Direct suppression of lymphocyte induction by the immunoregulatory human serum low density lipoprotein, LDL-In

Preparations of LDL-In, an immunosuppressive lipoprotein subfraction, were analyzed for the capacity to directly suppress the response of human lymphocytes to the representative stimulant PHA vis-a-vis indirect mechanisms mediated by soluble factors or cell:cell interactions. Serum lipoprotein subfraction enriched in LDL-In induced a suppressed state in lymphocytes during 18-hr induction cultures. These lymphocytes, whether partially or completely suppressed, when added to fresh responder lymphocytes in the presence of PHA did not suppress the response of the responder lymphocytes. In contrast, the major low density lipoprotein (LDL) did not suppress lymphocytes at equivalent concentration in the induction culture, nor did LDL-exposed lymphocytes suppress responder lymphocytes. The supernatant medium from LDL-In-suppressed lymphocytes did not contain a newly synthesized or released suppressive factor. Finally, LDL-In-suppressed lymphocytes were not rescued by normal lymphocytes. Each of these observations, and previous evidence that adherent cells do not mediate the biologic effects of LDL-In, support the hypothesis that the biologic manifestations of LDL-In suppression of lymphocyte function result from a direct effect on the lymphocyte that is exposed to this lipoprotein, possibly via the previously demonstrated LDL-In receptor.     

Mechanism of suppression of human peripheral blood lymphocyte proliferation by platelet activation factor

The influence of the phospholipid platelet activation factor (PAF) was studied on PHA-stimulated proliferation of peripheral blood lymphocytes in patients with bronchial asthma and normal subjects. It was found that influencing on the whole population of lymphocytes PAF suppressed proliferation mainly of T-cells. Besides, the influence of PAF on lymphocyte proliferation seemed to be mediated by monocytes since removal of monocytes from the whole population of mononuclear cells abolished the suppression of lymphocyte proliferation induced by PHA. Pretreatment of lymphocytes with PAF antagonist--BL 8705 almost completely blocked the suppression of lymphocyte proliferation induced by PHA.     

Accessory cells in immune suppression. I. Role of I-A+ accessory cells in effector phase idiotype-specific suppression of myeloma function

The suppression of MOPC 315 myeloma cells by idiotype-specific effector Ts requires the presence of non-immune AC. This requirement was demonstrated in cultures where myeloma targets and Ts were separated by cell-impermeable membranes or were in direct contact. The AC were adherent, radioresistant, and present in peritoneal exudates and in FcR+ as well as FcR- fractions of low density splenocytes; they bore cell surface I-A determinants and did not have to be H-2 compatible with myeloma cells and Ts. These studies demonstrate a novel role for Ia+ AC in immune regulation, and suggest that their accessory function may involve processing of T lymphocyte-derived suppressor factors or presentation of such factors to target cells.     

Accessory cells in immune suppression. III. Evidence for two distinct accessory cell-dependent mechanisms of T lymphocyte-mediated suppression

Suppression of antibody secretion by the 2,4,6-trinitrophenol (TNP)-binding BALB/c myeloma, MOPC 315, by idiotype- and hapten-reactive suppressor T cells is mediated by secreted factors (TsF) and requires the presence of accessory cells (AC). Idiotype-specific TsF functions only in the presence of Ia+ AC and is completely idiotype specific. Moreover, no suppression is observed when myeloma targets and AC are separated by cell-impermeable membranes, indicating that the role of AC may be to bind, focus, and/or present TsF to the myeloma cells. In contrast, TNP-specific TsF inhibits myeloma function in the presence of TNP-protein and activated macrophages that are not Ia+. This form of suppression is nonspecific at the effector stage; i.e., anti-TNP TsF inhibits a non-TNP binding cell line, TEPC 15, as long as TNP-protein and activated macrophages are present. Moreover, suppression occurs even when myeloma targets and AC are separated by cell-impermeable membranes. These results are consistent with the view that hapten-reactive TsF binds to antigen on the surface of macrophages and induces these cells to secrete nonspecific immunosuppressive molecules. Thus, different types of AC may play fundamentally different roles in TsF-mediated suppression; they may either bind and present TsF to targets (as in the case of idiotype-specific TsF) or secrete nonspecific immunosuppressants as a consequence of a TsF-antigen interaction (hapten-specific TsF). Autonomous, suppressible targets provide valuable experimental systems for analyzing the cellular interactions in T cell-mediated suppression.     

