Addition Of Exogenous Interleukin-2 Research Articles

Aging in Ghana

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

Cyclotides Suppress Human T-Lymphocyte Proliferation by an Interleukin 2-Dependent Mechanism.

Cyclotides are a diverse and abundant group of ribosomally synthesized plant peptides containing a unique cyclic cystine-knotted topology that confers them with remarkable stability. Kalata B1, a representative member of this family of mini-proteins, has been found to inhibit the proliferation of human peripheral blood mononuclear cells. Analysis of T-cell proliferation upon treatment with chemically synthesized kalata B1 mutants revealed a region comprising inter-cysteine loops 1 and 2 of the cyclotide framework to be important for biological activity. Cytokine signaling analysis using an ‘active’ kalata B1 mutant [T20K], and the reference drug cyclosporin A (CsA) demonstrated that treatment of activated T-lymphocytes with these compounds decreased the expression of the interleukin-2 (IL-2) surface receptor as well as IL-2 cytokine secretion and IL-2 gene expression, whereas the ‘inactive’ kalata B1 mutant [V10K] did not cause any effects. The anti-proliferative activity of [T20K] kalata B1 was antagonized by addition of exogenous IL-2. Furthermore, treatment with [T20K] kalata B1 led to an initial reduction of the effector function, as indicated by the reduced IFN-γ and TNF-α production, but the levels of both cytokines stabilized over time and returned to their normal levels. On the other hand, the degranulation activity remained reduced. This indicated that cyclotides interfere with T-cell polyfunctionality and arrest the proliferation of immune-competent cells through inhibiting IL-2 biology at more than one site. The results open new avenues to utilize native and synthetically-optimized cyclotides for applications in immune-related disorders and as immunosuppressant peptides.

Effect of activated human polymorphonuclear leucocytes on T lymphocyte proliferation and viability

Human polymorphonuclear leucocytes (PMN) are thought to be immunosuppressive. The suppressive mechanism(s) used by PMN are, however, not well defined and in this study they were analysed using T-cell responses to CD3(+) CD28 monoclonal antibodies (mAb) as a readout. We demonstrate that in vitro activated PMN (PMN(act)) can, without any T-cell interaction, induce apparent T-cell suppression by inhibiting the stimulatory capacity of the CD3 mAb. However, a cell-directed suppression of T-cell proliferation was observed when PMN(act) were added to pre-activated T cells that are already committed to polyclonal proliferation. This suppression was partially reversed by catalase addition (P < 0·01) and largely reversed by addition of exogenous interleukin-2 (P < 0·001) but was not significantly reduced by nitric oxide synthase inhibition, myeloperoxidase inhibition or addition of excess arginine. Following removal of PMN(act) , suppressed T cells could respond normally to further stimulation. In addition to suppressing proliferation, co-culture with PMN(act) also induced a significant decrease in T-cell viability that was reversed by catalase addition (P < 0·05). The addition of the arginase inhibitor N-hydroxy-nor-l-arginine induced both a further significant, catalase-sensitive, loss in T-cell viability and increased nitrite release (P < 0·001). These data demonstrate that PMN, when activated, can both induce T-cell death and reversibly inhibit proliferation of activated T cells. The mechanisms underlying these distinct processes and the effects of arginase inhibitors on PMN induced cytotoxicity merit further investigation.

Cell-Intrinsic NF-κB Activation Is Critical for the Development of Natural Regulatory T Cells in Mice

