Molecular characterization and functional analysis of murine interleukin 4 receptor allotypes.

The murine interleukin 4 receptor (IL-4R) exists as a transmembrane protein transducing pleiotropic IL-4 functions, or as soluble (s)IL-4-binding molecule with potent immunoregulatory effects. In this study we identified and characterized a murine IL-4R allotype. Sequence analysis of the IL-4R cDNA of BALB/c mice revealed 18 base substitutions leading to three extracellular and five cytoplasmic amino acid changes when compared with the published IL-4R sequence of C57BL/6 mice. Analyses with allotype-specific mAbs revealed that AKR/J and SJL/J mice possess the newly identified BALB/c IL-4R allotype whereas the IL-4Rs of C3H, CBA, DBA-2, and FVB/N mice are identical to that of the C57BL/6 mouse. The extracellular Thr49 to Ile substitution abrogates one N-glycosylation site in the naturally occurring BALB/c IL-4R as well as in the experimentally point mutated C57BL/6-T49I sIL-4R, and both molecules display a nearly threefold reduction in IL-4-neutralizing activity compared to the C57BL/6 sIL-4R. In line with this, a significantly enhanced dissociation rate of IL-4 was detected for the BALB/c IL-4R allotype by surface plasmon resonance and in radioligand binding studies with IL-4R-transfected cell lines. These findings suggest that the altered ligand binding behavior of the newly described IL-4R allotype may influence the IL-4 responsiveness, thus contributing to the diverse phenotypes of inbred mouse strains in IL-4-dependent diseases.

Crosslinking CD4 by human immunodeficiency virus gp120 primes T cells for activation-induced apoptosis.

During human immunodeficiency virus (HIV) infection there is a profound and selective decrease in the CD4+ population of T lymphocytes. The mechanism of this depletion is not understood, as only a small fraction of all CD4+ cells appear to be productively infected with HIV-1 in seropositive individuals. In the present study, crosslinking of bound gp120 on human CD4+ T cells followed by signaling through the T cell receptor for antigen was found to result in activation-dependent cell death by a form of cell suicide termed apoptosis, or programmed cell death. The data indicate that even picomolar concentrations of gp120 prime T cells for activation-induced cell death, suggesting a mechanism for CD4+ T cell depletion in acquired immune deficiency syndrome (AIDS), particularly in the face of concurrent infection and antigenic challenge with other organisms. These results also provide an explanation for the enhancement of infection by certain antibodies against HIV, and for the paradox that HIV appears to cause AIDS after the onset of antiviral immunity.

Differential signal transduction pathways regulating interleukin-2 synthesis and interleukin-2 receptor expression in stimulated human lymphocytes.

In human peripheral blood lymphocytes stimulated via the T-cell antigen receptor/CD3 complex IL-2 synthesis and cellular proliferation were effectively inhibited by a concentration of ouabain as low as 50 nM, whilst the expression of high affinity IL-2 receptors was not influenced. Binding of the monoclonal antibody, BMA 031 to the T-cell antigen receptor/CD3 complex resulted in a bimodal activation of protein kinase C. The activation of protein kinase C-alpha in the early phase of T-lymphocyte activation was not affected by 50 nM ouabain, in contrast sustained activation of protein kinase C-beta, between 90-240 min of stimulation was completely abolished by the cardiac glycoside. When protein kinase C was directly activated by PMA + ionomycin, 50 nM ouabain was ineffective in inhibiting protein kinase C activation, as well as subsequent IL-2 synthesis, suggesting that the glycoside interfered with signal transducing mechanism(s) upstream of the activation of protein kinase C. Ouabain had no influence on the elevation of intracellular calcium concentration in BMA 031 stimulated lymphocytes, ruling out the possibility that it interfered with the T-cell antigen receptor dependent phosphatidylinositol response. In contrast, lysophosphatide acyltransferase catalysed elevated incorporation of polyunsaturated fatty acids was effectively inhibited by low concentrations of ouabain in BMA 031-stimulated T-lymphocytes, whereas stimulation with PMA + ionomycin had no influence on the plasma membrane phospholipid fatty acid metabolism. These results suggest, that differential signal transduction pathways are involved in the activation of protein kinases C-alpha and -beta. They implicate that elevated incorporation of polyunsaturated fatty acids into plasma membrane phospholipids might contribute to sustained activation of protein kinase C-beta, and establish a link between activation of protein kinase C-beta and induction of IL-2 synthesis in human lymphocytes.

