Retrovirus-mediated immunosuppression. I. FeLV-UV and specific FeLV proteins alter T lymphocyte behavior by inducing hyporesponsiveness to lymphokines

Murine splenocytes were used to study the in vitro immunosuppressive effects of UV-inactivated feline leukemia virus (FeLV-UV). FeLV-UV blocks both alloantigen (DBA/2)-induced and Con A-induced proliferation of C57BL/6 splenocytes in a dose-dependent manner. Furthermore, C57BL/6 anti-DBA/2 mixed lymphocyte cultures containing FeLV-UV fail to develop detectable DBA/2-specific cytolytic activity, although FeLV-UV has no effect on the cytolytic activity of preformed C57BL/6 anti-DBA/2 cytolytic T cells (CTL). Disruption of lymphocyte proliferation and CTL generation by FeLV-UV could not be overcome by the addition of exogenous lymphokines. These data suggest that FeLV-UV can interfere with the lymphokine reactivity of alloactivated lymphocytes. In fact, FeLV-UV blocks the lymphokine-induced proliferation of the murine IL 2-dependent cell line CTLL-20. The CTLL-20 cells were subsequently used to study the mechanism(s) by which retroviruses alter T lymphocyte function. Normally, CTLL-20 cells undergo significant proliferation when cultured in EL4 SN, an IL 2-containing culture supernatant from PMA-stimulated EL4 cells. This lymphokine-induced CTLL-20 proliferation is abrogated in a dose-dependent manner by UV-inactivated murine leukemia virus (MuLV-UV), FeLV-UV, and a purified 15,000 dalton viral protein, p15, derived from FeLV. Suppression of CTLL-20 proliferation requires only brief contact (6 hr) with FeLV-UV or with p15, but is most efficient after prolonged (24 hr) contact with these agents. Furthermore, suppression of CTLL-20 proliferation by FeLV-UV and p15 is reversible, because CTLL-20 cells which have been pretreated for 24 hr with FeLV-UV or p15 are equally as efficient at responding to EL4 SN as untreated CTLL-20. Additional studies indicate that CTLL-20 cells continue to remove IL 2 activity from EL4 SN in the presence of suppressive concentrations of FeLV-UV, and that suppressive concentrations of FeLV-UV do not remove IL 2 activity from EL4 SN. This suggests that FeLV does not block CTLL-20 proliferation by absorbing or inactivating IL 2, or by occluding IL 2 receptors, and that T lymphocytes develop an insensitivity to lymphokines after contact with FeLV-UV, which may be caused by a metabolic, rather than an immunologic, defect. Because lymphokines are requisite signals for T cell function, considerable immunosuppression would be associated with acquired lymphokine insensitivity.     

Interleukin 2 enhancement of lymphokine secretion by T lymphocytes: analysis of established clones and primary limiting dilution microcultures

The effect of exogenous interleukin 2 (IL 2) on lymphokine production by T lymphocytes was examined in two systems: the secretion of macrophage-activating factor (MAF) and interferon (IFN) by cloned long-term T cell lines, and a limiting dilution system for estimating the frequency of precursors of MAF-secreting cells in normal spleen. An IL 2-containing, MAF- and IFN-free supernatant from the EL-4 thymoma (EL-4 SN) significantly enhanced release of MAF and IFN by mitogen- or antigen-stimulated, cytolytic or noncytolytic T lymphocyte clones directed against alloantigens or Moloney leukemia virus-associated antigens. Highly purified IL 2 produced equivalent enhancement as EL-4 SN in cultures of alloreactive clones stimulated with concanavalin A. Kinetics experiments showed that EL-4 SN increased both the rate and duration of MAF release by T cell clones. EL-4 SN also increased MAF production when added during restimulation of limiting dilution cultures of positively selected Lyt-2+ and Lyt-2- C57BL/6 splenic T lymphocytes activated against DBA/2 alloantigens. This enhancement resulted in a threefold increase in the apparent precursor frequency of MAF-secreting cells among Lyt-2+ lymphocytes, but did not affect the frequency among Lyt-2- cells. Additional analysis indicated that average MAF production in cultures of Lyt-2-+ cells was sixfold lower than in cultures of Lyt-2- cells, and hence that EL-4 SN allowed detection of a significant proportion of Lyt-2+ cell cultures secreting low levels of MAF. Under these improved conditions, the MAF assay detected the majority of responding Lyt-2+ and Lyt-2- lymphocytes.     

