Suppressive substance produced by T cells from mice chronically infected with Trypanosoma cruzi. III. Genetic restriction and further characterization

Earlier papers in this series reported that the culture supernatant of splenic L3T4+, Lyt-2- T cells from susceptible CBA mice chronically infected with Trypanosoma cruzi contain a SS3 that can inhibit the induction of delayed-type hypersensitivity to a wide range of Ag. The SS is a glycoprotein with an apparent molecular mass of 30 to 60 kDa and is distinct from T. cruzi Ag, IL-1, IL-2, IL-3 or IFN-gamma. It also has no effect on Th cells for antibody, cytotoxic T cells or immediate-type hypersensitivity. In this paper, we report that SS can suppress the induction of proliferating T cells but not the presentation of Ag to a cloned T cell line (D10). It also has no effect on the induction of delayed-type hypersensitivity in vitro. SS is produced by a number of inbred mouse strains irrespective of their susceptibility to infection with T. cruzi (BALB/c, highly susceptible; CBA, susceptible; C57BL/10, resistant) but only CBA mice are sensitive to the suppressive action of SS. This is so whether the SS is derived from CBA, BALB/c, or C57BL/10. The sensitivity to SS is not a feature of the H-2k haplotype inasmuch as B10.BR and BALB/k mice (also H-2k) do not respond to SS. Attempts to purify SS with a range of biochemical techniques substantially enriched the specific activity but failed to produce a substance visualizable by analytical gel electrophoresis. IEF chromatography revealed SS activity spanning a pH range from 6 to less than 4. This suggests that SS is likely to be a minor heterogeneous component of the suppressive supernatant. The genetically highly restricted nature of its action is intriguing and may explain some of the contradictory reports in the literature on this subject.     

Suppressive substance produced by T cells from mice chronically infected with Trypanosoma cruzi. I. Preferential inhibition of the induction of delayed-type hypersensitivity

Culture supernatants of spleen cells from susceptible CBA mice chronically infected with Trypanosoma cruzi were able to inhibit the induction of delayed-type hypersensitivity (DTH) to a wide range of antigens as measured by 24-hr footpad swelling, bone marrow homing, and radioactivity accumulation assays. The suppressive activity, which was also present in the serum of these chronically infected mice, appears to be specific for the induction of DTH and had no effect on the 3-hr immediate-type hypersensitivity. It also failed to modify the expression of DTH in presensitized mice. Furthermore, it did not affect the synthesis in normal recipients of specific antibody or the induction of helper T cells or cytotoxic T cells. It also failed to induce DTH tolerance as recipient mice with markedly reduced DTH were able to develop a normal DTH response after secondary immunization. The suppressive activity was produced by an Ig- macrophage-depleted splenic T cell population, whose capacity to secrete the suppressive substance was completely abrogated by treatment in vitro with anti-L3T4 antibody and complement, but not with anti-Lyt-2 antibody and complement. These results therefore demonstrate that L3T4+ T cells from mice chronically infected with T. cruzi can produce substances which interfere with the induction of DTH. This finding may help to identify the differential antigenic stimulatory requirement for the activation of the various subsets of T cells.     

Biological expressions of lymphocyte activation. V. Characterization of a soluble immune response suppressor (SIRS) produced by concanavalin A-activated spleen cells.

Supernatant fluids from murine spleen cell cultures incubated with concanavalin A for 48 hr contain a factor(s), soluble immune response suppressor (SIRS), which suppresses plaque-forming cell responses to sheep erythrocytes by murine spleen cells in vitro. In the present studies, some of the biochemical and biophysical properties of SIRS were investigated. SIRS was non-dialysable; the suppressive activity was stable at 56 degrees C for 30 min, but was destroyed by treatment at 70 degrees C for 30 min, 80 degrees C for 10 min, or at pH 2. The suppressive activity was not absorbed by the stimulating antigen, SRBC, or antisera against murine IgG or mu-chain, suggesting that SIRS does not contain immunoglobulin determinants. Murine spleen and thymus, but not kidney cells, however, absorbed SIRS activity. Enzyme treatments revealed that SIRS was resistant to DNase and RNase, but was destroyed by trypsin and chymotrypsin. In gel filtration with Sephadex G-100, SIRS activity eluted in the fraction corresponding to m.w. in the range between 48,000 and 67,000. With polyacrylamide gel electrophoresis, SIRS activity migrated in the region cathodal to albumin. Isopycnic centrifugation in a cesium chloride gradient suggested that SIRS is a glycoprotein. These supernatant fluids with SIRS activity were also found to contain macrophage migration inhibitory factor (MIF). In the experiments using gel filtration, polyacrylamide gel electrophoresis, and isopycnic centrifugation to fractionate supernatant fluids, SIRS and MIF activity were found in the same fractions, and to date we have been unable to dissociate definitively SIRS activity from MIF activity.     

