Possible 110 kDa receptor for interleukin 2 in the chicken

Chicken lymphoblasts were generated from spleen cells which had been incubated with concanavalin A (Con A) for 48 h. Monoclonal antibodies (mAbs) were produced by immunizing mice with 48-h Con A lymphoblasts. These antibodies were used to identify chicken lymphokine receptors on Con A-activated lymphocytes. The cellular enzyme immunoassay (EIA), with lymphoblasts and spleen cells as the target cells, was used to select for specific mAbs. One mAb was found by EIA to have a strong response against the lymphoblasts but weak against resting spleen cells. By immunofluorescence staining, it reacted strongly with lymphoblasts and weakly with resting spleen, erythrocytes, and bursal cells. This mAb was also shown to inhibit the lymphokine activity present in conditioned medium that was collected from 24-h cultures of Con A-activated spleen cells. By immunoprecipitation, this mAb precipitated a lymphoblast surface antigen with a molecular weight of about 110 kDa. This receptor may be analogous to the putative interleukin 2 γ subunit recently described in human and mouse cell lines.     

Transfer of experimental autoimmune thyroiditis to normal syngeneic mice by injection of mouse thyroglobulin-sensitized T lymphocytes after activation with concanavalin A

Induction of autoimmune thyroiditis in normal syngeneic CBA/J mice was achieved by injection of 72-hr concanavalin A (Con A)-induced lymphoblasts from donor mice which had been immunized with mouse thyroglobulin (MTg) emulsified with complete Freund's adjuvant (CFA). Injection of lymph node or spleen cells, or frequent injection of serum taken from mice with autoimmune thyroiditis failed to transfer appreciable thyroiditis to recipient mice. Selection by treatment of incubated cells with monoclonal antibody and complement revealed that effector cells in Con A-induced lymphoblast populations for the transfer of autoimmune thyroiditis were Thy-1.2+, Lyt-1.1+, and Lyt-2.1- lymphocytes. These results demonstrate that experimental autoimmune thyroiditis can be adoptively transferred into naive mice by activated Thy-1+, Lyt-2- lymphoblasts.     

OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor.

The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation.     

Concanavalin A-induced interferon gamma production by murine spleen cells and T cell lines. Lack of correlation with Lyt 1,2 phenotype

Concanavalin A(Con A)-induced interferon-gamma (IFN gamma) production by resting or preactivated murine spleen cells negatively selected with monoclonal antibodies specific for Lyt 1,2 antigens plus complement (C) and by interleukin 2(IL-2)-dependent T cell lines of different Lyt phenotype was studied. The data show that most of the IFN-gamma produced upon stimulation of resting spleen cells was a product of Lyt 1+2+ cells. Lyt 1-2+ cells were negative for IFN gamma production. When spleen cells that had been preactivated for 3 days with Con A were restimulated with Con A, Lyt 1+2+, Lyt 1+2- as well as Lyt 1-2+ cells produced IFN gamma in a relationship of approximately 5:3:1. In both cases the picture remained unaltered independently when the supernatants were harvested after 1, 3 or 5 days. Furthermore, two IL-2 dependent T cell lines were studied in regard to Con A-induced IFN gamma production. Line 1.3 was Thy 1+, Lyt 1-2+, whereas line 20.9. was Thy 1+, Lyt 1+2-. Both lines produced initially high titers of IFN gamma upon stimulation with Con A. After prolonged passage in vitro, however, they progressively lost the capacity to produce IFN gamma.     

