Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity.

Human blood mononuclear leukocytes stimulated with toxoplasma antigen, concanavalin A, mezerein plus lentil lectin, or staphylococcal enterotoxin A secreted a factor (macrophage-activating factor, or MAF) that enhanced the capacity of human macrophages to release H2O2 and to kill toxoplasmas. The same lymphoid supernatants contained IFN gamma but not IFN alpha or IFN beta. The MAF activity of six of seven unfractionated supernatants was completely eliminated by a monoclonal antibody that neutralizes IFN gamma, and MAF in the remaining supernatant was almost completely neutralized. Native IFN gamma partially purified by two independent protocols to specific activities of 1 X 10(6) and 10(7) U/mg protein was enriched in MAF activity at least as much as in antiviral activity. The capacity of macrophages to secrete H2O2 after incubation in partially purified native IFN gamma (mean peak stimulation, 8.8-fold) was greater than with unpurified lymphokines (3.8-fold) and sometimes equaled or exceeded the capacity of freshly harvested monocytes. The MAF activity of the partially purified native IFN gamma preparations was abolished by monoclonal anti-IFN gamma. Finally, IFN gamma of greater than 99% estimated purity was isolated (at Genentech, Inc.) from bacteria transformed with the cloned human gene for this lymphokine. Recombinant IFN gamma had potent MAF activity, stimulating the peroxide-releasing capacity of macrophages an average of 19.8-fold at peak response and enhancing their ability to kill toxoplasmas from 2.6 +/- 1.3% for untreated cells to 54 +/- 0.4% for treated cells. Attainment of 50% of the maximal elevation in peroxide-releasing capacity required a geometric mean concentration of 0.1 antiviral U/ml of recombinant IFN gamma, which is estimated to be approximately 6 picomolar for this preparation. Peroxide secretory capacity and toxoplasmacidal activity of macrophages peaked 2-4 d after exposure to IFN gamma. Peroxide-secretory capacity remained elevated during at least 6 d of continuous exposure, but the effect of IFN gamma was reversed within about 3 d of its removal. Activation was usually but not invariably accompanied by characteristic changes in cell morphology. Thus, IFN gamma activates human macrophage oxidative metabolism and antimicrobial activity, and appeared to be the only factor consistently capable of doing so in the diverse LK preparations tested.

Induction of cytotoxic T cell precursors in vivo. Role of T helper cells.

Strain AS rats respond with two populations of cytotoxic T lymphocytes to stimulation in vitro by the major histocompatibility complex (MHC)-incompatible strain HL rat tumor (HL-A2T2). One is specific for MHC alloantigens present on both HL-A2T2 and normal HL targets, the other is tumor specific. The activation of these killer cells requires helper T lymphocytes. The tumor-specific helper cells depend on syngeneic radioresistant accessory cells to present the tumor antigens in an immunogenic form. The appropriate helper-accessory cell interaction results in the production of soluble factors which then induce the maturation of precursor cells into effective killer cells. Studies with a procedure for inducing negative selection of T cells in vivo showed that short-term exposure to HL-A2T2 tumor induced selection only for TH but not cytotoxic T lymphocyte precursors (CTLp). Simultaneous injection of supernatants from concanavalin A-activated spleen cell cultures, however, did produce selection of CTLp. These and other findings suggest that under normal circumstances in vivo, both signals (recognition of antigen and acceptance of maturation factors) are provided in the vicinity of an antigen presenting macrophage-like accessory cell.

Factor-dependent in vitro growth of human normal bone marrow-derived basophil-like cells.

A factor(s) present in supernatants from lectin-stimulated peripheral blood mononuclear cells promoted the production of basophil-like cells in liquid cultures of normal human bone marrow cells. The cultured basophil-like cells had lobulated or round nuclei, and the cytoplasmic granules stained metachromatically with toluidine blue and azurophilic with Giemsa. 20% of the metachromatically staining cells were peroxidase positive but not positive for nonspecific esterase. The histamine content was 0.5-2 pg/cell. The basophil-like cells released histamine upon challenge with calcium ionophore A23187 but not with compound 48/80. They also released histamine with anti-IgE when passively sensitized with human myeloma IgE. The development of basophil-like cells was promoted in a dose-dependent fashion by a factor(s) in the conditioned medium. Blocking of cell proliferation with hydroxyurea or X irradiation inhibited the development of basophil-like cells. The production of the factor was dependent on the presence of T cells. The factor was different from interleukin 2 and its molecular weight was estimated to be 25,000-40,000 by gel filtration on a Sephacryl S-200 column. Thus, human basophil-like cells derived from normal bone marrow cells can grow and differentiate in vitro under the regulation of T cells.

