Study of the possibility to abolish the action of immunosuppressive factors of tumor cells]

We investigated the efficacy of IL-2, LPS, MDP, TRA, ionomycin and contrykal on proliferation of lymphocytes treated by tumor cell immunosuppressive factors (ISF). IL-2, LPS and/or MDP did not abolish the influence of P815 and B16 ISF on Con A or alloantigen-induced lymphocyte proliferation. TPA and in less extent ionomycin and combination of the above preparations totally abrogated the suppression of Con A-induced lymphocyte proliferation. In inverted experiments Con A abrogated ISF-mediated suppression of lymphocyte proliferation induced by TPA plus ionomycin.     

Study of the action of tumor cell immunosuppressive factors on the production of cytokines by lymphocytes and macrophages and the mitogenic activity of interleukin-2

The effect of immunosuppressive factors (ISF) derived from tumor cell lines (mastocytoma P-815, leukosis EL-4 and melanoma B16) on the production of interleukin-1 (IL-1) by macrophages and of interleukin-2 (IL-2) by splenocytes of BALB/C mice was investigated. We could show that treatment of above cells by the tumor products resulted in strong decrease of IL-2 production but did not affect IL-1 secretion. ISF inhibited the proliferation of BALB/C lymphoblasts caused by recombinant IL-2. Thus, one of the significant mechanisms of ISF action seems to be disturbance of monolymphokine cascade of lymphocyte activation.     

Lack of species specificity in the action of immunosuppressive factors of tumor cells and absence of competition between them and recombinant interleukin-2 and phytohemagglutinin for lymphocyte membrane receptors

We studied some possible mechanisms of action of immunosuppressor factors (ISF) produced by tumor cells on lymphocyte proliferation. ISF of murine tumor cell lines inhibited the mitogen induced proliferation of murine splenocytes as well as human mononuclear blood cells. Normal human mononuclear blood cells or concanavalin A-activated murine spleen cells preincubated with phytohemagglutinin (PHA) or interleukin 2 (IL-2) respectively, were strongly suppressed by ISF in response to these activators. When preincubated with splenocytes or blood cells for 2 h at 4 degrees C following washing, ISF suppressed the lymphocyte proliferation as effectively as when being with cells during all period of cultivation. ISF inhibited mitogen-induced lymphocyte proliferation at low dilutions. There was no competition for lymphocyte membrane receptors between these functionally heterogenic kinds of ISF. Collectively, these results show that ISF acted when being attached to some lymphocyte membrane receptors.     

Molecular targets for existing and novel immunosuppressive drugs

Anti-neoplastic cytostatic antiproliferative agents, such as methotrexate, 6-mercaptopurine and cyclophosphamide, were originally used as immunosuppressive drugs. Although these agents induced only modest anti-rejection activity, they caused serious non-specific bone marrow suppression, impairing host resistance and increasing the incidence of infections. Unlike these non-selective agents, cyclosporine A, tacrolimus and sirolimus act more selectively on different stages of the T-lymphocyte (T-cell) and B-lymphocyte (B-cell) activation cycles; however, cyclosporine and tacrolimus are nephrotoxic, whereas sirolimus causes hypertriglyceridaemia. Thus, despite this progress, continued efforts must be made to develop and test new, potentially very selective agents. The agent 15-deoxyspergualin moderately inhibits both mitogen-stimulated T-cell proliferation and the generation of cytotoxic T lymphocytes (CTLs) but does not affect the production of interleukin 2 (IL-2). Another drug, FTY720, has a unique action to prevent rejection, by altering the homing of lymphocytes to the lymphoid compartments. The newest members of the family of antiproliferative agents, namely mycophenolate mofetil, leflunomide and brequinar, are potentially more selective than their predecessors. However, the most promising agents are produced using antisense technology. This approach involves the design of antisense oligodeoxynucleotides; these novel drugs are designed to block allograft rejection by blocking selected messenger RNA (mRNA). This review outlines the mechanisms of action, the limitations of application and the molecular or cellular targets of traditional agents, newly developed drugs and also antisense technology, which is an example of a new application of molecular medicine.     

Partial purification of an immunosuppressive protein from a human tumor cell line and analysis of its relationship to transforming growth factor beta

The A673 human rhabdomyosarcoma cell line constitutively produces an acid-soluble, potent immunosuppressive factor (ISF), which inhibits T-cell proliferation. We have partially purified this factor from the culture supernatant of A673 cells by a sequence of acid extraction, gel filtration, cation exchange chromatography, and reverse-phase HPLC. Characterization studies indicate that ISF is similar or identical to transforming growth factor beta (TGF beta). ISF exhibits a molecular weight of 25 kDa in sodium dodecyl sulfate-polyacrylamide gels. ISF, like TGF beta, is a very basic protein (pI = 9.5) that is sensitive to reduction. Anti-TGF beta 1 antibodies completely block ISF activity in the thymocyte assay. Furthermore, ISF, like TGF beta, stimulated the anchorage-independent growth of normal rat kidney fibroblasts in soft agar.     