T cell hyperreactivity in obese strain (OS) chickens. Different mechanisms operative in spleen and peripheral blood lymphocyte activation

The enhanced T cell reactivity (ConA hyperresponsiveness and IL 2 hypersecretion) of spleen lymphocytes of Obese strain (OS) chickens with spontaneous autoimmune thyroiditis has recently been shown to be due to a defect in macrophage-derived non-specific suppressor factors that regulate IL 2 secretion and IL 2-promoted T lymphoblast proliferation in normal healthy animals. In the present study, we present several lines of evidence that the increased T cell response of peripheral blood lymphocytes (PBL) of OS chickens is due to mechanisms entirely different from the described dysregulation of splenic T cells: 1) In contrast to the splenic macrophages, peripheral blood monocytes of OS animals are not deficient in the production of IL 2 antagonistic activity (IAA); 2) therefore, cocultivation of PBL from OS and Normal White Leghorn (NWL) chickens in communicating culture chambers did not abrogate the difference in Con A response as previously observed with spleen lymphocytes. 3) Immunofluorescence with a monoclonal antibody (INN CH 16) against the chicken IL 2 receptor revealed enhanced numbers of mitogen activatable T cells in OS PBL but not OS spleen lymphocytes. 4) After prolonged Con A stimulation of PBL, OS and NWL lymphoblasts did not differ from each other in functional aspects. In contrast to this, Con A lymphoblasts from OS spleens exhibited enhanced staining with INN CH 16 in parallel with an increased proliferative response to IL 2. Thus, the primary T cell dysfunction involved in the development of autoimmune disease in OS chickens is the result of at least two separate regulatory defects.     

Inhibition of murine T cell-mediated cytolysis and T cell proliferation by a rat monoclonal antibody immunoprecipitating two lymphoid cell surface polypeptides of 94000 and 180000 molecular weight

The monoclonal antibody methodology was used to identify membrane structures involved in T cell functions. To optimize chances to produce and detect relevant antibodies, a xenogeneic sensitization protocol was utilized and hybridoma supernatants were screened, on functional rather than structural grounds, for their ability to inhibit a given function. The test function was T cell-mediated cytolysis. Mouse cytolytic anti-allogeneic cell populations were used to sensitize a rat, the spleen cells of which were fused to produce hybridomas; the supernatants of the latter were screened for their ability to inhibit mouse T cell-mediated cytolysis in vitro. Several inhibitory antibodies were obtained, one of which, H35-89.9 monoclonal antibody, was studied in more detail. It inhibited specific and concanavalin A (Con A)-mediated cytolysis by T cells, by acting on the effector cells. It reversibly inhibited soluble antigen-, alloantigen and Con A-induced T cell proliferation (but not LPS-induced B cell proliferation), after the production of interleukin 2, by acting on the responder cells. It also had a desagglutinating effect on Con A and LPS blasts and on EL4 cells. It immunoprecipitated from thymocyte membrane preparations two structures of 94000 and 180000 apparent molecular weight, and recognized cell surface determinants on both T and B lymphocytes. Our findings suggest that several antibodies directed against distinct effector cell membrane structures inhibit cytolysis. The case of H35-89.9 monoclonal antibody, which exerts multiple functional effects and immunoprecipitates two membrane polypeptides, raises the problem of the various possible relationships between these structures and functions.     

Role of T cells in the B-cell response: glutaraldehyde-fixed T-helper hybridoma cells synergize with the lymphokine IL-4 to induce B-cell activation and proliferation.

Antigen-unselected helper T-cell hybridomas (Th) which activate normal resting B cells to RNA synthesis and proliferation in the presence of concanavalin A (Con A) have been developed. The response is completely Th cell dependent, and not restricted by the haplotype of the B-cell major histocompatibility complex (MHC). Culture supernatants from the Con A-stimulated Th hybridomas contain interleukin-4 (IL-4) and IL-2, but undetectable level of IL-5. The supernatant alone, however, does not induce B-cell activation or proliferation. Although the Con A-mediated Th cell-dependent B-cell response occurs in an MHC-unrestricted manner, the response of resting B cells can be blocked by monoclonal Ia antibody specific for the surface class II molecules of the responding B cell. The response is also blocked by monoclonal antibody to L3T4. Significant activation and proliferation of resting B cells can also be triggered by glutaraldehyde-fixed Th hybridomas and Con A when exogenous IL-4 is added. The stimulation with fixed Th hybridomas plus IL-4 can be inhibited by monoclonal anti-L3T4 or anti-Ia. These results suggest that maximal B-cell activation requires a direct helper T cell-B cell interaction which depends on availability of Ia on the B cell and L3T4 on the T cell, even when Con A overcomes the requirement for MHC-restricted T-cell recognition. We suggest that this signal, in conjunction with T-cell produced lymphokine IL-4, is responsible for the activation and subsequent proliferation of the B cells which occurs following interaction with T cells.     

