Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). II. Kinetics of the production and characterization of the producer

Kinetics of the production of a stimulated T cell-derived inhibitory factor for cellular DNA synthesis (STIF) (1) from Con A-stimulated SD rat spleen cells, and the cells involved in the production of the factor, were examined comparatively with those of interleukin 2 (IL 2) and interferon (IFN). STIF activity in the culture supernatants reached a plateau 24 hr after the culture, and the plateau level was maintained during an additional culture for 72 hr. Characterization of the cells involved in STIF production by means of negative selection of unfractionated or nylon-fractionated spleen cells with anti-rat T cell serum or monoclonal antibodies plus complement revealed that the cells are nylon-nonadherent T cells bearing a suppressor T cell marker. The nylon-nonadherent T cell population did not require additional macrophages for STIF production. In vivo pretreatment of rats with cyclophosphamide (25 to 100 mg/kg) reduced or abolished the production of STIF from the Con A-stimulated spleen cells of the rats. The STIF production from Con A-stimulated spleen cells was inhibited by the addition of indomethacin at 10 ng/ml, indicating a regulatory role of prostaglandin(s) in STIF production. The above characteristics distinguish a T cell subset for STIF production from those for IL 2 and IFN production.     

Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). I. Isolation and characterization

A lymphokine inhibitory for cellular DNA synthesis (termed STIF) was isolated from the culture supernatants of concanavalin A (Con A)-stimulated SD rat spleen cells. STIF inhibited the DNA synthesis of mouse bone marrow cells as well as mouse leukemia cells. STIF has an apparent m.w. of 45,000 to 50,000 and is separable from IL 2, m.w. 20,000 to 25,000, by Sephacryl S-200 gel filtration, but not from immune interferon (IFN) having the same m.w. as STIF. Con A-Sepharose chromatography of the fraction containing STIF and IFN could separate these lymphokines into Con A-unbound and Con A-bound fractions, respectively. Further fractionation of the STIF fraction by DEAE-Sephadex A-50 or Mono Q-FPLC anion exchange chromatography indicated that the STIF fraction contained two components of STIF activity, both showing the same pI value (5.1 to 5.6) on flat-bed isoelectric focusing. STIF was characterized as a sugar-free lymphokine of trypsin-sensitive protein nature.     

Characterization and partial purification of a specific interleukin 2 inhibitor

To investigate the mechanisms that regulate the action of interleukin 2 (IL 2) and possibly limit its activity, we screened supernatants of mouse spleen cell cultures which had been stimulated with concanavalin A (Con A) for their ability to inhibit IL 2-mediated proliferation of a cloned IL 2-dependent line. Inhibitory activities with m.w. of 10,000 to 12,000 and 60,000 to 80,000 daltons could be identified in supernatants of both L3T4+ and Ly-2+ T cells, but not in supernatants of Con A or lipopolysaccharide-stimulated B cells. Maximal inhibitory activity was observed after 3 to 4 days of stimulation, and this inhibitory activity could be overcome by increasing the stimulatory concentration of IL 2. When the factor was further purified by reverse-phase high pressure liquid chromatography, it eluted as a single peak with an m.w. of 11,000 to 12,000 daltons which inhibited IL 2- but not IL 3-dependent proliferation. The mechanisms by which this new lymphokine might play in the control of the clonal expansion of T lymphocytes are discussed.     

Regulation of human T lymphocyte mitogenesis by antibodies to CD3

The inhibitory and mitogenic effects of anti-CD3 antibodies (anti-CD3) were examined in cultures of human peripheral blood T cells. Resting T cells required the presence of accessory cells (AC) or phorbol myristate acetate (PMA) to be stimulated by soluble anti-CD3 (OKT3 and 64.1). Anti-CD3 was unable to induce activation of AC-depleted T cells as determined by IL 2 receptor expression, IL 2 production, cell cycle analysis, or detectable DNA synthesis. Although T cell responses to PHA also required AC, far fewer were necessary to generate responses. Anti-CD3 inhibited PHA-stimulated T cell IL 2 production, IL 2 receptor expression and proliferation in partially AC-depleted cultures. Moreover, anti-CD3 was able to inhibit PHA responses when added to culture as late as 24 to 42 hr after the initiation of a 96-hr incubation. Increasing concentrations of PHA reduced the inhibitory effect of anti-CD3 on PHA-stimulated T cell proliferation, whereas IL 2 production remained suppressed. Anti-CD3 linked to Sepharose beads effectively inhibited PHA-stimulated T cell DNA synthesis, indicating that internalization of the CD3 molecule was not required for inhibition of PHA responses. Although inhibition of IL 2 production was a major effect of anti-CD3 in PHA-stimulated cultures, it was not the only apparent inhibitory effect because the addition of exogenous IL 2 could not prevent inhibition completely. Intact AC but not IL 1 also reduced anti-CD3-mediated inhibition of PHA responsiveness, whereas the addition of both IL 2 and AC largely prevented inhibition. Thus, anti-CD3 in the absence of adequate AC signals exerted a number of distinct inhibitory effects on mitogen-induced T cell activation. These results suggest that the CD3 molecular complex may play a role in regulating T cell responsiveness after engagement of the T cell receptor by a number of mechanisms, some of which involve inhibition of IL 2 production.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. II. Requisite role of the monoclonal antibody-defined antigen systems in activation and proliferation of human and rat lymphocytes

