Cytokine profiles of experimental autoimmune myasthenia gravis after treatment by the synthetic immunomodulator linomide.
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Adjuvants, Immunologic↗
Biomedical subjects
G Hedlund
Publications and source records attributed to G Hedlund.
The immune modulator Linomide prevents neuronal death in injured peripheral nerves of the mouse.
Neuronal death after injury or disease could result from imbalanced cytokine expression. Linomide (LS-2616, quinoline-3-carboxamide), a synthetic immunomodulator with effects on cytokine production, suppresses autoimmune diseases of the nervous system. Here adult mice were pre-treated with 200 mg/kg/day of Linomide for 9 days, after which the sciatic nerves were crushed. After another 10 days of Linomide treatment the dorsal root ganglia were dissected out and stained for apoptosis, either immediately or after 2 days in culture, which increases cell death. Superior cervical ganglia were also cultured for 2 days. The Linomide pretreatment profoundly reduced (approximately 60-80%) the injury-induced apoptotic death of neurons and satellite cells in both systems. The results suggest that modulation of the inflammatory cytokine cascade is a promising road to nerve cell rescue.
Adjuvants, Immunologic↗
Dendritic cells and macrophages are the first and major producers of TNF-alpha in pancreatic islets in the nonobese diabetic mouse.
The nonobese diabetic (NOD) mouse spontaneously develops autoimmune insulin-dependent diabetes mellitus (IDDM) and serves as an animal model for human type I diabetes. TNF-alpha is known to be produced by islet-infiltrating mononuclear cells during insulitis and subsequent beta cell destruction and has been implicated in the pathogenesis of IDDM. Previously, T cells have been suggested as the main source of TNF-alpha in the islet infiltrate. However, on immunohistochemical analysis of TNF-alpha expression in islets, we are able to show that the staining pattern of TNF-alpha resembles that of dendritic cells (DC) and macrophages (Mphi) rather than T cells and that TNF-alpha is expressed in islets at the very early stages of insulitis when no T cells are detected. On double staining for TNF-alpha and cell surface markers, we can demonstrate that TNF-alpha staining clearly correlates with DC and Mphi, whereas there is a poor correlation with T cells. This feature was observed at both early and late stages of insulitis. TNF-alpha expression was also seen in NOD-SCID islets, in addition to a peri-islet infiltration consisting of DC and Mphi, indicating that T cells are not required for the early DC and Mphi infiltration and TNF-alpha expression in islets. In conclusion, our results show that DC and Mphi are the major, early source of TNF-alpha in the NOD islet infiltrate and that TNF-alpha can be expressed independently of T cells, indicating that the early DC and Mphi infiltration and expression of TNF-alpha are crucial in initiation of diabetes.
Animals↗
ICAM-1 costimulation induces IL-2 but inhibits IL-10 production in superantigen-activated human CD4+ T cells.
We have previously reported that costimulatory pathways including B7-CD28 and lymphocyte function-associated antigen-3 (LFA-3)-CD2 shape distinct activation profiles in human CD4+ T cells. We now show that superantigen (SAg), in combination with intracellular adhesion molecule-1 (ICAM-1) costimulation drives a proliferative response accompanied by high levels of interleukin-2 (IL-2) and moderate levels of interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF). This response profile differs from that observed in B7 or LFA-3 costimulated T cells because our previous results showed that B7-CD28 costimulation was accompanied by high levels of IL-2, IFN-gamma and TNF, whereas LFA-3 was a potent inducer of IFN-gamma and TNF, but had little influence on IL-2 production. The ICAM-1-induced IL-2 production could efficiently be abrogated with monoclonal antibody (mAb) against ICAM-1 or LFA-1, showing that the activation is dependent of a functional ICAM-1-LFA-1 pathway. SAg-induced IL-2, IFN-gamma and TNF were detected in both CD4+ and CD8+ T cells, whereas production of IL-10 was restricted to CD4+ T cells. A major finding in the present study was that ICAM-1 costimulation strongly inhibits IL-10 production in CD4+ T cells. Our data demonstrate that ICAM-1 costimulation is sufficient to induce large amounts of IL-2. The presence of ICAM-1 results in suppression of IL-10 production in T helper (Th) cells, which may favour the development of Th1 and not Th2 T cells.
