Human Mixed Lymphocyte Reaction Research Articles

Effect of Antibodies Against Intercellular Adhesion Molecule-1, B7, and CD40 on Interleukin-18-Treated Human Mixed Lymphocyte Reaction

The interleukin (IL)-18 level in plasma is elevated during the acute rejection after organ transplantation. IL-18 elicits adhesion molecule expression as well as interferon-γ/IL-12 production and T-cell proliferation in the human mixed lymphocyte reaction, an in vitro model of acute rejection. We examined whether antibodies (Abs) against intercellular adhesion molecule (ICAM)-1, B7, CD40, and CD40, ligand (CD40L) affect the cytokine production and T-cell proliferation. Anti-ICAM-1 and B7 Abs suppressed the cytokine production, while all Abs inhibited T-cell proliferation. ICAM-1 and B7 as well as CD40 may play different roles in the acute rejection.

Characterization of immunosuppressive factors expressed in serum by rat tolerogenic liver transplantation

Background In a rat tolerogenic model of orthotopic liver transplantation (OLT), recipient serum after OLT (post-OLT serum) possesses strong immunosuppressive activity. This study aimed to identify immunosuppressive factors present in early post-OLT serum. Methods Immunosuppressive activity was evaluated in vitro by inhibition of the mixed-lymphocyte reaction (MLR). Autoantigens recognized by MLR-inhibitory IgG were identified by the internal protein sequencing. Results Recipient post-OLT serum inhibited MLR, and OLT-inducible IgG was the major immunosuppressive factor. IgG from post-OLT sera (2 to 3 weeks) specifically reacted to 31; 34; and 73-kd autoantigens on spleen cells. The internal sequences of the 31- and 34-kd antigens coincided completely with those of histone H1 molecules. Immunodepletion of anti-histone H1 antibodies (Abs) from early post-OLT serum abolished the MLR-inhibitory activity. Furthermore, rabbit polyclonal Ab-directed histone H1 not only significantly suppressed rat and human MLR but also prolonged survival of heart allografts. Flow-cytometric analysis revealed that some live PVG splenocytes were stained with antihistone H1 Abs, and that these positive cells increased on Con A stimulation. Western blot analysis indicated that several cross-reactive antigens against anti-histone H1 Abs were found in their membrane fraction. Conclusions In this study we provide evidence that autoreactive Abs, against histone H1 are a major OLT-induced graft survival factor, and may play at least a part in overcoming the acute rejection phase to establish solid allograft tolerance.

Treatment with β-SQAG9 prevents rat hepatic ischemia-reperfusion injury

Background Ischemia-reperfusion (I/R) injury occurs in various situations, including transplantation, trauma, and shock. We previously reported that the synthetic β-SQDG (18:0), which was derived from sulfoquinovosyl diacylglycerol of the sea urchin, possessed immunosuppressive effects, such as inhibition of T-cell responses in human allogenic human mixed lymphocyte reactions (MLR) and skin allograft survival in rats. β-SQAG9 was synthesized from β-SQDG (18:0) to improve structural stability in aqueous solution with the same biological activities to bind to CD62L (L-selectin) and CD62P (P-selectin) in vitro. We hypothesized that β-SQAG9 might attenuate leukocyte rolling on the endothelium and neutrophil infiltration in which L-selectin and P-selectin are key molecules. We investigated the protective effect of β-SQAG9 against hepatic I/R injury. Methods Male Lewis rats were divided into 6 groups: sham, control, and treatment. Rats in the control, and the treatment groups were subjected to hepatic ischemia for 30 minutes. They were injected with PBS or β-SQAG9 at doses of 5, 10, 25, and 50 mg/kg into the penile vein immediately before reperfusion. To assess the damage to the hepatic parenchyma, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured and histological evaluation was performed at 6 hours after reperfusion. Results In the group treated with β-SQAG9 at a dose of 10 mg/kg, AST, ALT, and LDH were significantly reduced, and the amount of neutrophil infiltration also was significantly reduced. Conclusions Our data suggest that SQAG-9 (10 mg/kg) reduces the warm hepatic I/R injury.