Apoptosis induced by HIV-gp120 in a Th1 clone involves the generation of reactive oxygen intermediates downstream CD95 triggering.

HIV-gp120 sensitizes Th1 clones from seronegative donors to apoptosis, which occurs through two distinct events: expression of CD95L followed by its interaction with CD95 to trigger cell death. gp120-apoptosis of the Th1 clone 103 was inhibited by Cyclosporin A, the PTK inhibitors Genistein and PNU152518, as well as the anti-oxidants Ascorbic Acid and Glutathione. Cyclosporin A interfered with CD95L expression, Ascorbic Acid and Glutathione inhibited cell death triggered by CD95/CD95L interaction; Genistein and PNU152518 acted on both steps. The occurrence of oxidative stress during CD95-dependent apoptosis was supported by the direct evidence of ROI production.

HIV/gp120 and PMA/ionomycin induced apoptosis but not activation induced cell death require PKC for Fas-L upregulation.

HIV protein gp120 in combination with T cell antigen receptor (TCR) triggering induces apoptosis (gp120-apoptosis) in Th1 cells. Gp120-apoptosis occurs by induction of Fas-L and subsequent triggering of the Fas apoptotic pathway. Here, through the use of several compounds inhibiting induction of Fas-L, we show that, in a Th1 clone, a protein kinase C (PKC) independent pathway activated by TCR stimulation is distinguishible from a PKC dependent pathway activated by either phorbol 12-myristate 13-acetate (PMA)/ionomycin or asynchronous stimulation of TCR and CD4 as occurs in gp120-apoptosis.

Synergistic immunosuppressive actions of cyclosporine with a mouse anti-rat alpha/beta-T cell receptor monoclonal antibody.

A mouse IgG1 mAb (R73) directed against the rat alpha/beta-TCR was documented not only to prolong the survival of allografts across major RT1 plus non-RT1 antigenic disparities, but also to display a synergistic immunosuppressive interaction with CsA. Heterotopic cardiac transplants from Buffalo (RT1b) rats survived significantly longer in Wistar-Furth (RT1u) hosts treated immediately after the operation with 0.25 mg/kg R73 i.v., with a mean survival time of 11.0 +/- 5.5 versus 6.8 +/- 1.2 days in the untreated group (P < 0.01). Administration of 0.5 or 5.0 mg/kg R73 displayed dose-dependent prolongation of survival to 17.0 +/- 8.3 days (P < 0.05) or 28.6 +/- 14.0 days (P < 0.01), respectively. One 0.5 mg/kg i.v. dose of R73 delivered to normal Wistar-Furth hosts produced peripheral T cell depletion that reversed after 16 days. Three injections of 0.5 mg/kg R73 on days 0, 2, and 4 prolonged allograft survival to 52.5 +/- 38.6 days compared with 17.0 +/- 8.3 days with a single dose (P < 0.01). Addition of 3 daily doses of 5 or 10 mg/kg CsA administered per oral gavage to a single dose of 0.05, 0.25, or 0.5 mg/kg R73 injected on day 0 produced a synergistic effect to prolong allograft survival, as determined by the rigorous median-effect analysis. The synergistic interaction, which may be explained by the inhibitory effect of CsA on Ca(2+)-dependent pathways triggered after activation of TCR, the target of R73, warrants clinical investigation in order to assess the potential impact of anti-alpha/beta-TCR mAb on CsA-based immunosuppressive regimens.

Clinical evaluation of induction immunosuppression with a murine IgG2b monoclonal antibody (BMA 031) directed toward the human alpha/beta-T cell receptor.