Desensitization of delayed-type hypersensitivity in mice: suppressive environment

The systemic injection of high doses of antigen into a preimmunized animal results in transient unresponsiveness of cell-mediated immune responses. This phenomenon is known as desensitization. Serum interleukin 2 (IL-2) activity was found transiently in desensitized mice at 3 h after the antigen challenge. These mice could not reveal antigen nonspecific delayed-type hypersensitivity (DTH) 1 d after the challenge. Specific suppression of DTH was observed at later stages. Sera from 3 h desensitized mice showed suppressive effects on DTH in preo immunized mice. Administration of recombinant IL-2 into preimmunized mice led to the failure of development of DTH to antigens. These observations suggest that IL-2 plays an important role in the suppressive environment.     

Antigen-specific augmentation factor involved in murine delayed-type footpad reaction. IV. Effect of delayed-type hypersensitivity augmentation factor on in vitro induction of DTH

We found an antigen-specific factor capable of augmenting delayed-type hypersensitivity (DTH) in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR), or in the culture supernatant of the mixture of sensitized T cells and specific antigens. This factor (DTH augmentation factor; DAF) was confirmed to augment DTH in transferred recipients. In this paper, such an activity of DAF was further investigated using the system with in vitro induction and local transfer of DTH. DAF also augmented the primary in vitro induction of DTH, when spleen cells from mice transferred with the DAF-containing serum 12 hr previously or spleen cells incubated with the DAF-containing serum on ice for 2 hr were cultured with heterologous erythrocytes. DAF acted on the induction phase of DTH and augmented a typical DTH which was dependent on Thy-1-positive T cells. DAF showed antigen specificity, but was not assigned to conventional immunoglobulin. The activity of DAF was detected when nylon-wool nonadherent cells were incubated with DAF prior to the culture of those cells and antigens, but not detected when only nylon-wool adherent cells were incubated with DAF. Thus, DAF exerted its effect through binding to acceptor cells which were included in nylon-wool nonadherent spleen cells from normal mice.     

Thymic stroma-derived T cell growth factor (TSTGF). I. Functional distinction of TSTGF from interleukins 2 and 4 and its preferential growth-promoting effect on helper T cell clones

A recently established thymic stroma-derived cell line (TSCL) supported the growth of the interleukin (IL) 2-dependent, antigen-specific helper T cell (Th) clone, 9-16, without requirement for IL-2 and antigen, and such growth was substituted by a factor produced into cultures by this established TSCL. This substance, thymic stroma-derived T cell-growth factor (TSTGF), was capable of inducing the proliferation of various Th clones including 9-16 Th clone, but not of cytotoxic T cell clones. TSTGF-induced growth promotion was obtained in a dose-dependent fashion and in maintaining antigen specificity of Th clones. The culture supernatant from the TSCL did not contain detectable level of IL-1, IL-2, IL-3, IL-4, or interferon activity. The proliferation of 9-16 Th clone was stimulated by recombinant IL-2 and IL-4 as well as TSTGF, but not by IL-1, IL-3, or interferons. However, the proliferation of this Th clone by IL-2 or IL-4 was almost completely inhibited by anti-IL-2 receptor or anti-IL-4 monoclonal antibody, respectively, whereas TSTGF-induced growth of 9-16 Th clones was not affected by either type of antibody, demonstrating that TSTGF is functionally distinct from IL-2 and IL-4. In addition, TSTGF activity was also obtained from the culture supernatant of the primary thymic explant, which was freshly prepared. These results indicate that the primary thymic explant as well as an established TSCL produce factors capable of promoting the growth of helper but not cytotoxic type of T cells in the absence of T cell growth factors thus far defined.     

Lymphokine control of type 2 antigen response. IL-10 inhibits IL-5- but not IL-2-induced Ig secretion by T cell-independent antigens.