The anti-CD2 monoclonal antibody BTI-322 generates unresponsiveness by activation-associated T cell depletion.

The antihuman CD2 MoAb BTI-322 (Lo-CD2a) effectively inhibits T cell responses in vitro to allogeneic cells, which is followed by unresponsiveness to the original stimulator in secondary stimulation. We studied the xenogeneic human antiporcine mixed lymphocyte reaction (MLR), and utilized anti-T cell receptor (TCR) Vbeta family antibody-induced cell proliferation to determine the specificity and mechanism. BTI-322 and its humanized version, MEDI-507, effectively inhibited the primary xenogeneic MLR. After suboptimal primary stimulation using lower numbers of xenogeneic stimulator cells, the unresponsiveness in secondary culture was apparent only for xenogeneic stimulator cells of the original SLA haplotype, and not for third-party stimulators or allogeneic cells. The inhibition of primary MLR was not observed for nylon-wool-purified T cells, but was seen after reconstitution of purified T cells with monocytes. Similarly, anti-Vbeta family-specific stimulation showed family-specific unresponsiveness in secondary culture. This required the presence of the whole BTI-322 molecule: a F(ab')2 fragment was not effective. T cells of a distinct Vbeta family were depleted after stimulation with an anti-Vbeta family-specific antibody and BTI-322. We conclude that the inhibition by BTI-322 of a primary xenogeneic MLR or the response to an anti-TCR Vbeta antibody is associated with unresponsiveness upon restimulation, due to activation-associated cell depletion. In this process, the interaction between monocytes and the Fc part of the antibody is involved. This unique characteristic of BTI-322 suggests the potential of the antibody for tolerance induction in vivo, besides the potential use as a T cell depleting agent.

Inhibition of CD62L + T-cell response in vitro via a novel sulfo-glycolipid, β-SQAG9 liposome that binds to CD62L molecule on the cell surface

We previously reported that synthetic sulfo-glycolipid, 3- O-(6-deoxy-6-sulfono-β- d-glucopyranosyl)-1,2-di- O-acylglycerol (β-SQDG(18:0)) which was deduced from sulfonoquinovosyl-diacylglycerols of sea urchin possessed immunosuppressive effects, such as human mixed lymphocyte reaction (MLR) and skin allograft in rat, and that these effects were caused by contact inhibition between T-cells and antigen presenting cells (APCs). Here, we investigated the mechanism of these immunosuppressive effects on human MLR by β-SQAG9 which had been newly synthesized from β-SQDG(18:0) to improve structural stability in water solution. CD62L + T-cells in peripheral blood predominantly respond to APCs, and β-SQAG9 inhibited the response of CD62L + T-cell subset in human allogeneic MLR. Surprisingly, it was demonstrated that β-SQAG9 bound to L- and P-selectin (CD62L and P) molecule in vitro. Meanwhile, β-SQAG9 efficiently formed liposome structure and bound to L-selectin on the cell surface of CD62L + T-cell subset but might not be incorporated into the cells. Because the immunosuppressive effects of β-SQAG9 disappeared when β-SQAG9 liposome was changed to soluble form by detergent, the liposome structure of β-SQAG9 was presumed to be essential for these effects. These findings suggested β-SQAG9 to be a novel drug with a unique immunosuppressive action.

Efficacy and safety of ABI793, a novel human anti-human CD154 monoclonal antibody, in cynomolgus monkey renal allotransplantation.