A murine monoclonal antibody (MoAb) B3 to rat cells and MoAb HBJ127 and HBJ98 to human cells were found previously to recognize the homologous antigen systems (gp130 in the rat and gp125 in the human) which are predominantly distributed on the cell surface of proliferating cells of the respective species, and the expression of the antigen systems in lymphocytes were indicated previously to correlate closely with the activation and proliferation of the lymphocytes. In this respect, the in vitro effects of these MoAb on the nucleic acid synthesis, cell cycles, or proliferation of stimulated rat and human lymphocytes were examined by use of T cell-enriched and B cell-enriched cell populations. The addition of B3 MoAb to cultures diminished Con A-induced or allogeneic mixed lymphocyte culture-induced rat T cell proliferation and lipopolysaccharide-induced rat B cell proliferation, whereas B31 MoAb, which is unreactive with the gp130 antigen, did not inhibit these lymphocyte responses. Similarly, both HBJ127 and HBJ98 MoAb could inhibit the human lymphocyte proliferation in vitro, although HBJ127 MoAb showed about eight times greater inhibitory activity than did HBJ98 MoAb; HBJ127 MoAb almost completely inhibited the DNA synthesis of the Con A-stimulated lymphocytes at concentrations higher than 13 micrograms/ml. The flow cytometric analysis of the cellular nucleic acid contents with acridine orange-stained cells showed that when B3 MoAb and Con A were simultaneously added to unstimulated rat T cells, progression of the cell cycle was blocked at the G0 to G1 transition. In this culture condition, the appearance of the B3-defined antigen was arrested in a moderate level, as determined with fluorescein-stained cells. On the addition of B3 MoAb to the culture of the T cells after 24-hr Con A stimulation, the MoAb also strongly inhibited the cellular DNA synthesis, but it did not arrest the cell cycle at a certain phase and did not modulate the corresponding antigen. These data suggest that the B3 MoAb-defined antigen on the rat lymphocytes and the HBJ127/HBJ98 MoAb-defined antigen on the human lymphocytes may play some requisite roles not only in lymphocyte activation but also in the subsequent progression through the cell cycle to proliferate.     

Concanavalin A triggers T lymphocytes by directly interacting with their receptors for activation

Anti-HLA-DR antibodies did not inhibit concanavalin A-(Con A) induced T cell proliferation or the generation of suppressor cells capable of inhibiting immunoglobulin synthesis in autologous mononuclear cells after pokeweed mitogen stimulation. Nylon-wool purified T cells (pretreated with anti-HLA-DR antibody and C) exposed to Con A acquired responsiveness to interleukin 2 (IL 2) and were able to absorb this growth factor, whereas nonlectin-treated cells did not respond to IL 2 and could not absorb it. In the presence of interleukin 1 (IL 1), Con A stimulated the synthesis of IL 2 in purified OKT4+ lymphocytes but not OKT8+ cells. However, in the absence of IL 1, neither resting OKT4+ nor Con A-treated OKT4+ cells produced IL 2. Con A by itself did not directly stimulate macrophages to synthesize IL 1, although it could do so in the presence of OKT4+ but not OKT8+ lymphocytes. In addition, Con A induced proliferation of purified T cells provided IL 1 was supplied to the cultures. Cyclosporin A rendered Con A-treated T cells unresponsive to IL 2, made lectin-stimulated OKT4+ lymphocytes unable to respond to IL 1, and inhibited the synthesis of IL 2. Furthermore, this drug abrogated the Con A-stimulated synthesis of IL 1 by acting on OKT4+ lymphocytes and not on macrophages. Finally, cyclosporin-A suppressed the proliferative response and the generation of suppressor T cells induced by Con A. The following are concluded: 1) HLA-DR antigens do not seem to play any role in the triggering of T cells by Con A, and macrophages participate in lectin-induced activation of T cells mainly by providing IL 1. 2) Cyclosporin-A inhibits activation of T cells by interfering with the mechanism by which Con A stimulates T lymphocytes. 3) Con A triggers T lymphocytes by directly interacting with their receptors for activation.     

