anti-CD40 Ligand Research Articles

Prolonged survival of rat islet and skin xenografts in mice treated with donor splenocytes and anti-CD154 monoclonal antibody.

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and a brief course of anti-CD154 (anti-CD40 ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for approximately 50 days without further intervention. We now report adaptation of this protocol to the transplantation of islet and skin xenografts. We observed prolonged survival of rat islet xenografts in mice treated with donor-specific spleen cell transfusion and anti-CD154 monoclonal antibody (mAb). Challenge islet xenografts placed on these animals indicated that graft acceptance was species-specific but not strain specific. Spleen cells from recipients bearing intact grafts led to rejection of rat islet xenografts in scid mice, suggesting that graft acceptance was not due to complete clonal deletion of xenoreactive cells. We also observed prolonged survival of rat skin xenografts in mice treated with donor-specific transfusion and anti-CD154 mAb. Prolonged survival of skin xenografts was also species specific. We conclude that treatment with appropriately timed donor-specific transfusion and anti-CD154 mAb induces durable survival of both islet and skin xenografts in mice. Because this procedure is targeted directly at CD154, a co-activation molecule expressed predominantly by activated CD4+ T-cells, the results suggest that CD4+ cells have a major role in the cellular immune response to xenografts.

CD4(+) T cells tolerized ex vivo to host alloantigen by anti-CD40 ligand (CD40L:CD154) antibody lose their graft-versus-host disease lethality capacity but retain nominal antigen responses.

A major goal of the transplant field is to tolerize donor T cells to prevent graft-versus-host disease (GVHD) (1). We describe an ex vivo approach in which the blockade of CD40 ligand (CD40L:CD154):CD40 interactions, a pathway required for optimal T cell expansion, induces donor CD4(+) T cells to become tolerant to host alloantigens (2). High doses of tolerized cells did not cause GVHD lethality in vivo. T cells had intact responses to antigens not present during tolerization. Tolerance was long lived and not readily reversible in vivo. These data have significant implications for the use of tolerization approaches to prevent human GVHD.

Prolonged survival of rat islet and skin xenografts in mice treated with donor splenocytes and anti-CD154 monoclonal antibody

Treatment of C57BL/6 mice with one transfusion of BALB/c spleen cells and a brief course of anti-CD154 (anti-CD40 ligand) antibody permits BALB/c islet grafts to survive indefinitely and BALB/c skin grafts to survive for ∼50 days without further intervention. We now report adaptation of this protocol to the transplantation of islet and skin xenografts. We observed prolonged survival of rat islet xenografts in mice treated with donor-specific spleen cell transfusion and anti-CD154 monoclonal antibody (mAb). Challenge islet xenografts placed on these animals indicated that graft acceptance was species-specific but not strain specific. Spleen cells from recipients bearing intact grafts led to rejection of rat islet xenografts in scid mice, suggesting that graft acceptance was not due to complete clonal deletion of xenoreactive cells. We also observed prolonged survival of rat skin xenografts in mice treated with donor-specific transfusion and anti-CD154 mAb. Prolonged survival of skin xenografts was also species specific. We conclude that treatment with appropriately timed donor-specific transfusion and anti-CD154 mAb induces durable survival of both islet and skin xenografts in mice. Because this procedure is targeted directly at CD154, a co-activation molecule expressed predominantly by activated CD4+ T-cells, the results suggest that CD4+ cells have a major role in the cellular immune response to xenografts.

B cell-mediated down-regulation of IFN-gamma and IL-12 production induced during anti-tumor immune responses in the tumor-bearing state.

Unfractionated spleen cells taken from tumor-bearing mice contained tumor-primed T cells which produced lymphokines such as IFN-gamma and IL-2 through collaboration with antigen-presenting cells (APC) binding tumor antigens when cultured in vitro. Here, we investigated the regulatory mechanisms underlying IFN-gamma production by T-APC interactions. Elimination of B cells from a splenic population of tumor-bearing mice resulted in enhanced IFN-gamma production. Adding B cells back into cultures down-regulated IFN-gamma production to almost the same levels as those induced by unfractionated spleen cells. IL-2 production was not enhanced by B cell depletion, but rather was significantly suppressed. IFN-gamma-selective up-regulation was due to an enhancement of IL-12 production because IL-12 was detected in B cell-depleted cultures and enhanced IFN-gamma production was prevented by addition of anti-IL-12 mAb or anti-CD40 ligand (CD40L) mAb capable of inhibiting CD40L-induced IL-12 production. These results indicate that B cells interfere with IFN-gamma production induced through interactions between anti-tumor T cells and APC, and this suppressive effect is based on the capacity of CD40+ B cells to down-regulate the CD40L-induced IL-12 production by APC.

Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance.

A reliable, nontoxic method of inducing transplantation tolerance is needed to overcome the problems of chronic organ graft rejection and immunosuppression-related toxicity. Treatment of mice with single injections of an anti-CD40 ligand antibody and CTLA4Ig, a low dose (3 Gy) of whole body irradiation, plus fully major histocompatibility complex-mismatched allogeneic bone marrow transplantation (BMT) reliably induced high levels (>40%) of stable (>8 mo) multilineage donor hematopoiesis. Chimeric mice permanently accepted donor skin grafts (>100 d), and rapidly rejected third party grafts. Progressive deletion of donor-reactive host T cells occurred among peripheral CD4(+) lymphocytes, beginning as early as 1 wk after bone marrow transplantation. Early deletion of peripheral donor-reactive host CD4 cells also occurred in thymectomized, similarly treated marrow recipients, demonstrating a role for peripheral clonal deletion of donor-reactive T cells after allogeneic BMT in the presence of costimulatory blockade. Central intrathymic deletion of newly developing T cells ensued after donor stem cell engraftment had occurred. Thus, we have shown that high levels of chimerism and systemic T cell tolerance can be reliably achieved without myeloablation or T cell depletion of the host. Chronic immunosuppression and rejection are avoided with this powerful, nontoxic approach to inducing tolerance.

PROLONGATION OF MURINE CARDIAC AND SKIN ALLOGRAFT SURVIVAL BY ANTI-CD40 MONOCLONAL ANTIBODY

512 CD40/CD40L interactions are crucial for the development of CD4+ T cell dependent immune responses such as allograft rejection. For example, simultaneous administration of a CD40L monoclonal antibody (mAb) and the CTLA4-Ig fusion protein synergistically prolongs the survival of skin and cardiac allografts. In this report we establish the efficacy of an anti-CD40 mAb alone and in combination with CTLA4-Ig as an alternative to anti-CD40 ligand as a strategy to inhibit rejection. Immunohistochemical analysis demonstrates that CD40 is strongly induced on both donor and recipient cells in the rejecting allograft. Furthermore, transplants using CD40-/- mice indicate that CD40 on the recipient is functionally more important than on donor tissues. Hearts from CD40-/- donors are rejected promptly, whereas CD40-/- recipients reject wild type (wt) cardiac allografts more slowly than wt B6,129 F1 control recipients. The ability of an anti-CD40 mAb, 7E1, to inhibit allograft rejection was tested in the BALB/c to C3H/HeJ strain combination. Recipient mice were treated with 7E1 (250µg/dose) intraperitoneally (IP) on days 0, 2, 4& 6. 7E1 significantly prolonged cardiac allograft survival relative to untreated controls. However, histological assessment of the 7E1 treated animals demonstrated significant interstitial fibrosis and coronary vasculopathy by postoperative day (POD) 140. As previously reported with a CD40L mAb (MR1), 7E1 synergized with CTLA4-Ig in inhibiting these pathologic changes. C3H/HeJ recipients of BALB/c skin grafts were treated with anti-CD40 mAb, alone and in combination with CTLA4-Ig (250µg/dose IP on days 0, 2, 4& 6). Anti-CD40 mAbs alone prolonged skin graft survival (MST=30d) compared to MR1 treatment (MST-14d). Untreated controls rejected the skin grafts promptly by POD11. As in the cardiac allograft model, 7E1 and CTLA4-Ig synergize to improve skin graft survival (MST=93d). 7E1 was found to prevent the formation of donor specific alloantibodies in the cardiac allograft recipients. Alloantibodies were undetectable on POD 29, 35 & 57, whereas alloantibodies were detected in the control BALB/c cardiac allograft recipients at a dilution of 1:512 at POD 29, 34 & 57. Flow cytometric analysis of peripheral blood of cardiac allograft recipients treated with 7E1 demonstrated partial B cell depletion on POD 8 and 21 which returned to normal by POD55. 7E1 treatment had no effect on T cell and macrophage compartments. These studies suggest that the use of anti-CD40 mAbs is an effective alternative to MR1 in the blockade of the gp39-CD40 pathway alone or in combination with CTLA4Ig.