BackgroundNaturally occurring CD4+CD25+Foxp3+ regulatory T (Treg) cells develop in the thymus and represent a mature T cell subpopulation critically involved in maintaining peripheral tolerance. The differentiation of Treg cells in the thymus requires T cell receptor (TCR)/CD28 stimulation along with cytokine-promoted Foxp3 induction. TCR-mediated nuclear factor kappa B (NF-κB) activation seems to be involved in differentiation of Treg cells because deletion of components of the NF-κB signaling pathway, as well as of NF-κB transcription factors, leads to markedly decreased Treg cell numbers in thymus and periphery.Methodology/Principal FindingsTo investigate if Treg cell-intrinsic NF-κB activation is required for thymic development and peripheral homeostasis of Treg cells we used transgenic (Tg) mice with thymocyte-specific expression of a stable IκBα mutant to inhibit NF-κB activation solely within the T cell lineage. Here we show that Treg cell-intrinsic NF-κB activation is important for the generation of cytokine-responsive Foxp3− thymic Treg precursors and their further differentiation into mature Treg cells. Treg cell development could neither be completely rescued by the addition of exogenous Interleukin 2 (IL-2) nor by the presence of wild-type derived cells in adoptive transfer experiments. However, peripheral NF-κB activation appears to be required for IL-2 production by conventional T cells, thereby participating in Treg cell homeostasis. Moreover, pharmacological NF-κB inhibition via the IκB kinase β (IKKβ) inhibitor AS602868 led to markedly diminished thymic and peripheral Treg cell frequencies.Conclusion/SignificanceOur results indicate that Treg cell-intrinsic NF-κB activation is essential for thymic Treg cell differentiation, and further suggest pharmacological NF-κB inhibition as a potential therapeutic approach for manipulating this process.

B7-H4 mediates inhibition of T cell responses by activated murine hepatic stellate cells

Liver fibrosis is mediated by the transformation of hepatic stellate cells (HSC) from a quiescent to an activated state. To understand the role of HSC in liver immunity, we investigated the effect of this transition on T cell stimulation in vitro. Unlike quiescent HSC, activated HSC did not induce proliferation of antigen-specific T cells. Phenotypic analysis of quiescent and activated HSC revealed that activated HSC expressed the coinhibitory molecule B7-H4. Silencing B7-H4 by small interfering RNA (siRNA) in activated HSC restored the ability of T cells to proliferate, differentiate, and regain effector recall responses. Furthermore, expression of B7-H4 on HSC inhibits early T cell activation and addition of exogenous interleukin (IL)-2 reversed the T cell anergy induced by activated HSC. These studies reveal a novel role for activated HSC in the attenuation of intrahepatic T cell responses by way of expression of the coinhibitory molecule B7-H4, and may provide fundamental insight into intrahepatic immunity during liver fibrogenesis.

CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling

CD95 is a multifunctional receptor that induces cell death or proliferation depending on the signal, cell type, and cellular context. Here, we describe a thus far unknown function of CD95 as a silencer of T cell activation. Naive human T cells triggered by antigen-presenting cells expressing a membrane-bound form of CD95 ligand (CD95L) or stimulated by anti-CD3 and -CD28 antibodies in the presence of recombinant CD95L had reduced activation and proliferation, whereas preactivated, CD95-sensitive T cells underwent apoptosis. Triggering of CD95 during T cell priming interfered with proximal T cell receptor signaling by inhibiting the recruitment of ζ-chain–associated protein of 70 kD, phospholipase-γ, and protein kinase C-θ into lipid rafts, thereby preventing their mutual tyrosine protein phosphorylation. Subsequently, Ca2+ mobilization and nuclear translocation of transcription factors NFAT, AP1, and NF-κB were strongly reduced, leading to impaired cytokine secretion. CD95-mediated inhibition of proliferation in naive T cells could not be reverted by the addition of exogenous interleukin-2 and T cells primed by CD95 co-stimulation remained partially unresponsive upon secondary T cell stimulation. HIV infection induced CD95L expression in primary human antigeen-presenting cells, and thereby suppressed T cell activation, suggesting that CD95/CD95L-mediated silencing of T cell activation represents a novel mechanism of immune evasion.

Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells

AbstractIt has been shown that mesenchymal stem cells (MSCs) induce T cells to become unresponsive. We characterized the phenotype of these T cells by dissecting the effect of MSCs on T-cell activation, proliferation, and effector function. For this purpose, an in vitro murine model was used in which T-cell responses were generated against the male HY minor histocompatibility antigen. In the presence of MSCs, the expression of early activation markers CD25 and CD69 was unaffected but interferon-γ (IFN-γ) production was reduced. The inhibitory effect of MSCs was directed mainly at the level of cell proliferation. Analysis of the cell cycle showed that T cells, stimulated in the presence of MSCs, were arrested at the G1 phase. At the molecular level, cyclin D2 expression was profoundly inhibited, whereas p27kip1 was up-regulated. When MSCs were removed from the cultures and restimulated with the cognate peptide, T cells produced IFN-γ but failed to proliferate. The addition of exogenous interleukin-2 (IL-2) did not restore proliferation. MSCs did not preferentially target any T-cell subset, and the inhibition was also extended to B cells. MSC-mediated inhibition induces an unresponsive T-cell profile that is fully consistent with that observed in division arrest anergy.