Helper signals in the plaque-forming cell response to protein-bound haptens.

We have demonstrated the ability of a series of murine T cell hybridomas to deliver an antigen-specific, B cell I-region-restricted helper signal in the generation of specific PFC responses to protein-bound haptens. With some hybridomas the elicitation of optimal PFC responses required the addition of nonspecific factors provided by culture supernatants of concanavalin A-stimulated (Con A SN) spleen cells. Using hapten-primed B cells depleted of both T cells and macrophages (Mphi) we have now demonstrated a requirement for three nonspecific factor preparations to substitute for spleen Con A SN in the elicitation of optimal PFC responses. The first preparation was the interleukin 1 containing culture supernatant of the Mphi tumor cell line P388D1, the second the interleukin 2 (IL-2) and B cell growth factor containing Con A SN of the T cell hybridoma FS6-14.13, and the third, the gamma interferon containing Con A SN of the T cell hybridoma FS7-20.6.18. The P388D1 and FS6-14.13 factor preparations were most effective when added at the initiation of culture, while the FS7-20.6.18 factor preparation was most effective when added at 24 h of culture. The activity of FS6-14.13 Con A SN was depleted by incubation with the IL-2-dependent T cell line HT-2. The activity of FS7-20.6.18 Con A SN was abrogated by incubation at pH 2. The results suggest that the generation of PFC responses to protein-bound haptens require at least three nonspecific factors in addition to an antigen/Ia specific helper signal.

Murine gamma interferon activates the release of a macrophage-derived Ia-inducing factor that transfers Ia inductive capacity.

In this report we demonstrate that when the murine macrophage tumor cell line P- 388D1 is incubated for 48-72 h with either concanavalin A-stimulated rat spleen cell supernatant or cloned murine immune interferon (IFN-gamma), the cultured cells release a cell-free factor activity that in turn induces the cell surface expression of Ia antigen on the murine monocyte cell line WEHI-3. This IFN-gamma-stimulated, Ia-inducing activity cannot be blocked with an anti-IFN-gamma heteroantiserum that does block the induction of Ia expression on WEHI-3 by both cloned murine IFN-gamma and rat Con A supernatant. The Ia-inducing factor ( IaIF ) generated from P- 388D1 after stimulation by IFN-gamma does not demonstrate any antiviral activity. The P- 388D1 -derived IaIF is not shed plasma membrane Ia glycoprotein molecules, as demonstrated by the inability of the active component to bind specifically to an anti-I-Ad affinity column or to a protein A column after the active supernatant is first treated with an excess of anti-I-E/Cd,k monoclonal antibody.

Decreased T lymphocyte migration in patients with malignancy mediated by a suppressor cell population.

The migration and concentration of lymphocytes at sites of antigenic challenge are an integral part of the expression of delayed cutaneous hypersensitivity, as well as of tumor and graft rejection. In this study, we have analyzed the migration of T lymphocytes from patients with malignancy. We used casein and concanavalin A (Con A)-stimulated mononuclear cell supernatants to stimulate T cell locomotion. Peripheral blood T lymphocytes from 30 patients with established malignancy, 10 patients with indolent malignancy or benign tumor, and 42 normal adult controls were tested. Data are expressed as a migration index (MI), which represents the difference in micrometers between the distance migrated in response to a stimulus and the distance migrated in response to media alone. We observed a marked depression in casein-stimulated T lymphocyte migration in patients with established malignancy (mean MI +/- 1 SD = 17.0 +/- 9 microns) as compared with normal adult controls (mean MI +/- 1 SD = 35.3 +/- 10 microns). Similar results were observed with migration in response to Con A supernatants. T cells from patients with established malignancy had a mean MI of 5.8 +/- 4 microns to Con A supernatants as compared with 24.5 +/- 5 for controls. This depressed migration was apparent both in the distance that cells migrated and in the number of cells that migrated into the membrane. Of 10 patients with indolent malignancy or benign tumor, T cell migration in 8 was not significantly decreased as compared with controls. When we mixed equal concentrations of normal control T lymphocytes with T lymphocytes from patients with cancer and added the mixture directly to the upper compartment of the chemotaxis chamber, the response of the normal T cells to casein was inhibited by an average of 48%. We observed inhibition of this migration of normal cells when we added as little as 10% of patient cells to normal cells. When we mixed normal control T lymphocytes from different donors and added them directly to the upper compartment of the chemotaxis chamber, T lymphocyte migration in response to casein was not significantly altered. If T cells from patients with cancer were cultured overnight, the suppressive effect on lymphocyte locomotion was lost. Our results indicate that there is a population of T lymphocytes in patients with cancer that suppress normal T lymphocyte migration. This suppressor activity may partially explain the subversion of immunosurveillance in established neoplastic states, as well as the defective inflammatory reaction to intradermal injection of antigen observed in many patients with malignancy.