Anti-CD154 monoclonal antibodies (mAbs) cause long-term graft survival in preclinical allotransplantation experiments. This is the first report on the efficacy and safety of ABI793, a novel human anti-human CD154 mAb, in Cynomolgus renal transplant recipients. ABI793 (human immunoglobulin-G1:kappa) was derived from a hybridoma generated after immunization of human immunoglobulin transgenic mice (HuMAb-Mouse, Medarex Inc., Annandale, NJ). Cynomolgus monkey recipients of major histocompatibility complex-mismatched, life-supporting renal allografts were treated repeatedly with intravenous ABI793 for a 3-month period posttransplantation. Graft function was monitored by serum creatinine, and rejection was confirmed histologically. ABI793 binds to human, Cynomolgus and Rhesus monkey CD154; it inhibits dose dependently in vitro CD154:CD40 binding and human mixed lymphocyte reaction. ABI793 is comparable to the mouse anti-human CD154 mAbs 5c8 and 24-31 with respect to affinity, inhibitory capacity, and species specificity; however, ABI793 binds to a different CD154 epitope. With 20 mg/kg of ABI793, five of nine recipients showed substantially prolonged graft survival after cessation of treatment, whereas four of nine recipients were killed because of high serum creatinine while still receiving treatment. ABI793 treatment was associated with episodes of severe acute tubular necrosis (which was unrelated to rejection and responded to fluid and diuretic treatment) and a decrease in platelet numbers. Chronic and acute thromboembolic vascular lesions with hemorrhages were observed in the lung and brain of two allograft recipients. None of these side effects were observed in animals that underwent autotransplantation, thus excluding direct toxicity of ABI793. ABI793 treatment effectively prevents graft rejection in Cynomolgus monkeys. Evidence for rare thromboembolic events, as also previously observed with different anti-human CD154 mAbs, suggests that thromboembolic complications may be a class effect of anti-CD154 mAbs, unrelated to their epitope specificity.

Effect of ??2-adrenergic receptor agonists on intercellular adhesion molecule (ICAM)-1, B7, and CD40 expression in mixed lymphocyte reaction

The plasma interleukin (IL)-18 level is elevated in acute rejection after organ transplantation. Although beta2-adrenergic receptor (AR) agonists suppress the rejection of organ and tissue transplants, little is known about their action mechanisms. We examined the effects of endogenous catecholamines and beta2-AR agonists on the expression of intercellular adhesion molecule (ICAM)-1, B7.1, B7.2, CD40, and CD40 ligand (CD40L) in human mixed lymphocyte reaction (MLR) and in an in vitro model of acute rejection in the presence or absence of IL-18. ICAM-1, B7.1 B7.2, CD40, and CD40L expression on monocytes was measured by flow cytometry, and the production of interferon (IFN)-gamma and IL-12 was determined by enzyme-linked immunosorbent assay. Lymphocytes proliferation in MLR was measured by [3H]-thymidine uptake. The relevant AR subtypes were characterized using subtype-selective agonists and antagonists. beta2-AR agonists inhibited the expression of ICAM-1 and CD40 during MLR in the absence of IL-18. Among IL-18-induced expression of ICAM-1, B7.1, B7.2, CD40, and CD40L, beta2-AR agonists inhibited ICAM-1 and CD40 expression. beta2-AR agonists prevented the production of IFN-gamma and IL-12 in the presence of IL-18 but had no effect in the absence of IL-18. beta2-AR agonists inhibited lymphocyte proliferation in IL-18-treated MLR. We found that beta2-AR agonists strongly inhibited the expression of ICAM-1 and CD40, irrespective of the presence or absence of IL-18, which is different from that of histamine and prostaglandin E2.

Expression of the negative co-stimulatory ligand sCD152 in the yeast, Pichia pastoris, and its regulation of antigen specific immune responses

CD152, a ligand expressed on the surface of the activated T cells that inhibits co-stimulatory signals, is associated with negative regulation of T-cell activation in the antigen-presenting process. In order to interfere with immune signal transmission, obtain functional proteins with specific immunosuppressive effects, and regulate the immune response, we cloned the full-length extracellular domain of CD152 into the expression vector pPICTLA and transformed Pichia pastoris GS115 cells by electroporation. Yeast colonies expressing and secreting large quantities of the extracellular soluble fragment of CD152 (sCD152) were isolated, and the protein was purified and used in assays designed to investigate the ability of sCD152 to regulate the immune system. In a series of antigen specific immune response interference tests in mice, human allogeneic mixed lymphocyte reaction and antigen specific lymphocyte transformation tests, we found that sCD152 had a marked immunosuppressive effect. This protein reduced antibody titer in mice immunized with BSA, significantly inhibited the killing of antibody-dependent cytotoxicity and CTL-mediated donor target cells, and reduced the expression of IL2 in mice. In the absence of exposure of sCD152, long-term stimulation (>5 days) by the same antigen in vivo partially restored the immune response-correlated stimulation index (SI), showing the characteristics of a primary immune response. Following the deletion of sCD152, however, there was no obvious inhibitory effect on the primary immune response triggered by the nonrelevant antigen OVA, indicating that sCD152 had an antigen-specific immunosuppressive effect and that the inhibitory effect of sCD152 required the co-administration of antigen. These findings suggest that sCD152 may have clinical potential as an immunosuppressive agent.