Ryanodine receptor signaling is required for anti-CD3-induced T cell proliferation, interleukin-2 synthesis, and interleukin-2 receptor signaling

Ryanodine receptors (RyR) are involved in regulating intracellular Ca(++) mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR-selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor-mediated activation events and cytokine-dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)-2 by T lymphocytes responding to mitogenic anti-CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation-induced expression of the alpha chain (CD25) of the high affinity IL-2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL-2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL-2-dependent proliferation of CTLL-2 T cells. These data indicate that RyR are involved in regulating IL-2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR-associated Ca(++) signaling regulates T cell proliferation by promoting both IL-2 synthesis and IL-2-dependent cell cycle progression.     

Glioblastoma cells release interleukin 1 and factors inhibiting interleukin 2-mediated effects

Studies were designed to investigate whether the cellular immunodeficiency state observed in human glioblastoma patients could be due to inhibitory factors released by the tumor cells. Cultured human glioblastoma cells were found to secrete an interleukin 1-like factor (m.w. 22,000) and a factor (m.w. 97,000) that inhibits interleukin 2 (IL 2)-dependent T cell mechanisms. This is demonstrated by its inhibitory effect on the IL 2-induced proliferation of T cell clones and on the induction of alloreactive cytotoxic T cells in mixed lymphocyte cultures. Additionally the glioblastoma cell-derived 97,000-m.w. factor inhibited growth of neuroblasts but not of fibroblasts and thus shares the characteristics of the neuroblast growth inhibition factor (NGIF) previously detected in the supernatant of fetal rat glia cell cultures. If released by glioblastoma cells in vivo, the factor may contribute to impaired immunosurveillance and to the cellular immunodeficiency state detected in the patients.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.     

Inhibition of interleukin 2 production by factors released from tumor cells

In previous studies, cultured melanoma cells were shown to have a suppressive influence on the induction of cytotoxic T cells. Our investigation of the mechanism of these effects revealed that supernatants from certain cultures of melanoma cells contained inhibitory activity against the production of interleukin 2 (IL 2) from phytohemagglutinin (PHA)-stimulated cultures of lymphocytes. These supernatants did not inhibit interleukin 1 production, and also did not inhibit the mitogenic activity of performed IL 2 on IL 2-dependent target cells. Production of the inhibitory activity could be reduced by inhibitors of protein synthesis, but this activity was not inhibited by digestion with the proteolytic enzymes trypsin or pronase. Gel filtration analysis of tumor supernatants revealed that the majority of the inhibitory activity was detected in fractions of approximately 44 and 7 Kd. The addition of supernatants with inhibitory activity to PHA-stimulated cultures of lymphocytes was associated with reduced transition of cells from G1 to S phase of cell division, which could be reversed by the addition of IL 2. Preliminary studies suggest that the release of the factor(s) from melanoma cells may be related to rapid progression of tumor growth in patients, and therefore may be of prognostic significance in tumor host relationships.     

Soluble interleukin 2 receptors are released by long term-cultured insulin-specific T cells transiently after contact with antigen

An enzyme-linked immunosorbent assay was used to quantitate soluble interleukin 2 receptors (IL 2R) released by antigen-dependent, insulin-specific murine T cells into the culture supernatant, as well as cell-associated IL 2R present in cell lysates. IL 2R were released solely after T cell activation by antigen. The release of IL 2R was transient, reaching optimal levels within 72 hr after antigen challenge and gradually declining to background levels thereafter, when the cells were subcultured in IL 2-enriched medium. The decrease in the amount of IL 2R released during culture in IL 2-containing medium paralleled the decrement in cellular IL 2R detected in cell lysates, in cell surface-expressed IL 2R as determined by cytofluorometry, as well as in high-affinity IL 2R. In contrast, IL 2R were constitutively released by an IL 2-dependent T cell clone. Soluble IL 2R might exert an immunoregulatory function by competing with cellular IL 2R for IL 2 binding.     