Effect of mitogen concentration on glucocorticoid suppression of normal and cystic fibrosis lymphocyte activation

It has previously been demonstrated that glucocorticoid suppression of mitogen-induced lymphocyte activation is a function of mitogen dose. Glucocorticoids suppress lymphocyte activation more at low doses, which induce suboptimal lymphocyte activation, than at higher doses which are optimal for lymphocyte activation. This observation suggests that glucocorticoid suppression of lymphocyte activation might be greater than normal in disease states which are associated with depressed mitogen-induced lymphocyte activation. To test this hypothesis, lymphocytes from normal individuals and patients with cystic fibrosis were activated by a full range of concentrations of concanavalin A (Con A) in the presence or absence of dexamethasone. Con A activation of cystic fibrosis lymphocytes was markedly depressed compared to the activation of normal lymphocytes at all doses of Con A, but the suppressive effect of dexamethasone on the activation of normal and cystic fibrosis lymphocytes was the same. We conclude that glucocorticoid suppression of lymphocyte activation is more a function of mitogen dose than of the level of lymphocyte activation and is not necessarily greater than normal in disease states which are associated with depressed mitogen-induced lymphocyte activation.     

CEACAM1 dynamics during neisseria gonorrhoeae suppression of CD4+ T lymphocyte activation

Neisseria gonorrhoeae colony opacity-associated (Opa) proteins bind to human carcinoembryonic antigen cellular adhesion molecules (CEACAM) found on host cells including T lymphocytes. Opa binding to CEACAM1 suppresses the activation of CD4(+) T cells in response to a variety of stimuli. In this study, we use primary human CD4(+) T cells isolated from peripheral blood to define the molecular events occurring subsequent to Opa-CEACAM1 binding. We establish that, in contrast to other cell types, T cells do not engulf N. gonorrhoeae upon CEACAM1 binding. Instead, the bacteria recruit CEACAM1 from intracellular stores and maintain it on the T cell surface. Upon TCR ligation, the co-engaged CEACAM1 becomes phosphorylated on tyrosine residues within the ITIMs apparent in the cytoplasmic domain. This allows the recruitment and subsequent activation of the src homology domain 2-containing tyrosine phosphatases SHP-1 and SHP-2 at the site of bacterial attachment, which prevents the normal tyrosine phosphorylation of the CD3zeta-chain and ZAP-70 kinase in response to TCR engagement. Combined, this dynamic response allows the bacteria to effectively harness the coinhibitory function of CEACAM1 to suppress the adaptive immune response at its earliest step.     

Interaction between T cells and non-T cells in suppression of cytotoxic lymphocyte responses.

Generation of cytotoxic T lymphocytes (CTL) in mixed leukocyte cultures was suppressed by a factor elaborated by alloantigen-activated T cells. This suppressor factor, CTL-TsF, in contrast to a factor that suppresses proliferative responses in mixed leukocyte reactions (MLR-TsF), was effective only when added during the first 24 hr of a 6-day-culture period. Moreover, removal of CTL-TsF 24 hr after culture initiation failed to restore CTL responses. CTL activity could be rescued from suppressed cultures, however, by addition of 2-mercaptoethanol on days 3 or 4. Similarly, transfer of nonadherent cells at 3 or 4 days from cultures treated with CTL-TsF to cultures of adherent cells initiated in control factor restored CTL responses. Mixing experiments with cells pulsed with CTL-TsF for 4 hr at culture initiation identified a target of CTL-TsF as a Thy-1 negative cell that was adherent to plastic and to Sephadex G-10. Suppression was not due to interference with physiologic accessory cell function, but more likely was accomplished via a negative signal from CTL-TsF-pulsed cells. The results thus suggest that CTL-TsF acts early, but reversibly, in the CTL differentiative process via a second suppressor effector cell, possibly a macrophage.     

Histamine and serotonin suppression of lymphocyte response to phytohemagglutinin and allogeneic cells

Histamine added to murine spleen cells suppressed in vitro proliferation of lymphocytes induced by PHA or allogeneic spleen cells. Another vasoactive amine, serotonin (5-hydroxytryptamine), exerted a similar inhibitory activity on PHA- or allogeneic cell-induced lymphocyte proliferation. Anti-H2 histamine antagonists, cimetidine, metiamide, and ranitidine, blocked the histamine and serotonin suppressive effect. Cyproheptadine, an anti-H1 histamine and anti-serotonin antagonist, and methysergide, an anti-serotonin antagonist, also blocked histamine and serotonin inhibitory activities. These data suggest the presence, on lymphocytes, of receptors for serotonin which might be related to histamine receptors.     