A supernatant derived from the Th2 clone D10.G4.1 (D10 supernatant) stimulated high numbers of Ig-secreting cells when added to dextran-conjugated anti-delta-antibody (anti-delta-dextran)-activated B cells but stimulated only marginal Ag-specific responses when added to B cells cultured with TNP-Ficoll. When anti-IL-10 antibody was added to cultures containing D10 supernatant, IL-5, and TNP-Ficoll, there was a significant increase in the numbers of anti-TNP-antibody producing cells, suggesting that at least a part of the inhibitory activity of D10 supernatant is mediated by IL-10. Addition of rIL-10 inhibited both TNP-Ficoll- and anti-delta-dextran-mediated Ig secretion that was stimulated in the presence of IL-5 but had no suppressive effect on IL-2-stimulated responses, indicating that its inhibitory effect was selective for a specific mode of B cell activation. Addition of IL-10 did not, however, inhibit anti-delta-dextran-stimulated B cell proliferation. The IL-10-induced-inhibition of Ig secretion was not due to suppression of IFN-gamma production, because the addition of IFN-gamma did not reverse the inhibition, nor did the addition of anti-IFN-gamma mimic the IL-10-mediated inhibition. These data suggest that a composite of lymphokines secreted by Th cells may contain both inhibitory and stimulatory activities. Sorting out the conditions under which stimulation or inhibition is seen may reveal additional diversity in Ag-stimulated pathways of B cell activation.     

Novel mechanism for Trypanosoma cruzi-induced suppression of human lymphocytes. Inhibition of IL-2 receptor expression

Co-culture of blood forms of Trypanosoma cruzi, the causative agent of Chagas' disease, with human PBMC impaired the capacity of T lymphocytes to express surface receptors for IL-2. This effect was evidenced by marked reductions in both the proportion of Tac+ cells and the density of Tac Ag on the surface of the positive cells, determined by flow cytometry. The extent of the inhibition increased with parasite concentration. Under optimal or suboptimal conditions of stimulation with either PHA or monoclonal anti-CD3, specific for an epitope of the T3-Ti human T cell Ag receptor complex, the presence of T. cruzi curtailed the capacity of T lymphocytes to proliferate and express Il-2R but did not affect IL-2 production. Furthermore, the addition of exogenous IL-2 did not restore the responsiveness of suppressed human lymphocytes but did when mouse lymphocytes were used instead. Therefore, unlike mouse lymphocytes, human lymphocyte suppression by T. cruzi did not involve deficient IL-2 production and was accompanied by impaired IL-2 utilization. Co-culture of human monocytes/macrophages with suppressive concentrations of T. cruzi increased IL-1 production, and the parasite did not decrease IL-1 secretion stimulated by a bacterial LPS. Therefore, the suppression of IL-2R expression and lymphoproliferation is not likely to have been an indirect consequence of insufficient IL-1 production due to infection of monocytes or macrophages. We have shown that suppression of human lymphocyte proliferation by T. cruzi is not caused by nutrient consumption, absorption of IL-2, lymphocyte killing, or mitogen removal by the parasite. Therefore, these results uncover a novel suppressive mechanism induced by T. cruzi, involving inhibited expression of IL-2R after lymphocyte activation and rendering T cells unable to receive the IL-2 signal required for continuation of their cell cycle and mounting effective immune responses.     

Proliferation of hematopoietic cell lines induced by a soluble factor derived from human squamous cell carcinomas of the head and neck