Monoclonal antibodies that inhibit activation and proliferation of lymphocytes. I. Expression of the antigen on monocytes and activated lymphocytes

It has been shown previously that HBJ127 and HBJ98 monoclonal antibodies raised against a human bladder cancer cell line, and B3 monoclonal antibody against a rat bladder cancer cell line recognized unique cell surface antigens abundant in proliferating cells of the corresponding species. Distribution of the antigens and kinetics of the appearance on human and rat lymphoid cells were examined by means of flow cytometry. Rat macrophages and human peripheral blood monocytes were stained strongly with the B3 and HBJ127 monoclonal antibodies, respectively. With regard to lymphocytes, the expression of the B3-defined antigen on rat lymphocytes was found to have a negative correlation with the maturation of the lymphocytes; the antigen was most abundant in bone marrow cells, less abundant in thymocytes, and least abundant in spleen, lymph node, and peripheral blood lymphocytes. Similarly, the HBJ127-defined antigen on human peripheral lymphocytes was negligible. On activation with Con A or alloantigens, however, both rat and human T lymphocytes did strongly express these antigens. Activation of human or rat B cells with lipopolysaccharide also resulted in the augmented expression of these antigens. Kinetics studies revealed that the antigen expression was readily manifested within 12 hr on activation of rat or human T cells with Con A, was augmented progressively with culture time, and reached a plateau within 36 hr. This somewhat earlier appearance of these antigens apparently preceded the manifestations of the IL 2 receptor (Tac antigen) and the augmented DNA synthesis. The B3-defined antigen on Con A-stimulated T cells was more rich on the lymphocytes in S and G2/M phases than those in G1 phase, and the expression was not significantly affected by the addition of hydroxyurea, but was moderately inhibited by the addition of sodium butylate. These results suggest that the appearance and expression of the B3-defined antigen and probably also those of the HBJ127/HBJ98-defined antigen are correlated with lymphocyte activation and subsequent progression through the cell cycle.     

Inhibition of ongoing myeloma IgE synthesis in vitro by activated human T cells

The ability of activated T cells to suppress ongoing IgE synthesis in vitro was assessed using U266--a human myeloma cell line spontaneously producing IgE. T cells were able to inhibit U266 IgE synthesis in the presence of 10 micrograms/ml of Con A by 41.8% (p less than 0.01). T cells preincubated with 10 or 50 micrograms/ml of Con A and washed extensively were still able to inhibit U266 IgE synthesis in the absence of Con A by 41 and 46% (p less than 0.05 and p less than 0.02, respectively). The decrease in IgE measured was due to inhibition of newly formed IgE by U266, as shown by control experiments with cycloheximide. The inhibition was not due to the simple depletion of nutrient growth factors by the activated T cells, as it did not occur with MOLT-4, T cells that are very active metabolically; nor could it be reversed with medium containing IL 2 and B cell growth factors. Culture supernatants of Con A-activated T cells were also able to suppress IgE synthesis by U266 (21%; p less than 0.01), which suggests that upon appropriate activation, T cells secrete material(s) with inhibitory properties for IgE synthesis. Activation of T cells by mixed lymphocyte culture using puromycin-treated lymphoblastoid cell lines as stimulators also generated T cells that had suppressive activity for IgE synthesis. T cells activated with Con A and subsequently incubated with IgE demonstrated a diminished ability to suppress IgE synthesis. This observation is in agreement with the finding that patients with high levels of IgE may lack isotype-specific suppressor T cells for spontaneous IgE secretion. However, T cells from such patients have so far shown variable loss of IgE suppressive function. These results suggest that human IgE synthesis is susceptible to inhibition at a very differentiated stage, and this may be important in expression of allergic diseases.     

Interleukin-2 mRNA expression, lymphokine production and DNA synthesis in glutathione-depleted T cells