Accessory cell competence of ovine oligodendrocytes in mitogenic activation of human peripheral T cells

The multiple sclerosis (MS) plaque is characterized by mononuclear inflammatory cell infiltration, demyelination, loss of oligodendrocytes (OGC), and proliferation of astrocytes. Although antigen-specific, Ia-dependent cellular immune mechanisms have been sought in plaque pathogenesis, Ia-independent T cell activation has not been actively investigated. We examined a potential role of OGC in accessory cell-dependent T cell mitogenesis with the anti-T3 monoclonal antibody OKT3. OGC isolated from ovine white matter on sucrose density gradients were uniformly negative for esterase activity, unlike ovine monocytes. Purified human T cells did not exhibit significant proliferation in 3-day cultures with OKT3, autologous peripheral blood adherent cells (PBAC), or ovine OGC. When T cells were cultured with either PABC or OGC in the presence of OKT3, brisk mitogenesis was observed. Thus, OGC have the capacity to function as accessory cells in the mitogen-induced proliferation of T cells.     

Measles virus-induced suppression of lymphocyte proliferation

The mechanism by which measles virus induces immunosuppression was investigated using an in vitro system employing phytohemagglutinin (PHA)-induced human peripheral mononuclear cell (PBMC) proliferation. At a multiplicity of infection of 1.0 or greater measles virus significantly inhibited (45%) the proliferation of PBMC. This inhibition was not due to an alteration in the kinetics of proliferation. PHA-stimulated PBMC were then infected with measles virus for 72 hr and irradiated (3200 rad) to prevent further proliferation. These infected, irradiated PBMC when added to fresh autologous PBMC caused significant inhibition of lymphoproliferation over a wide range of infected:fresh cell ratios (maximum inhibition seen at a 1:1 ratio, 85% inhibition). Virus recovered from the irradiated, infected cells was 100-fold lower than the virus titer needed to cause inhibition by direct addition of measles virus. However, antibody to measles virus reversed the inhibition. Virus-free supernatant fluids from the infected irradiated cells caused immunosuppression of the PHA response. This immunosuppressive material induced by the measles virus was maximally produced after 72 hr and did not appear to require viral replication. This factor was not prostaglandin E or interferon-alpha or -gamma. The production of such suppressive factors during viral infection may explain some of the profound immunosuppression seen in situations in which little or no infectious virus can be detected.     

Splenic I-J-bearing antigen-presenting cells in activation of suppression: further characterization

A set of I-J-bearing murine splenic antigen-presenting cells (APC) has been found to be responsible for first order suppressor cell (Ts1, afferent suppressor cell) activation in the azobenzenearsonate (ABA) hapten system after intravenous administration. Suppressor cells induced by this set of hapten-coupled cells do not function in the efferent phase of the delayed hypersensitivity (DTH) response. The functional activity of this novel APC to activate afferent suppressor cells was resistant to a dose of ultraviolet radiation (UVR) sufficient to largely abrogate the ability of splenic APC to immunize for a DTH response. It was also found that the previously described splenic I-J-bearing APC needed for third-order suppressor cell (Ts3, effector-suppressor cell) activation is adherent and UVR resistant. The sets of I-J-bearing APC appear to be crucial elements in the activation of suppression and thus in determining the balance between immunologic reactivity and unresponsiveness. Furthermore, the UVR resistance of this set of novel APC may be relevant to the in vivo effects of UVR exposure to mice.     