The supernatant of a cell line of squamous cell carcinoma of the head and neck (SCCHN), PCI-50, was previously shown to induce activation, promote proliferation and increase antitumor cytotoxicity of freshly purified human natural killer (NK) cells and CD4⁺ T lymphocytes [Arch Otolaryngol Head Neck Surg (1994) in press]. This supernatant was found also to promote the growth of a variety of hematopoietic cell lines, including Jurkat, THP-1, K562, NK-92 or Epstein-Barr-virus-transformed B cell lines. The Jurkat cell line was selected as a reporter cell in an 18-h proliferation assay established to measure the growth-promoting activity of PCI-50 supernatant. The presence of soluble tumor-derived factors able to induce proliferation of Jurkat cells was demonstrated in the supernatant produced by several other SCCHN cell lines but not in that produced by a gastric cancer cell line (HR) or renal cell carcinoma line (5117G8). The growth-promoting PCI-50 supernatant was shown to contain 28±0.5 pg/ml interleukin-6, (IL-6) in vitro but was negative for interferon , IL-1, IL-2, IL-4, tumor necrosis factor , granulocyte/macrophage-colony-stimulating factor and IL-12. The addition of any of these recombinant cytokines to Jurkat cell cultures did not significantly promote growth, while PCI-50 supernatant was consistently growth-stimulatory. This supernatant neither enhanced intracellular Ca²⁺ concentration in Jurkat cells nor induced up-regulation of activation antigens on the cell surface, although it supported growth of Jurkat cells in the absence of IL-2. The growth-promoting activity in the PCI-50 supernatant was acid-labile at pH 2 for 4 h, heat-resistant at 96 °C for 1 h and sensitive to treatments with trypsin and pepsin. Preincubation of the PCI-50 producer cells with tunicamycin or cyclohexamide reduced the level of growth-promoting activity in the supernatant. A partial purification of this activity was achieved using Amicon filtration, chromatography on concanavalin-A-Sepharose and then a hydroxyapatite column and high-pressure liquid chromatography gel filtration. The partially purified glycoprotein had a molecular mass of 50–70 kDa, as detemined by gel filtration.This work was supported in part by the Pathology Education and Research Foundation and American Cancer Society grant IM-696 to TLW     

Accessory function of human fibroblasts in mitogen-stimulated interferon-gamma production by T lymphocytes. Inhibition by interleukin 1 and tumor necrosis factor

Highly purified human T cells from peripheral blood fail to produce interferon (IFN)-gamma in the absence of accessory cells. The ability of T cells to produce IFN-gamma upon stimulation with phytohemagglutinin (PHA) or concanavalin A could be restored by the addition of cultured allogeneic human foreskin fibroblasts. Addition of antibodies specific for HLA-DR, DQ, and DP antigens failed to block this accessory function of the fibroblasts. In contrast, antibodies to HLA-DR and DQ antigens inhibited the accessory cell activity of autologous monocytes. Allogeneic fibroblasts failed to exert accessory activity when exogenous interleukin 2 (IL-2) was used as the stimulus for IFN-gamma production. In contrast, autologous monocytes were active as accessory cells for IL-2-stimulated T cells. Addition of recombinant human interleukin 1 alpha (IL-1 alpha) or IL-1 beta to PHA-stimulated T cells co-cultured with fibroblasts stimulated IFN-gamma production. In contrast, preincubation of fibroblasts with IL-1 alpha or IL-1 beta caused a dose-dependent suppression of the ability of fibroblasts to augment PHA- and concanavalin A-induced IFN-gamma production by T cells. Preincubation of fibroblasts with recombinant human tumor necrosis factor (TNF) also reduced their accessory activity. Incubation of fibroblasts with IFN-gamma produced some reduction in their accessory activity and the inhibitory effect of TNF was further enhanced in the presence of IFN-gamma. A 4- to 10-hr incubation of fibroblasts with IL-1 or TNF was sufficient to produce a maximal suppression of accessory activity. Fixation of fibroblasts with formaldehyde decreased their accessory activity, but fixation did not abolish the suppression of accessory function induced by earlier incubation with IL-1. Supernatants of IL-1-treated fibroblast cultures had less suppressive activity than the IL-1-treated fibroblasts per se, and no suppressive activity at all was detected in the supernatants of TNF-treated fibroblasts. Enhanced prostaglandin synthesis may play a role in the IL-1- and TNF-induced suppression of accessory cell function, but other factors are likely to be involved. Our results show that fibroblasts can have a marked effect on T cell function and that IL-1 and TNF can exert immunoregulatory activities indirectly by altering the interactions of fibroblasts with T cells.     

Immunity to herpes simplex virus type 2. Suppression of virus-induced immune responses in ultraviolet B-irradiated mice

Ultraviolet B irradiation (280 to 320 nm) of mice at the site of intradermal infection with herpes simplex virus type 2 increased the severity of the herpes simplex virus type 2 disease and decreased delayed-type hypersensitivity (DTH) responses to viral antigen. Decrease in DTH resulted from the induction of suppressor T cells, as evidenced by the ability of spleen cells from UV-irradiated mice to inhibit DTH and proliferative responses after adoptive transfer. Lymph node cells from UV-irradiated animals did not transfer suppression. DTH was suppressed at the induction but not the expression phase. Suppressor T cells were Lyt-1+, L3T4+, and their activity was antigen-specific. However, after in vitro culture of spleen cells from UV-irradiated mice with herpes simplex virus type 2 antigen, suppressor activity was mediated by Lyt-2+ cells. Culture supernatants contained soluble nonantigen-specific suppressive factors.     