Autocrine stimulation by prolactin of hormone-responsive breast cancer growth in culture.

Evidence obtained in human breast cancer cell lines in culture suggests that estradiol stimulates the synthesis of secretory proteins which may, in turn, mediate its mitogenic effect. We questioned whether a similar mechanism could mediate the growth-promoting effect of PRL in the N-nitrosomethylurea-induced rat mammary tumor grown in soft agar, where PRL exerts a dose-dependent colony-stimulating effect. Conditioned medium obtained from PRL-treated tumors, but not from control tumors, was found to exert a significant dose-dependent colony-stimulating effect when added to N-nitrosomethylurea-induced mammary tumors plated in soft agar under serum-free medium conditions. The growth-promoting action of conditioned medium from PRL-treated tumors was abolished by pretreatment with heat, trypsin, and Concanavalin-A, suggesting the possible glycoprotein nature of the oPRL-induced growth factor(s). These results provide support for the novel hypothesis that estradiol and PRL may support the growth of hormone-responsive breast cancer through a similar mechanism.

Regulation of plasminogen-activator in mastocytoma cells by lymphokines.

Culture supernatants from mitogen-stimulated splenocytes were found to stimulate protease production in P815Y mastocytoma cells. Such supernatants increased cell-associated plasminogen activator levels in a dose-dependent fashion, and under serum-free conditions. In contrast to peritoneal exudate cells, tumor-cell plasminogen activator was not enhanced by the mitogen ConA alone. The tumor cell line P815Y may, thus, be used as a homogeneous cell source for the quantitation of lymphocyte factors which activate or inhibit plasminogen activator activity.

Killer-cell lines derived from mouse thymus, resembling large granular lymphocytes and expressing natural killer-like cytotoxicity.

Murine thymus cells were maintained in vitro with supernatant-factors derived from Concanavalin-A-stimulated spleen cells. After an initial phase of vigorous proliferation, large granular cells (GC), which were not observed in fresh thymus cell preparations, appeared in these cultures. GC, derived from C3Hf/Tif-, BALB/c-, and C57BL/10-thymus cultures, could be slowly expanded and have been maintained as increasingly homogeneous (oligoclonal) lines for up to six months. During this time, other types of thymus cells died or were diluted out. Thymic GC differ functionally and histochemically from macrophages and mast cells. They do not phagocytize zymosan particles, bind opsonized SRBC, express nonspecific esterases or contain detectable amounts of histamine. GC share many features with natural killer (NK) cells and large granular lymphocytes (LGL). One morphologically representative line (C3Hf/Tif) had the following surface phenotype: Thy-1+, Lyt-1-, Lyt-2-, H-2K+, I-A-, asialo Gm1+. GC bind peanut agglutinin (PNA) on their surface and contain azurophilic granules. These cytoplasmic granules are considerably larger than those in LGL. Cells of a GC line derived from mouse strain C3Hf/Tif (H-2k) lysed the NK-sensitive YAC-1 (H-2a) and EL-4 (H-2b), but not the NK-insensitive P815 cells.

Induction of Ia and H-2 antigens on a macrophage cell line by immune interferon.