Biomarkers↗
The effects of leflunomide and cyclosporin A on rejection of cardiac allografts in the rat.
Leflunomide is a new low molecular weight immunosuppressive drug which inhibits the enzymes dehydroorotate-dehydrogenase and protein tyrosine kinase, both of which are important components in the immune response. As the mechanisms of action of leflunomide and cyclosporin A (CsA) are different, we postulated a synergistic effect of the two drugs and tested graft survival following leflunomide administration alone or in combination with CsA in a rat cardiac transplantation model. Low- and high-responder rat strain combinations were used in parallel and the experiments were performed both with and without challenge with Linomide, an immunomodulator which promotes graft rejection in this model. In the low-responder rat strain combination (Piebald Virol Glaxo graft to Dark Agouti recipient; PVG to DA), graft survival appeared to be a dichotomous variable, being characterized by tolerance or early rejection. Leflunomide (10 or 5 mg/kg) given for 10 days induced tolerance and CsA did likewise; the addition of Linomide abolished the immunosuppressive effect of leflunomide but not that of CsA. In the high-responder combination (DA to PVG), no tolerance was seen and graft survival was moderately prolonged both after leflunomide and after CsA treatment; the addition of Linomide to CsA or to leflunomide (5 mg/kg) abolished the immunosuppressive effect of the drugs. However, when CsA-Linomide or leflunomide-Linomide were supplemented with the second immunosuppressive drug, leflunomide or CsA respectively, graft survival was significantly prolonged (P < 0.001 in both cases). This suggests leflunomide and CsA have additive potential.
Animals↗
Linomide (roquinimex) affects the balance between pro- and anti-inflammatory cytokines in vitro in multiple sclerosis.
OBJECTIVES: Multiple sclerosis (MS) is characterized by high levels of circulating mononuclear cells (MNC) that respond to myelin proteins like myelin basic protein (MBP) in vitro by expressing mRNA of both pro-inflammatory cytokines, e.g. interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT) that may make MS worse, and anti-inflammatory cytokines like IL-4, IL-10 and transforming growth factor-beta (TGF-beta) that may act beneficially. Substances that down-regulate cytokines such as TNF-alpha or promote IL-10 or TGF-beta can be anticipated to affect MS beneficially. MATERIAL AND METHODS: In situ hybridization to detect and enumerate IFN-gamma, TNF-alpha, LT, IL-4, IL-10 and TGF-beta mRNA expressing blood MNC after stimulation with myelin basic protein (MBP), control antigens and without antigen in presence and absence of Linomide (roquinimex, LS-2616) was employed. In parallel, ELISPOT assay to detect MBP- and PHA-reactive IFN-gamma secreting blood MNC+/-Linomide was used. RESULTS: Here we report that Linomide, a synthetic immunomodulator, at concentrations effective in vivo reduces the number of MBP-reactive TNF-alpha and increases MBP-reactive IL-10 and TGF-beta mRNA expressing MNC from MS patients' blood when analysed in vitro. Compared to dexamethasone, Linomide up-regulated levels of blood MNC expressing mRNA of TGF-beta after culture in presence of MBP. CONCLUSIONS: Changes of cytokine balance towards a production of anti-inflammatory cytokines could be a desirable effect to be evaluated in future drug studies of Linomide-like substances. At present, Linomide is not evaluable in MS clinical trials due to side-effects.
Adjuvants, Immunologic↗
The inhibitory effect in experimental autoimmune encephalomyelitis by the immunomodulatory drug Linomide (PNU-212616) is not mediated via release of endogenous glucocorticoids.
The immunomodulatory drug Linomide (PNU-212616) is an efficient inhibitor of experimental autoimmune encephalomyelitis (EAE) and a variety of other models of autoimmunity. The mechanism of action of the drug is, however, incompletely resolved. It was recently suggested that Linomide might exert its immunomodulatory activity by stimulation of the hypothalamic-pituitary-adrenal axis. To investigate the relevance of this mechanism of action, we monitored the plasma levels of endogenous corticosterone after treatment with Linomide, and also directly compared the inhibitory activity of the drug on acute EAE induced in sham or adrenalectomized SJL/N mice. Treatment with Linomide resulted in a dose related inhibition of EAE in line with previously reported results. Upon onset of clinical signs of EAE, there was a 7-10 fold elevation of plasma corticosterone from the normal baseline level. Administration of Linomide did however not by itself result in any change in plasma corticosterone levels, neither at the pre-symptomatic phase of the disease nor during acute short term treatment. In adrenal ectomized animals immunized for EAE, paralytic disease developed rapidly and was of a more severe and fatal nature as compared to sham-operated controls. Treatment with Linomide had a profound inhibitory effect on development of paralytic disease in both the ectomized and sham-operated groups. These results strongly suggest that Linomide does not exert its immunomodulatory activity via the release of endogenous glucocorticoids.