The stimulation of DNA synthesis in lymphocyte populations was previously shown to depend strongly on the intracellular glutathione (GSH) level. Since T cell growth is known to depend on interleukin 2 (IL-2), the experiments in this report were designed to determine whether intracellular GSH depletion may inhibit IL-2 production or the IL-2 dependent DNA synthesis. Our experiments revealed that IL-2 production and DNA synthesis of mitogenically stimulated splenic T cells have indeed different requirements for GSH. The addition of relatively high concentrations of GSH (5 mM) to cultures of concanavalin A (Con A)-stimulated splenic T cells was found to augment strongly the DNA synthesis but inhibited the production of IL-2. Moderate intracellular GSH levels, however, are apparently not inhibitory for IL-2 production, since intracellular GSH depletion by cysteine starvation or by graded concentrations of DL-buthionine sulfoximine (BSO) had virtually no effect on IL-2-specific mRNA expression and the production of T cell growth factor (TCGF). The DNA synthesis activity, in contrast, was strongly suppressed after GSH depletion with either method. As in cultures of splenic T cells, GSH depletion had no substantial effect on the induction of IL-2 mRNA and TCGF production in several mitogenically stimulated T cell clones. Taken together, our experiments suggest that complex immune response may operate best at intermediate GSH levels that are not too high to inhibit IL-2 production but sufficient to support DNA synthesis.     

Activation of cytotoxic T lymphocytes requires at least two spleen cell-derived helper factors besides interleukin 2

The dependency of induction of T cell cytotoxicity on lymphokines was studied. 1 X 10(5) nylon wool-purified thymic lymphocytes or 10(4) spleen cells were cultured with TNP-haptenated syngeneic UV-irradiated spleen cells in the presence of a variety of lymphokine preparations. Concanavalin A-induced spleen cell supernatants mediated strong cytotoxic responses in this system. Three other preparations, namely, a partially purified IL 2 preparation from PMA-stimulated EL-4 thymoma cells, a Con A-induced spleen cell supernatant that was absorbed with an IL 2-dependent cell line, and a Con A-induced supernatant that was dialyzed at pH 2 were all ineffective in mediating a cytotoxic response. In reconstitution experiments, cytotoxic responses were only obtained when either the absorbed preparation or the pH 2-treated preparation was mixed with the IL 2 preparation from EL-4 cells. No reconstitution occurred after mixing of the absorbed with the pH 2-treated preparation. pH 2 treatment of the absorbed preparation did not abolish its synergistic effect when added to the IL 2 preparation from EL-4 cells. These results led to the conclusion that activation of cytotoxic lymphocyte precursors requires at least two other lymphokines in addition to IL 2. One T cell cytotoxicity-inducing factor (TCF1) remained in Con A-induced supernatants after absorption with IL 2 receptor-bearing T cell line cells. It was pH 2-resistant and was not found in EL-4 supernatants. A second T cell cytotoxicity-inducing factor (TCF2) was pH 2-sensitive and was found in Con A-induced spleen cell supernatants as well as in interferon-free supernatants of PMA-stimulated EL-4 cells. This activity co-purified with IL 2. It was absorbed by the IL 2-dependent T cell line together with IL 2. IL 2 differs from TCF2 since it is pH 2-resistant.     

Role of the transferrin receptor in lymphocyte growth: a rat IgG monoclonal antibody against the murine transferrin receptor produces highly selective inhibition of T and B cell activation protocols

We have produced a new rat IgG monoclonal antibody against the murine transferrin receptor (TR). This antibody (C2F2) exhibits a surprisingly selective pattern of inhibition of murine lymphocyte activation protocols. C2F2 inhibits the mixed lymphocyte reaction (MLR) and the generation of cytotoxic T cells. Interestingly, although interleukin 1 (IL 1)-dependent thymocyte co-stimulatory activity is strongly inhibited by C2F2, interleukin 2 (IL 2)-dependent thymocyte co-stimulation is only marginally reduced. IL 2-dependent growth of CTLL cells is also not inhibited by C2F2. These data suggest that IL 1-dependent helper T cell activation is very sensitive to C2F2-mediated inhibition. Studies with phytohemagglutinin, Concanavalin A, and lipopolysaccharide induced activation also indicate that the inhibitory effects of C2F2 are selective, and T cell activation may be more sensitive to inhibition than B cell activation. Although there is little published information about the functional effects of other rat anti-mouse TR antibodies, the available data suggest that the patterns of inhibition produced by anti-TR antibodies may be individually distinct. Anti-TR antibodies may constitute a new set of highly selective probes for the study of lymphocyte activation.     