Immunosuppressive activity of human seminal plasma. I. Inhibition of in vitro lymphocyte activation.

A high molecular weight fraction prepared from human seminal plasma by gel filtration chromatography suppresses human lymphocyte transformation and DNA synthesis induced by mitogens (PHA, Con A, PWM), antigens (Candida albicans, tetanus toxoid), and allogenic cells. This same fraction also suppresses the stimulated response of mouse lymphocytes to allogenic cells and to various mitogens, including T cell-dependent and T cell-independent mitogens. The induction, but not the expression, of cell-mediated cytotoxicity is also suppressed. Similar high molecular weight fractions suppress the in vitro humoral response of mouse spleen cells to both a T cell-dependent (SRBC) and a T cell-independent (DNP-F) antigen. The high m.w. fraction exhibited in vitro suppressive activity at concentrations of 0.1 to 1.0 mg/ml which corresponds to a 1/50 or greater dilution of human seminal plasma. These observations support the concept that a local immune response against sperm in the female reproductive tract is actively suppressed by a component in seminal plasma.     

Regulation of lymphocyte blastogenesis and antibody production by soluble factor released by a human B-lymphoblastoid cell line

Yam 1B, a human B lymphoblastoid cell line, spontaneously produced an immunoregulatory factor, which suppresses blastogenesis and antibody formation by human lymphocytes. The Yam 1B cells, which were derived from the peripheral blood of an adult T-cell leukemia patient, have been established and maintained in our laboratory since 1985. This cell line expressed mature B-cell surface antigens including surface immunoglobulin M (IgM), CD23, and HLA-DR; had cytoplasmic IgM; and secreted small amounts of IgM in the culture supernatants. Yam 1B was positive for Epstein-Barr virus-associated antigen (EBNA) but negative for adult T-cell-associated antigen (ATLA). The serum-free Yam 1B culture supernatants (SN) inhibited the expression of transferrin R, but neither the expression of interleukin 2 (IL-2) R(CD25) nor the production of IL-2 in the lymphocytes stimulated with phytohemagglutin. Yam 1B SN also inhibited DNA synthesis by human T and B lymphocytes and immunoglobulin generation by normal B cells as well as by Epstein-Barr virus-transformed human B lymphoblastoid cell lines. The inhibitory activity of Yam 1B SN was inactivated at 56 degrees C and at pH 10 but was relatively stable at pH 2. It was abrogated by digestion with pronase and was partially stable by digestion with trypsin. Fractions collected from a Sephacryl S-300 gel filtration column (Pharmacia Fine Chemicals, Uppsala, Sweden) were found to have a peak of inhibitory activity of cell proliferation associated with molecules of apparent MWr of 43,000 to 67,000. The inhibitory activity of Yam 1B SN was not blocked by the anti-transforming growth factor beta antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preferential suppression of delayed-type hypersensitivity by L-156,602, a C5a receptor antagonist.

In the course of our screening for in vivo immunomodulating substances in which sheep red blood cells (SRBC) and heat-killed Brucella abortus cells (thymus dependent and independent antigens, respectively) for antibody production assays, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) assay were adopted as antigens, we detected a DTH-specific suppressive activity. The producing organism was isolated from a soil sample collected in Ushiku City, Ibaraki, Japan and identified with Streptomyces sp. A1502 (FERM P-12448). The active component was identified with L-156,602, a C5a receptor antagonist. L-156,602 suppressed both TNBS-induced and TNP-SRBC-induced DTH while it enhanced antibody production against SRBC, Brucella abortus, and TNP-SRBC. L-156,602 significantly suppressed DTH induced by direct injection of type 1 helper T cells and its relevant antigen into hind-footpads, indicating that the efferent phase of DTH was affected by L-156,602. The results demonstrated that L-156,602 preferentially suppressed the DTH response.     

