Proliferative responses of murine lymphoid cells were elicited in vitro with supernatant fluid from cultures of EL-4 thymoma cells stimulated with phorbol ester. It was demonstrated that such responses depend on IL-2 contained in the supernatant fluid, but reflect co-stimulation with IL-2 and phorbol ester. Striking differences in the magnitude of proliferative responses of spleen, splenic T lymphocytes, thymus and bone marrow cells from various strains were observed. Three classes of responders could be identified. The differences in responsiveness, at least in part, reflected differences in the frequency of responsive cells and were genetically controlled by codominant alleles of two independent somatic genes.

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SUMMARY Methodologies are described for the production and characterization of monoclonal antibodies to human haemoglobin. Three monoclonal antibodies are described, two of which recognize distinct determinants on the α‐chain subunit. A third monoclonal antibody, Hb‐2d, recognizes a determinant expressed on human β‐chain. The Hb‐2d determinant is shared by human and baboon haemoglobins, but is not expressed by haemoglobins from beef, goose, pig, rabbit, sheep, dog, rat or mouse. Monoclonal antibody Hb‐2d will bind to haemoglobin A2 but not to foetal haemoglobin suggesting that δ‐ but not γ‐chain also expresses the Hb‐2d determinant. The results of testing a limited panel of human haemoglobin variants is presented.

Proliferation of normal (not immunized intentionally) murine spleen cells was elicited with concanavalin A, supernatant fluid from cultures of EL-4 cells, human recombinant interleukin 2 (IL-2), or a mixture of phorbol ester and calcium ionophore A23187. IL-2-induced proliferation was inhibited by membrane-permeable dibutyryl cyclic adenosine monophosphate (cAMP) or by the adenylate cyclase activator forskolin. Consistent with these observations was the finding that stimulation with IL-2 decreased and forskolin increased the intracellular content of cAMP. IL-2-induced proliferation, as well as that induced by concanavalin A or phorbol-ionophore mixture, was inhibited by monoclonal antibodies specific for L3T4 or Lyt-2 cell surface markers. This inhibition was observed even when antibodies were added several hours after exposure of cells to IL-2. Notably, antibodies did not alter the intracellular content of cAMP. Thus, the experimental data failed to establish a functional linkage between the inhibitory effect of antibodies and the regulatory effect of the adenylate cyclase system. However, our results provide a rational basis for the postulation that antibodies, upon binding to their corresponding ligands, generate a negative signal that interferes with IL-2-induced proliferation. Therefore, L3T4 and Lyt-2 molecules appear to play an important role in the regulation of lymphocyte proliferation.

The primary immune response of mice to the Thy-1 antigens was elicited by intravenous injection of thymocytes from H-2 compatible donors. The magnitude of the ensuing response judged by the number of plaque forming cells producing antibodies lytic for the Thy-1 bearing cells was influenced by some non-H-2 antigens present on the immunizing thymocytes. Absence of non-H-2 incompatibility resulted in a poor response whereas the presence of such an incompatibility allowed a good response. Genetic analysis revealed that the non-H-2 incompatibility affecting the anti-Thy-1 response is controlled by 1-3 independently segregating genes presumably determing molecules acting as carrier determinants for otherwise poorly immunogenic Thy-1 antigens. The carrier effect appeared to be influenced by the gene dosage of the carrier molecules on the immunizing cells. The data presented are discussed in the context of the current concepts of the mechanisms controlling various immune responses.