Immunomodulation by peptide analogs of retroviral envelope protein

The mechanism by which retroviral proteins exert their immunosuppressive influence has remained enigmatic. Early studies have demonstrated that retroviral infection suppresses cellular and humoral immune responses. A hydrophilic 26 amino acid region of the otherwise hydrophobic transmembrane envelope protein of murine and feline leukemia viruses, p15E, is conserved among the transmembrane envelope proteins of numerous animal retroviruses (e.g. murine, feline, bovine and simian) as well as in human T-cell leukemia virus, and to a lesser extent, in human immunodeficiency virus (HIV). We evaluated the immunomodulatory properties of various synthetic retroviral envelope peptides synthesized as overlapping fragments to this conserved sequence. We report that two small peptides inhibit human mixed lymphocyte reaction (MLR), interleukin-2 (IL-2) and tumor necrosis factor (TNF-α) production. These peptides did not affect human natural killer (NK) cell cytotoxicity in vitro, and nitric oxide (NO) production in mouse macrophage cells, RAW264.7. Our observations suggests immunomodulatory potential of two retroviral peptide analogs.

Human autologous mixed lymphocyte reaction as an in vitro model for autoreactivity to apoptotic antigens.

Recent studies have indicated that cells undergoing apoptosis are the source of autoantigens which drive autoimmune responses in systemic lupus erythematosus (SLE). It has been recognized for many years that in vitro stimulation of T cells with irradiated major histocompatibility complex (MHC) class II-bearing autologous cells results in T-cell proliferation with immunological specificity and memory, namely the autologous mixed lymphocyte reaction (AMLR). The nature of the major stimulants in the AMLR is still unclear. We investigated whether apoptotic fragments from irradiated cells act as antigenic stimulators for AMLR or nucleohistone-primed T cells. T-cell proliferation in the primary AMLR was significantly suppressed by the presence of a caspase inhibitor Z-Val-Ala-Asp-CH2F (Z-VAD.fmk), indicating that apoptotic antigens released from irradiated autologous feeder cells act as stimulators of AMLR T cells. This inhibitory effect of Z-VAD was not caused by toxic effects, because the T-cell response to the mitogen phytohaemagglutinin (PHA) was not inhibited by Z-VAD. A nucleohistone preparation was shown to contain antigens that are important in the AMLR, as culture with nucleohistone (but not with thyroglobulin or hen-egg lysozyme) primed T cells to respond with secondary kinetics in a subsequent AMLR that was also suppressed by Z-VAD. Our data provide evidence that the AMLR constitutes a model for the evaluation of cellular and molecular mechanisms that may be relevant to the pathogenesis of SLE and similar autoimmune diseases.

Effect of histamine on intercellular adhesion molecule-1 expression and production of interferon-gamma and interleukin-12 in mixed lymphocyte reaction stimulated with interleukin-18.