Adjuvants, Immunologic↗
Activation of alloreactive natural killer cells is resistant to cyclosporine.
BACKGROUND: We have previously shown that cytotoxic T lymphocytes (CTL) with alloreactivity were induced when Wistar Furth (WF; RT1u) rats were immunized with allogeneic Brown Norway (BN; RT1n) cells. In contrast, when BN rats were immunized with WF cells, the allospecific response was confined to alloreactive natural killer (NK) cells, and no CTL activity was observed. In this study, the effect of cyclosporine (CsA) on the activation of alloreactive NK cells in vivo was analyzed. METHODS: Distinct peritoneal effector cells from rats immunized with allogenic cells with or without concomitant CsA and/or interleukin (IL) 2 treatment were tested for specific cytolytic activity. Furthermore, the presumptive role of NK cells in rejection immunity was addressed in a cardiac graft model. RESULTS: The results showed that doses of CsA that completely inhibited the activation of alloreactive CTL, only marginally affected the activation of alloreactive NK cells. We also showed that CsA treatment failed to prolong graft survival in BN recipients of WF hearts. Treatment of BN rats with CsA/IL-2 during immunization with allogeneic WF cells resulted in concomitant induction of alloreactive NK cells and alloreactive CTL. CONCLUSIONS: We have demonstrated that CsA failed to suppress the activation of alloreactive NK cells. Consequently, the cardiac graft survival in the donor-recipient combination known to activate alloreactive NK cells was not significantly prolonged by CsA treatment, emphasizing the involvement of NK cells as effectors in organ rejection. Furthermore, the parallel emergence of alloreactive NK cells and CTL only in the presence of CsA/IL-2 indicated that CsA interfered with alloreactive NK cell-associated suppression of CTL activated by allogeneic tissue.
Animals↗
The role of B7-1 and LFA-3 in costimulation of CD8+ T cells.
This study compares the ability of LFA-3 (CD58) and B7-1 (CD80) ligands to provide costimulatory signals for superantigen (SAg)-stimulated CD8+ and CD4+ T cells. We show that B7-1 and LFA-3 costimulation activate CD8+ T cells to proliferation, cytokine production (IL-2, TNF, and IFN-gamma), and cytotoxicity. A long-lasting proliferative response was observed after combined DR/B7-1/LFA-3 costimulation. Detailed analysis of SEA-activated CD8+ T cells revealed that maximal production of IFN-gamma was seen in LFA-3-costimulated cells, while production of IL-2 was mainly induced after B7-1 costimulation. A fivefold increase in the IFN-gamma production was observed when activated CD8+ T cells were costimulated with Chinese hamster ovary (CHO)-DR/LFA-3 cells compared with the secretion induced by CHO-DR/B7-1. In contrast, SEA-treated CD4+ T cells costimulated with B7-1 or LFA-3 gave rise to a similar production of IFN-gamma, suggesting a preferential function for the CD2/LFA-3 pathway in the regulation of IFN-gamma in CD8+ T cells. Moreover, the generation of CTL was supported similarly by B7-1 and LFA-3 costimulation, but not by CHO-DR cells. We conclude that ligation of the CD28 and CD2 receptors mediate distinct effect on CD8+ and CD4+ T cell effector functions.
Animals↗
Allogeneic heart transplantation activates alloreactive NK cells.