Effect of glucocorticoids on the development of suppressive activity in human lymphocyte response to a polysaccharide purified from Candida albicans

The action of glucocorticoids on the proliferative response of human lymphocytes stimulated in vitro by MPPS has been investigated. The effect of Dex was dependent on the time of steroid addition to the cultures. Dex added at the beginning of the culture period inhibited, cell proliferation and IL 1/IL 2 synthesis, although not completely. However, a delayed addition of 24 to 48 hr resulted in an enhancing effect on cell proliferative responses that was maximal at day 4. The effect of Dex on T suppressor cell activity was then investigated. Dex added 1 day before the appearance of suppressor cells resulted in a marked decrease or disappearance of the suppressive activity. Moreover, primed T lymphocytes treated with Dex in the presence of exogenous IL 2 enhanced the proliferative responses of fresh autologous PBMC stimulated by MPPS. Taken together, our data suggest that glucocorticoids inhibit the differentiation of T suppressor cells and that IL 2 is unable to reverse this inhibitory effect.     

Expression of interleukin 1 receptors on human peripheral T cells

The expression of interleukin 1 receptors (IL 1R) on human peripheral T cells was studied by the binding assay with 125I-labeled recombinant human interleukin 1 (IL 1) alpha and IL 1 beta and by the flow cytofluorometry with the fluorescein isothiocyanate (FITC)-conjugated IL 1 alpha. Peripheral blood lymphocytes expressed only few IL 1R without any stimulations. When they were stimulated with concanavalin A (Con A), IL 1R-positive cells began to increase by 4 hr, reached the maximum level at 48 hr, and then gradually decreased. The kinetics of the expression of IL 1 alpha R and IL 1 beta R showed the same pattern. Furthermore the binding of 125I-labeled IL 1 alpha to IL 1R on T cells was inhibited by the addition of either cold IL 1 alpha or IL beta, but not by interleukin 2 or interferons. The similar results were observed in the binding of 125I-labeled IL 1 beta. These results suggest that IL 1R on human peripheral T cells reactive for IL 1 alpha and IL 1 beta were identical. By Scatchard plot analysis, the numbers of IL 1R were estimated as 40 and 350 molecules per cell before and after Con A stimulation, respectively, and their Kd values were 3.1 X 10(-10) M and 2.8 X 10(-10) M. When purified T cells alone were stimulated with Con A, IL 1R were only marginally expressed. However, by the addition of monocytes, IL 1R were expressed on T cells in a dose-dependent manner. The maximum response was induced in the presence of 10% monocytes. The maximum IL 1R-positive T cells were approximately 30% by the detection of the flow cytofluorometry with FITC-conjugated IL 1 alpha. This enhancing activity of IL 1R expression on T cells by monocytes was inhibited by the addition of an anti-HLA-DR antibody or by the treatment of monocytes with the anti-HLA-DR antibody and complement. Furthermore T cell proliferative responses induced with IL 1 and Con A were also enhanced by the addition of HLA-DR-positive monocytes. These results suggest that IL 1R are expressed as the result of monocyte-T cell interaction in the early stage of T cell activation, and the expression of IL 1R on T cells and the responsiveness of T cells for IL 1 require the accessory function of HLA-DR-positive monocytes.     

Regulation of IFN-gamma induction in human peripheral blood cells by exogenous and endogenously produced interleukin 2

Interferon (IFN)-gamma production, stimulated by the addition of exogenous interleukin (IL) 2, T cell mitogens, or tuberculin purified protein derivative (PPD) was studied in cultures of separated human mononuclear cells or unseparated peripheral blood leukocytes (PBL). IFN-gamma was induced by the addition of IL 2 to cultures of otherwise unstimulated cells. The minimal concentration of exogenous IL 2 required to cause a reproducible stimulation of IFN-gamma was about 10 U/ml, i.e., approximately 50 times the minimal concentration required to stimulate proliferation in an IL 2-dependent murine cytotoxic T cell line. Approximately 500 to 1000 IL 2 U/ml were required to produce maximal stimulation of IFN-gamma production in otherwise unstimulated cultures. Monoclonal antibody anti-Tac, specific for an epitope associated with the IL 2 receptor (IL 2 R), inhibited IFN-gamma induction by exogenous IL 2 less strongly than induction by phytohemagglutinin (PHA) or concanavalin A (Con A). The highest degree of inhibition was exerted by anti-Tac on IFN-gamma production stimulated with PPD. Stimulation of IFN-gamma induction by exogenous IL 2 and the inhibitory action of anti-Tac on IFN-gamma production were also seen in cultures of irradiated (2000 R) cells. Treatment of cells with subinducing doses of Con A or phorbol myristate acetate increased IFN-gamma induction by exogenous IL 2. Taken together, the data suggest that endogenously generated IL 2 is a major mediator of IFN-gamma induction in PBL cultures stimulated with antigens or T cell mitogens.