Involvement of CD40 Ligand-CD40 and CTLA4-B7 Pathways in Murine Acute Graft-Versus-Host Disease Induced by Allogeneic T Cells Lacking CD28

The blockade of B7, using B7 antagonists such as anti-CD80 and/or -CD86 mAbs or CTLA4Ig in vivo, has been shown to induce an efficient suppression of T cell-mediated immune responses in allograft, allergy, and autoimmune models. However, this treatment does not result in complete tolerance. In this study, we examined CD28-B7-independent activation pathways in the pathogenesis of graft-vs-host disease (GVHD) using allogeneic T cells from CD28-deficient mice. Acute GVHD was induced in the absence of CD28 on donor T cells and its manifestations were obvious in the lymphoid tissues. The CD28-independent GVHD was significantly improved by treatment with anti-CD40 ligand (CD40L) mAb. In contrast, treatment with anti-CD80 plus anti-CD86 mAbs exacerbated the clinical manifestations of GVHD and increased the T cell response against host alloantigen, resulting in the expression of CTLA4, CD40L, and CD25 on splenic T cells. These data suggested that the CD40L-CD40 pathway significantly contributed to the CD28-independent pathogenesis of acute GVHD, whereas the CTLA4-B7 pathway acted protectively in the development of GVHD. These results imply that selectively blockading CD28, instead of disrupting both CD28 and CTLA4, would be a better therapeutic strategy for GVHD. Additionally, the simultaneous use of CD40 antagonists may be advantageous.

PROLONGATION OF MURINE CARDIAC AND SKIN ALLOGRAFT SURVIVAL BY ANTI-CD40 MONOCLONAL ANTIBODY

512 CD40/CD40L interactions are crucial for the development of CD4+ T cell dependent immune responses such as allograft rejection. For example, simultaneous administration of a CD40L monoclonal antibody (mAb) and the CTLA4-Ig fusion protein synergistically prolongs the survival of skin and cardiac allografts. In this report we establish the efficacy of an anti-CD40 mAb alone and in combination with CTLA4-Ig as an alternative to anti-CD40 ligand as a strategy to inhibit rejection. Immunohistochemical analysis demonstrates that CD40 is strongly induced on both donor and recipient cells in the rejecting allograft. Furthermore, transplants using CD40-/- mice indicate that CD40 on the recipient is functionally more important than on donor tissues. Hearts from CD40-/- donors are rejected promptly, whereas CD40-/- recipients reject wild type (wt) cardiac allografts more slowly than wt B6,129 F1 control recipients. The ability of an anti-CD40 mAb, 7E1, to inhibit allograft rejection was tested in the BALB/c to C3H/HeJ strain combination. Recipient mice were treated with 7E1 (250μg/dose) intraperitoneally (IP) on days 0, 2, 4 & 6. 7E1 significantly prolonged cardiac allograft survival relative to untreated controls. However, histological assessment of the 7E1 treated animals demonstrated significant interstitial fibrosis and coronary vasculopathy by postoperative day (POD) 140. As previously reported with a CD40L mAb (MR1), 7E1 synergized with CTLA4-Ig in inhibiting these pathologic changes. C3H/HeJ recipients of BALB/c skin grafts were treated with anti-CD40 mAb, alone and in combination with CTLA4-Ig (250μg/dose IP on days 0, 2, 4 & 6). Anti-CD40 mAbs alone prolonged skin graft survival (MST=30d) compared to MR1 treatment (MST=14d). Untreated controls rejected the skin grafts promptly by POD11. As in the cardiac allograft model, 7E1 and CTLA4-Ig synergize to improve skin graft survival (MST=93d). 7E1 was found to prevent the formation of donor specific alloantibodies in the cardiac allograft recipients. Alloantibodies were undetectable on POD 29, 35 & 57, whereas alloantibodies were detected in the control BALB/c cardiac allograft recipients at a dilution of 1:512 at POD 29, 34 & 57. Flow cytometric analysis of peripheral blood of cardiac allograft recipients treated with 7E1 demonstrated partial B cell depletion on POD 8 and 21 which returned to normal by POD55. 7E1 treatment had no effect on T cell and macrophage compartments. These studies suggest that the use of anti-CD40 mAbs is an effective alternative to MR1 in the blockade of the gp39-CD40 pathway alone or in combination with CTLA4Ig.