Human urine deoxyribonuclease increases endogenous thymidine in the mouse thymocyte interleukin 1 assay: an artificial interleukin-1 inhibitor

Molecular size chromatography of urine from normal individuals showed two peaks of apparent IL-1 suppressive activities when tested by the murine thymocyte comitogenic assay (mol wt greater than 600 kD and 20 to 60 kD). However, the high molecular weight inhibitory activity disappeared if the concentration of PHA was increased during assay, and the low molecular weight inhibitory activity subsided in the presence of a high concentration of [3H]thymidine. The 20 to 60 kD fractions contained DNase activity which acted on DNA liberated from the considerable number of dying thymocytes during the course of the assay. Thus, incubating the urine fractions with freeze-killed murine T cells, whose DNA was prelabeled with [3H]thymidine, showed the appearance of supernatant [3H]thymidine correlating quantitatively with the DNase activity in the fractions. This indicates that urine DNase together with phosphatase(s) in the thymocyte cultures increase the level of extracellular, unlabeled thymidine, thereby diluting the specific activity of the tracer. These artificial IL-1-inhibitors may explain why urine from both normal and febrile individuals 'inhibits' IL-1 only when tested for thymocyte-activating activity but not when tested for other biological activities.     

Two types of murine helper T cell clone. II. Delayed-type hypersensitivity is mediated by TH1 clones

We have previously shown that at least two types of Lyt-1+, Lyt-2-, L3T4+ helper T cell clones can be distinguished in vitro by different patterns of lymphokine secretion and by different forms of B cell help. Evidence is presented here to show that one type of helper T cell clone (TH1) causes delayed-type hypersensitivity (DTH) when injected with the appropriate antigen into the footpads of naive mice. The antigen-specific, major histocompatability complex (MHC)-restricted footpad swelling reaction peaked at approximately 24 hr. Footpad swelling was induced by all TH1 clones tested so far, including clones specific for soluble, particulate, or allogeneic antigens. In contrast, local transfer of TH2 cells and antigen did not produce a DTH reaction, even when supplemented with syngeneic spleen accessory cells. Similarly, local transfer of an alloreactive cytotoxic T lymphocyte clone into appropriate recipients did not produce DTH. The requirements for the DTH reaction induced by TH1 cells were investigated further by using TH1 clones with dual specificity for both foreign antigens and M1s antigens. Although these clones responded in vitro to either antigen + syngeneic presenting cells, or M1s disparate spleen cells, they responded in vivo only to antigen + MHC and did not cause footpad swelling in an M1s-disparate mouse in the absence of antigen. Moreover, in vitro preactivation of TH1 or TH2 cells with the lectin concanavalin A was insufficient to induce DTH reactions upon subsequent injection into footpads. From these results, we conclude that the lack of DTH given by TH2 clones in vivo could be due to the inability of the TH2 cells to produce the correct mediators of DTH, or to a lack of stimulation of TH2 clones in the footpad environment.     

Spontaneous production of a suppressor factor by a human macrophage-like cell line U937. II. Suppression of antigen- and mitogen-induced blastogenesis, IL 2 production and IL 2 receptor expression in T lymphocytes

U937, a human macrophage-like cell line, spontaneously produces a factor which inhibited blastogenic responses of human blood T lymphocytes stimulated with tuberculin-purified protein derivative (PPD) or phytohemagglutinin (PHA). We investigated the mechanism of suppressor action of the U937 factor. The U937 suppressor factor inhibited interleukin 2 (IL 2) production by human blood T lymphocytes stimulated with PPD or PHA. IL 1 did not overcome the inhibitory action of the U937 factor on PPD-induced IL 2 production by human blood T lymphocytes. The U937 factor also inhibited the production of IL 2 by a human leukemic cell line, JURKAT, stimulated with PHA. The U937 suppressor factor interfered with the expression of Tac antigen (IL 2 receptor) on PPD- or PHA-stimulated blood T lymphocytes. The inhibitory activity of the U937 factor on Tac expression was not affected by the addition of IL 2 or a crude lymphokine-containing T cell supernatant. Tac expression was more sensitive than IL 2 production to inhibition by U937-conditioned medium. The U937 suppressor factor was precipitable by 33 to 67% saturated ammonium sulfate and was inactivated at pH 2 or pH 11. Sephacryl S-200 Gel filtration analysis of U937 culture supernatants revealed that the inhibitory activities for blastogenesis, IL 2 production, and Tac expression co-purified in fractions with an apparent m.w. between 67,000 and 130,000. These data indicate that U937 spontaneously produces a macromolecular suppressive factor with major locus of action on the production of IL 2 and the expression of the IL 2 receptor.     