Mouse mAbs directed against the alpha/beta-TCR were tested in clinical phase II and in triple-blind, randomized phase III studies. A clinical phase II trial administered 50 mg of murine anti-human alpha/beta-TCR mAb (BMA 031) intravenously on the day of, as well as 2 and 4 days after, cadaveric donor renal transplantation in combination with a CsA/prednisone regimen. None of 12 patients showed even moderately adverse side effects. A phase III, triple-blind randomized trial enrolled 24 patients in the BMA 031 group and 22 patients in a placebo control group. BMA 031 treatment significantly reduced the incidence of rejection events within the first 10 posttransplant days to 1 patient versus 9 episodes in the placebo group (P < 0.01). By the end of 30 days, 6 rejection episodes had occurred in the BMA 031 group and 11 in the control cohort (P = NS). After a minimum of 30 months follow-up, the actual allograft survival rate was 87% in the BMA-treated group compared with 68% in the control cohort.

alpha/beta-T cell receptor-directed therapy in rat cardiac allograft recipients. Treatment prior to alloantigen exposure prevents sensitization and abrogates accelerated rejection.

An mAb directed to the alpha/beta-heterodimer of the rat T cell receptor was used to prevent rejection of cardiac allografts in sensitized (accelerated rejection) recipients. Over a wide dose range, alpha/beta-TCR-directed therapy abrogated accelerated rejection at 24-36 hr and extended cardiac allograft survival in a dose-dependent fashion, both when given after heart transplantation as well as during or before the sensitizing skin transplants (8.9 +/- 1.0 days, 12.7 +/- 0.6 days, or 8.7 +/- 1.5 days, respectively). Pretreatment with R73 completely abrogated host sensitization induced by skin grafting. As a result, post-heart transplant cyclosporine course (15 mg/kg for 7 day) has led to long-term graft acceptance (> 90 days vs. 15.2 +/- 1.6 days with postoperative CsA therapy alone). Administration of R73 mAb produced incomplete depletion (CD5+ cells) and partial modulation (alpha/beta-TCR/CD5 double-positive cells) in the peripheral blood. It suppressed in situ protein expression of many cytokines to background levels, in particular that of IL-2 and IFN-gamma, both when given after as well as before cardiac transplantation. However, only pretransplant mAb application was associated with augmented in situ elaboration of IL-4. alpha/beta-TCR-directed therapy induced strong host anti-idiotypic and, to a lesser degree, anti-isotypic antibody responses. Taken together, these results provide the rationale for a novel immunosuppressive strategy involving induction of hyporesponsiveness by alpha/beta-TCR-directed therapy before the alloantigenic exposure.

alpha/beta-T cell receptor-directed therapy in rat allograft recipients. Long-term survival of cardiac allografts after pretreatment with R73 mAb is associated with upregulation of Th2-type cytokines.

Rejection of vascularized allografts still poses the major problem in organ transplantation. Therefore, transplant rejection of cardiac allografts was investigated in a rat model (BN-to-LEW) under alpha/beta-TCR (R73) mAb-targeted therapy. Two protocols were studied: posttransplant ("immunosuppressive") and pretransplant conditioning therapy. In posttransplant therapy over a wide dose range, R73 mAb only marginally improved cardiac allograft survival (7.8 +/- 0.8 days vs. 12.5 +/- 0.8 days at 0.1 mg/kg for 7 days). In contrast, conditioning treatment with low-dose (0.1 mg/kg) anti-alpha/beta-TCR mAb given 3 to 7 days prior to organ transplantation was highly effective and resulted in 50% permanent acceptance of cardiac allografts. R73 mAb-treated rats were monitored with respect to peripheral lymphocyte populations and intragraft cytokine levels. A temporary, incomplete reduction (CD5+ cells) and partial modulation (alpha/beta-TCR/CD5 double+ cells) in the peripheral blood was observed. In contrast to untreated controls, intragraft production of IL-2 and IFN-gamma at the mRNA and protein level was abolished in both post- and pretreated recipients. Interestingly, pretransplant mAb application was associated with augmented in situ elaboration of the Th2-type cytokines, IL-4 and IL-10, together with up-regulated TGF-beta and PGE. Increased expression of IL-4 and IL-10 continued to be observed in long-term surviving allografts. In conclusion, the mechanism of conditioning therapy with alpha/beta-TCR mAb prior to alloantigen exposure appears to be a switch from Th1 to Th2 response allowing long-term acceptance of allogeneic grafts.