Involvement of CTLA-4 in T-cell anergy induced by staphylococcal enterotoxin A in vitro

Superantigens, like staphylococcal enterotoxin A (SEA), induce a strong proliferative response followed by clonal deletion of a substantial portion of defined V β T-cells. The remaining cells display in vitro anergy. Anergy is a major mechanism to ensure antigen-specific tolerance in T-lymphocyte in the adult. Co-stimulatory molecules B7-1 (CD80)/B7-2 (CD86) and their counter-receptors CD28/CTLA-4 play pivotal roles in T-cell activation and immune regulation. While increasing data further suggested a role for CTLA-4 in regulating T-cell tolerance in vivo, the mechanism by which CTLA-4 influences T-lymphocyte tolerance is unclear. In the present study, we established an in vitro anergy model using superantigen SEA as the anergizing agents and examined CD3, CD28 and CTLA-4 expression of anergic T-cells in response to SEA rechallenge. It is found that anergic T-cell fails to produce the autocrine growth factor interleukin-2 (IL-2) upon stimulation, and addition of exogenous IL-2 can reverse the anergic state. Both TCR/CD3 complex and CD28 expression is not reduced in anergic cells during whole immune response, but the expression of CTLA-4 on the cell surface is enhanced dramatically in the late stages of an immune response. Using CTLA-4/B7-blocking agent, we found T-cell anergy was aborted and anergic T-cells restored the ability to proliferate and produce IL-2, suggesting that CTLA-4 may play a critical role in the induction of T-cell anergy.

CD30 is involved in inhibition of T-cell proliferation by Hodgkin's Reed-Sternberg cells.

CD30 is expressed on Hodgkin's Reed-Sternberg (H-RS) cells, the tumor cells in Hodgkin's disease. Increased levels of serum CD30 are observed in Hodgkin's disease patients and are a good marker for predicting a poor prognosis and a poor response to therapy. In this study, we addressed the effect of CD30 on T cells. We showed that CD30, either as a membranous protein on H-RS cells and Chinese hamster ovary cells or as a plate-bound chimeric protein, inhibited T-cell proliferation. Anti-CD3-stimulated T cells in the presence of CD30 failed to increase tritium uptake and failed to express CD25 and CD26 and to produce interleukin 2. The inhibition of T-cell proliferation was, however, reversed with addition of exogenous interleukin 2 or pretreatment of H-RS cells with anti-CD30. Inability of T cells to express CD25 and CD26 in cocultures with H-RS cells or a plate-bound CD30 chimeric protein is in accordance with the results of immunohistochemistry on disease-involved tissues. We conclude that H-RS cells are able to inhibit the proliferation and activation of T cells through CD30-related interaction. The outcome of CD30-related interaction is an ineffective antitumor immunity, which is clearly in favor of the growth and survival of the tumor cells.

Diminished proliferation of human immunodeficiency virus-specific CD4+ T cells is associated with diminished interleukin-2 (IL-2) production and is recovered by exogenous IL-2.

Virus-specific CD4(+) T-cell function is thought to play a central role in induction and maintenance of effective CD8(+) T-cell responses in experimental animals or humans. However, the reasons that diminished proliferation of human immunodeficiency virus (HIV)-specific CD4(+) T cells is observed in the majority of infected patients and the role of these diminished responses in the loss of control of replication during the chronic phase of HIV infection remain incompletely understood. In a cohort of 15 patients that were selected for particularly strong HIV-specific CD4(+) T-cell responses, the effects of viremia on these responses were explored. Restriction of HIV replication was not observed during one to eight interruptions of antiretroviral therapy in the majority of patients (12 of 15). In each case, proliferative responses to HIV antigens were rapidly inhibited during viremia. The frequencies of cells that produce IFN-gamma in response to Gag, Pol, and Nef peptide pools were maintained during an interruption of therapy. In a subset of patients with elevated frequencies of interleukin-2 (IL-2)-producing cells, IL-2 production in response to HIV antigens was diminished during viremia. Addition of exogenous IL-2 was sufficient to rescue in vitro proliferation of DR0101 class II Gag or Pol tetramer(+) or total-Gag-specific CD4(+) T cells. These observations suggest that, during viremia, diminished in vitro proliferation of HIV-specific CD4(+) T cells is likely related to diminished IL-2 production. These results also suggest that relatively high frequencies of HIV-specific CD4(+) T cells persist in the peripheral blood during viremia, are not replicatively senescent, and proliferate when IL-2 is provided exogenously.

