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The mechanism of interleukin-2 (IL-2) signal transduction was analyzed by use of an inducible B lymphoma. Like normal antigen-activated B lymphocytes, the lymphoma cells respond to IL-2 by proliferating and differentiating into antibody-secreting cells; both responses are blocked by a second interleukin, IL-4. Analyses of the signaling pathway showed that IL-2 stimulated the rapid hydrolysis of an inositol-containing glycolipid to yield two possible second messengers, a myristylated diacylglycerol and an inositol phosphate-glycan. The myristylated diacylglycerol response exhibited the same IL-2 dose dependence as the growth and differentiative responses, and the generation of both hydrolysis products was inhibited by IL-4. These correlations implicate the glycosyl-phosphatidylinositol system in the intracellular relay of the IL-2 signal.

Provision of beta-galactosidase (GZ) under defined conditions of dose and time can either help or suppress a subsequent response to trinitrophenyl (TNP)-GZ in CBA/J mice. The optimal helper effect occurs when 10(7) spleen cells from mice primed 9 or more days previously with 10 mug GZ are adoptively transferred to irradiated recipients which are than challenged with 10 mug TNP50GZ. Optimum suppression results from the transfer of spleen cells from mice primed 3 days previously with 100 mug GZ and challenge of recipients with TMP150GZ. Both help and suppression are carrier-specific and mediated by T cells. In experiments where helper or suppressor cells were mixed with normal cells, the anti-TNP response was proportional to the number of primed cells transferred. The results point to a wave of suppression as the initial event after immunization, which is succeeded by period in which the helper effect dominates.

Spleen cells from neonatal animals, placed in culture for 6 days spontaneously develop the ability to block the activity of suppressor T cells, a phenomenon that is referred to as contrasuppression. The effector cell which is derived from the interactions among the cells which comprise a contrasuppressor "circuit" is an Ly-1 T cell. It can be separated from Ly-1 helper cells by three criteria other than function: its generation is dependent on Ly-2+ cells, it is I-J+, and it sticks to the Vicia villosa lectin. Those cells which deliver help to B cells under the experimental conditions studied are not dependent on Ly-2+ cells for generation and neither express determinants that our anti-I-J antisera recognize nor stick to V. villosa. The mechanism by which these Ly-1 contrasuppressor cells function was elucidated by adding them to "'intermediate cultures" containing activated Ly-2 suppressor cells and in vivo immunized Ly-1.1-congenic helper cells. After 48 h in these intermediate cultures, the neonatal Ly-1.2 contrasuppressor cells and the Ly-2 suppressor cells were removed by treatment with the appropriate antiserum plus complement. The remaining activity of the in vivo generated Ly-1.1 helper cells was assayed in fresh cultures of B cells. The contrasuppressor cells not only diminished suppression of the Ly-1 helper cells by the Ly-2 suppressor cells in the intermediate culture, but actually conferred a state of relative resistance to suppression upon the helper cells. This state persisted after the contrasuppressor cells were removed. Why such a cellular circuit, which confers resistance to suppression, might be beneficial to neonatal mice and how considering its attributes might help explain some immunological paradoxes is the subject of discussion.

Anti-murine interleukin 2 (IL-2) receptor monoclonal antibodies (mAb) were made from rats immunized with murine cytotoxic lymphocytes. One mAb, designated M7/20, strongly inhibited the proliferation of both IL-2 dependent CTLL-2 cells and concanavalin A (Con A)-induced T-cell blasts. Inhibition was linearly dependent on the concentrations of both M7/20 and IL-2. Utilizing FACS analysis, M7/20 was shown to bind selectively to mitogen-activated T lymphocytes and, to a lesser degree, to activated B lymphocytes. 125I-Labeled M7/20 binding assays indicated that 48-hr Con A-induced T-cell blasts possessed 89,000 binding sites/cell with a Kd of 1.2 X 10(-9) M. Competitive binding analyses indicated that M7/20 and IL-2 occupy the same or overlapping cell surface sites. Preliminary biochemical characterization of M7/20 immunoprecipitates of detergent extracts from both surface-iodinated and internally D-[3H]glucosamine-labeled T lymphoblasts indicated that the murine IL-2 receptor is an N-glycosylated 58,000-Da glycoprotein. Together these results suggest that mAb M7/20 binds at or near the IL-2-binding epitope on the murine IL-2 receptor and, thus, upon manipulation may act as an IL-2 agonist.