Suppression of mitogen-induced lymphocyte proliferation by syringomycin-E

Abstract Syringomycin E (SR-E) is low molecular weight bacterial lipodepsipeptide with antifungal properties. Owing to immunosuppressive activities of such compounds as cyclosporine, FK506 and rapamycin, we studied the effect of SR-E on proliferation of human blood lymphocytes in vitro. SR-E, by itself, had no effect but the mitogen-induced lymphocyte proliferation was significantly suppressed. The suppressive effect was more pronounced with pokeweed mitogen (PWM) as compared to phytohemagglutinin (PHA) or monoclonal antibody to CD3 (anti-CD3). Since these mitogens induce cellular immunity (T cell-dependent), SR-E may potentially be a novel immunosuppressive compound.     

Anti-Pim-1 mAb inhibits activation and proliferation of T lymphocytes and prolongs mouse skin allograft survival

Pim-1 is an important signaling molecule mediating cell proliferation and survival. Our previous study identified a Pim-1 specific monoclonal antibody, P9, with significant inhibitory effect on cell proliferation. Herein, we report that P9 inhibited the activation and proliferation of PHA-stimulated human PBMC and induced them to undergo apoptosis. In contrast, P9 showed little effect on freshly isolated human blood T lymphocytes which poorly expressed Pim-1. P9 also detected an up-regulation of Pim-1 in mouse lymphocytes after mitogen stimulation, and showed similar selective inhibition on stimulated cells as observed with hPBMC. Furthermore, P9 inhibited the in vitro mixed lymphocyte reaction and P9 treatment significantly prolonged the survival of mouse skin allografts (P < 0.001). It is concluded that Pim-1 expression correlates with lymphocyte proliferation and activation. P9 functions as a Pim-1 antagonist and is potential for immunosuppressive therapy.     

Impairment of T-helper function by a Plasmodium berghei-derived immunosuppressive factor

Mice injected with an immunosuppressive factor (ISF) extracted from Plasmodium berghei-infected rat erythrocytes have a reduced antibody response to unrelated antigens. T-cells from ISF-treated mice failed to provide adequate help to naive, syngeneic B-cells in the primary IgM response in vitro to sheep red blood cells and to dinitrophenylated keyhole limpet hemocyanin. The same T-cells, however, were able to cooperate with memory B-cells in the secondary IgG response. No other cellular deficit was detected in ISF-treated mice; B-cells and macrophages behaved normally, and there was no detectable excess of suppressor cells. The T-cell impairment was not reflected in decreased production of interleukin 2, but was also shown by the diminished delayed type hypersensitivity reaction to sheep red blood cells of ISF-treated mice.     

The immunosuppressive effect of Wharton's jelly stromal cells depends on the timing of their licensing and on lymphocyte activation

BackgroundMesenchymal stromal cells (MSC) have been proven to have potent immunosuppressive action and hence have been proposed for the treatment of severe Graft Versus Host Disease. However, in most models, MSC were added at the same time of lymphocyte stimulation, which is quite different from what occurs in vivo.AimsTo investigate how the timing of lymphocyte activation and the exposure to activation-related cytokines (licensing) can influence the immunosuppressive action of Wharton's jelly stromal cells (WJSC).MethodsWJSC, licensed or not with activation-related cytokines, were added lymphocytes the same time or 24 hours after their stimulation with phytohaemoagglutinin. Proliferation of lymphocytes and cytokines production was measured after three days co-culture.ResultsLymphocytes stimulated in the presence of WJSC displayed a dramatic decrease in proliferation and production of cytokines, in spite of normal expression of activation markers. The suppression was weakened when targeted lymphocytes were seperated by a membrane and partially rescued by the addition of exogenous l-tryptophan, suggesting a major role for indoleamine 2,3-dioxigenase with a probable paracrine effect. Licensing of WJSC increased the immunosuppressive effect, in both contact and non-contact settings. The timing of WJSC licensing was crucial for the immunosuppressive action. Lymphocytes pre-stimulated alone for 24 h, and added afterwards to non-licensed WJSC, showed normal or even increased proliferation. On the other hand, their proliferation was strongly inhibited by licensed WJSC.ConclusionsWJSC have a potent immunosuppressive function best realized with direct contact, and increased by licensing signals before and during lymphocyte stimulation. Our results could contribute to the set up of new WJSC-based therapies for severe autoimmuno disorders.     