Costimulatory molecule requirement for bovine WC1+gammadelta T cells' proliferative response to bacterial superantigens.

We have previously shown that the proliferation of freshly isolated bovine WC1+gammadelta T cells to superantigens (SAgs) including staphylococcal enterotoxin A (SEA), and staphylococcal enterotoxin B (SEB) or toxic shock syndrome type-1 (TSST-1) required the presence of antigen-presenting cells (APC) and the addition of exogenous interleukin (IL)-2. The costimulatory activity provided by molecules expressed on professional APC for the proliferation of gammadelta T cells has not been addressed hitherto. In the present study, we investigated the ability of two selected APC populations, the dendritic cells (DCs) highly expressing CD80 and CD86 molecules (CD80highCD86high) and the monocytes expressing the same molecules at a rather low level (CD80lowCD86low), to stimulate the proliferation of purified bovine WC1+gammadelta T cells to SAgs. DCs were more efficient than monocytes in inducing gammadelta T-cell proliferation, and this response was dependent on exogenous IL-2 in both presentation modes. Stimulating gammadelta T cells with gradual doses of SAgs or concanavalin A (ConA) resulted in similar dose-dependent reaction profiles suggesting a minimal role of the major histocompatibility complex (MHC). However, significant proliferation was already obtained with the starting doses in the presence of DC compared with monocytes, and higher proliferation was reached with DC at optimal doses. Finally, the addition of monoclonal antibody (MoAb) anti-CD86 markedly inhibited SAgs- and ConA-mediated proliferation, whereas MoAb anti-CD80 had no effect. The combination of both anti-CD80 and anti-CD86, however, suppressed this response. These results suggest that bovine gammadelta T-cell proliferation response requires indubitably CD86 costimulation. The role of CD80 molecule seems less clear.

Importance of dose of type II collagen in suppression of collagen-induced arthritis by nasal tolerance.

To determine the influence of the dose of collagen given nasally on the induction of specific mucosal tolerance in collagen-induced arthritis. The severity of clinical arthritis induced in DBA/1 mice was studied after the nasal administration (before disease induction) of 1 of 4 doses (across a 2-log range) of bovine type II collagen (CII). Parameters of immunity included lymphocyte proliferation and cytokine production in vitro in response to antigen stimulation, and the production of anticollagen IgG antibody subclasses. The 3 highest doses (20, 80, and 320 microg) ameliorated disease severity, whereas the lowest dose (5 microg) aggravated disease. These findings correlated well with antigen-specific T cell proliferation and cytokine and antibody production. T cell proliferation was suppressed by the higher doses of CII, whereas the low dose enhanced T cell proliferation, indicating it primed the T cells. Suppression of T cell proliferation could be overcome by the addition of exogenous interleukin-2 (IL-2) to these cultures. Decreased T cell proliferation was associated with suppression of both Th1 (interferon-gamma [IFNgamma]) and Th2 (IL-4) cytokines and all the subclasses of anticollagen IgG in mice receiving 20, 80, or 320 microg of collagen. Overall, the highest dose of collagen (320 microg) was less effective at suppressing the immune response and disease than the 20-microg or 80-microg doses. There was an increased production of antibodies of all IgG isotypes, and of the Th1-associated cytokines IFNgamma and IL-2, in animals that had received the lowest dose of 5 microg collagen nasally. Nasal administration of antigens is effective in inducing tolerance and reducing disease severity, but the effects are dose dependent. Low doses can prime the immune system and aggravate disease; high doses may not suppress disease. Suppression of the immune response, which correlates with suppression of disease, is not obviously associated with a type I to type II T cell switch, but rather with an overall suppression of both forms of T cell response, with a potential role for anergy of T cells in this process.