Mechanistic study of saikosaponin‐d (Ssd) on suppression of murine T lymphocyte activation

Saikosaponin‐d (Ssd) is a triterpene saponin derived from the medicinal plant, Bupleurum falcatum L. (Umbelliferae). Previous findings showed that Ssd exhibits a variety of pharmacological and immunomodulatory activities including anti‐inflammatory, anti‐bacterial, anti‐viral and anti‐cancer effects. In the current study we have investigated the effects of Ssd on activated mouse T lymphocytes through the NF‐κB, NF‐AT and AP‐1 signaling pathways, cytokine secretion, and IL‐2 receptor expression. The results demonstrated that Ssd not only suppressed OKT3/CD28‐costimulated human T cell proliferation, it also inhibited PMA, PMA/Ionomycin and Con A‐induced mouse T cell activation in vitro. The inhibitory effect of Ssd on PMA‐induced T cell activation was associated with down‐regulation of NF‐κB signaling through suppression of IKK and Akt activities. In addition, Ssd suppressed both DNA binding activity and the nuclear translocation of NF‐AT and activator protein 1 (AP‐1) of the PMA/Ionomycin‐stimulated T cells. The cell surface markers like IL‐2 receptor (CD25) were also down‐regulated together with decreased production of pro‐inflammatory cytokines of IL‐6, TNF‐α and IFN‐γ. These results indicate that the NF‐κB, NF‐AT and AP‐1 (c‐Fos) signaling pathways are involved in the T cell inhibition evoked by Ssd, so it can be a potential candidate for further study in treating T cell‐mediated autoimmune conditions. J. Cell. Biochem. 107: 303–315, 2009. © 2009 Wiley‐Liss, Inc.     

Maturation and activation of dendritic cells induced by lymphocyte activation gene-3 (CD223)

Lymphocyte activation gene-3 (LAG-3) is an MHC class II ligand expressed on activated T and NK cells. A LAG-3Ig fusion protein has been used in mice as an adjuvant protein to induce antitumor responses and specific CD8 and CD4 Th1 responses to nominal Ags. In this work we report on the effect of LAG-3Ig on the maturation and activation of human monocyte-derived dendritic cells (DC). LAG-3Ig binds MHC class II molecules expressed in plasma membrane lipid rafts on immature human DC and induces rapid morphological changes, including the formation of dendritic projections. LAG-3Ig markedly up-regulates the expression of costimulatory molecules and the production of IL-12 and TNF-alpha. Consistent with this effect on DC maturation, LAG-3Ig disables DC in their capacity to capture soluble Ags. These events are associated with the acquisition of professional APC function, because LAG-3Ig increases the capacity of DC to stimulate the proliferation and IFN-gamma response by allogeneic T cells. These effects were not observed when using ligation of MHC class II by specific mAb. Class II-mediated signals induced by a natural ligand, LAG-3, lead to complete maturation of DC, which acquire the capacity to trigger naive T cells and drive polarized Th1 responses.     

Accessory muscle activation during the superimposed burst technique

Quadriceps muscle activation is assessed using the superimposed burst technique. This technique involves percutaneous muscle stimulation superimposed during maximal isometric volitional knee extension. It is unknown whether accessory muscle activation during maximal knee extension influences estimates of quadriceps muscle activation. Our aim was to compare accessory muscle activation while performing the superimposed burst technique using investigator delivered verbal instruction to constrain the system (CS) and a participant preferred (PP) technique. Twenty five healthy, active individuals (13M/12F, age=23.8 ± 3.35, height=72.73 ± 14.51 cm, and weight=175.29 ± 9.59 kg) were recruited for this study. All participants performed superimposed burst testing with (CS) and without (PP) verbal instruction to encourage isolated quadriceps activation during maximal isometric knee extension. The main outcome variables measured were knee extension torque, quadriceps central activation ratio and mean EMG of vastus lateralis, biceps femoris, and lumbar paraspinal muscles. There were significant differences in knee extension torque (CS=2.87 ± 0.93 Nm/kg, PP=3.40 ± 1.12 Nm/kg, p<0.001), superimposed burst torque (CS=3.40 ±0.98 Nm/kg, PP=3.75 ± 1.11 Nm/kg, p=0.002) and quadriceps CAR (CS=84.1 ± 12.0%, PP=90.2 ± 9.9%, p<0.001) between the techniques. There was also a significant difference in lumbar paraspinal EMG (CS=6.40 ± 8.52%, PP=11.86 ± 14.89%, p=0.043) between the techniques however vastus lateralis EMG was not significantly different. Patient instruction via verbal instruction to constrain proximal structures may help patient minimize confounders to knee extension torque generation while maximizing quadriceps activation.     

Biological expressions of lymphocyte activation. IV. Concanavalin A-activated suppressor cells in mouse mixed lymphocyte reactions.