Monoclonal antibodies to the murine Ly-2.1 cell surface antigen.

Six monoclonal anti-Ly-2.1 antibodies are described that arose from a single fusion using the spleen from a 129/ReJ mouse immunized with CBA/H thymus cells. The specificity was determined from the strain distribution, and testing of congenic strains where all six monoclonal antibodies detected the Ly-2.1 alloantigen. Furthermore, the antibodies fall into two groups: Group I (four antibodies) detected the expected number of Ly-2+ thymocytes and peripheral T cells, whereas Group II (two antibodies) detected 10% fewer peripheral T cells. Functional studies demonstrated that the Ly-2.1 determinant detected by a Group I antibody (49-31.1) was presented on cytotoxic T cells (Ly-1+2+), on concanavalin A (Con A)-induced suppressor T cells (Ly-1-2+), but absent from helper T cells (Ly-1+2-). One antibody (49-11.1) precipitated a 68,000-75,000 mol.wt glycoprotein, which on reduction yielded 30,000 and 35,000 mol.wt moieties. Thus, by functional, genetic and biochemical criteria these antibodies detected the Ly-2.1 specificity. In addition, a monoclonal, noncytotoxic, IgGl, anti-Thy-1.2 antibody is described.     

Delayed-type hypersensitivity induced in immunodeficient mice with syngeneic modified self antigens: a suggestive model of autoimmune response

Previous studies suggested that trinitrophenyl (TNP)-modified syngeneic red cells induced humoral autoimmune response in mice with defective T cell function but not in normal mice. The ability of modified self antigen to induce autoimmune response in immunodeficient mice was further explored using the delayed-type hypersensitivity (DTH) as an assay system. Mice were immunized with syngeneic TNP-modified spleen cells (TNP-SC) and challenged by syngeneic nonmodified conconavalin A (Con A) or lipopolysaccharide (LPS)-stimulated spleen cells injected into their footpads. The DTH response was assessed 24, 48 and 72 h later by measuring the footpad swelling and was transferred to naive recipients with enriched T cells from TNP-SC-immunized irradiated A mice but not with serum or non-T cells. Adult thymectomized, X-irradiated (250 rds) and cyclophosphamide-treated mice injected with syngeneic TNP-SC generated a DTH response when subsequently challenged with syngeneic lymphoblasts (induced with Con A or LPS) but not when challenged with allogeneic blast cells. In contrast, normal mice treated in a similar manner exhibited a much less significant DTH response. SC incubated 1 to 3 h with Con A failed to elicit the DTH response of immunodeficient mice previously injected with TNP-SC. Both lymphoblasts that were induced in vitro with Con A diluted in fetal calf serum or in normal mouse serum-containing media, and lymphoblasts that were induced in vivo by interleukin 2 elicited DTH responses in X-irradiated, TNP-SC immunized mice. The syngeneic DTH response of the immunodeficient mice injected with TNP-SC was abrogated when they were simultaneously transplanted with syngeneic SC or nylon wool-passed syngeneic SC. If the transplanted splenocytes had been treated with anti-Thy-1 antiserum and complement they failed to abrogate the syngeneic-DTH response of the above mentioned mice. This result suggests that suppressor cells are programmed to control the autoimmune response induced with modified self antigens.     