Glucocorticoid inhibition of lymphokine secretion by alloreactive T lymphocyte clones

The effect of glucocorticoids on lymphokine production by T lymphocytes was examined by using long-term alloreactive T cell clones that secreted one or more of the lymphokines interleukin 2 (IL 2), interferon-gamma, macrophage-activating factor (MAF), and colony-stimulating factor when stimulated by an antigen or a mitogen. Production of all of these four lymphokines was inhibited when glucocorticoids were added at physiologic concentrations (10(-8) to 10(-6) M) to clones stimulated with concanavalin A (Con A). Clones were heterogeneous with respect to their sensitivity to glucocorticoid inhibition of MAF production; cytolytic clones were generally more resistant than noncytolytic clones. The glucocorticoid dexamethasone (Dex) and an IL 2-containing supernatant exerted opposing effects on clonal MAF production. Kinetics experiments showed that Dex inhibited MAF production by reducing the rate of secretion without causing a compensatory increase in the duration of secretion, whereas the IL 2 source increased the rate and the total amount of MAF secretion. Dex abrogated the effect of IL 2. Inhibition by Dex was apparent from the earliest time of detectable MAF production (about 4 hr after stimulation) and increased with longer exposure until production ceased (12 to 24 hr). Pre-exposure and removal of Dex before Con A stimulation also inhibited MAF release. Effects of Dex on lymphokine secretion by clones could be dissociated from effects on their growth in response to stimulator cells and IL 2. Factor production by the 16 clones tested was inhibited to some degree. Proliferation, however, by two of these clones (both cytolytic) was unaffected by Dex, whereas proliferation of two noncytolytic clones was strongly inhibited even in the presence of a saturating dose of IL 2.     

Mechanism of lymphocyte activation. II. Requirements for macromolecular synthesis in the production of lymphokines

Reversible inhibitors of protein synthesis, cycloheximide and puromycin, and an irreversible inhibitor of RNA synthesis, actinomycin D, were employed to study the kinetics and types of macromolecular synthetic events required for the production of migration inhibitory factor (MIF) and macrophage activating factor (MAF) by Con A-stimulated lymphocytes. Reversible inhibition of protein synthesis during the first 2 hr of stimulation completely inhibited MIF and MAF production. The same treatment, performed 4 hr after the beginning of the stimulation, had no effect. When the inhibitors of protein synthesis were left in the cultures, a block of lymphokine production was observed when the drugs were added at 6 hr as well as at time 0. In contrast, irreversible inhibition of RNA synthesis at 6 hr was ineffective and only treatment at the beginning of culture blocked lymphokine production. These data suggest that a critical protein is synthesized during the first few hours of stimulation, which is required for subsequent production of lymphokines. After this special early requirement, however, continued protein synthesis is needed for lymphokine production. In contrast, the RNA required for MIF and MAF production seemed to be completely synthesized within 4 to 6 hr of stimulation. The possibility that suppressor macrophages inhibit lymphokine production via modulation of macromolecular synthesis is discussed.     