BMA031, a monoclonal antibody suited to identify the T-cell receptor alpha beta/CD3 complex on viable human T lymphocytes in normal and disease states.

Two types of T lymphocytes can be discriminated on the basis of expression of either the classical T-cell receptor (TCR) alpha beta or the more recently identified TCR gamma delta. Whereas TCR alpha beta + lymphocytes are known to respond to recognition of antigen in the context of major histocompatibility complex molecules by proliferation, lymphokine secretion, and/or cytotoxicity, the potential ligand specificities and functions of TCR gamma delta + cells have not been completely unraveled. Antibodies specific for either receptor type are important tools to elucidate the role TCR gamma delta + cells play in the immune system. They can be used to quantify TCR gamma delta + cells and TCR alpha beta + cells in normal and disease states, to isolate both T-cell subsets, and to perform in vitro functional assays. Only few antibodies reactive with common determinants on either TCR alpha beta or TCR gamma delta are available. Generally, the monoclonal antibody (mAb) WT31 is used for definition of viable human TCR alpha beta + cells. However, WT31 has recently been shown to cross-react with TCR gamma delta. We describe an mAb, BMA031, that combines the unique features of reactivity with intact viable cells and true specificity for a common determinant on the TCR alpha beta/CD3 complex. Its performance in immunofluorescence staining and immunochemistry has been compared with that of WT31 and anti-TCR gamma delta mAbs, using TCR alpha beta and TCR gamma delta expressing cells isolated from blood and bone marrow of healthy individuals and immunodeficient patients.

ICE/Caspase-1 inhibitors as novel anti-inflammatory drugs.

In recent years, several strategies that selectively inhibit pro-inflammatory cytokines, have yielded effective protein-based therapies for inflammatory disorders, validating the therapeutic hypothesis that intervention in cytokine signalling can provide clinical benefit. However, these protein-based products must be administered by injection, a constraint associated with inconvenience, adverse effects and expense for patients, caregivers and insurers. Besides interfering with the effects of cytokines such as TNF-alpha or IL-1beta that have already been produced, inhibition of pro-inflammatory cytokine production or signalling with low-molecular weight orally-active drugs would combine the convenience of conventional pharmaceuticals with the focused efficacy of the protein therapies. Reducing IL-1beta and IL-18 production by inhibition of IL-1beta converting enzyme (ICE, caspase-1) is one promising strategy because of the key roles of these cytokines in many inflammatory diseases. Pralnacasan, the first orally available, potent and selective ICE inhibitor to enter clinical trials, is currently under investigation in rheumatoid arthritis.

Monoclonal antibodies against human astrocytomas and their reactivity pattern.

The establishment of hybridomas after fusion of X63-Ag8.653 mouse myeloma cells and splenocytes from BALB/c mice hyperimmunized against human astrocytomas is presented. The animals were primed with 5 X 10(6) chemically modified uncultured or cultured glioma cells. Six weeks after the last immunization step an intrasplenal booster injection was administrated and 3 days later the spleen cells were prepared for fusion experiments. According to the specificity analysis of the generated antibodies 7 hybridoma products (MUC 7-22, MUC 8-22, MUC 10-22, MUC 11-22, MUC 14-22, MUC 15-22 and MUC 2-63) react with gliomas, neuroblastomas and melanomas as well as with embryonic and fetal cells but do not recognize non-neurogenic tumors. The selected monoclonal antibodies (McAbs) of IgG1 and IgG2a isotypes are not extensively characterized but these antibodies have been demonstrated to be reactive with a panel of glioma cell lines with varying patterns of antigen distribution. Using the McAbs described above and a series of cryosections of glioma biopsies and paraffin sections of the same material as well as glioma cultures established from these, variable antigenic profiles among glioma cell populations could be demonstrated. From these results it is evident that there is not only a distinct degree of antigenic heterogeneity among and within brain tumors, but also that the pattern of antigenic expression can change continuously. Some of the glioma associated antigens recognized by the selected antibodies persist after fixation with methanol/acetone and Karnovsky's fixative and probably are oncoembryonic/oncofetal antigen(s). The data suggest that the use of McAbs recognizing tumor associated oncofetal antigens in immunohistochemistry facilitates objective typing of intracranial malignancies and precise analysis of fine needle brain/tumor biopsies in a sensitive and reproducible manner.