Lack of CD80 expression by fibroblast-like synoviocytes leading to anergy in T lymphocytes.

To investigate whether contact with HLA-DR+, but CD80-, fibroblast-like synoviocytes (FLS) in the presence of antigen leads to the induction of anergy in, rather than stimulation of, T cells. Cell surface expression of activation and costimulatory markers on FLS were studied by flow cytometry. Functional changes were investigated by T cell proliferation to tuberculin purified protein derivative or allogeneic responses to FLS, in the presence or absence of DAP3.B7 cells, a human CD80-transfected mouse fibroblast cell line. Induction of anergy was investigated by a 2-stage culture system. T cells were cocultured with allogeneic FLS in the primary culture, rested, and restimulated in the secondary culture by FLS in the presence or absence of DAP3.B7 cells or interleukin-2 (IL-2). Direct contact between T cells and FLS caused up-regulation of CD69 on T cells and HLA-DR on FLS in both the allogeneic and autologous cultures. The addition of DAP3.B7 cells to FLS-T cell cocultures restored the depressed allogeneic responses of T cells. The allogeneic response by T cells to FLS in the presence of DAP3.B7 cells could be completely inhibited by blocking CD80 with CTLA-4 Ig. Indirect evidence that T cells cocultured with FLS were anergic was the up-regulation of CD25, negligible T cell proliferation, and the restoration of proliferation by the addition of exogenous IL-2. Direct evidence of anergy was obtained when T cells from the primary cultures with FLS remained unresponsive to secondary culture with FLS even in the presence of DAP3.B7 cells. In contrast, primary culture of T cells with FLS plus DAP3.B7 cells initiated a good allogeneic response in all subsequent cultures. It is possible that T cells within the synovium may be anergized by contact with HLA-DR+ CD80- FLS.

Different Roles of the CD2 and LFA-1 T-Cell Co-receptors for Regulating Cytotoxic, Proliferative, and Cytokine Responses of Human Vγ9/Vδ2 T Cells

Human V gamma 9/V delta 2 T lymphocytes recognize nonpeptidic antigens in a manner distinct from the classical antigen recognition by alpha beta T cells. The apparent lack of major histocompatibility (MHC) restriction and antigen processing allows very fast responses against pathogenic insults. To address the potential functional requirement for accessory molecules, we investigated the roles of the CD2 and lymphocyte function-associated antigen (LFA)-1 T-cell co-receptors in antigen-induced activities of human V gamma 9/V delta 2 T-cell clones. Human peripheral blood V gamma 9/V delta 2 T lymphocytes were cloned and their cytotoxicity against Daudi lymphoma was measured by a standard 51Cr-release assay. The responses of V gamma 9/V delta 2 T lymphocytes to nonpeptidic antigens were assessed using DNA synthesis and cytokine ELISA assays. Monoclonal antibodies specific for various molecules with potential T-cell accessory functions were utilized in blocking assays. All of our V gamma 9/V delta 2 T-cell clones displayed the Th1 phenotype. The anti-LFA-1 antibody strongly inhibited the cytotoxicity of V gamma 9/V delta 2 T cells against Daudi B-cell lymphoma; whereas, it had no influence on the antigen-induced cytokine release or proliferation. In contrast, antibodies against CD2 and LFA-3 had no effect on the lytic activity of V gamma 9/V delta 2 T cells, but strongly inhibited the cytokine release and proliferation. However, the CD2-LFA-3 interaction was not an absolute requirement for the cytokine release and the DNA synthetic activity of antigen-stimulated V gamma 9/V delta 2 T cells, since the inhibitory effect could be reversed by addition of exogenous interleukin 2 (IL-2). These novel observations indicate that the signals generated by different accessory molecules and IL-2 can contribute in an integrated fashion to the regulation of V gamma 9/V delta 2 T cells. These interactions may be important for the effectiveness of V gamma 9/V delta 2 T-cell responses.

The B7/CD28/CTLA4 T-cell activation pathway. Implications for inflammatory lung disease.