Alloreactive cloned T cell lines. VI. Multiple lymphokine activities secreted by helper and cytolytic cloned T lymphocytes

Culture supernatants generated by alloantigenic or lectin stimulation of a cloned helper T lymphocyte, designated L2, contain interleukin 2 (IL 2), granulocyte/macrophage colony-stimulating factor (CSF), B cell stimulating factor (BCSF), macrophage (Ia+)-recruiting factor (MIRF), (Ia+)-inducing activity, gamma-interferon, Fc receptor-enhancing activity, macrophage migration inhibitory factor (MIF), macrophage activation factor (MAF), interleukin 3 (IL 3), and a factor responsible for prolonging the synthesis and secretion of the fourth and second components of complement by guinea pig peritoneal macrophages. Erythropoietin was not detected. A spontaneously arising variant of L2, designated L2V, produces much lower quantities of macrophage-stimulating activities, IL 2, and interferon. However, when compared to L2, L2V produces much higher levels of BCSF, equivalent amounts of IL 3, and slightly smaller amounts of CSF. Unlike L2V, a cytolytic clone, designated L3, secretes lymphokines that primarily affect macrophage function. The time course of lymphokine production by L2 cells indicates that for the six lymphokine activities studied there are three different times at which maximal or near maximal levels are reached, as follows: 1) IL 2, 12 to 24 hr; 2) IL 3 and CSF, 24 to 48 hr; and 3) (Ia+)-inducing activity, MAF, and interferon, 48 hr or later. Only IL 2 activity disappears during the 8-day culture cycle. The time course data and the differential production of activities by the three types of lymphocyte clones suggest that at least four terminal effector lymphokine molecules account for the ten biologic activities tested.     

Enhanced response to Con A and production of TCGF by lymphocytes of obese strain (OS) chickens with spontaneous autoimmune thyroiditis

The mitogenic response to Con A and the production of T cell growth factor or interleukin 2 (IL 2) by splenic and peripheral blood lymphocytes of obese strain (OS) chickens with spontaneous autoimmune thyroiditis have been investigated. By using an optimized method with Con A-coated chicken erythrocytes (MRC), lymphocytes of OS chickens were found to exhibit significantly elevated mitogenic responses as compared with cells from either Normal White Leghorn chickens (NWL) or animals of the Cornell C-Strain (CS), from which the OS has originally been developed. This difference was observed throughout ontogeny up to 15 mo of age, and was associated with increased levels of IL 2 activity in the culture supernatants. The elevated responsiveness of OS T lymphocytes was also found to be manifested in the expression of receptors for IL 2, because Con A-stimulated lymphocytes of OS birds were significantly more effective than those from normal controls in absorbing IL 2 activity from conditioned media (CM) of stimulated spleen cells. High concentrations of CM were suppressive in IL 2 assays, signaling the presence of an inhibitory factor(s) in addition to IL 2. An additional indication for defective immunoregulation was that CM from OS lymphocyte cultures showed significantly less of this suppressive activity in comparison with CM of normal (NWL and CS) lymphocyte cultures. Finally, the spontaneous uptake of 125IUdR of embryonic and early post hatching OS spleen lymphocytes was consistently and significantly enhanced. This difference, however, in contrast to the one observed in Con A responses, was found to decrease with age. The data are discussed in view of the contradictory results concerning T cell functions reported for several autoimmune states in mammals.     

Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). III. Effect on cell proliferation and immune responses

Stimulated T cell-derived inhibitory factor for cellular DNA synthesis (STIF), a lymphokine produced from concanavalin A (Con A)-stimulated rat suppressor T cells, was examined for its inhibitory effect on various cultured cells and on in vitro immune reactions. STIF could inhibit the DNA synthesis of a variety of normal and neoplastic cells from rats, mice, and humans in a dose-dependent fashion. Kinetics studies revealed that STIF selectively inhibited cellular DNA synthesis after incubation for 12 hr, but after 36 hr, it also inhibited RNA and protein syntheses. The inhibited cellular DNA synthesis by 12-hr incubation with STIF was recovered after culturing the cells in STIF-free medium. The inhibitory effect of STIF on the DNA synthesis was not blocked by addition of a sugar (alpha-methyl-D-mannoside, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, L-fucose, or L-rhamnose) in culture, as determined by using rat bone marrow cells. STIF inhibited proliferative responses of rat lymphocytes to T cell mitogens, Con A and phytohemagglutinin, and a B cell mitogen, lipopolysaccharide, as well as IL 2-dependent growth of cloned T572 cells. It could also inhibit both blastogenesis and cytotoxic T cell generation in allogeneic mixed lymphocyte reaction. The release of IL 2 from Con A-stimulated T cells was also inhibited by the added STIF in culture, as demonstrated from the finding that IL 2 activity was not detected in the supernatants even after an anion-exchange column chromatography. These results indicate that STIF could inhibit cellular DNA synthesis in a species-unrestricted manner and thus inhibits the proliferation of various normal and neoplastic cells, and that it could also inhibit lectin- or IL 2-dependent T cell proliferation as well as IL 2 production from T cells in in vitro immune reactions.     