Effects of macrophage depletion and anti-CD40 ligand on transgene expression and redosing with recombinant adenovirus.

The anti-CD40 ligand antibody MR-1, and macrophage-depleting liposomes were tested for their ability as transient immunosuppressive agents to: (1) prolong transgene expression; and (2) permit redosing after recombinant adenovirus infusion of mice. To test for effect on transgene duration, mice were infused with recombinant adenovirus coding for human factor IX (AdFIX), and plasma FIX levels monitored over time. Treatment with either agent significantly prolonged transgene expression. Persistence was accompanied by inhibition of anti-adenovirus (anti-Ad) IgG, and decreased IL-10 and IFN-gamma production from splenic lymphocytes re-exposed to virus particles in vitro. To test for effect on redosing, mice were given a primary infusion of recombinant adenovirus coding for bacterial beta-galactosidase (Ad beta gal), followed by secondary and tertiary infusions of AdFIX on days 24 and 63. Mice that had received MR-1 had low to undetectable anti-Ad on day 24, and efficient transduction occurred. Furthermore, FIX levels endured in these mice, with 40% retention of FIX on day 63, in contrast to rapid loss in naive controls. On day 63, the continuance of negligible anti-Ad levels correlated with successful tertiary transduction. These results suggest that both macrophage depletion and CD40 ligand blockade inhibit immune responses to recombinant adenovirus to slow decline of transgene expression, while only CD40 ligand blockade inhibits anti-Ad antibody generation sufficiently to allow redosing to the liver.

Underproduction of Interleukin-12 in Susceptible Mice during Progressive Leishmaniasis Is Due to Decreased CD40 Activity

Interleukin-12 promotes Th1 lymphocyte responses necessary for the cure of murineLeishmania majorinfection. We found that IL-12 p40 mRNA expression peaked at 4 weeks of infection in resistant C57BL/6 mice at levels threefold greater than in BALB/c mice. Peak IL-12 p40 expression in both strains was reduced threefold following treatment with neutralizing anti-CD40 ligand antibody and disease worsened in C57BL/6 mice. Direct activation of cultured lymph node cells by anti-CD40 MAb or soluble CD40 ligand failed to restore deficient IL-12 production by infected BALB/c mice unless recombinant IFN-γ was added to culture. Infected BALB/c lymph nodes also contained two- to threefold fewer low-density CD40+accessory cells compared to that in C57BL/6 mice. We conclude that CD40-dependent responses are continually required for healing of leishmaniasis and that progressive disease is associated with decreased CD40-stimulated IL-12 synthesis as a consequence of either altered cytokine environment or inadequate accessory cell number.

Anti-CD40 Ligand Antibody Treatment of SNF1 Mice with Established Nephritis: Preservation of Kidney Function

Prior studies have demonstrated that treatment of young, prenephritic lupus-prone mice with Ab directed against CD40 ligand (CD40L) prolongs survival and decreases the incidence of severe nephritis. In this report, we show that for (SWR x NZB)F1 (SNF1) animals with established lupus nephritis, long-term treatment with anti-CD40L beginning at either 5.5 or 7 mo of age prolonged survival and decreased the incidence of severe nephritis. "Older" mice were chosen for these studies to more closely resemble the clinical presentation of patients with established renal disease. We show that age at the start of treatment, which typically correlates with severity of disease, is an important factor when determining an efficacious therapeutic protocol since animals that began treatment at 7 mo of age required a more aggressive treatment protocol than animals at 5.5 mo of age. Remarkably, several anti-CD40L-treated animals beginning treatment at age 5.5 mo demonstrated a decline in proteinuria, as opposed to increasing proteinuria levels seen in hamster IgG (HIg)-treated controls, and histologic examination of kidneys from anti-CD40L-treated mice revealed dramatically diminished inflammation, sclerosis/fibrosis, and vasculitis, in marked contrast to the massive inflammation and kidney destruction observed in control animals that received hamster IgG. Spleens from anti-CD40L-treated mice also exhibited markedly reduced inflammation and fibrosis compared with controls. Together, these results show that treatment of older, nephritic SNF1 animals with long-term anti-CD40L immunotherapy significantly prolongs survival, reduces the severity of nephritis, and diminishes associated inflammation, vasculitis, and fibrosis.