Thymus-derived lymphocytes (T cells) from mouse spleen, activated in vitro or in vivo with concanavalin A (Con A), suppress proliferative responses of syngenic lymphocytes in mixed lymphocyte reactions (MLR). Replication in vitro was not required for expression of suppressor activity by Con A-activated cells and was blocked in MLR by treating suppressor cells with mitomycin C or irradiation. Kinetics of MLR responses and viability of cultures were not altered by addition of activated suppressor cells. The data are consistent with a direct inhibitory effect of suppressor T cells on antigen-induced DNA replication. These observations extend a model previously described for regulation of antibody synthesis by Con A-activated T cells to control of cell-mediated immune responses. This model should be particularly useful in further definition of regulatory T cell subpopulations, and in investigation of interactions and relationships between such populations.     

Mechanism of mastocytoma-mediated suppression of lymphocyte reactivity.

Previously we have shown that mitomycin-treated P-815 (H-2d) cells (P-815m) inhibit the in vitro response of C57BL/6 spleen cells to mitogens and DBA/2 alloantigens. The present data indicate that P-815m cells also inhibit the response of C57BL/6 spleen cells to AKR (H-2k)-stimulating cells and that the inhibition does not appear to be the result of crowding. Subpopulations of spleen cells obtained by density gradient centrifucation or after renoval of glass-adherent cells are all sensitive to the inhibitory effects of P-815m. We also show that P-815m cells appears to operate both in vitro and in vivo. The experiments characterize the suppression of MLC reactivity by cell-free preparations of sonicated P-815 mastocytoma cells. The results suggest that the inhibition is not removed by ultracentrifugation, ultra-violet irradiation, or dialysis of the sonicate. P-815 sonicates subjected to chloroform extraction, heating at 56 degrees C, 0.1 mu filtration, or treatment with anit-minute virus of mice serum are still inhibitory. This inhibition is resistant to RNAase treatment but destroyed by pronase. P-815 sonicate suppresses the response of C57BL/6 spleen cells to both DBA/2 and AKR-stimulating spleen. Inhibition of MLC reactivity is only seen if P-815 sonicate is added within the first 48 hr after the initiation of culture. These results indicate that the inhibition of MLC by sonicates of P-815 cells is due to a nondialyzable protein whose effects are not H-2 restricted and are acting on the early phase of sensitization.     

Measles virus-induced suppression of lymphocyte reactivity in vitro

Measles virus (Edmonston strain B), in various multiplicities of infection, was added to human lymphocytes which were cultured in medium containing fetal bovine serum. Live measles virus was found to cause an almost complete inhibition of [³H]-thymidine incorporation in lymphocytes cultured in the presence of phytohemagglutinin, pokeweed mitogen, tuberculin purified protein derivate (PPD), or allogeneic lymphocytes. Analysis of cell size in the lymphocyte cultures revealed that blast transformation was inhibited as well. Measles virus, inactivated by heat or ultraviolet irradiation, did not cause inhibition. The inhibitory effect of measles virus was only measurable in the initial stages of culture; when added later, i.e., 24 hr before measuring [³H]-thymidine incorporation, it had no effect. The diminished reactivity of measles virus-infected lymphocytes cannot be explained by cytopathologic effects or by altered kinetics of lymphocyte transformation. When lymphocytes were cultured at 39 °C the extent of virus-induced suppression was significantly reduced. Very small amounts of pooled normal human serum, as well as IgG, prepared from the serum of a patient with subacute sclerosing panencephalitis, were able to prevent the inhibitory effect of measles virus.     

Regulatory T cells inhibit dendritic cells by lymphocyte activation gene-3 engagement of MHC class II

Lymphocyte activation gene-3 (LAG-3) is a CD4-related transmembrane protein expressed by regulatory T cells that binds MHC II on APCs. It is shown in this study that during Treg:DC interactions, LAG-3 engagement with MHC class II inhibits DC activation. MHC II cross-linking by agonistic Abs induces an ITAM-mediated inhibitory signaling pathway, involving FcgammaRgamma and ERK-mediated recruitment of SHP-1 that suppresses dendritic cell maturation and immunostimulatory capacity. These data reveal a novel ITAM-mediated inhibitory signaling pathway in DCs triggered by MHC II engagement of LAG-3, providing a molecular mechanism in which regulatory T cells may suppress via modulating DC function.