Lymphokine regulation of activated (G1) lymphocytes. I. Prostaglandin E2-induced inhibition of interleukin 2 production

PGE2-induced inhibition of the proliferatory response of PHA-stimulated human PBL and Con A-stimulated murine thymocytes was analyzed by flow cytometry. It was found that the activation process (G0-G1a transition) was not influenced by PGE2 over a wide range of concentrations (10(-10) to 10(-6) M), nor was the formation of IL 2 receptors inhibited. Similarly, the viability of human lymphocytes was practically unaltered. In contrast, the IL 2-dependent cell cycle event (G1a-G1b transition), which is required for proliferation, was inhibited in a dose-dependent fashion. The addition of IL 2-containing supernatants to such cultures prevented the PGE2-mediated block in the G1a phase and reconstituted a normal lymphocyte proliferation. Furthermore, lower IL 2 titers were measured in supernatants from PHA-stimulated human PBL treated with PGE2. These findings strongly suggest that PGE2 primarily exerts its inhibitory effect on lymphocyte proliferation through an inhibition of IL 2 production.     

Stages of T cell activation: continued antigen dependence of IL 2-producing cells after IL 2 receptor expression

Alloantigen stimulation leads, within 48 to 72 hr, to the expression of IL 2 receptors (IL 2R) on the surface of most of the helper T cells with specificity for the stimulating antigens. The IL 2R-bearing cells, separated by flow cytometry from 3-day human or mouse mixed lymphocyte cultures, were found by limiting dilution methods to be enriched 10- to 20-fold (compared to IL 2R- cells) for antigen-specific helper T cells detected by IL 2 production. Although these cells have been activated to an IL 2R+ stage, most of them are unable to produce detectable IL 2 unless they are cultured together with the original, activating antigen. Even when IL 2 is supplied, and lymphokine production measured by assay of a second factor, IL 3, the large majority of individual activated helper T cells remain dependent on further antigenic exposure for their continued maturation into lymphokine-secreting effectors. Helper T cells at this early stage of activation can therefore proliferate when given IL 2 alone, but lymphokine secretion involves a second antigen-dependent step.     

Secretion of interleukin 2, macrophage-activating factor, interferon, and colony-stimulating factor by alloreactive T lymphocyte clones

Sixty-four murine alloreactive, cytolytic and noncytolytic , T lymphocyte clones were tested for the production of interleukin 2 (IL 2), macrophage-activating factor (MAF), interferon (IFN), and colony-stimulating factor (CSF). Approximately 90% of both cytolytic and noncytolytic clones secreted MAF and IFN upon antigen or mitogen stimulation. IL 2, in contrast, was only released in detectable amounts by 50% of noncytolytic clones and 50% of a subclass of cytolytic clones in which the proliferation was independent of exogenous IL 2 ("antigen-driven" clones); IL 2-dependent cytolytic clones did not release measurable IL 2. CSF was secreted by approximately 90% of noncytolytic and IL 2-independent cytolytic clones and 40% of IL 2-dependent cytolytic clones. The analysis reported here revealed a strong quantitative correlation between the titers of MAF and IFN released by the clones, suggesting that these two assays may measure the same lymphokine. Although the other activities measured were not directly correlated, a broad association was noted between IL 2 secretion and the production of high titers of MAF, IFN, and CSF. Thus, noncytolytic and IL 2-independent cytolytic clones on average released significantly higher titers of these factors than IL 2-dependent cytolytic clones.     