The ability of natural killer (NK) cells to recognize and reject transplants has so far been shown in hematopoietic grafts only. This study was designed to ascertain whether NK cells may also be involved in the rejection of transplanted organs. In most rat strain combinations, immunization with allogeneic cells induces a T cell response with cytotoxic T lymphocyte (CTL) activation. We have previously found one exception to this. In contrast to Wistar Furth rats (WF, RT1u), which manifest allospecific CTL activation in response to immunization with Brown Norway (BN, RT1n) cells, BN rats immunized with repeated intraperitoneal (i.p.) injections of allogeneic WF spleen cells manifest activation of alloreactive NK effector cells. The alloreactive NK cells were of the TCR-, CD3-, CD8+, and NKR-P1 intermediate phenotype and killed target cells with alloselectivity. In this study we used a heart transplantation model to study the rejection response of BN rats receiving WF grafts. NK cell infiltration was greater in WF hearts transplanted to BN recipients than in BN hearts transplanted to WF recipients. Furthermore, the extent of T cell infiltration was less in BN recipients. In WF rats transplanted with allogeneic BN hearts, CTL were activated in response to i.p. challenge with allogeneic BN cells, whereas BN rats transplanted with allogeneic WF hearts and i.p. challenged with allogeneic WF cells, manifested activation of alloreactive NK cells but no measurable activation of classic CTL. The alloreactive NK cells killed their allogeneic targets with specificity and with potency comparable to that of CTL. Furthermore, WF grafts were rejected in BN recipients as efficiently as were BN grafts in WF recipients. These results not only show cardiac allografts to be able to activate alloreactive NK cells, but also suggest that NK cells may be involved in the rejection of solid organ transplants and function as classic CTL in certain donor-recipient combinations.
Animals↗
Prevention of superantigen-induced tolerance in vivo by interleukin-2 treatment.
Injection of the superantigen staphylococcal enterotoxin A (SEA) activates both CD4+ and CD8+ T cells expressing certain families of T cell receptor (TCR) variable-region beta (V beta) chain. T cells respond with profound cytokine production and induction of cytotoxicity. Repeated injections, however, cause deletion and anergy of both CD4+ and CD8+ T cells, resulting in reduced frequency of SEA-responsive cells TCR-V beta11+ as well as reduced cytokine levels in serum upon challenge with SEA. Exogenous interleukin-2 (IL-2) in vivo rescued SEA-responsive CD4+ and CD8+ cells from SEA-induced deletion and/or increase expansion of SEA-primed cells as well as preventing downregulation of endogenous IL-2 production in vivo. Combined treatment with SEA and IL-2 also superinduced production of important cytokines for the cytotoxic function of T cells, tumour necrosis factor alpha, interferon gamma and IL-6, on a cellular level. These studies show that continuous stimulation with IL-2 in vivo could be useful for superantigen-based immunotherapy by induction of excessive T cell activation and by prevention of the development of T cell deletion and anergy.
Animals↗
Linomide suppresses both Th1 and Th2 cytokines in experimental autoimmune myasthenia gravis.
Suppressive effects of the synthetic immunomodulatory drug Linomide have been shown in several autoimmune models, including antibody-mediated experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia gravis (MG). To define the mechanisms underlying EAMG suppression, we injected Linomide subcutaneously at different doses into Lewis rats immunized with Torpedo acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA), and investigated AChR-specific T and B cell responses, and the levels of lymph node cells expressing mRNA of different cytokines after AChR stimulation in vitro. Both 160 and 16, but not 1.6, mg/kg/day of Linomide effectively suppressed clinical muscle weakness, accompanied by decreased AChR-induced T and B cell responses. Linomide also suppressed the mRNA expression of the Th1 cytokines IFN-gamma, IL-12 and TNF-alpha as well as the Th2 cytokines IL-4 and IL-10, which are important in the immunopathogenesis of EAMG by promoting antibody production. There were no differences for IL-1beta, IL-6, lymphotoxin or TGF-beta expression in Linomide-treated vs nontreated control EAMG rats. We conclude that Linomide suppresses clinical EAMG as well as B and T cell responses to AChR by counteracting the production of AChR-induced Th1 and Th2 cytokines.
Adjuvants, Immunologic↗
Linomide-induced suppression of experimental autoimmune neuritis is associated with down-regulated macrophage functions.