Recent work from a number of laboratories has suggested that Ia antigen expression on macrophages can be modulated by soluble factors released by T cells after antigen or mitogen activation. The murine macrophage tumor cell line, WEHI-3, provides an in vitro system for the study of this type of lymphokine regulation of Ia antigen expression. Previous work from our laboratory has shown that supernates from Concanavalin A-activated rat or mouse spleen cells (Con A sup) can stimulate increased levels of biosynthesis and cell surface expression of both Ia and H-2K,D antigens on this cell line. The experiments described in this report were designed to identify the regulatory factor responsible for this inducing activity, and in particular to determine whether the inducing factor present in crude supernatant fluids is immune interferon (IFN-gamma). These experiments show that: 1) murine IFN-gamma, produced free of other lymphokines by recombinant DNA technology and DNA-mediated gene transfer, can induce Ia antigen expression and increase H-2 antigen expression on WEHI-3 cells; 2) the biochemical characteristics of partially purified inducing factor from crude supernate fluids are similar to those previously described for murine IFN-gamma by other investigators; and 3) both crude Con A sup and IFN-gamma-containing supernates have inducing activity and antiviral activity which exhibit very similar dose-related responses. These results support the conclusion that the previously described ability of Con A sup to regulate MHC antigen expression on WEHI-3 cells is due to the activity of immune interferon in those supernates.

Changes in concanavalin A agglutinability during development of the inner cell mass and trophoblast of mouse blastocysts in vitro.

Mouse blastocyst cultures were analysed for Concanavalin A (Con A) agglutinability by microhaemadsorption methods and for Con A binding capacity with Rhodamine-Con A stain. The inner cell mass, which was not agglutinable in the early culture stages, became agglutinable when it started to develop. The trophoblast, which was initially agglutinable, lost this property as the cells matured. There was no apparent correlation between changes in agglutinability and capacity to bind Rhodamine-Con A. The pattern of change in Con A agglutinability which characterized development of the inner cell mass and trophoblast is consistent with an interpretation that agglutinability was related to the migratory activities of these cells. The loss of agglutinability associated with trophoblast maturation may have been due to alterations in Con A receptor accessibility.

Interleukin 2 is not sufficient for the continuous growth of cloned NK-like cytotoxic cell lines.

Interleukin 2 (IL 2) has been strongly implicated as the agent responsible for the continuous growth of T cell lines in vitro. In the present study we confirmed that IL 2 alone could support the growth of a widely used cytotoxic T cell line. In contrast, we found that IL 2 was not sufficient to support the long-term growth of cloned NK-like cytotoxic lymphocyte cell lines. Whereas such lines would grow indefinitely in concanavalin A-induced mouse spleen cell supernatant, they would only grow for short periods (2 to 3 days) in the IL 2-containing supernatant of phytohemagglutinin-stimulated LBRM-33 tumor cells, or in IL 2 partially purified from spleen cell or LBRM-33 supernatants. The addition of concanavalin-A or interferon (type beta or gamma) to these supernatants did not improve growth. By contrast, the NK-like cells proliferated equally well in a short-term (24-hr) assay, irrespective of the source of IL 2 (spleen or LBRM-33 supernatant, or partially purified IL 2). Furthermore, the NK-like cells readily depleted IL 2 from the medium, either during growth at 37 degrees C or by absorption at 4 degrees C. It is concluded that at least some cytotoxic cell lines require both IL 2 and other, as yet unidentified, spleen cell-derived factors for long-term growth.

In vitro induction of cytotoxic polymorphonuclear leukocytes by supernatant from a concanavalin A-stimulated spleen cell culture.

Rat polymorphonuclear leukocytes (PMN) pretreated with supernatant of a spleen cell culture stimulated with concanavalin A (Con A sup) inhibited 3H-thymidine uptake of various syngeneic, allogeneic, and xenogeneic tumor cells. Con A sup-treated PMN were cytostatic not only to tumor cells but also to embryonal fibroblasts and normal Con A blasts. Stimulated PMN also killed some tumor cells, as shown by 3H-uridine release assay. Cytostasis by Con A sup-treated PMN began at a very early phase of incubation, but in the cytolysis assay, incubation for more than 12 hr was required to obtain significant killing. The time required for activation of cytostatic PMN by Con A sup was short (5 min was enough) if high titers of Con A sup were used for activation. PMN-stimulating activity of Con A sup was not abrogated by treatment at 60 degrees C for 30 min or by change of pH from 1 to 11. The mechanisms of cytotoxicity by Con A sup-treated PMN are discussed.

Antigenically activated helper T cells are required as stimulator cells for optimal activation of cytotoxic T lymphocytes under conditions of limiting helper factors.