Effects of N-acyl-2-hydroxymethyl aziridines on in vitro proliferative responses of human lymphocytes stimulated by mitogens

Aziridines have been shown to possess marked immunotropic activity. The aim of this work was to study the in vitro effects of different concentrations of three novel aziridines, 2-hydroxy-methyl-1-(N-phtaloylglycyl) aziridine (aziridine 1), 2-hydroxy-methyl-1-(N-phtaloylalanyl) aziridine (aziridine 2) and 2-hydroxy-methyl-1-(N-phtaloylphenylalanyl) aziridine (aziridine 3), on the proliferative responses of human lymphocytes stimulated by mitogens (concanavalin A (Con A) and lipopolysaccharide (LPS)), and interleukin-2 (IL-2), interleukin-6 (IL-6) secretion. The results showed that aziridines 1 and 3 significantly stimulated the resting and Con A or LPS lymphocyte proliferation at concentrations between 1 micromol/l and 1 mmol/l, in a dose-dependent manner, the action of aziridine 3 being the highest. They also increased IL-2 and IL-6 secretion. However, aziridine 2 had no effect on the resting lymphocyte proliferation in the absence of mitogens, at any concentration used, reduced Con A-stimulated T lymphocyte proliferation and LPS- stimulated B lymphocyte proliferation in a dose dependent manner and diminished IL-2 and IL-6 production. None of the three aziridines affected cell viability. In conclusion, the three aziridines used in this study displayed immunomodulatory properties. Aziridines 1 and 3 are potentially immunostimulant while aziridine 2 is immunosuppressive and could be used to provide nonspecific cell-mediated immune responses.     

In vitro T cell hyperreactivity in Obese strain (OS) chickens is due to a defect in nonspecific suppressor mechanism(s)

Spontaneous autoimmune thyroiditis of OS chickens is associated with a marked hyperreactivity of the T cell system. The purpose of the present study was to investigate the underlying regulatory mechanisms. Co-cultivation experiments between Con A-stimulated OS and NWL lymphocytes in communicating cultures revealed soluble regulatory factors to be responsible for the observed functional differences: the high proliferative response to Con A and hyperproduction of IL 2 of OS cells was found to be due to a deficiency in the conditioned medium of dialyzable inhibitory factor(s) that regulate IL 2 secretion of NWL lymphocytes. Furthermore, sera of young NWL chickens were found to profoundly inhibit the IL 2-promoted lymphoblast proliferation. This IL 2 antagonizing activity is lost with age (3 to 6 yr) and was found to be significantly diminished in OS birds throughout ontogeny, thus pointing to possible parallels between immune regulatory dysfunction in autoimmunity and in physiologic aging. Both enhanced T cell response and the defect in serum suppressor were inherited by (OS X CB)F1 animals, indicating that these two aberrations may be related to each other.     

Production of and responsiveness to interleukin 2 in autoimmune BXSB mice

BXSB male mice serve as one of several murine models of human systemic lupus erythematosus. T-cell abnormalities in these mice involve decreased production of and responsiveness interleukin 2 (IL-2) and are age-related. The studies presented here investigated the mechanism of these T-cell defects. The results suggest that excessive suppressor-T-cell activity as well as soluble inhibitors of IL-2 production and activity, including PGE, are not responsible for the low levels of IL-2 observed in culture supernatants of Con A-stimulated lymphocytes from "old" (3-6 months) BXSB male mice. Supplementation of Con A-stimulated lymphocyte cultures from BXSB male mice with human IL-1 or normal murine accessory cells did not augment IL-2 production. Reduced proliferative responses were observed in bulk cultures of Con A- or alloantigen-stimulated "old" BXSB male lymphocytes, which were not enhanced by exogenous IL-2. Limiting dilution analysis revealed reduced frequencies of Con A- and alloantigen-inducible IL-2-reactive T cells in these mice. These results suggest intrinsic defects in the ability of T cells from "old" BXSB male mice to be activated to produce and respond to IL-2.     