Effects of alveolar surfactant aggregates on T-lymphocyte proliferation

The effects of alveolar large aggregate (LA) and small aggregate (SA) surfactant subfractions isolated from healthy adult rats on mitogen-stimulated proliferative responses of human peripheral blood mononuclear cells (PBMC) was examined. Various concentrations of total surfactant suppressed proliferation of stimulated lymphocytes by up to 95% of mitogen-stimulated cells alone. LA subfractions of total surfactant had no effect on proliferation, whereas SA significantly enhanced the lymphocyte proliferation at lower concentrations (7.8 microg/ml) compared to mitogen-stimulated cells alone. Higher concentrations of SA (62.5 microg/ml) inhibited lymphocyte proliferation. This concentration-dependent effect of SA on proliferation of PBMC was also present when cells were stimulated with various lectins including anti-CD3, concanavalin A and phytohemagglutinin. Analysis of the supernatant of mitogen-stimulated cell cultures treated with inhibitory concentrations of SA showed decreased amounts of interleukin (IL)-2, compared to cells alone, which could be reversed by adding exogenous IL-2 to the cell cultures with the SA. These results suggest that alveolar surfactant subfractions have distinct functions within the alveoli, both biophysically and with respect to their effects on the host's immunomodulatory responses.     

Principles of antibody therapy.

The success of monoclonal antibodies in clinical practice is dependent on good design. Finding a suitable target is the most important part as other properties of the antibody can be altered by genetic engineering. Antibodies that target lymphocyte antigens offer less toxic immunosuppressive treatment than currently available drugs and the first monoclonal antibody approved for human use is an immunosuppressive agent for treating rejection of renal transplants. Human trails of monoclonal antibodies to treat septic shock have been done and antibodies are also being developed to target common pathogens such as herpes simplex virus. Although monoclonal antibodies against cancer have been much heralded, their success has been limited by the poor access to the inside of tumours. Treatment of blood cancers has been more successful and a human antibody against B cell malignancies is being clinically tested. As knowledge about natural immune responses and antibody engineering increases many more monoclonals are likely to feature in clinical practice.     

Potent immunosuppressive activities of cytomegalovirus-encoded interleukin-10.

Cytomegalovirus (CMV) has highly evolved mechanisms for avoiding detection by the host immune system. Recently, in the genomes of human and primate CMV, a novel gene comprising segments of noncontiguous open reading frames was identified and found to have limited predicted homology to endogenous cellular interleukin-10 (IL-10). Here we investigate the biological activities of the CMV IL-10-like gene product and show it to possess potent immunosuppressive properties. Both purified bacterium-derived recombinant CMV IL-10 and CMV IL-10 expressed in supernatants of human cells were found to inhibit proliferation of mitogen-stimulated peripheral blood mononuclear cells (PBMCs), with specific activity comparable to that of recombinant human IL-10. In addition, CMV IL-10 expressed from human cells inhibited cytokine synthesis, as treatment of stimulated PBMCs and monocytes with CMV IL-10 led to a marked decrease in production of proinflammatory cytokines. Finally, CMV IL-10 was observed to decrease cell surface expression of both major histocompatibility complex (MHC) class I and class II molecules, while conversely increasing expression of the nonclassical MHC allele HLA-G. These results demonstrate for the first time that CMV has a biologically active IL-10 homolog that may contribute to immune evasion during virus infection.     

Peritoneal macrophages exposed to purified macrophage colony-stimulating factor (M-CSF) suppress mitogen- and antigen-stimulated lymphocyte proliferation

The effect of M-CSF-exposed macrophages on murine splenic lymphocyte responses was determined. Resident peritoneal macrophages incubated with purified M-CSF for 48 hr inhibited lymphocyte proliferation to Con A, PHA, and listerial antigen as determined by [3H]TdR uptake, and inhibited Con A-stimulated lymphocyte IL 2 production. The inhibition was similar to that observed with macrophages from BCG-infected mice. Maximal suppression occurred at M-CSF concentrations of 500 U/ml or greater and when the incubation time with M-CSF was 48 hr or more. M-CSF effect was specific because rabbit anti-M-CSF IgG blocked the suppression whereas control rabbit IgG did not. Secretory products of macrophages could not be implicated in this interaction. Catalase and indomethacin, alone or together, did not reverse the inhibition. In addition, putative suppressive factors were not detected in supernatants of M-CSF-stimulated macrophages. Lymphocytes that were removed from macrophage monolayers and were recultured in medium plus Con A were able to proliferate. Macrophages stimulated by M-CSF therefore appear to have inhibitory activity for proliferating lymphocytes, and may play a role in immunoregulatory mechanisms.     