Detection of Individual Interleukin 4- and Gamma Interferon-Producing Murine Spleen Cells after Activation with T-Cell Mitogens

Murine spleen cells were activated with concanavalin A (Con A), pokeweed mitogen (PWM), or phorbol myristate acetate (PMA) and the calcium ionophore A23187. Cells producing gamma interferon (IFN-gamma) or interleukin 4 (IL-4) could be detected by lymphokine-specific monoclonal antibodies and indirect immunofluorescence. The frequency and kinetics of the lymphokine-producing cells were examined and were approximately the same after stimulation with Con A or PMA and A23187. Thirty hours after activation, 3-9% of the cells produced IFN-gamma. There were few IL-4-producing cells, and the maximal frequency was 1 out of 400 spleen cells 48 h after activation. When the cells were activated with PWM, the frequency of IFN-gamma-producing cells was still high 72 h after culture. The majority of the IFN-gamma-producing cells were CD8+ and expressed receptors for IL-2.     

Effects of K-76COOH (MX-1) on immune response: induction of suppressor T-cells by MX-1.

K-76COOH (MX-1), isolated from the cultured supernatant of a species of fungi imperfecti, Stachybotrys complement nov. sp. K-76, is an inhibitor of the complement component, C5. The effects of MX-1 on various immune responses were investigated. MX-1 enhanced the response of spleen cells to PHA and LPS: MX-1 at 0.01-250 micrograms/ml for PHA and at 10-250 micrograms/ml for LPS. In contrast, it inhibited the response to Con A: MX-1 at 0.01-500 micrograms/ml for spleen cells and at 100-500 micrograms/ml for thymocytes. MX-1 and IL-1 synergistically acted to enhance the Con A response of spleen and thymus cells from which accessory cells and Ia-positive cells had been removed by passing through Sephadex G-10 columns and treating with anti-Ia monoclonal antibody plus complement. T-cells pretreated with MX-1, IL-1 and Con A for 3 days suppressed not only the response of B-cells to LPS but also the production of anti-SRBC antibodies. In addition, MX-1 was found to increase CD8+ T-cells. These results suggest that MX-1 acts on T-cells to induce suppressor T-cells.     

B cells as accessory cells in a Con A response of a T cell clone

Accessory cell (AC) function of B cells was examined in Con A response of a cloned T cell line, 22-9D, which is Thy 1+,L3T4+,Lyt2-,H-2KbDb+ and I-Ab-.22-9D cells produced IL 2 in the presence of Con A without participation of AC. For the initiation of a proliferative response to Con A, the addition of spleen cells or spleen adherent cells was required. B cells as AC were unable to induce the proliferative response. In the presence of culture supernatant of spleen cells stimulated with Con A (CAS), 22-9D cells showed proliferative response to Con A with B cell AC. The response was inhibited by a relevant monoclonal anti-I-A antibody. Although irradiated spleen cells as AC induced IL 2 receptor expression of 22-9D cells in the presence of Con A, B cells were shown to require the addition of unknown factor(s) in CAS, which was suggested to be different from IL 1, IL 2, IL 3, or IFN-gamma, for the induction of the receptor expression on 22-9D cells.     

H-2 and viral haemagglutinin expression by influenza-infected cells; the proteins are close but do not cocap.

The proximity of H-2K and D antigens and influenza virus haemagglutinin (HA) molecules on the surface of infected target cells was assessed by a topographical study using monoclonal antibodies to H-2 and to HA. The effect of pretreatment of fixed, infected cells with excess of one monoclonal antibody on the subsequent binding of a second radiolabelled antibody was measured. Using CBA mouse B lymphoblasts which were paraformaldehyde fixed 5 hr postinfection with influenza virus (A/USSR/90/77), pretreatment with monoclonal antibody 30/3 to H-2Kk and Dk partially blocked (Approximately equal to 37%) the binding of one radiolabelled monoclonal anti-HA antibody (264/2). A different monoclonal IgG (W18/1) directed to the same HA molecule was not blocked by similar pretreatment of cells with the anti-H-2 antibody. Interaction of monoclonal antibodies with their sites is highly specific, and mutual blocking of two antibodies requires very closely located sites even if the antibodies are directed to the same molecule. We therefore have evidence for proximity of H-2 and HA molecules; however, we were unable to demonstrate cocapping of H-2K and D antigens with influenza HA.     