This paper deals with the question of how antigenically activated helper cells interact with cytotoxic T-lymphocyte (CTL) precursor cells in an environment where helper factor is limiting. Experiments in culture systems with limiting concentrations of helper factor indicated that the (optimal) activation of CTL required antigenically activated helper T cells as stimulator cells. These experiments were performed partly in macrocultures which contained prostaglandin E2 (PGE2) and partly in microcultures with small numbers of responder cells and without additional helper factors. The results showed that a strong activation of CTL against TNP-haptenated syngeneic or semiallogeneic cells occurred only if the cultures contained TNP-haptenated stimulator cells from euthymic but not athymic donors and if the haptenated stimulator cells were exposed to allogeneic determinants. Moreover, combinations of F1-hybrid stimulator cells and parental responder cells generated no substantial cytotoxic responses against determinants of the other parent, unless the cultures were supplemented with a source of I-region determinants which were foreign to the semiallogeneic stimulator cells. Strong responses against haptenated syngeneic or semiallogeneic stimulator cells were obtained, however, when helper factors were added to the cultures. It was concluded that our cultures with limiting concentrations of helper factors required a close proximity between helper T cells and CTL precursor cells; and this proximity was obviously provided by the receptors of the CTL precursor cells with no detectable contribution from the helper-T-cell receptors. Allogeneically activated helper T cells in the responder cell population or in a second irradiated spleen cell population which did not bear the target antigens delivered no substantial helper effect to the CTL precursor cells under test.

Antigen-reactive cloned helper T cells. I. Unresponsiveness to antigenic restimulation develops after stimulation of cloned helper T cells.

Murine helper T lymphocyte (HTL) clones reactive to ovalbumin (OVA) were maintained in continuous culture in vitro. Clones were propagated by weekly stimulation in the presence of irradiated splenic filler cells, antigen, and supernatant fluid (SF) containing IL 2. By varying the quantity of these reagents in cultures of HTL cells, the reactivity to antigen of the cloned cells was altered markedly. After stimulation by antigen or SF, HTL clones became profoundly unresponsive to antigenic restimulation. Cells remained unresponsive for 2 to 9 days after stimulation, depending on the culture conditions that were chosen for their maintenance. The addition of SF containing a high concentration of IL 2 prolonged the duration of unresponsiveness by 3 days, and the presence of a non-T splenic filler cell increased the period of unresponsiveness by an additional 4 days. The use of a high concentration of OVA in cell cultures also prolonged the time of unresponsiveness. The results described here demonstrate that the response to antigen of HTL cells is down-regulated after stimulation, and appears to be correlated with exposure to SF that contains IL 2.

Role of interleukin 1 in antigen-specific T cell proliferation.

The role of interleukin 1 (IL 1) in human antigen-specific T cell proliferation was examined. Nylon wool-purified T cells proliferated in the presence of autologous monocytes (Mo.) pulsed for 18 h with tetanus toxoid (TT) antigen (Mo.TT). Irradiation of Mo.TT with ultraviolet (UV) light (72 J/m2) abolished their capacity to support T cell proliferation and drastically reduced their capacity to secrete IL 1 after stimulation with Staphylococcus albus. The defect in antigen presentation induced by UV irradiation of Mo.TT was reversed in a dose-dependent manner by the addition of two different preparations containing human interleukin 1 (IL 1). The first preparation consisted of supernatants of Mo. stimulated with Con A for 18 hr and in which Con A activity was blocked by alpha-D-methyl-mannoside (Mo.-Con A-Sup). The second preparation consisted of human IL 1 partially purified from supernatants of human peripheral blood mononuclear cells stimulated with S. albus. This IL 1 copurified with human leukocyte pyrogen (LP) and was termed IL 1/LP. Both IL 1-containing preparations enhanced the response of C57BL/6 mouse thymocytes to phytohemagglutinin. A rabbit antibody to human IL 1/LP inhibited the capacity of T cells to proliferate in response to Mo.TT and inhibited the capacity of Mo.-Con A-Sup to reconstitute the T cell response to UV-irradiated Mo.TT. IL 1/LP was not necessary for T cells to recognize the immunogenic moiety presented by Mo., because monolayers of UV-irradiated Mo.TT were equivalent to monolayers of unirradiated MO.TT in their capacity to adsorb TT-reactive T cells specifically. Furthermore, the addition of rabbit antibody to IL 1/LP did not interfere with the capacity of UV-irradiated Mo.TT to adsorb TT-reactive T cells. The results obtained in this study indicate that IL 1 is involved in optimal antigen-driven proliferation of human T lymphocytes.