Haematological effects of rhGM-CSF in dogs exposed to total-body irradiation with a dose of 2.4 Gy.

It was the specific aim of this study to test the stimulatory effects of recombinant human GM-CSF (rhGM-CSF) on haemopoietic regeneration in dogs which had received total-body irradiation (TBI) with a dose of 2.4 Gy. In normal dogs rhGM-CSF given subcutaneously at 10 microgram/kg per day or 30 microgram/kg per day for 21 days caused strong but transient increases in the peripheral blood neutrophils. The monocyte counts also showed a transient rise during treatment in a dose-dependent fashion, whereas the lymphocyte counts increased only at the higher dose of rhGM-CSF and the platelet counts were transiently depressed during the course of the treatment. In the irradiated animals treatment with rhGM-CSF decreased the severity and shortened the duration of neutropenia but had no significant influence on monocyte or lymphocyte recovery. The granulocyte values showed a characteristic pattern of fluctuations with the first peak occurring at the same time (day 10 to day 13) when the abortive rise was observed in the untreated dogs. In contrast the GM-CFC in the peripheral blood remained depressed during the whole treatment course, similar to the untreated irradiated controls. These results indicate that treatment with GM-CSF can be an effective biological monotherapy for radiation-induced bone marrow failure, but that for higher radiation doses the number of GM-CSF responsive target cells will become a critical determinant of therapeutic efficacy.

The new immunosuppressants, the malononitrilamides MNA 279 and MNA 715, inhibit various graft-vs.-host diseases (GvHD) in rodents.

The use of inbred mouse strains of defined genetic background has allowed for the development of systems capable of reproducibly generating either an acute or chronic graft-versus-host disease (GvHD). The malononitrilamides MNA 279 and MNA 715, analogues of the main metabolite of leflunomide, have been shown to directly inhibit T-cell proliferation and B-cell functions. Therefore, they have been studied in a local GvH reaction in the popliteal lymph node (PLN) assay in LBN rats, on the development of an acute and lethal GvHD in B6C3F1 mice and on a chronic autoimmune GvHD in BDF1 hybrid mice. In the PLN assay an oral administration of various concentrations (7.5 to 50 mg/kg) of both MNAs inhibited the localized GvH reaction dose-dependently and suppressed the lymph node hyperplasia. Both MNAs also acted therapeutically in this assay when they were given as late as day 4 or 5 after challenge. In the model of an acute lethal GvHD the treatment of the GvH-B6C3F1 hybrid mice with the MNAs (2.5 to 20 mg/kg/day) shortly after disease induction on days 3 to 12 resulted in a dose-dependently improved survival rate. With 20 mg/kg of drugs, mortality of this life-threatening GvHD was completely prevented and also other parameters like splenomegaly, erythrocyte counts and hematocrit values were strongly suppressed. Treatment of sensitized GvH-BDF1 hybrid mice in the chronic autoimmune-like model with the MNAs (30 mg/kg/day), given on days 3 to 36 by oral gavage, resulted in an improved survival rate, inhibited lymphadenopathy and splenomegaly, reduced the levels of autoantibodies and other immunoglobulins like IgE and IgG1, prevented proteinuria and the development of glomerulonephritis. Both MNA 279 and MNA 715 can inhibit ongoing aberrant immune responses in animals suffering from GvHD.

A rabies-specific human monoclonal antibody that protects mice against lethal rabies.

According to a recommendation from WHO (World Health Organisation) for prevention of a possible rabies infection, active vaccination has to be combined with application of immunoglobulin to get a fast protective effect. At present, preparations of purified human or equine rabies-specific immunoglobulin are used. We have generated a human rabies-specific monoclonal antibody (huMAb) by immortalization of human B-cells with Epstein Barr Virus (EBV), followed by fusion with a mouse myeloma cell. The resulting clone TW-1 secrets an IgG1 lambda huMAb which specifically reacts in ELISA with 5 laboratory rabies virus strains of serotype 1 and DUV3 (Duvenhage, serotype 4). Western Blot analysis revealed fine specificity for the G glycoprotein (gp67) of rabies virus. HuMAb TW-1 neutralizes rabies virus in vitro (RFFIT) as well as in vivo and protects rabies infected mice. Compared to polyclonal human rabies immunoglobulins, huMAb TW-1 is advantageous, because of its defined specificity and the very low amounts of total protein needed for therapeutic effects.