Prevention of chronic rejection in mouse aortic allografts by combined treatment with CTLA4-Ig and anti-CD40 ligand monoclonal antibody.

In this study, using a murine model of aortic allotransplantation, the role of blockade of signaling through CD28/B7 and CD40/CD40 ligand costimulatory pathways in the evolvement of posttransplant vasculopathy was examined. Aortic allografts were transplanted across C57BL/1OJ (H2b)-->C3H (H2k) strain combinations. Transient or more stable blockade of second signaling was achieved by either a single injection or multiple injections of CTLA4-Ig fusion protein (200 microg/dose i.p.) and/or anti-CD40 ligand (CD40L) monoclonal antibody (250 microg i.m.). At day 30 after transplantation, the grafts were harvested for histopathological and immunohistochemical examination. Similar to allografts of untreated animals, aortic allografts obtained from recipients treated with either CTLA4-Ig or anti-CD40L monoclonal antibody alone exhibited marked narrowing of the lumen primarily due to concentric intimal thickening caused by proliferation of alpha-smooth muscle actin-positive cells. Contemporaneous treatment, however, with either a single injection or multiple injections of CTLA4-Ig and anti-CD40L monoclonal antibody resulted in marked diminution of intimal thickening. Interestingly, concurrent prolonged inhibition of CD28/B7 and CD40/CD40L pathways resulted in complete abrogation of the development of posttransplant arteriopathy. These data suggest that a more stable disruption of signaling through costimulatory pathways may be required to obviate the development of posttransplant vasculopathy.

Efficient adenovirus-mediated gene transduction of normal and leukemic hematopoietic cells.

We evaluated the efficiency of adenovirus-mediated gene transfer into normal and malignant human hematopoietic cells. An E-1 and E-3 deleted, replication-defective recombinant Ad.RSV beta gal vector was used and the transduction efficiency was studied at a multiplicity of infection of 13 p.f.u. per cell. Approximately 40-50% of normal monocytes were transduced, whereas purified normal resting T cells and B cells were resistant to infection. We showed that 50-80% of primary chronic myeloid leukemia cells (CML, n = 12) were efficiently transduced in contrast to CML, successful transduction of resting primary chronic B lymphocytic leukemia cells required appropriate preactivation of targeted cells. A novel protocol for the efficient transduction of adenovirus into B-CLL cells was presented. We showed that anti-CD40 mAb or CD40 ligand acts in synergy with rhIL-4 to enable the transduction of approximately 50-75% of B-CLL cells (B-CLL, n = 6). Expression of beta-galactosidase in transduced CML cells and B-CLL cells was detected for at least 15 days after transduction. The present studies underline the utility of adenovirus vectors for the construction of cytokine gene-modified tumor vaccines for the treatment of hematopoietic malignancies such as CML and B-CLL.

Normal B cells fail to secrete interleukin-12.