Antigen-specific T helper clones in a nonresponder strain require augmentation for expression of helper activity. Evidence for a possible antigen presentation defect in B cells.

Hen egg-white lysozyme (HEL)-specific Thy-1+, Lyt-1+2- T cell lines and clones were derived from the nonresponder C57BL/6 strain. Although the antigen-specific proliferative response of these T cells in the presence of syngeneic irradiated spleen cells as a source of antigen-presenting cells (APC) was normal, the same cells were incapable of stimulating B cells to secrete antibody in vitro. This deficiency could, however, be corrected by the addition of an excess of normal T cells or a supernatant from concanavalin A-stimulated rat spleen cells. Alternatively, the use of highly cross-reactive ring-necked pheasant lysozyme in the cultures allowed expression of efficient help, ruling out any inherent deficiency in the T cells. The antibody response was specific and required MHC compatibility between the T lines and responding B cells. By using (H-2b X H-2d)F1 B cells and another H-2d-restricted HEL-specific T line, it was shown that only the H-2b-restricted T-B collaboration required exogenous factors, and the H-2d-restricted collaboration did not. Because both proliferative and helper responses are dependent upon MHC-restricted antigen presentation by macrophage-APC and B cells, respectively, these results suggest that the defect in the nonresponder H-2b-restricted T-B collaborative pathway may relate to the inability of B cells to adequately process and present HEL to clonal T cells.

In vitro production of anti-histone antibodies by spleen cells from young autoantibody negative NZB/NZW mice.

Anti-histone antibodies (AHA) are spontaneously produced in NZB/NZW mice as part of their autoimmune disease. IgM AHA are usually not detected until after 4 mo of age, and older female mice switch to the production of IgG AHA. We studied the in vitro production of AHA by spleen cells from young (less than or equal to 12-wk-old) NZB/NZW mice. Despite the absence of elevated serum AHA activity, spleen cells from these mice demonstrated marked spontaneous autoantibody production in culture. In kinetic studies, little in vitro production was detectable after 1 day of culture, and maximal accumulation occurred on day 5. Elevated AHA production was apparent by cells from 2-wk-old NZB/NZW mice, and an age-dependent increase in autoantibody production was also noted. Only AHA of the IgM class were detected in cultures of young spleen cells. The in vitro production of IgM AHA in culture was T cell dependent, depletion of T cells resulting in a 70 to 90% reduction in production, which was corrected by the readdition of T cells. In cultures where both IgM AHA and total IgM secretion were measured, a much greater T cell dependence for AHA production was apparent. The requirement for T cells could also be partially replaced by factors present in concanavalin A supernatant. AHA secretion was induced by lipopolysaccharide by using cells from both NZB/NZW and non-autoimmune mice. Although production was greater with NZB/NZW cells, the difference was much less than that for spontaneous production. Thus, AHA-secreting cells that are dependent on in vitro T cell help are present in young NZB/NZW mice. These studies may help define the mechanisms responsible for selective autoantibody secretion in lupus-like disease.

Carbohydrates on human Fc gamma receptors. Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils.

To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.

A T cell activity that enhances polyclonal IgE production and its inhibition by interferon-gamma.

The addition of concanavalin A-stimulated supernatants of the helper T cell clone, D9.1, to cultures of lipopolysaccharide (LPS)-stimulated T-depleted mouse spleen cells caused more than a 100-fold increase in immunoglobulin (Ig) E production. These supernatants cause a 10-fold to 15-fold increase in IgG1, a fivefold to 10-fold increase in IgA, and a fivefold to 10-fold decrease in IgG3. These effects are optimal when the supernatants are added 1 to 2 days after stimulation with LPS. Cells from mouse strains that normally give little or no IgE response in vivo give normal IgE levels in response to LPS plus the supernatant of Concanavalin A-stimulated D9.1 cells in vitro. The enhancement of both IgE and IgG1 can be completely inhibited by relatively low concentrations of interferon-gamma (IFN-gamma). Both the IgE-enhancing activity and IFN-gamma act directly upon purified B cells.