Synergistic activity of malononitrilamides with cyclosporine to control and reverse xenograft rejection.

Several studies in xenotransplantation have stressed the role of humoral mechanisms of graft rejection and have emphasized the role of xenophile antibodies as well as T-cell activation in the rejection of xenografts. Malononitrilamides (MNAs), derivatives of the primary metabolite of leflunomide, were found to be potent inhibitors of the IgM and IgG antibody response in vitro and were able to reduce auto- and allo-antibody formation in vivo. Here we tested the immunosuppressive activities of MNA 279 and MNA 715 in a concordant mouse-to-rat skin xenotransplantation model. Mouse skin xenograft survival in untreated Lewis rats (5.4 +/- 1.1 days) and those given a vehicle solution only by gavage (6.1 +/- 0.9 days) were statistically indistinguishable. In our model treatment with cyclosporine (10 mg/kg/day) as a single drug showed no significant prolongation of xenograft survival (6.4 +/- 0.9 days) as compared to controls. When MNAs were given alone to xenograft recipients, they were able to demonstrate a significant and dose-dependent prolongation of graft survival time. Even a short-term application of these new immunosuppressive agents showed efficacy in the prevention of hyperacute xenograft rejection. Both MNA 279 and MNA 715 also have therapeutic activity and can reverse ongoing acute skin xenograft rejection. With these drugs a remarkable suppression of donor-specific IgM and IgG xenoantibody production in vivo was also clearly demonstrated by flow cytometry. Interestingly, whereas cyclosporine alone was unable to prolong xenograft survival, MNA-combination therapy, even a short-term application with an ineffective dose of CyA, was synergistically effective and significantly prolonged xenograft survival. This synergistic effect of MNAs with CyA was seen also when they were given therapeutically on days 5 to 9 and they reversed hyperacute skin xenograft rejection in this mouse-to-rat model.

The alloreactivity in the popliteal lymph node (PLN) assay is regulated by malononitrilamides (MNAs).

Malononitrilamides (MNA 279 and MNA 715) represent a new class of low molecular weight immunosuppressants and belong to the derivatives of the primary metabolite of leflunomide A7771726. They have been shown to prevent and reverse established acute allograft rejection and effectively prolong xenograft survival, and have also been found to be potent inhibitors of B- and T-cell mediated autoimmune processes. The MNAs mediate their effects by binding specifically to dehydro orotate-dehydrogenase (DHODH) and inhibiting de novo pyrimidine biosynthesis, thereby blocking T- and B-cell proliferation and strongly suppressing the IgM and IgG antibody production. In this study we evaluated the effects of MNA 279 and MNA 715 on the in vivo lymphoproliferation that occurs after challenge with allogeneic cells in a local graft-versus-host (GvH) reaction in Lewis x Brown-Norway (LBN) F1-hybrid rats by measuring the enlargement of the PLN draining the site of allogeneic cell injection. Oral administration of one of the two MNAs (7.5 to 50 mg/kg) on day 0, dose-dependently prevented the localized lymphoproliferative response and suppressed the lymph node hyperplasia. The MNAs even acted therapeutically when they were given during an ongoing alloreactivity as late as day 4 or 5 after challenge. Consistent with the mode of action that MNAs inhibit de novo pyrimidine biosynthesis, a complete reversal of the immunosuppression on the lymphoproliferation in vivo was attempted in this protocol by addition of exogenous uridine during days 0 to 5. These data suggest that MNA 279 and MNA 715 mediate their antiproliferative and immunosuppressive effects in the PLN-assay in vivo by decreasing the activity of DHODH in the lymph node cells and thereby inhibiting pyrimidine biosynthesis.