Spleen cells from B6 mice injected with fetal calf serum (FCS) could be kept proliferating as a continuous cell line in vitro provided they were culture in the presence of irradiated syngeneic spleen cells and FCS. Cells in this cell line showed a strong proliferative response when stimulated with concanavalin A (Con A), and they were able to mediate the following functions: (1)they helped the generation of alloantigen-specific cytotoxic T lymphocytes (CTL) from thymocyte-spleen cell mixed lymphocyte cultures (MLC), (2)they induced the generation of CTL from normal syngeneic spleen cells in the absence of allogeneic stimulator cells, and (3)they induced normal spleen cells to differentiate into anti-sheep erythrocyte (SRBC) plaque-forming cells (PFC), in the absence of SRBC in the cultures. The use of this cell line (called line 12) may thus provide an interesting approach for the study of cellular and molecular requirements for cell-cell interactions and for the differentiation of T and B effector functions.

Thirteen monoclonal antibodies to ppp(A2'p5')nA oligonucleotides have been produced by fusing Y3 myeloma cells with spleen cells of rat hyperimmunized with A2'p5'A succinyl albumin as immunogen. 125I-labeled A2'p5'A succinyl tyrosine methyl ester was used as a labeled probe. Antibodies were detected by their ability to bind the labeled analog and were selected for their affinity for A2'p5'A. There were no significant differences between the properties of the monoclonal antibodies and of the antiserum. They discriminated between 2'-5' and 3'-5' phosphodiester bonds: they crossreacted poorly with (A3'p5')2A (crossreactivity ratio, greater than 10(4)) and even less with ATP an adenosine (crossreactivity ratio, greater than (6)). The affinity was high (Kd = 6 x 10(-12) M) for succinyl A2'p5'A, which is the best ligand, and also high for A2'p5'A (crossreactivity ratio, 3) and (A2'p5')2A, (A2'p5')3A, and (As'p5')4A (crossreactivity ratio, 7.5). The binding to triphosphorylated isomers, ppp(A2'p5)nA, was affected by the presence of the triphosphate groups, and the affinity increased as the length of the isomer increased (crossreactivity ratio, 10,000 for n = 1 to 200 for n = 4). Thermodynamic analysis of these data demonstrated that, in dephosphorylated isomers, the binding site of highest affinity was located at the 5'-OH end of the molecule whereas in phosphorylated isomers, the favorable binding sites were in the middle and at the 2'-OH end of the chain. Use of monoclonal antibodies of such a specificity together with the 125I-labeled 2-5A analog allows quantification of (A2'p5')nA directly and of ppp(A2'p5')nA after removal of the terminal phosphates by alkaline phosphatase treatment.

Sensitivity to the inhibition of division during cell crowding in vitro was determined for hybrids between normal and tumor cell populations of human and mouse origin. Interspecies hybrids were more sensitive to cell crowding than intraspecies hybrids. The results suggest that the inhibition of cell division due to crowding was more dependent upon the species of origin rather than on the normal or transformed character of the parental cell lines of the hybrid cell populations.

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