Subcutaneous transplantation of the syngeneic P815 mastocytoma in DBA/2J mice induced an activation of splenic T cells which resulted in a hyperresponsiveness of the tumor-bearing animal to the unrelated antigens pneumococcal polysaccharide (Pn) and sheep red blood cells (SRBC). These tumor-activated T cells appeared to increase the plaque-forming cell (PFC) potential of suboptimal numbers of spleen cells, caused normal spleen cells to express increased numbers of PFC, and produced lymphokine(s) which also increased PFC responses of normal splenocytes. The tumor-activated T cells responsible for stimulating normal splenocytes in an in vitro antibody response were shown to be Ly+2- cells. The activity of the tumor-activated T-cell supernatants was not genetically restricted and required additional Ly1 T cells in order to induce rigorously clean B cells to produce antibody. The T cells capable of stimulating non-specific antibody responses were also capable of slowing tumor growth when injected with tumor cells in normal recipient mice. These results suggest that T cells activated by tumor antigens release immunostimulatory lymphokines and, at the same time, are capable of leading to inhibition of tumor growth.

Within 2 to 3 days of infection with Trypanosoma brucei strain S42, the ability of spleen cells from infected CBA mice to mount a primary in vitro antibody response to sheep red blood cells (SRBC) is profoundly reduced, and suppressor cells are generated as detected by cell mixture experiments. Suppressor cell activity lies in the T and adherent cell compartments of spleens from infected mice, but not in the B cell compartment, although antibody responses to a thymus-independent antigen, DNP-Ficoll, are significantly reduced. Suppression of antibody responses of normal spleen cells depends on viable cells from infected mice. The trypanosome, itself, plays no direct role in suppression, and we have ruled out the possibility of antigenic competition as a mechanism of suppression. Our data is consistent with the model of suppressor T cells induced by concanavalin A mitogenesis. We hypothesize that trypanosome antigens may directly stimulate T cells with the concomitant release of factors with affinity for macrophage surfaces thus becoming suppressive for T and B cell responses.

A "feedback suppressor T cell" highly dependent on signals from Ly 1 T helper cells for activation is described. The signal from the Ly 1 cell binds to Fc-like receptors on macrophage membranes. The feedback suppressor cell expresses all three Ly antigens as well as the Qa 1 antigen on its surface, is very sensitive to low doses of cyclophosphamide, disappears relatively rapidly after adult thymectomy, and cannot be demonstrated in NZB mice 6 weeks or older.

The basic elements of the feedback suppression circuit of T cell interactions are summarized. Ly1 T cells can be stimulated directly to become inducers of Ly2+ T suppressor cells or helper cells for B cells that produce antibody to erythrocyte antigens. Ly2 cells can be activated to become suppressors only in the presence of Ly1 cells. An Ly1,2,3, cell is the acceptor of the Ly1 inducer signal. Using an intermediate culture technique, it can be shown that Ly1 inducers are a target of suppressor T cell activity.

Clones of inducer cells activated by antigen and class II major histocompatibility complex (MHC) gene products synthesize large amounts of several distinct mRNA species not detected in other activated cell types, including antigen-activated suppressor or killer cell clones. Although inducer cells continued to synthesize and secrete peptides at about the same rate for 5 days after activation by adherent cells and antigen, they expressed a new set of mRNAs approximately 3 days after activation. We therefore tested the functional activity of purified Ly1 cells at different days after activation by adherent cells and antigen. We wished to find out (a) whether there was a qualitative or quantitative change in the level of inducer activity, and if so, (b) whether this functional change was an intrinsic property of Ly1 cells or, rather, was dependent on signals from the cellular environment, in particular from adherent antigen-presenting cells. We incubated purified Ly1 cells and splenic adherent cells for 1-7 days in vitro, and tested inducer activity in cultures containing highly purified B cells and sheep erythrocytes (SRBC). Anti-SRBC plaqueforming cells were counted 5 days later. We found that inducer cells that have been incubated with adherent cells in culture for more than 72 h (a) did not induce B cells to produce antibody, and (b) prevented virgin but not immune B cells from receiving T-helper signals. We term this latter phenomenon "paralysis." Acquisition of the ability to paralyze virgin B cells required an I-E gene-regulated interaction between inducer cells and in vitro-activated adherent cells. This interaction did not require antigen and was associated with transition from Ly1:Qa1- to Ly1:Qa1+ cell-surface phenotype. Taken together these findings indicate that (a) interactions between inducer cells and adherent antigen-presenting cells result first in classical inducer ("helper") activity and later in expression of paralytic activity and (b) sequential expression of these inducer activities depends on two distinct signals, supplied by resting and activated adherent cells, respectively. The signal supplied by autologous activated adherent cells is regulated by I-E gene products and is independent of corecognition of foreign proteins. The physiologic significance of this paralytic inducer activity in the prevention of potentially harmful immune reactions to foreign microbial agents is discussed.