Composite activities on B cells of products released by T cells activated by concanavalin A.

Supernatants from murine spleen cells cultured for 48 h in the presence of 1.0 μg of concanavalin A (Con A) induced polyclonal antibody synthesis in cultures of spleen cells from both normal and athymic mice in the absence of exogeneous antigen. Moreover, the Con A-induced supernatant rescued B cells which had been rendered unresponsive by a tolerogen [haptenated poly(D Glu,D Lys)]. The capacity of the supernatant to induce cell proliferation was studied under both high and low-density culture conditions. In contrast to antibody secretion, proliferation was only detectable in low-density cultures. The Con A-induced supernatant also contained suppressive components, since the primary anti-sheep red blood cell (SRBC) response was markedly suppressed when the antigen added to cultures consisted of SRBC which had previously been used for absorbing the supernatants. Absorbed supernatants displayed enhanced helper activity indicating that only the suppressor component was removable by antigen. The suppressive component was eluted from erythrocytes with ammonium thiocyanate and was itself strongly suppressive when added to cultures with fresh erythrocytes. Furthermore, the suppressive component proved to be highly antigen-specific, as the SRBC-absorbed factor did not affect the response to horse RBC. The results indicate that supernatants from Con A-activated spleen cells contain both helper and suppressor factors, the latter having easily demonstrable antigen specificity. They further suggest that a nonantigen-derived signal is sufficient to trigger both proliferation and antibody synthesis by B cells.     

Mechanisms by which accessory cells contribute in growth of resting T lymphocytes initiated by OKT3 antibody

This study was undertaken to determine how accessory cells (AC) participate in growth of normal resting T cells initiated by anti-T3 monoclonal antibodies. Highly purified peripheral blood resting T cells were obtained by sequentially using three procedures (adherence to plastic surface, adherence to nylon wool columns and treatment with four monoclonal antibodies against antigens on AC and activated T cells plus complement). The assays for T cell growth were carried out at low cell density (10(4) cells/well) and with T cell populations where we could not detect cells bearing OKM1 and Ia antigens. Soluble OKT3 antibody, concanavalin A, recombinant interleukin 2 (IL 2) or purified interleukin 1 (IL 1) alone did not induce proliferation of purified resting T cells. Recombinant IL2 together with soluble OKT3 antibody stimulated significant growth whereas purified IL1 and two distinct preparations derived from AC containing IL 1 activity did not. Nevertheless, purified IL 1 amplified the proliferation of T cells induced by soluble OKT3 antibody in the presence of a small number of irradiated AC (3%). Phorbol myristate acetate (PMA) together with soluble OKT3 antibody activated purified resting T cells to proliferate, but PMA alone had little growth-promoting activity only. Soluble OKT3 antibody did not by itself induce a detectable number of resting T cells to express receptors for IL2 as determined by direct immunofluorescence staining and FACS analysis with monoclonal anti-IL2 receptor antibody. Cloned IL2 or purified IL1 alone did not induce resting normal T cells to express receptors for IL2 either. In contrast, T cells exposed to both soluble OKT3 antibody and IL2 exhibited IL2 receptors. PMA alone stimulated some resting T cells to express IL2 receptors and this response was significantly increased when the drug was used together with soluble OKT3 antibody. Studies were performed with unfractionated mononuclear cells from a donor whose cells respond to OKT3 (IgG2) but not to Leu 4 (IgG1) anti-T3 antibodies. Recombinant IL 2 but not purified IL 1 corrected the defective response to Leu 4 antibody. Finally, OKT3 antibody linked to beads, but not in soluble form, and purified IL1 replaced AC in growth of purified resting T cells. Based on these data I conclude the following: (a) AC participate in growth of resting normal T cells initiated by anti-T3 antibodies through their Fc receptors in two ways, namely, by providing a matrix to favour cross-linking of the T3 complex and simultaneously by secreting IL1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Con-A-activated T Cells Secrete Factors with Polyclonal B-Cell-activating Properties