Differential effect of monoclonal anti-DR antibody on monocytes in antigen- and mitogen-stimulated responses: Mechanism of inhibition and relationship to interleukin 1 secretion

A differential role for DR antigens on monocytes in antigen-stimulated as opposed to mitogen-stimulated human lymphocyte responses has been observed. A monoclonal anti-DR antibody used to treat monocytes caused inhibition of antigen-induced T-cell responses and of T-cell-dependent B-cell responses. However, anti-DR antibody treatment of monocytes did not inhibit mitogen-induced responses. Anti-DR treatment of monocytes did not induce suppression, as antigen-induced responses could be reconstituted with untreated monocytes. Anti-DR treatment of monocytes did not merely block interleukin 1 (IL-1) secretion since addition of IL-1 could not restore antigen-induced responses. Monoclonal anti-DR antibody did not directly inhibit monocyte secretion of IL-1. DR-negative monocytes, selected by antibody and complement, could not present antigen, even though they were capable of secreting IL-1. Thus, this monoclonal anti-DR antibody sterically blocks antigen presentation by monocytes without induction of suppression or inhibition of IL-1 secretion. Monocyte DR antigens appear essential for stimulation of antigen-induced responses, but DR antigens on monocytes may not be essential for mitogen-stimulated responses and do not appear to be related to the ability of monocytes to secrete IL-1.     

Augmentation of prostaglandin and thromboxane production in vitro by monocytes exposed to histamine-induced suppressor factor (HSF)

A possible mechanism to explain the suppression of mitogen-induced lymphocyte proliferation in vitro by histamine-stimulated mononuclear cells was investigated. In initial experiments, the inhibitory action of histamine-induced suppressor factor (HSF) on lymphocyte proliferation was documented to be reduced by the addition of indomethacin (1 μg/ml). Moreover, the addition of exogeneous PGE₂ (10^?7-10^?8 M) to mononuclear cell cultures reconstituted HSF activity in the presence of indomethacin. In order to ascertain the nature of the target cell responding to HSF, control and suppressor supernatants were incubated with human lymphocytes or monocytes (5 × 10⁶ cells/ml) for 24 hr. Following incubation, the supernatants were assayed for their content of prostaglandin E₂, F_2α, and thromboxane B₂. Monocytes (but not lymphocytes) incubated with supernatants containing HSF increased their production of prostaglandin E₂, F_2α, and thromboxane B₂ by 169, 53, and 49%, respectively. Suppressor supernatants were generated with histamine or an H-2 agonist (dimaprit) and chromatographed by gel filtration on Sephadex G-100. The elution profiles for the factor(s) inducing suppression of lymphocyte proliferation (25–40,000 daltons) and augmenting PGE₂ production (25,000 daltons) overlapped but were not identical. Collectively, these data suggest that HSF-mediated inhibition of lymphocyte proliferation may occur in part through the augmented production of prostaglandins and/or thromboxane B₂ by human monocytes.     

Characterization of a low molecular weight suppressor of lymphocyte proliferation from guinea pig L2C leukemia cells

Conditioned medium (CM) from 24-hr culture of guinea pig L2C B lymphoblastic leukemia cells contained an inhibitor(s) of mitogen- and antigen-stimulated proliferation of syngeneic (strain 2 guinea pigs), allogeneic (Hartley guinea pigs), and xenogeneic (Balb/c mouse, NZW rabbit) lymphocytes. The proliferation of several lymphoid and nonlymphoid cell lines also was inhibited in the presence of CM. The inhibitor(s) in CM was not toxic to any of the cultures studied. CM inhibited the mitogen-stimulated proliferation of lymphocytes when added to cultures up to 52 hr after addition of mitogen. Normal responsiveness to mitogens could be restored by washing the CM-treated lymphocytes with medium during the first 6 hr of culture. The addition of exogenous IL-2 to lymphocyte cultures did not overcome the CM-mediated suppression of mitogen- or antigen-stimulated proliferation. CM also inhibited the IL-2-dependent proliferation of murine CTLL-2 cells. Preincubation of guinea pig lymphocytes in CM did not inhibit the capacity of these cells to release IL-2 after exposure to mitogen. The antiproliferative activity of CM was stable to heating at low pH (100 degrees C, 10 min, pH 4.0), was resistant to treatment with papain, pronase, DNase, and RNase and did not bind to Con A-Sepharose. Incubation of the L2C cells in indomethacin did not inhibit the release of the inhibitor(s). The inhibitor(s) in CM had an apparent molecular weight of 500-3500 Da as determined by dialysis and ultrafiltration analysis. The inhibitory activity was recovered in the organic phase after extraction with chloroform:methanol and eluted distinct from the thymidine standard after gel filtration on Sephadex-G 25. These data suggest that the inhibitor(s) in CM is a nonspecific, low molecular weight, lipid-like component (not prostaglandin) that exerts its antiproliferative effects subsequent to cell activation. The inhibitor(s) did not appear to suppress other biologic functions associated with activation, such as IL-2 secretion. The inhibitor in CM may be important in promoting tumor survival in vivo by suppressing potential anti-tumor cellular immune responsiveness.     