We have described a number of the parameters involved in the in vitro induction of specific SRBC-binding T cells (T rosette-forming cells, T-RFC). Although T-RFC precursors pass through nylon, most of the induced cells do not; nor do detectable numbers of Ly 1+2, 3- cells bind antigen with sufficient stability to form rosettes. The ratio of Ly 2,3:Ly 1,2,3 T-RFC varies with time after immunization and with the dose of antigen used for stimulation. Relatively high or low doses of antigen selectively induce Ly 1,2,3 T-RFC. Ly 2,3 T-RFC, when they appear, follow Ly 1,2,3 T-RFC. Pretreatment of T cells with anti-Ly sera before RFC induction prevents formation by Ly2+ T-RFC. Since anti-Ly 1 treatment blocks RFC formation and since Ly 1,2,3, T-RFC always precede the appearance of Ly 2,3, T-RFC, our results suggest that some Ly 1+ cells (Ly 123 at least, but perhaps also Ly 1) may act as inducers, precursors, and/or amplifiers for Ly 2,3 RFC as they appear to do for Ly 2,3 suppressor and killer cells. Thus, our results confirm and extend the observed similarities between T-RFC and other Ly 2+ cells such as killer and suppressor cells as well as their differences from Ly 1+ helper cells.

The role of the products of prostaglandin synthetase in the suppression of in vitro secondary antibody responses by rat spleen cells was examined. By including cells from both germfree and conventional rats in these studies, the effect of in vivo microbial stimulation on this suppression was also determined. We found that 1) the suppression that is normally present in the spleen cells of rats can be eliminated by the use of glass wool or indomethacin; 2) germfree rats do not display this suppressor activity, and 3) this "deficit" in the germfree rat may be due to a lack of a microbial-induced signal from a nonadherent cell to an adherent prostaglandin-containing macrophage, implying that at least two cells are involved in the prostaglandin-synthetase dependent suppressor effect. It is possible that large amounts of some or all microbial products activate "suppressor" macrophages and that inbred rats behave like "chronically infected" mice because of an inability to regulate their "normal" microbial flora.

Strains of Staphylococcus aureus isolated from patients with toxic shock syndrome (TSS) make a characteristic protein known as toxic-shock-syndrome toxin-1 (TSST-1), but the role of this protein in the pathogenesis of TSS is not certain. We have purified TSST-1 by using a combination of alcohol precipitation, isoelectric focusing, and gel chromatography. TSST-1 has an isoelectric point of 7.2 and a molecular weight of 23,100, in accordance with previously published determinations for this protein, and is serologically identical to pyrogenic exotoxin C and staphylococcal enterotoxin F. In highly purified form, TSST-1 is a potent inducer of interleukin-1 production by human monocytes, as quantitated in a thymocyte-proliferation assay. This capability is not attributable to contamination by other staphylococcal products or gram-negative endotoxin and can be blocked by hydrocortisone. Many features of TSS suggest that induction of interleukin-1 by TSST-1 in vivo may play a central role in the elaboration of this disease.

A radioimmunoassay for circulating soluble antigen-antibody complexes is described. The assay detects complexes either in antigen or antibody excess. Soluble complexes were found in the sera of chickens tolerant to bovine serum albumin (BSA). The complexes appeared in the serum as soon as 7 days following neonatal induction of tolerance. The amount of complexes reached a peak between 1 to 2 weeks of age and disappeared by 6 weeks when responsiveness returned. The complexes were found in the bottom third of a 10 to 40% sucrose density gradient and by analytical ultracentrifugation indicated a size of 22.8S. If tolerant chickens were challenged with BSA at 2, 4 or 6 weeks, the disappearance of complexes was not accelerated, and a proportion of the previously tolerant chickens exhibited a heightened antibody response.