Concanavalin A induced polyclonal antibody synthesis in normal spleen cells in vitro. Optimal responses were obtained by Con A concentrations lower than those optimal for induction of DNA synthesis. T cells, but not macrophages, were necessary for the effect. Spleen cells from nude mice were not activated, whereas cells from the LPS non-responder stain C3H/HeJ were activated to polyclonal antibody synthesis by Con A. Supernatants from Con A activated spleen cells could by themselves induce polyclonal antibody synthesis in untreated spleen cell cultures, even when Con A had been removed by absorption with Sephadex G-50 and when alpha-methyl-mannoside was present in the secondary cultures. T cells produced the active supernatants, which were competent to induce polyclonal antibody synthesis, but not DNA synthesis, in both H-2-incompatible and compatible strains. When the supernants were absorbed with erythrocyte antigens, they specifically induced an enhanced response, in secondary cultures, to the antigen used for absorption. Possible mechanisms of this specific effect are discussed.     

Monoclonal antibodies that recognize a broad range of mammalian terminal deoxynucleotidyl transferases

Terminal deoxynucleotidyl transferase (TdT) is a useful marker for lymphocyte precursors in the bone marrow and thymus and for lymphoblastic leukemia and lymphoma cells. To simplify and enhance the detection and phenotypic analysis of these cells, we sought to develop monoclonal antibodies to this enzyme. In order to obtain antibodies that bind a variety of mammalian TdTs, mice were immunized with bovine TdT and the hybridoma secretions were screened by immunofluorescence assays on cultured TdT-positive and negative human lymphoblasts. Four monoclonal antibodies which bound specifically to TdT-positive lymphoblasts were characterized in detail. All four antibodies immunoprecipitated the native 60 kd TdT molecule from extracts of TdT-positive human lymphoblasts and bound specifically in immunoblot assays to the 43.8 and 11 kd proteolytic fragments of bovine thymus TdT. To assess whether the antibodies bound to related or distinct epitopes on bovine TdT, we measured the displacement of radiolabeled antibody from the immobilized enzyme by an excess of unlabeled heterologous antibody. These studies revealed that three of the antibodies competed for the same determinant on bovine TdT, while one antibody reacted with a distinct epitope. Antibody binding to either epitope, however, partially inhibited the enzymatic activity of bovine TdT. Specificity for TdT was tested by immunofluorescence and competition radioimmunoassays. In these assays, the antibodies did not stain a variety of known TdT-negative human hematopoietic cells and cell lines. both normal and neoplastic, nor were the antibodies displaced from purified bovine TdT by extracts of these TdT-negative cells. These results confirmed the cross-reactivity of the antibodies with human and bovine TdT. To assess cross-reactivity with TdT from other species, extracts of rabbit, mouse, and rat thymus were prepared and shown to specifically displace the antibodies from bovine TdT. Thus, these antibodies bound to TdT derived from at least five mammalian species. To determine whether these antibodies could be used to detect small subpopulations of TdT-positive cells, mixtures of TdT-positive and negative cells were prepared and stained with fluorescein conjugates of the antibodies. When assayed by flow cytometry, a population of 1% TdT-positive cells was easily detectable. We conclude that these monoclonal antibodies should be useful for the enumeration and analysis of TdT-positive cells in normal and neoplastic hematopoietic tissues from several mammalian sources, including man.     