Isolation of a low molecular weight inhibitor of lymphocyte proliferation from tumorous ascites

Intraperitoneal growth of P-815 mastocytoma cells in syngeneic DBA/2 mice produces ascites fluid which strongly inhibits mitogen-stimulated lymphocyte proliferation. The less than 10,000 m.w. fraction from gel filtration chromatography of tumorous ascites on Sephadex G-150 showed no inhibition of proliferation when eluted under physiologic conditions but was inhibitory when eluted with a high ionic strength, acidic buffer. The organic phase of a chloroform/methanol extract of the low m.w. fraction contained all the inhibitory activity. Purification of the inhibitor to relative homogeneity was achieved by reverse phase, HPLC with a gradient of acetonitrile in dilute acetate buffer. Inhibitory activity eluted between 30 and 35% acetonitrile. The active fraction contained less than 30 pg/ml PGE by RIA which was insufficient to inhibit proliferation and may actually have been stimulatory. Inhibition comparable to that produced by the ascites fraction required greater than 300 pg/ml of PGE. This low m.w. (less than 10,000), lipid-like inhibitor of lymphocyte proliferation is acid stable, not sensitive to proteolytic enzymes, soluble in both aqueous and organic solvents and occurs normally bound to a higher m.w. carrier molecule.     

Enhancement of interleukin 2 production in human and Gibbon T cells after in vitro treatment with lithium.

The effect of trace elements lithium, selenium, and zinc on interleukin 2 (IL-2) production by peripheral blood mononuclear cells (PBMC), MLA144, and Jurkat cell lines has been studied. Lithium markedly enhanced IL-2 production by MLA144 and PBMC, but not by Jurkat. Selenium could only enhance IL-2 production by MLA144, whereas in none of these three systems was IL-2 production altered by zinc. The enhancing effect of lithium on IL-2 production showed some differences from that of tetradecanoylphorbol acetate (TPA) in the following aspects: (i) TPA could reverse the inhibitory effect of anti-CD2 monoclonal antibody on IL-2 production, whereas lithium could not; and (ii) lithium was unable to synergistically induce IL-2 production with anti-CD3 monoclonal antibody as TPA did. The effect of lithium on IL-2 production was in the early phase of lymphocyte activation. The addition of cholera toxin or theophylline to phytohemagglutinin-stimulated PBMC culture suppressed IL-2 production. However, IL-2 production could be restored by lithium. There was a corresponding increase in cAMP in cholera toxin or theophylline-treated PBMC, which could be reversed by lithium. Therefore, lithium restores IL-2 production via a decrease in cAMP.     

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.     

The antiproliferative effect of mesenchymal stem cells is a fundamental property shared by all stromal cells

Although it has been widely demonstrated that human mesenchymal stem cells exert potent immunosuppressive effects, there is little information as to whether more mature mesenchymal stromal cells (SC) share the same property. Accordingly, we set out to test the ability of SC from different human tissues to inhibit the proliferation of PBMC following polyclonal stimuli. Chondrocytes, as well as fibroblasts from synovial joints, lung, and skin, were used as a source of SC. Irrespective of their differentiation potential and/or content of progenitor cells, SC from all tissues exhibited antiproliferative functions. This was in marked contrast to parenchymal cells. Although SC did not interfere with early T lymphocyte activation, they arrested stimulated T cells in the G(0)/G(1) phase of the cell cycle and rescued them from apoptosis. In addition, IFN-gamma and TNF-alpha production were reduced. We observed that the inhibitory effect is ultimately mediated by soluble factors, the production of which requires SC to be licensed in an inflammatory environment by cell contact. We conclude that the immunosuppressive effect of mesenchymal cells is not confined to multipotent stem cells, but is a fundamental characteristic of all stroma. Our data suggest that SC, appropriately licensed, regulate T cell homeostasis.