Interleukin (IL)-18 was identified as an interferon (IFN)-gamma-inducing factor and was demonstrated to up-regulate the expression of intercellular adhesion molecule (ICAM)-1 on human monocytes. In organ transplantation, elevation of plasma IL-18 levels has been reported during acute rejection. In the present study, we examined the effect of IL-18 on human mixed lymphocyte reaction (MLR), an in vitro model of acute rejection after organ transplantation. We also investigated the modulatory effects of histamine on IL-18 action because histamine has been demonstrated to be a modulator of IL-18 effect and a mediator of inflammation. We measured the expression of ICAM-1 on human monocytes in MLR in the presence or absence of IL-18 by flow cytometer and determined the associated production of IFN-gamma and IL-12 by ELISA. The modulatory effects of histamine and the relevant histamine receptor subtypes were characterized pharmacologically. The expression of ICAM-1 on monocytes in MLR was markedly enhanced by the addition of IL-18 in a concentration- and time-dependent manner. In parallel to ICAM-1 up-regulation, IL-18 significantly enhanced the production of IFN-gamma and IL-12 in MLR. Histamine concentration-dependently inhibited ICAM-1 expression and cytokine production in MLR stimulated with IL-18, whereas histamine alone did not show any effects on these responses in the absence of IL-18. The effects of histamine on both ICAM-1 expression and cytokine production were mimicked by the selective H2-receptor agonists 4-methylhistamine and dimaprit and were antagonized by the H2-receptor antagonist famotidine but not by H1- and H3-receptor antagonists. IL-18 strongly enhanced human MLR with respect to ICAM-1 expression and cytokine production. The fact that histamine could inhibit the IL-18-stimulated MLR implies that immunomodulation by histamine and selective H2-receptor agonists may have an important role in future immunosuppressive strategies.

An immunosuppressive effect by synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin.

It is important to develop new immunosuppressive agents without clinical drawbacks. In this article, we reveal the possibility of a chemically synthetic sulfonolipid that acts as a novel immunosuppressive drug. We evaluated the immunosuppressive effect of 3-O-(6-deoxy-6-sulfono-beta-D-glucopyranosyl)-1,2-di-O-acylglycerol (beta-SQDG) that contains a saturated C18 fatty acid, which is designated as beta-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of beta-SQDG(18:0). beta-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. beta-SQDG(18:0) did not inhibit the direct activation of responder T. This reagent could not affect the expression of either major histocompatibility antigen complex (MHC) class I or class II molecules on the cell surface of the stimulator cells, antigen-presenting cells. In contrast, beta-SQDG(18:0) was demonstrated to inhibit the binding among allogeneic lymphocytes. However, the expression of known cell surface accessory and adhesion molecules, such as CD4, CD28, leukocyte function-associated antigen 1, intercellular adhesion molecule 1, and CTLA-4, was not affected by beta-SQDG(18:0) treatment. beta-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by beta-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.

Immunosuppressive and anticancer effect of a mammalian ribonuclease that targets high-affinity interleukin-2-receptors.

To target high-affinity interleukin (IL)-2 receptors involved in lymphocyte proliferation processes such as allograft rejection, autoimmune disorders, and certain haematological malignancies, using a minimally immunogenic mammalian-derived enzyme, bovine RNaseA, which becomes cytotoxic on entering cytoplasm. Laboratory study. Teaching hospital, Japan. Human lymphocytes isolated from healthy histoincompatible donors in mixed lymphocyte cultures or stimulated with phytohemagglutinin (PHA) to promote IL-2Ralpha expression. MJ, an HTLV-1-infected malignant T-cell line that overexpresses IL-2Ralpha, and the IL-2Ralpha-negative cell lines MOLT-4F and MT-1, were used as controls. Bovine RNaseA was chemically conjugated to 7G7B6, a monoclonal antibody to the alpha-chain of human IL-2 receptors, and several concentrations of the conjugates were added to the lymphocyte cultures. Inhibition of cell proliferation as a percentage of 3H-thymidine incorporation in 24 hours. 7G7B6-RNaseA dose-dependently inhibited cell proliferation in PHA-stimulated human lymphocytes at a 50% inhibitory concentration (IC50) of 2 x 10(-7) M. whereas RNase alone and RNase plus antibody had no inhibitory effect. 7G7B6-RNaseA also dose-dependently inhibited the human mixed lymphocyte reaction at an IC50 of 2 x 10(-6) M, whereas RNase alone did not. The conjugate also inhibited cell proliferation in MJ cells, a cell line that is infected with HTLV-I and overexpresses the high-affinity IL-2 receptor, at an IC50 of 5 x 10(-7) M. However the conjugate had no inhibitory effect on the IL-2 receptor non-expressing human T-cell lymphoblastic leukaemia cell lines MOLT-4F or MT-1. 7G7B6-RNaseA can inhibit cell proliferation in antigen- or mitogen-stimulated lymphocytes that overexpress high-affinity IL-2 receptors, and it may be safer than conventional chemotherapy or immunotoxins in the treatment of transplant rejection, certain lymphocytic malignancies, and other IL-2R-associated diseases, because it contains a mammalian cytotoxic enzyme.