Role of interleukin 2 on enhancement of concanavalin A-induced human peripheral blood lymphocyte proliferation by murine B cell mitogens

Murine B cell mitogens such as bacterial lipopolysaccharide (LPS), butanol-extracted water soluble adjuvant (Bu-WSA), dextran sulfate (DS), synthetic muramyl dipeptide (MDP), and its analog MDP-Lys (L18) do not show any mitogenic ability in vitro on human peripheral blood lymphocytes or mixed cell populations of purified T and B cells obtained from the lymphocytes in an ordinary culture system. However, these mitogens are capable of enhancing the mitogenic effect of concanavalin A (Con A) in the cultures. In the presence of one of these mitogens, the activity of interleukin 2 (IL 2), but not interleukin 1, in the supernatants obtained from cultures containing Con A-stimulated T cell and B cell populations was higher than that of control cultures. The role of the newly produced IL 2 in the synergistic effect of the mitogens in human lymphocyte cell cultures was discussed.     

Coordinate production by a T cell hybridoma of gamma interferon and three other lymphokine activities: multiple activities of a single lymphokine?

The T cell hybridoma FS7-20, produced by the fusion of normal B10.BR T cells to the AKR thymoma BW5147, was found when stimulated with concanavalin A (Con A) to produce the lymphokines: interleukin 2 (IL 2), interferon-gamma (IFN gamma), macrophage-activating factor (MAF), Ia induction factor IaIF), and the B cell helper factor interleukin X (IL X). The clones and subclones of FS7-20 varied dramatically in their ability to produce these lymphokines, presumably because of karyotypic variations. The ability to produce IL 2 segregated independently from the ability to produce the four other lymphokine activities; however, production of the latter activities showed a strong correlation. This coordinate production of IFN gamma, MAF, IaIF, and IL X was also observed with a cloned normal cytotoxic T cell line, cr15. These results suggest either that IFN gamma, MAF, IaIF, and IL X are all manifestations of a single molecular species or that, although these activities are different structurally, their production is controlled by a common genetic mechanism. In support of the first possibility, the IFN gamma, MAF, IaIF, and IL X activity produced by FS7-20 were all found to be equally sensitive to inactivation at pH 2. These results illustrate the usefulness of using T cell hybridomas for the study of lymphokines.     

Interleukin 2 inhibits antigen-stimulated lymphokine synthesis in helper T cells by inhibiting calcium-dependent signalling

Certain L3T4+, Lyt-2- cloned murine helper T lymphocytes (HTL), when cultured with a high concentration of interleukin 2 (IL 2), become temporarily unresponsive to antigenic stimulation, as indicated by failure to proliferate and by reduced secretion of lymphokines when challenged with antigen. Exposure of cloned HTL to IL 2 also renders these cells less responsive to concanavalin A (Con A). Here we demonstrate that antigen-unresponsive HTL also accumulate reduced levels of lymphokine mRNA, thus indicating a pretranslational block of the response to antigen. However, HTL which had been pretreated with IL 2 and were unresponsive to antigen responded strongly to antigen + A23187 or to A23187 + PMA but failed to respond to antigen + PMA. With HTL made unresponsive to antigen or to Con A by exposure to IL 2, increases in intracellular calcium ion levels stimulated by Con A also were reduced. Thus, for mouse HTL clones, the IL 2-induced state of unresponsiveness to antigen or Con A appears to reflect an inability of such HTL to increase intracellular free calcium to a level sufficient for activation of lymphokine genes.     