The molecular events that modulate the T-cell response to antigen are key determinants in the outcome of pulmonary immune responses. Over the past several years, it has become evident that the B7:CD28 costimulation pathway is a critical regulator of T-cell responses both in vitro and in vivo . Given its importance in T-cell activation and its potential as a target for immune-based therapies, this perspective will review the current data on the regulation of T-cell activation by the B7 costimulatory pathway. In 1970 Bretscher and Cohn put forth the two-signal model of lymphocyte activation to explain self/nonself discrimination (1). This model proposes that T-cell activation requires two independent signals. The first is transduced through the T-cell receptor (TCR) after engagement by antigen; and the second, costimulatory signal is delivered by ligation of a distinct receptor present on the surface of the T cell. This model predicts that engagement of the TCR in the absence of costimulation will fail to activate the T cell. A substantial body of evidence has accumulated in support of this hypothesis. Early studies demonstrated that antigen presented by chemically fixed antigen-presenting cells (APCs) resulted in a failure of T-cell activation and rendered them unresponsive to further antigenic stimulation, a process termed induction of anergy. This was shown to be the result of the inability of the fixed APCs to engage costimulatory molecules, in particular CD28 (2). The anergic T cell fails to produce the autocrine growth factor interleukin-2 (IL-2) upon stimulation, and addition of exogenous IL-2 can reverse the anergic state (3). The molecular basis for costimulation remained elusive until the cloning of CD28 by Arrufo and Seed in 1987 (4). First termed Tp44, crosslinking of CD28 was initially shown to augment the proliferative response of T cells in 1984 (5). CD28 enables T cells to proliferate in the presence of the immunosuppressant cyclosporine A (6). Cytokine expression is markedly enhanced after CD28 costimulation through both transcriptional activation and posttranscriptional stabilization of messenger RNA (mRNA) (7). In addition to these effects, CD28 also regulates cell survival by induction of the anti-apoptotic protein Bcl-X L and activation of the protein kinase Akt (8). CD28 has two ligands, B7-1 (CD80) and B7-2 (CD86). B7-1 was identified as an adhesion receptor on B cells that interacted specifically with CD28 (9, 10). Subsequent work led to the identification of a second member of the B7 family, B7-2, which also bound CD28 (11–13). The greater complexity of the system became evident with the identification of an additional counter-receptor on T cells, cytotoxic T-lymphocyte antigen 4 (CTLA4), which could bind both B7-1 and B7-2 (14, 15). In contrast to CD28, CTLA4 is expressed only on activated T cells and is a negative regulator of T-cell function (16). CTLA4-deficient mice manifest massive lymphoproliferative disease, which is lethal by 3 wk of age (17, 18). Recently, new members of the B7/CD28 family have been identified. The search for new tumor necrosis factor (TNF)–inducible, nuclear factor k B (NF k B)–dependent genes led to the cloning of B7h (19). This protein shares sequence homology with both B7-1 and B7-2 but does not bind to either CD28 or CTLA4. Instead, it binds to a newly identified CD28 homolog, inducible costimulator (ICOS), which is expressed on a subset of activated T cells (20). In addition, Dong and colleagues report the cloning of another B7 homolog, B7-H1, which may be the human ortholog of B7h (21). The role of these new B7/CD28 family members in the immune response remains to be explored. The importance of CD28 in in vivo immune responses was highlighted by early studies examining transplant rejection. Blockade of B7 with CTLA4Ig, a soluble inhibitor of B7-1 and B7-2, led to prolonged graft survival after both heterotopic cardiac transplantation in rats and islet-cell xenografts in mice (22, 23). Intriguing results have also been found in the manipulation of B7:CD28:CTLA4 interactions in tumor immunity. Transfection of B7 into murine melanoma cells led to effective antitumor responses in vivo (24). Prevention of negative signaling through CTLA4 resulted in regression of primary tumors, as well as augmented secondary responses upon tumor rechallenge in mice (25). In addition to its role in the initial activation of naive T cells, recent work has examined whether CD28 influences the subsequent differentiation of CD4 1 T cells. Blockade of B7-1 in experimental autoimmune encephelomyelitis led to a reduction in disease severity and promotion of T helper (Th)2-cell development (26). In contrast, blockade ( Received in original form January 3, 2000 )

Inhibition of proliferation of human peripheral blood mononuclear cells by calcium antagonists. Role of interleukin-2.

