John J. Farrar

The biochemistry, biology, and role of interleukin 2 in the induction of cytotoxic t cell and antibody-forming b cell responses.

Interleukin 2 (IL 2) (Aarden et at. 1979) is a genetically unrestricted, soluble immunoenhancing factor which is produced by helper T cells following stimulation with either T cell mitogens or allogeneic cells. Presumably, IL 2 is also produced and secreted following specific antigen stimulation, although this question has not been carefully evaluated. The definitive biological assay for IL 2 measures the factors ability to maintain the growth of factor-dependent cytotoxic T cell lines. However, a broad spectrum of biological activities has been ascribed to IL 2. The factor has been reported to induce thymocyte proliferation either in the presence (Chen & DiSabato 1976, Paetkau et al. 1976) or absence (Farrar et al. 1978) of a suboptimal concentration of T cell mitogen. Additionally, IL 2 has been shown to augment the proliferation and generation of cytotoxic cells by alloantigenstimulated T cell populations (Wagner & Rollinghoff 1978, Farrar et al. 1978) and, in the process, induce the synthesis of immune interferon (IFNji) by the alloantigen-stimulated T cells (Farrar et al. 1981a). IL 2 has also been reported

Evidence for the identification of lymphocyte activating factor as the adherent cell-derived mediator responsible for enhanced antibody synthesis by nude mouse spleen cells

Abstract Human adherent peripheral blood leukocytes spontaneously elaborate both a thymocyte proliferative factor and a factor which augments the in vitro anti-sheep erythrocyte (SRC) plaque-forming cell (PFC) response of nu/nu mouse spleen cells. Nonadherent leukocytes do not spontaneously elaborate either factor. The adherent cell-derived factors appear to have an identical molecular weight (approximately 14,500 Daltons) as determined by Sephadex gel filtration. The data support the hypothesis that the molecule(s) mediating both enhancing activities is identical to the previously described adherent leukocyte product, LAF.

Role of mitogenic factor, lymphocyte-activating factor and immune interferon in the induction of humoral and cell-mediated immunity.

Although the humoral and cell-mediated immune responses have traditionally been thought to be clearly separated, there is increasing evidence that they may be extensively interdigitated and interdependent and in fact, share common regulatory mechanisms. This view is based, in part, on observations indicating that macrophages and T-cells are capable of exerting both enhancing and suppressive effects on both branches of the immune r e s p o n ~ e . ~ ~ For example, it has been demonstrated that the induction of the antibody response to thymicdependent antigens and the cytotoxic T-cell response to alloantigens are both dependent on “helper” T-cells that bear the same surface antigenic markers (Ly 1+2-3-).4e5 Moreover, the Ly 1’2-3T-cells have been shown to be required for the production of the antigen-nonspecific soluble mediators (lymphokines), which replace the helper T-cells during the induction of both humoral and cytotoxic The results presented in this report will summarize our characterization studies of three such immunoenhancing soluble mediators (Lymphocyte-activating factor, LAF; thymocyte mitogenic factor, TMF; and immune interferon, IF). The data indicate that macrophage-derived LAF and T-cell-derived TMF are capable of enhancing antibody responses as well as cytolytic T-cell responses whereas the I F enhances only the cytotoxic T-cell response.8-12

Partial purification of a bone-resorbing factor elaborated from human allogeneic cultures.

Abstract Human mixed leukocyte culture supernatants contain a factor(s) capable of effecting resorption of fetal rat bones in vitro . We have investigated procedures for the large-scale generation and partial purification of this bone-resorbing factor(s). Human mononuclear leukocytes obtained by leukapheresis from two healthy donors (average of 1 × 10 10 cells/donor) were cultured together (2.5 × 10 6 cells/ml) for 40 hr, the supernatants harvested and assayed for bone-resorbing activity by measurement of 45 Ca release from fetal rat bones in vitro . These supernatants consistently induced significant bone resorption when compared to supernatants from a single donors cells. Neither the release of this bone-resorbing factor nor its biologic activity were abrogated by addition of indomethacin. The bone-resorbing factor(s) eluted from Sephadex G-75 at an apparent molecular weight of 11,000 to 18,000 and appeared only in the breakthrough when fractionated on DEAE-cellulose (0.005 M phosphate, 0.02 M NaCl, pH 7.5). A concentrated eluate containing bone-resorbing activity after two cycle Sephadex G-75 gel filtration and DEAE-cellulose chromatography was applied to anionic polyacrylamide disc gels. A single peak of bone-resorbing activity was eluted, although this activity could not be attributed to a single stainable protein band. The results indicate that human mixed leukocyte cultures elaborate a low-molecular-weight boneresorbing factor(s) resembling the previously-described osteoclast-activating factor. Methods presented for the large-scale generation and partial purification of this activity should facilitate clarification of the role of this mediator in tissue destruction.

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

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.