Experimental autoimmune neuritis (EAN) is a T-cell mediated autoimmune disease of the peripheral nervous system, in which macrophages and T-cells feature prominently in nerve lesions. EAN represents a counterpart to Guillain-Barré syndrome in humans. In the present study, we investigated the in vitro and in vivo effects of Linomide (LS-2616, quinoline-3-carboxamide), a synthetic immunomodulatory compound, on macrophages in relation to EAN. Linomide strongly suppressed IFN-gamma and lipopolysaccharide (LPS)-induced IL-1 beta, TNF-alpha and IL-6 mRNA expression in macrophages in vitro as demonstrated by in situ hybridisation. Linomide administered daily subcutaneously from the day of inoculation completely prevented the development of clinical symptoms of EAN. Linomide administered from day 9 post immunisation (p.i.) significantly suppressed clinical EAN. Macrophages from Linomide-treated EAN rats showed decreased IL-1 beta, TNF-alpha and IL-6 mRNA expression in response to IFN-gamma and LPS. LPS-induced nitric oxide production by macrophages was also suppressed by Linomide in vitro. Linomide, however, does not affect macrophage death and release of lactate dehydrogenase. We conclude that Linomide may exert its actions in EAN and perhaps also in other autoimmune disease models, by suppressing macrophage functions.
Adjuvants, Immunologic↗
Prevention of superantigen-induced down-regulation of T-cell mediated cytotoxic activity by IL-2 in vivo.
Administration of staphylococcal enterotoxin A (SEA) to mice induces profound activation, cytokine production and cytotoxic activity of both CD4+ and CD8+ T cells, but subsequently activated cells are deleted or become anergic. This study demonstrates that administration of interleukin-2 (IL-2) can prevent sea-induced hyporesponsiveness in CD8+ cytotoxic T lymphocytes (CTL). Repeated injections with sea every fourth day resulted in severely reduced cytotoxic activity in the spleen, which correlated with a reduced number of sea-responsive T-cell receptor (TCR)-V beta 11+ CD8+ cells. Studies of purified TCR-V beta 11+ CD8+ cells showed that they possessed intact cytotoxic activity per cell compared with cells from mice given a single injection of SEA, indicating that deletion was the main mechanism for the reduced cytotoxic activity. Combined treatment with SEA and IL-2 increased the number of cytotoxic cells in the spleen after each SEA injection and prevented the down-regulation of cytotoxic activity. Increased cytotoxic activity could be related to increased number and proliferation of CD8+ IL-2R alpha + cells, suggesting that administration of IL-2 maintained IL-2 responsiveness among CD8+ cells. Studies of sorted TCR-V beta 11+ CD8+ cells demonstrated that combined treatment with SEA and IL-2 also increased cytotoxic activity per cell compared with treatment with SEA alone. Taken together, IL-2 administration in vivo augmented SEA-induced expansion of T cells as well as the cytotoxic activity per CTL, and prevented SEA-induced cell deletion.
Animals↗
The tumour associated cell surface antigen A6H is costimulatory for human CD4+ but not CD8+ T cells.
The A6H monoclonal antibody (mAb) recognizes a 120,000-140,000 MW antigen that is expressed at similar densities on 85-90% of human CD4+ and CD8+ T cells and on renal cell carcinomas. The binding of the A6H mAb induced a costimulatory signal in anti-CD3 activated T cells. In the present report, we show that A6H costimulated cell proliferation and cytokine production in purified CD4+ T cells. Unexpectedly, the CD8+ T-cell subpopulation failed to respond. CD4+ T cells costimulated with the A6H mAb upregulated CD80, CD86, CD71, interleukin-2 (IL-2)R alpha, IL-2R beta and IL-2R gamma, while no corresponding up-regulation of these cell surface molecules was seen in CD8+ T cells. In order to investigate the nature of the A6H mAb costimulus at the transcriptional level we have examined induction of the transcription factors OCT-1, AP-1 and NF-kappa B which are known to be transcriptional regulators of several cytokine and cytokine receptor genes, including the IL-2 and IL-2R genes. Co-ligation of the A6H antigen and the CD3 complex induced expression of the transcription factor AP-1 in CD4+ T cells, whereas no increase in NF-kappa B and octamer-binding (Oct) proteins was seen compared to T cells stimulated with anti-CD3 alone. Furthermore, no induction of AP-1 was seen in A6H costimulated CD8+ T cells. These results suggests that both proximal steps in CD8+ T-cell activation as well as the later phases are unresponsive to A6H ligation. Molecular differences of the A6H molecule or distinct regulation of the A6H transduced AP-1 activation pathway may exist in CD4+ and CD8+ T cell subpopulations.