To evaluate the role of interleukin-2 (IL-2) in the inhibition of the proliferation of human peripheral blood mononuclear cells (PBMC) by calcium channel blockade, the effect of nifedipine, which blocks the L-type calcium channel on the proliferation, the IL-2 expression and the IL-2 production in human PBMC, was compared with the effect of mibefradil, which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels. The rate of [3H]-thymidine incorporation into control and concanavalin A-induced PBMC in the presence or absence of the calcium channel blockers nifedipine or mibefradil (1, 10 or 50 microM) was assayed in the cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures was also determined in nifedipine- or mibefradil-treated control or stimulated cells. Restoration of the proliferative response in nifedipine- or mibefradil-treated cells was investigated by addition of exogenous IL-2. IL-2 receptor expression in the cells was monitored using antiactivated T-cell antigen (Tac) antibody, and the IL-2 production in the cell supernatants of the cultures was determined by an enzyme amplified sensitive immunoassay. Nifedipine and mibefradil concentration-dependently reduced the cell number and [3H]-thymidine incorporation or the do novo DNA synthesis in control and concanavalin A-stimulated human PBMC. The proliferative response of nifedipine- or mibefradil-treated cells was restored by addition of exogenous IL-2. The normal expression of IL-2 receptors was preserved while the IL-2 production was blocked in the presence of nifedipine or mibefradil.

Stealth Cells: Prevention of Major Histocompatibility Complex Class II-Mediated T-Cell Activation by Cell Surface Modification

AbstractTransfusion or transplantation of T lymphocytes into an allogeneic recipient can evoke potent immune responses including, in immunocompromised patients, graft-versus-host disease (GVHD). As our previous studies demonstrated attenuated immunorecognition of red blood cells covalently modified with methoxy(polyethylene glycol) (mPEG), we hypothesized that T-cell activation by foreign antigens might similarly be prevented by mPEG modification. Mixed lymphocyte reactions (MLR) using peripheral blood mononuclear cells (PBMC) from HLA class II disparate donors demonstrate that mPEG modification of PBMC effectively inhibits T-cell proliferation (measured by 3H-thymidine incorporation) in a dose-dependent manner. Even slight derivatization (0.4 mmol/L mPEG per 4 × 106 cells) resulted in a ≥75% decrease, while higher concentrations caused ≥96% decrease in proliferation. Loss of PBMC proliferation was not due to either mPEG-induced cytotoxicity, as viability was normal, or cellular anergy, as phytohemagglutinin (PHA)-stimulated mPEG-PBMC demonstrated normal proliferative responses. Addition of exogenous interleukin (IL)-2 also had no proliferative effect, suggesting that the mPEG-modified T cells were not antigen primed. Flow cytometric analysis demonstrates that mPEG-modification dramatically decreases antibody recognition of multiple molecules involved in essential cell:cell interactions, including both T-cell molecules (CD2, CD3, CD4, CD8, CD28, CD11a, CD62L) and antigen-presenting cell (APC) molecules (CD80, CD58, CD62L) likely preventing the initial adhesion and costimulatory events necessary for immune recognition and response.

Stealth Cells: Prevention of Major Histocompatibility Complex Class II-Mediated T-Cell Activation by Cell Surface Modification

Transfusion or transplantation of T lymphocytes into an allogeneic recipient can evoke potent immune responses including, in immunocompromised patients, graft-versus-host disease (GVHD). As our previous studies demonstrated attenuated immunorecognition of red blood cells covalently modified with methoxy(polyethylene glycol) (mPEG), we hypothesized that T-cell activation by foreign antigens might similarly be prevented by mPEG modification. Mixed lymphocyte reactions (MLR) using peripheral blood mononuclear cells (PBMC) from HLA class II disparate donors demonstrate that mPEG modification of PBMC effectively inhibits T-cell proliferation (measured by 3H-thymidine incorporation) in a dose-dependent manner. Even slight derivatization (0.4 mmol/L mPEG per 4 × 106 cells) resulted in a ≥75% decrease, while higher concentrations caused ≥96% decrease in proliferation. Loss of PBMC proliferation was not due to either mPEG-induced cytotoxicity, as viability was normal, or cellular anergy, as phytohemagglutinin (PHA)-stimulated mPEG-PBMC demonstrated normal proliferative responses. Addition of exogenous interleukin (IL)-2 also had no proliferative effect, suggesting that the mPEG-modified T cells were not antigen primed. Flow cytometric analysis demonstrates that mPEG-modification dramatically decreases antibody recognition of multiple molecules involved in essential cell:cell interactions, including both T-cell molecules (CD2, CD3, CD4, CD8, CD28, CD11a, CD62L) and antigen-presenting cell (APC) molecules (CD80, CD58, CD62L) likely preventing the initial adhesion and costimulatory events necessary for immune recognition and response.

