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Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.

Studies were conducted on the mechanism of action of a soluble mediator which was generated in human mixed lymphocyte cultures and assayed for helper activity in T cell-deficient murine spleen cell cultures. The mediator, termed xenogeneic reconstitution factor or XFR, restored the anti-sheep erythrocyte plaque-forming cell response of spleen cells from thymectomized, lethally irradiated, and syngeneic bone marrow transplated (TxB) mice. It was found that in order to obtain a maximal antibody response, the XRF had to be present sometime during the first 24 to 40 hr of the induction process. XRF was also required during the last 24 hr of the incubation period. A striking synergistic effect was obtained by combined exposure of the T cell-deficient spleen cell cultures to XRF during the first and last days of culture. These data suggested a bi-modal mechanism of action of XRF, and raised the possibility that in addition to providing a signal to the B cells, XRF-mediated activation of the residual TxB splenic T cells was crucial to the successful restoration of the antibody response. Treatment of the nonadherent splenocytes from the TxB mice with anti-T cell serum and guinea pig complement completely abrogated the antibody response of these cells in the presence of adherent spleen cells, sheep erythrocytes, and the helper factor XRF. The antibody response of this combination of cells, antigen, and XRF was reconstituted by a population of unimmunized murine T cells which, in the absence of XRF, were totally unable to restore responsiveness. These results suggest that B cell activation to the formation of antibody involves the mandatory co-participation of two functionally distinct helper activities.

Concanavalin A and alloantigen-stimulated mouse spleen cells release into the supernatant at least two distinct antigen-nonspecific factors that enhance the generation of cytotoxic cells in vitro. As previously reported, analysis of the supernatant by ammonium sulfate fractionation, gel filtration, hydroxylapatite chromatography, and hydrophobic chromatographic showed one of the two killer cell helper factors (KHF) to be associated with thymocyte mitogenic factor. This report demonstrates the second KHF to be separable from thymocyte mitogenic factor but inseparable from type II (immune) interferon. In addition, this KHF exhibits the same sensitivity to exposure to pH-2 buffer as does immune interferon. The KHF activity of an unfractionated supernatant, which is greater than of either the TMF-associated or interferon-associated KHF alone, is the result of the additive activities of the two independently acting helper factors.

Modified Marbrook culture vessels with two chambers separated by a 0.2-mu porosity membrane have been utilized to show that antigen-stimulated human lymphocytes produce a soluble factor(s) which restores the ability of thymectomized, irradiated, and bone marrow-protected mice to mount a primary IgM plaque-forming cell response in vitro. In the initial experiments, the human lymphocytes plus antigen (sheep erythrocytes) were cultured in the lower chambers of the Marbrook vessels and the T cell-deficient mouse spleen cells plus sheep erythrocytes were cultured in the upper chambers. The response of the spleen cells was shown to be enhanced as a function of the number of human lymphocytes in the lower chambers. In subsequent experiments, the human lymphocytes were challenged with allogeneic lymphocytes or activated with a variety of T cell mitogens. Supernatants from these cultures, when placed in the lower chambers of the Marbrook vessels, were also capable of reconstituting the antibody-forming cell response of the mouse B cells. The results of the experiments are discussed in relation to a model of B cell induction which incorporates a non-antigen-specific "helpher" T cell.