IL-2 receptor(p55)/Tac-inducing factor. Purification and characterization of adult T cell leukemia-derived factor

Many human T cell lymphotropic virus-I (HTLV-I) transformed T cells from adult T cell leukemia (ATL) patients continuously produce a humoral factor called ATL-derived factor (ADF) which induces IL-2R/Tac expression on T and NK cells. Using gel filtration, procion red Sepharose, DEAE, and reverse phase chromatography, we have purified ADF protein to homogeneity from 15 liters of serum-free culture supernatant of an HTLV-I(+) T cell line ATL-2. Purified ADF protein had the m.w. of 14,000 by SDS-PAGE and gel filtration, and its isoelectric point is around 5.0. ADF did not react with heteroantibodies against IL-1 alpha and IL-1 beta, which have also IL-2R/Tac-inducing activity on suitable target cells. Partial N-terminal amino acid sequence of ADF is different from other cytokines such as, IFN, BSF-2, and various IL whose cDNA has been cloned. Western blot analysis using rabbit antibodies against N-terminal 10mer synthetic peptide of ADF showed that IL-1 alpha and ADF are different proteins. ADF had its IL-2R/Tac-inducing activity not only on human NK-like cell line YT, but also on HTLV-I(+) T cells, such as ED. In contrast, macrophage-derived IL-1 lacked IL-2R/Tac-inducing activity on ED cells despite their IL-2R/Tac induction on YT, indicating that ADF and IL-1 have their effect via different receptors.     

Effects of suramin on the immune responses to sheep red blood cells in mice. I. In vivo studies

The effect of Suramin on the cell-mediated delayed-type hypersensitivity (DTH) and the humoral immune responses elicited in mice by sheep erythrocytes was studied. The results show that administration of Suramin, at various times before or after antigenic sensitization, results in a profound inhibition of cell-mediated responses but has no adverse effect on antibody production. Suramin was particularly effective when given during the effector phase of DTH: mice which were treated with this drug, 4 days after immunization, at the time of skin testing, exhibit negative or low DTH responses compared to control mice. Evidence is presented that this short-term Suramin-induced suppressive effect on the expression of DTH is related to a defective recruitment, by sensitized T lymphocytes, of phagocytic cells at the site of the inflammatory reaction. In addition, when treatment with Suramin precedes by 8 days (Day -8) or by 1 hr sensitization with sheep erythrocytes for DTH, decreased DTH reactions over controls were observed. The inhibitory effect exerted by Suramin administered on Day -8 can be reversed by increasing the dose, from 10(6) to 10(8) sheep erythrocytes, of the sensitizing antigen. The possibility is discussed that, in this case, Suramin may interfere with the generation of DTH-mediating cells through a rapid degradation of antigen related to the Suramin-induced hyperplasia of the mononuclear phagocyte system. In contrast, DTH anergy in mice treated with Suramin 1 hr before sensitization is maintained regardless of the sensitizing antigen dose. Analysis of the sensitized lymphocyte population in these mice indicates that Suramin does not prevent the induction of DTH-mediating cells and suggests that the expression of these latter is inhibited by suppressive cells which are generated as a result of drug treatment.     

T cell dependence and factor reconstitution of in vitro antibody responses to TNP-B. Abortus and TNP-Ficoll: restoration of depleted responses with chromatographed fractions of a T cell-derived factor

In vitro anti-trinitrophenyl (TNP) antibody responses to TNP conjugates of killed Brucella abortus organisms (TNP-BA), an antigen previously designated as a type 1 thymus-independent (TI-1) antigen, are markedly diminished after vigorous depletion of T cells, as are the responses to the type 2 TI (TI-2) antigen, TNP-Ficoll. We, therefore, propose that these antigens be redesignated as type 1 and type 2, respectively, to reflect their T cell dependence but to differentiate them from classical T cell-dependent (TD) antigens. T cell-depleted responses to type 1 and type 2 antigens can be restored by the addition of a) EL4 supernatant, b) phenyl-sepharose-purified fractions of EL4 supernatant that are rich in interleukin 2(IL2), and c) pl 4.5-5.5 isoelectric focused (IEF) fractions of EL4 supernatant which are also rich in IL2 activity. Removal of IL2 activity from EL4 supernatant by absorption on IL2-dependent T cells substantially reduced its restorative ability. Whether the active principle in EL4 supernatant activity responsible for restoring responses to type 1 and type 2 antigens is IL2, and whether it acts directly on B cells or by acting on contaminating T cells, is unresolved.