Identification of rat prostatic steroid binding protein (PSBP) as an immunosuppressive factor

Prostatic steroid binding protein (PSBP) is the major protein produced (∼20% of the total cytosolic protein) and secreted into the seminal fluid by the rat ventral prostate but its physiological function has not been elucidated yet. Since PSBP is secreted into the seminal fluid (which is itself a potent immunosuppressor) and has strong homology with uteroglobin (which possess an important anti-inflammatory function) our aim was to determine what effect, if any, PSBP would have on the immune system. With that purpose in mind we performed mononuclear cell cultures in the presence or absence of purified PSBP and analysed the effect of this protein on different functional parameters. PSBP inhibits the mitogen-induced proliferation of normal rat spleen mononuclear cells (MNC) specifically and in a dose-dependent manner. It reduces the production of IL-2 and the expression of its receptor (analysed by flow cytometry) which are important events for lymphocyte proliferation. Also, PSBP was able to inhibit OVA-specific proliferation of lymph node cells from previously primed animals. The immunosuppressive effect of PSBP is not due to an inherent toxic effect to the cells, since the cell viability was kept intact at the different times of culture studied. We also analysed the effect of rat PSBP on mitogen-induced proliferation of mouse spleen and human blood MNC. The proliferation was strongly abolished in a dose-dependent and non-species specific fashion. Moreover, PSBP strongly inhibits the human mixed lymphocyte reaction. Taken together, the present data support evidence for a new type of function for PSBP. We report that PSBP is a potent immunosuppressor factor and we describe its effect on the immune function in vitro. Here, we discuss the possible implications of these findings in the protection of sperm from immunologic damage in the feminine reproductive tract.

Differential kinetics of intracellular caspase-1-like and caspase-3-like enzyme activity in human alloreactive CD4(+) and CD8(+) T cells undergoing apoptosis.

The ICE-like families of serine proteases (caspases) have integral roles in apoptosis. These studies were performed to further understand the role of two critical caspases in relation to apoptotic regulation of the alloimmune response. A novel three-color cytofluorographic technique was utilized for measuring intracellular (in situ) caspase-1-like and caspase-3-like enzyme activity in responding CD4(+) and CD8(+) T cells over several time points of human mixed lymphocyte reactions (MLR). We found that activity levels of caspase 3 in both CD4(+) and CD8(+) responder cells began rising at day 10 of the MLR and peaked at day 14. By comparison, caspase 1 demonstrated the highest activity at day 7 in both cell subpopulations. These results coincided with the appearance of apoptotic cells among the alloreactive cells in the MLR. These findings demonstrate that intracellular caspase-1- and -3-like enzyme activity increases in both CD4(+) and CD8(+) alloreactive T cells as the primary response to allostimulatory cells progresses. While the kinetic profiles for these enzymes differed, both had a temporal association with the appearance of apoptosis in the MLR-generated cells. In all cases, the highest enzyme activity and presence of apoptosis was seen subsequent to the peak proliferative period. These results support the concept that changes in the rate and amount of apoptosis in alloreactive T cells is one mechanism by which the response to alloantigens is attenuated (i.e., tolerance) or sustained.

Calcineurin antagonists inhibit interferon-gamma production by downregulation of interleukin-18 in human mixed lymphocyte reactions.