Induction of CTL responses to alloantigens by a Db-specific T helper clone

A T cell helper clone was derived 2 yr ago from a mixed lymphocyte culture. This clone, referred to as clone 9, was propagated in interleukin 2 (IL 2)-containing medium in the presence of irradiated stimulator and irradiated syngeneic spleen cells. Clone 9 was of H-2d origin and was found to be Thy-1+ and Lyt-1-2-. Clone 9, as well as supernatant factor(s) derived from it, were able to enhance the primary cytotoxic responses of Db responder cells to alloantigens. Furthermore, clone 9 cells or its factor(s) were only active when added during the first 24 hr of a 5-day culture period. When a low stimulator cell dose (10(4) cells per 0.2 ml culture) was used, it was possible to demonstrate that clone 9 also required a source of irradiated allogeneic splenic accessory cells to exert its helper action. Under these conditions, clone 9 or its factor(s) could also synergize with IL 2-containing medium in mounting cytotoxic responses to alloantigens. Synergy between IL 2-containing medium and clone 9 or its factor(s) was observed only when Db responder cells were used. The helper activity in clone 9 supernatant was also specifically absorbed out by Con A-stimulated Db spleen cell blasts. Preincubation with clone 9 supernatant for 1 hr at room temperature also led to enhanced cytotoxic responses of Db responder cells to alloantigens. Clone 9 supernatant was also found to be devoid of detectable IL 2 activity. Thus, clone 9 or its helper factor(s) appear to exert its helper activity by an early interaction with Db cytotoxic T lymphocyte precursors (CTL-P).     

Requirement for three distinct lymphokines for the induction of cytotoxic T lymphocytes from thymocytes

The induction of cytotoxic T lymphocytes (CTL) from CTL precursors requires a combination of antigen and lymphokine signals. To investigate lymphokine requirements for CTL generation, we used an assay in which helper T cell and accessory cell-depleted spleen cells or whole thymocytes were cultured with lectin (Con A) and lymphokines. This culture was followed by assessment of lectin-dependent cytolysis. High concentrations of recombinant interleukin 2 (R-IL 2) (100 U/ml) alone were not sufficient for lectin-mediated CTL induction from thymocytes, whereas 20 to 100 U/ml of R-IL 2 alone could induce a significant lectin-mediated CTL response from accessory cell-depleted spleen cells. Using thymocytes as responders, we found purified or recombinant interferon-gamma (IFN-gamma) did not cause cytolytic activity either in the absence of or in the presence of R-IL 2. However, supernatant from Con A-stimulated rat spleen cells (rat Con A SN) in combination with R-IL 2 could induce cytolytic activity, suggesting that several factors are required for CTL induction. Con A SN was fractionated by gel filtration and the fractions were tested for ability to induce CTL. In the presence of a low level of R-IL 2 (5 U/ml), fractions with a Mr of approximately 31,000 could induce CTL, and this activity was referred to as CTL differentiation factor (CDF). The peak fractions containing CDF activity did not have detectable IL 1, IL 2, IFN-gamma, or CSF activity. However, by add-back experiments and the use of blocking antibodies, a monoclonal antibody against the IL 2 receptor or antibodies against murine IFN-gamma, we demonstrated that CTL induction from mature thymocytes (L3T4-, Lyt-2+) requires CDF activity in addition to IL 2 and IFN-gamma.     

Functional analysis of macrophage hybridomas. III. Inhibition of lymphocyte blastogenesis and tumor proliferation

Four cloned macrophage hybridoma cells prepared by fusion of splenic adherent cells with a P388D1 macrophage tumor markedly inhibited the growth of lymphocytes and tumor cells. Macrophage clones 5, 8, 63, and 64 are strong inhibitors of B-cell blastogenesis, T-cell blastogenesis, and tumor proliferation, while the P388D1, tumor line and clones 13, 59, and 67 demonstrated little inhibitory activity in all three systems. The inhibitory effect of macrophage hybridomas can be detected within 8 hr, although greater inhibition was noted following longer incubation. The correlations among these three assay systems suggest that similar mechanisms may be involved. The data indicate that the inhibition of cell proliferation was not due to cell lysis. Furthermore, the inhibition of lymphocyte proliferation in Con A-stimulated cultures was not accompanied by inhibition of lymphokine production in the same cultures. Neither prostaglandins nor hydrogen peroxide appear to be primarily responsible for growth inhibition. The inhibitory properties of these macrophage hybridoma lines represent a stable phenotype and provide a homogeneous source of cells for further analysis of the phenomenon.