Mouse spleen cells primed in vivo with beta-galactosidase (GZ), and cultured in vitro with trinitrophenyl beta-galactosidase (TNP-GZ), make a very poor anti-TNP response compared to cultures of normal spleen cells. The lack of response is caused by active suppression as indicated by cell mixture experiments. At 2-months following priming, while the helper effect predominates, suppression can be recalled by rechallenge of the mice with GZ. Both primary suppression (after a single injection) and secondary suppression (after two injections) reduce the response of normal or carrier-primed helper cells at equivalent cell ratios in mixture experiments. Both kinds of suppression are carrier specific, slectively suppress high avidity antibody production, and require cell division to suppress the response of normal cells. The only difference between primary and secondary suppression is that primary suppression is obliterated in cultures which are not challenged with antigen until 24 hr after cultures are established. Secondary suppression is still observed when cultures receive TNP-GZ at 24 hr. Suppression appears to be an early component of both primary and secondary responses. The regulatory function of such suppression may be the delay of antibody secretion.

T cells involved in the generation of suppressor activity bear an I-J-subregion controlled determinant (e.g., J1) which is distinct from that (e.g., J2) found on non-T:non-B accessory cells. T-cell subsets examined include Ly-1 inducer and Ly-1,2 acceptor cells which collaborate to generate suppressor activity in the in vitro sheep red blood cell antibody system. Non-T:non-B accessory cells examined include accessory cells involved in concanavalin-A induced, T-cell proliferative responses and in in vitro antibody responses to sheep red blood cells. These results provide evidence for serologic and genetic complexity of the I-J subregion of the murine H-2 gene complex.

The growth of the P815 mastocytoma in syngeneic DBA/2 mice led to an activation of Ly1+2- T cells. These T cells produced a soluble factor or factors in culture which, when added to normal spleen cells or B cells in the presence of syngeneic Ly1 cells, caused a genetically unrestricted augmentation of the plaque-forming response toward sheep red blood cells (SRBC). The culture supernatant of the activated T cells did not support the proliferation of an interleukin-2 (IL-2)-dependent cell, nor exhibit properties of late-acting TRF. Active supernatants appeared to affect directly B cells during the first 48 hr of culture with SRBC in such a way as to make them more responsive to antigen-specific Ly1-cell help.

Spleen cells taken from mice soon after infection with Trypanosoma brucei S 42 enhance the primary in vitro antibody response of normal spleen cells to sheep red blood cells (SRBC), but do not affect their response to DNP-Ficoll. Spleen cells harvested later in the infection (day 6 onwards) suppress the antibody response of normal spleen cells to both SRBC and DNP-Ficoll. The enhancing and suppressive effects of "infected" spleen cells are sensitive to treatment with anti-Thy 1.2 anti-serum and complement, and can be mediated by nylon wool-purified populations of T cells. The enhancing T cell is sensitive to ALS, not lost within 4 weeks of adult thymectomy, and bears the Ly-1+, 23- phenotype. The suppressor T cell is insensitive to ALS, lost within 20 weeks of adult thymectomy, and bears the Ly-1+, 23+ phenotype. The significance of the activation of distinct helper and suppressor T cells is discussed in relation to the pathogenesis of trypanosomiasis.

We describe conditions for generating sheep red blood cell-specific suppressor T cells in Mishell-Dutton cultures. The production of specific suppressor cells is favored by increasing antigen dose in the initial culture but can be produced by transferring more cells when lower doses of antigen are used. Transfer of small numbers of cells cultured with low doses of antigen leads to a specific helper effect. Transfer of large numbers of educated cells leads to nonspecific suppression. Suppression can be effected by the effluent cells from nylon wool columns which do not make detectable PFC. A fraction of these cells become resistant to treatment with anti-T cell sera and complement after culture. The suppressor cells are radiation sensitive and must be able to synthesize protein to suppress. They take 2 to 3 days of education to reach maximum suppressive efficiency and will not suppress cultures if added 2 to 3 days after culture initiation. Their production is favored by the absence of mercaptoethanol, suggesting that the observed suppression is not "too much help". The ability to generate specific suppressor cells in vitro should be of great benefit in determining the factors that regulate their appearance in vivo.