Tacrolimus (FK-506) and cyclosporin A (CsA) are calcineurin antagonists used widely as T-cell immunosuppressants; however, their relative efficacy on the production of interleukin-18 (IL-18) remains undefined. We have examined the effects of FK-506 and CsA on the cytokine generation of human peripheral blood mononuclear cells (PBMCs) in mixed lymphocyte reaction (MLR) with lipopolysaccharide (LPS). We studied the levels of interleukin-18 (IL-18), IL-12, IL-10, IL-6, IL-2 and interferon-gamma (IFN-gamma) in the supernatant in allo-MLR by ELISA assay. Supernatant levels of IFN-gamma, IL-2, IL-6, IL-10 and IL-12 were detected 12 h after MLR and markedly increased thereafter. In contrast, production of IL-18 was detected at 12 h, reached a near maximum level at 24 h and decreased at 72 h. These results suggested that IFN-gamma production depended on IL-18, IL-12 and IL-2 in the early phase of MLR and depended mainly on IL-12 and IL-2 in the late phase. Both calcineurin antagonists inhibit the generation of IL-18, which plays a large role in allogeneic cell interactions, in macrophages and they also promote an equivalent down-regulation of T helper 1 (Th1) and Th2 responses in a concentration-dependent manner. About 90% of IFN-gamma production induced by MLR was inhibited by an anti-IL-18 antibody, showing that IL-18 can trigger IFN-gamma production in MLR. These results suggest that dual signaling consisting of antigen-driven nuclear factor of activated T cells (NFAT) activation and LPS-mediated NF-kappaB activation is crucial for IL-18 production in macrophages, and that IL-18 can trigger IFN-gamma production in T-cells by MLR.

EFFECTS ON HUMAN AND NONHUMAN PRIMATE IMMUNE RESPONSE OF A NEW RAT ANTI-CD2 MONOCLONAL ANTIBODY1

Nonhuman primate models are highly clinically relevant in transplantation. The development of immunosuppressive tools or a tolerogenic regimen for primate models therefore represents an important goal of transplantation immunological research. Hence, we have developed a rat monoclonal antibody (mAb) that recognizes the CD2 molecule (LO-CD2b) on both human and nonhuman primate cells. The LO-CD2b mAb has been characterized by flow cytometry, E-rosetting inhibition, and Western blotting. In vitro inhibition of immune responses by LO-CD2b was assessed after both mitogenic and allogeneic stimulation in mixed lymphocyte reactions (MLR). Several LO-CD2b dose and time responses were tested. In vivo, peripheral and lymph node T-cell depletion was examined both by flow cytometry and immunohistology in 10 baboons that received intravenous injection of LO-CD2b at different doses and time courses. Xenosensitization (anti-rat) was assessed by ELISA. Renal allograft survival was followed in two baboons treated with iterative LO-CD2b injections. In vitro, LO-CD2b binds a lymphocyte antigenic determinant of 52 kDa that is recognized by other well-characterized anti-CD2 mAbs (T11, Leu5b). LO-CD2b recognized natural killer CD2+ cells. Administration of 200 ng/ml LO-CD2b almost completely inhibited human and baboon mitogenic stimulation. Allogeneic baboon and human MLR were completely inhibited by the addition of LO-CD2b (at 312 ng/ml) on the day of the initiation of culture; when added after 1 or 2 days, LO-CD2b still provided a significant MLR inhibition (>50%). Incubation of LO-CD2b with baboon peripheral blood mononuclear cells produced very low cytokine levels (interferon-y, tumor necrosis factor-alpha, interleukin 2). In secondary MLR, baboon peripheral blood mononuclear cells previously incubated with LO-CD2b were unable to respond to a second allogeneic stimulation but were able to react to mitogens. In vivo, within the first hour after LO-CD2b injection (at 0.15, 0.5, and 2 mg/kg), an 85-90% peripheral depletion of CD2+ cells was observed. A partial T-cell depletion in inguinal lymph nodes was seen after 1 week. The mechanism of peripheral T-cell depletion could have been antibody-dependent cell cytotoxicity or opsonization but was complement independent. Iterative LO-CD2b injections (12 days at 0.35 mg/kg) slightly prolonged the renal allograft survival in two baboons. LO-CD2b is a nonactivating rat anti-CD2 mAb able to strongly inhibit both mitogenic and allogeneic responses in human and nonhuman primates. In vivo, LO-CD2b provides a rapid peripheral T-cell depletion, which is reversible within days after the cessation of injections. This rat mAb represents a very important tool for in vivo experimental investigation in nonhuman primates because it similarly reacts against human T cells in vitro.