Interferon-gamma inhibits serotonin release from mouse peritoneal mast cells

Conditioned medium (CM) from concanavalin A-activated mouse spleen cells inhibits antigen-induced 5-hydroxytryptamine (5-HT) release from mouse peritoneal mast cells when added for 24 or 48 h to cell cultures containing monoclonal IgE sensitizing antibody. We present the following evidence that the spleen cell-derived inhibitory factor is interferon-gamma (IFN-gamma): (a) CM from Con A-activated spleen cells and from Chinese hamster ovary (CHO) cells transfected with the murine (Mu)IFN-gamma gene both produced a graded inhibition of 5-HT release when added to peritoneal cell cultures containing IgE and subsequently challenged with antigen; (b) the relative inhibitory potency of the two preparations corresponded to their relative IFN content as determined by antiviral bioassay; (c) the inhibitory activity of spleen cell CM and of CM from IFN-gamma gene-transfected CHO cells was abolished by treatment with a rat monoclonal MuIFN-gamma-neutralizing antibody, i.e. 5-HT release returned to control levels, and (d) a highly immunopurified preparation of recombinant MuIFN-gamma retained the inhibitory activity, and was active at concentrations as low as 2 U/ml. The inhibitory activity of IFN-gamma appeared to be a direct effect of the cytokine on mast cells, since CM generated for different time periods from unfractionated peritoneal cells treated with purified recombinant MuIFN-gamma contained no inhibitory activity other than IFN-gamma itself, as demonstrated by neutralization of the cytokine with monoclonal antibody. Furthermore, purified recombinant MuIFN-gamma was equally active on mast cells enriched by density centrifugation up to 95% purity as on unfractionated peritoneal cells (1%-2% mast cells). Purified recombinant MuIFN-gamma inhibited 5-HT release induced by a range of different cell activators, namely antigen, anti-IgE, compound 48/80 and calcium ionophore A23187. Hence inhibition is not specific to IgE-dependent activation of mast cells, but seems to be directed more generally at the secretory process of these cells.     

Lymphocyte activation: IV. The ultrastructural pattern of the response of mouse T and B cells to mitogenic stimulation in vitro

Thymocytes from cortisone-treated mice (`T' cells), `B' spleen cells (B lymphocytes from thymectomized, irradiated, marrow reconstituted mice) and normal spleen (T + B) cells were examined by electron microscopy after 60 hours stimulation by Concanavalin A (a T cell specific mitogen), endotoxin (B cell specific mitogen), and pokeweed mitogen (which stimulates both T and B cells). Stimulation of T cells by Con A or PWM induced the appearance of lymphoblasts (Type I) and only PWM or endotoxin stimulated B cells developed `plasmablast' features (dilated, vesicular rough endoplasmic reticulum; Type II). A few stimulated B cells also had lymphoblast morphology. Large cells from normal (T + B) spleen stimulated by PWM were heterogeneous consisting of 55–60 per cent plasmablasts and 40–45 per cent lymphoblasts. It was concluded that the ultrastructure of stimulated lymphocytes depended on whether T or B cells were stimulated and not primarily on the mitogen used. In general, the response evoked by mitogens paralleled at the ultrastructural level that induced by antigens. It was also found that multivesicular bodies and glycogen particles occurred predominantly in the cytoplasm of stimulated T cells (lymphoblasts).     

Production of high titers of interferon-gamma by prestimulated murine spleen cells

Concanavalin A (Con A)-induced interferon-gamma (IFN-γ) production by murine spleen cells prestimulated with Con A for different periods of time has been studied. Highest titers of antiviral activity were obtained by restimulation of cells that had been prestimulated with Con A for 3 days. These cells produced up to 30 times more IFN than freshly isolated spleen cells stimulated under the same conditions. In an effort to explain this rise in the capacity to produce IFN-γ, the possibility that it was due to the inactivation of a suppressor cell was excluded. The addition of freshly isolated spleen cells at different concentrations to prestimulated cells did not affect subsequent Con A-induced IFN-γ production. Separation of freshly isolated or prestimulated spleen cells by velocity sedimentation at unit gravity showed that in the latter case most of the IFN-γ was produced by a population of large-sized cells not present in the former population. It was concluded from these experiments that prestimulation of spleen cells with Con A gives rise to a population of large-sized cells that produce, upon restimulation with the same mitogen, much higher titers of IFN-γ than those obtained upon primary stimulation of small resting lymphocytes.