts) were occasionally also used as supplements. Culture supernatant fluids were collected and tested by an enzyme-linked immunosorbent assay for the presence of murine immunoglobulins (Engral and