Antibodies, Monoclonal↗
Linomide enhances apoptosis in CD4+CD8+ thymocytes.
Linomide, a quinoline-3-carboxamide, has a pleiotropic immune modulating capacity and inhibits development as well as progression of disease in animal models of autoimmunity. Linomide treatment of mice resulted in a dramatic, dose-dependent decrease of the thymic cell number shortly after the start of administration. Flow cytometric analysis revealed that the major thymocyte subset, the early immature type CD4+CD8+ thymocytes, were reduced in number by 75%, mature CD4+CD8- or CD4-CD8+ thymocytes were less sensitive to treatment. The polyclonal T cell activator Con A (Concanavalin A) was used together with IL-2 to evaluate the potential proliferative responsiveness of ex vivo thymocytes. Thymocytes from mice treated with Linomide exhibited a more vigorous proliferation than control cultures. An effect shown to not only be due to the enrichment of mature thymocytes in the cultures from Linomide treated animals, but also when purified, mature thymocytes (CD4+CD8- and CD4-CD8+) were cultured with Con A and IL-2, these cells responded with a significantly enhanced proliferation. In vivo Linomide treatment did not result in increased plasma concentrations of corticosterone and treatment of adrenalectomized mice resulted in a reduction of thymocytes which was comparable to the effect in intact mice, indicating that glucocorticoids (GC) are not major mediators of Linomide-induced thymocyte deletion. In addition to this, and supporting a glucocorticoid independent mode of action, Linomide treatment of thymocytes in vitro resulted in a significant increase in the number of apoptotic cells, specifically in the CD4+CD8+ subset, implicating apopotosis as one component in the course of thymocyte reduction. In addition to this, in vivo treatment with Linomide resulted in an identical pattern to that seen in vitro in that there was significantly increased apoptosis only in the CD4+CD8+. These data indicate that Linomide modifies thymocyte development using a glucocorticoid independent pathway and results in the increased apoptosis of the CD4+CD8+ subset.
Adjuvants, Immunologic↗
Selective cytotoxicity of two rodent T cell lymphomas to rat yolk sac tumours involves a retroviral envelope protein expressed by the lymphoma.
A Gross virus induced rat T cell lymphoma G1-Tc1 and a Moloney virus induced mouse T cell lymphoma YAC-1 are shown to exert a strong cytotoxic activity against rat yolk sac tumours but not to various types of rat, mouse or human normal cells or tumour cell lines including carcinomas, sarcomas, lymphomas and gliomas. Both lymphomas are CD3+, CD4-, CD8- and T-cell receptor (TCR) alpha beta +. The cytotoxicity was not MHC restricted or dependent on the density of MHC class I of the target cells, and the mouse lymphoma killed the rat yolk sac tumour target. The cytotoxic action was fast and up to 80% specific killing was observed in 4-h 51Cr release assays. A rat B cell hybridoma was established from a Wistar/Furth (WF) rat immunized with the syngeneic lymphoma G1-Tc1 producing an immunoglobulin (Ig)G2c monoclonal antibody (MoAb) 1F2. This binds to the lymphomas G1-Tc1 and YAC-1 and also to a murine non-cytolytic Rauscher lymphoma RMA, but not to any other of several rat, mouse or human cell types tested. The 1F2 completely inhibited the killing of rat yolk sac tumours by the two cytolytic lymphomas, but did not interfere with the killing mediated by natural killer (NK) cells or cytolytic lymphokine-activated killer (LAK) cells. Immunochemical analysis of solubilized cell membranes of the lymphoma G1-Tc1 demonstrates that the 1F2 antibody recognizes an epitope on a retroviral gp 70 envelope protein. This indicates that a retroviral protein is involved in the lytic activity of the two lymphomas.
AKR murine leukemia virus↗
Costimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding.
We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-kappaB complexes induced in B7-1- and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position -500 to +60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-kappaB, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancer-promoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA-3 was unaffected. Our data strongly suggest a selectivity in induction of nuclear factors by the CD2-LFA-3 and CD28-B7-1 pathways. This selectivity may contribute to regulation of the levels of IL-2 induced by LFA-3 and B7-1 costimulation and favor autocrine and paracrine T-cell responses, respectively.
B7-1 Antigen↗