CTLA-4 Ligation Suppresses CD28-induced NF-κB and AP-1 Activity in Mouse T Cell Blasts

The effects of cytotoxic lymphocyte antigen 4 (CTLA-4) on CD3/CD28 monoclonal antibody (mAb) activation of CD4(+)/CTLA-4(+) blastoid T cells were studied in an in vitro model system. As previously reported, coligation of CTLA-4 mAb results in suppression of T cell proliferation and cytokine production. The proliferation but not the interleukin 2 (IL-2) production could be restored by addition of exogenous IL-2, suggesting that the inhibitory effect occurred at the level of IL-2 production rather than at the regulation of the IL-2 receptor pathway. To study the effects on nuclear factors critical for T cell activation, we analyzed the levels of the transcription factors NF-kappaB and AP-1. These were potently induced in CD3/CD28 mAb-restimulated T cells. In contrast, CTLA-4 ligation strongly suppressed the induction of both transcription factors. The compositions of NF-kappaB and AP-1 family members were similar, irrespective of stimulation conditions. Analyses of the NF-kappaB regulator IkappaB-alpha revealed similar levels of IkappaB-alpha protein in the preparations. However, a reduced phosphorylation of IkappaB-alpha in CTLA-4 coengaged T cell blasts compared with T cells ligated with CD3/CD28 was found. Previous studies have concluded that CTLA-4 ligation regulates T cell activation by inhibiting the T cell receptor-mediated signals. However, the present findings propose that the major impact of CTLA-4 ligation is inhibition of signals mediated by CD28.

Mibefradil-induced inhibition of proliferation of human peripheral blood mononuclear cells.

To evaluate the role of intracellular calcium and particularly Ca2+-uptake in the initiation of lymphocyte mitogenesis, the effect of mibefradil, which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels, on the proliferation of human peripheral blood mononuclear cells (PBMCs) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of [3H]thymidine incorporation into control and concanavalin A-stimulated PBMCs in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 microM), and of the intracellular calcium antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8; 1, 10, 25, or 50 microM) was assayed in the cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures also was determined in mibefradil- or nifedipine-treated control or stimulated cells. Restoration of the proliferative response in mibefradil- or nifedipine-treated cells was investigated by addition of exogenous interleukin-2. Interleukin-2-receptor expression in the cells was monitored by using anti-activated T-cell antigen (Tac) antibody, and the interleukin-2 production in the cell supernatants of the cultures was determined by an enzyme-amplified sensitive immunoassay. Mibefradil and nifedipine concentration-dependently reduced the cell number and the [3H]thymidine incorporation or the de novo DNA synthesis in control and concanavalin A-stimulated human PBMCs. Mibefradil exhibited a more pronounced inhibition of the proliferation of human PBMCs than did nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent on the timing of treatment with the drugs. The inhibitory effect of mibefradil or nifedipine on the lymphoproliferative response was nearly abolished if the drugs were added 20 h after cell stimulation. A markedly reduced inhibitory effect was found when mibefradil or nifedipine was added 1-7 h after cell stimulation. However, regardless of time of addition, TMB-8 caused a persistent inhibition of the proliferation of human PBMCs. The inhibitory effect of mibefradil or nifedipine on the proliferation of human PBMCs is nearly abolished by addition of the calcium channel activator Bay K 8644. The proliferative response of mibefradil- or nifedipine-treated cells is restored by addition of exogenous interleukin-2. The normal expression of interleukin-2 receptors was preserved, whereas the interleukin-2 production was blocked in the presence of mibefradil or nifedipine. Our data show that mibefradil has a more pronounced inhibitory effect on the proliferation of human PBMCs than nifedipine and that this inhibitory effect on DNA synthesis is dependent on the timing of treatment with both drugs.