Immunosuppressive Effect on T Cell Activation by Interleukin- 16-cDNA-Transfected Human Squamous Cell Line

It is well known that it is difficult to induce an immunotolerance with allogeneic skin transplantation. We attempted to find the immunosuppressive protocol for prolonging skin allograft rejection by using interleukin-16 because IL-16 is considered one of the natural ligands to CD4 molecules. First we examined whether synergistic immunosuppressive effects of recombinant IL-16 plus anti-CD4 mAbs are induced in mixed lymphocyte reaction (MLR). Next we used IL-16-cDNA-transfected OSC-20 (human oral squamous cell carcinoma cell line) as an in vitro model of the epidermal keratinocyte equivalent and examined whether this transfectant could inhibit the activation of allogeneic T cells. Our data indicated that IL-16 clearly inhibited human MLR and that IL-16 increased synergistically the immunosuppressive effect of anti-CD4 mAb. We also used IL-16 transfectant and this produced more than 50 ng/ml of IL-16 in the supernatant by which human MLR was significantly inhibited. Furthermore, this transfectant also inhibited the activation of allogeneic lymphocytes stimulated directly with transfectant cells. These results indicated that the IL-16-producing allogeneic skin graft might have a local immunosuppressive action that would prolong graft survival.

Inhibitory effect of 1alpha,25-dihydroxyvitamin D3 on allogeneic lymphocyte stimulation and Langerhans cell maturation.

1alpha,25-Dihydroxyvitamin D3 (calcitriol) has been shown to inhibit the proliferation of peripheral blood mononuclear cells induced by allogeneic Langerhans cells in a human mixed epidermal cell lymphocyte reaction. The potent antigen-presenting function of Langerhans cells is gained during culture. We tried to dissect the effect of calcitriol on lymphocyte proliferation and Langerhans cell maturation in a murine mixed epidermal cell lymphocyte reaction using unfractioned epidermal cells as a source for Langerhans cells. First, calcitriol was added upon setup of the mixed epidermal cell lymphocyte reaction using cultured epidermal cells as antigen-presenting cells, and this was found to inhibit the proliferation of lymphocytes in a time- and concentration-dependent manner. When calcitriol was added only during preculture of freshly isolated epidermal cells, the subsequent mixed epidermal cell lymphocyte reaction was also inhibited. In addition, the growth of keratinocytes and the production of granulocyte/macrophage colony-stimulating factor during preculture of epidermal cells was completely inhibited. Supplementation with this growth factor only partially restored the proliferative response of lymphocytes. These results suggest that calcitriol inhibits both the alloantigen-driven stimulation of naive T cell and Langerhans cells maturation. Further experiments with purified Langerhans cells are required to elucidate the mechanism of action of calcitriol on Langerhans cell maturation.

Initiation of the Autologous Mixed Lymphocyte Reaction Requires the Expression of Costimulatory Molecules B7-1 and B7-2 on Human Peripheral Blood Dendritic Cells

The human autologous mixed lymphocyte reaction (AMLR) consists of a proliferative response of primarily CD4+ T lymphocytes stimulated by autologous non-T cells expressing class II MHC-encoded gene products and is thought to represent a self-recognitive mechanism that might be important in regulating the cellular interactions involved in the generation of normal immune responses. To further define appropriate stimulator cell populations, as well as the molecular mechanism responsible for the initiation of AMLR, we compared the T cell-stimulatory capacity of highly purified populations of peripheral blood dendritic cells (DCs) and monocytes (Mos) under serum-free conditions, thus carefully avoiding the presence of xenogeneic Ags. Whereas both freshly isolated Mos and DCs were found to be poor stimulators of autologous T cell proliferation, preactivation of DCs, but not of Mos, for 48 h with granulocyte-macrophage CSF led to a 113-fold increase in DC stimulatory capacity. AMLR was inhibited by mAbs against HLA-DR and CD4 molecules, and, in addition, showed a higher dependence on the granulocyte-macrophage CSF-induced up-regulation and/or de novo expression of the costimulatory molecules B7-2 and, in particular, B7-1 as compared with an Ag-specific or allogeneic MLR. Thus, our data suggest that the high density of costimulatory molecules together with MHC class II molecules on competent APCs appear to be the major triggers for the initiation of AMLR.