Interleukin-12 is a key regulatory cytokine produced by antigen-presenting cells (APC) which drives the development of interferon-gamma (IFN-gamma)-producing cells and promotes cell-mediated immunity. Following subcutaneous immunization with protein antigen in adjuvant, dendritic cells (DC) but not small nor large B cells in immune lymph nodes express antigenic complexes and secrete substantial amounts of bioactive IL-12 p75 upon antigen-specific interaction with T cells. We have analyzed secretion of IL-12 p40 and p75 by cell populations enriched in DC, macrophages or B cells in response to nonspecific stimulation or to interaction with antigen-specific CD4+ cells. These APC populations do not produce IL-12 constitutively but, upon stimulation with heat-fixed Staphylococcus aureus and IFN-gamma, IL-12 p40 and p75 are secreted by DC and macrophages, whereas B cells fail to produce IL-12. B cells also fail to secrete IL-12 in response to stimulation with LPS and IFN-gamma. Co-culture with CD4+ T hybridoma cells and antigen induces IL-12 secretion by DC. Up-regulation of IL-12 secretion by interaction with antigen-specific CD4+ T cells is abrogated by anti-class II monoclonal antibodies (mAb), by soluble CD40 molecules and by anti-CD40 ligand mAb, demonstrating a positive feedback between T cells and DC mediated by TCR-peptide/class II and by CD40-CD40 ligand interactions. Expression of class II and CD40 molecules is comparable in B cells and DC, and both APC types activate CD4+ T cells. Yet, even upon interaction with antigen-specific T cells, B cells fail to secrete IL-12. The capacity of B cells to present antigen but not to secrete IL-12 may explain their propensity to selectively drive T helper type 2 cell development.

Transient immunomodulation with anti-CD40 ligand antibody and CTLA4Ig enhances persistence and secondary adenovirus-mediated gene transfer into mouse liver.

Although recombinant adenovirus vectors offer a very efficient means by which to transfer genetic information into cells in vivo, antigen-dependent immunity limits the duration of gene expression and prevents retreatment. Recombinant murine CTLA4Ig and anti-CD40 ligand antibody block costimulatory interactions between T cells and antigen presenting cells. We previously reported that murine CTLA4Ig prolongs adenoviral-mediated gene transfer, but does not allow for secondary expression after readministration of the vector. In studies described here, when anti-CD40 ligand and recombinant murine CTLA4Ig were coadministered around the time of primary vector administration (i) prolonged adenovirus-mediated gene expression (length of experiment up to 1 year) from the livers of >90% of treated mice was observed, and (ii) secondary adenovirus-mediated gene transfer was achieved in >50% of the mice even after the immunosuppressive effects of these agents were no longer present. Nearly two-thirds of these mice had persistent secondary gene expression lasting for at least 200-300 days. Neither agent alone allowed transduction after secondary vector administration. Treated mice had decreased immune responses to the vector as shown by markedly decreased production of neutralizing antibodies, diminished spleen proliferation responses and IFN-gamma production in vitro, and reduced T cell infiltrates in the liver. These results suggest that it may be possible to obtain persistence as well as secondary adenoviral-mediated gene transfer with transient immunosuppressive therapies.

Increased interleukin 12 production in progressive multiple sclerosis: induction by activated CD4+ T cells via CD40 ligand.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system postulated to be a cell-mediated autoimmune disease in which interferon gamma (IFN-gamma) plays an important role. There is increased IFN-gamma secretion in MS, and IFN-gamma administration induces exacerbations of disease. We found that interleukin 12 (IL-12) was responsible for raised IFN-gamma secretion in MS as anti-IL-12 antibodies reversed raised anti-CD3-induced IFN-gamma in MS patients to normal levels. Furthermore, we found a marked increase in T cell receptor-mediated IL-12 secretion in progressive MS patients vs. controls (24.8 +/- 7.7 pg/ml vs. 1.5 +/- 1.0 pg/ml, P = 0.003) and vs. relapsing-remitting patients (3.7 +/- 1.4 pg/ml, P < 0.05). Investigation of the cellular basis for raised IL-12 demonstrated that T cells from MS patients induced IL-12 secretion from non-T cells, and that T cells from MS patients could even drive non-T cells from normal subjects to produce increased IL-12. Anti-CD40 ligand antibody completely blocked IL-12 secretion induced by activated T cells, and we found increased CD40 ligand expression by activated CD4+ T cells in MS patients vs. controls. The CD40 ligand-dependent Th1-type immune activation was observed in the progressive but not in the relapsing-remitting from of MS, suggesting a link to disease pathogenesis and progression and providing a basis for immune intervention in the disease.

T cell-derived IL-4 and dendritic cell-derived IL-12 regulate the lymphokine-producing phenotype of alloantigen-primed naive human CD4 T cells.

Previous studies on human Th subset development were restricted to the analysis of naive T cells activated with anti-CD3 mAb in the absence of physiologic APC. In this study, we have analyzed the role of cytokines and physiologic APC on T cell maturation in an Ag-specific system, in which naive neonatal CD4 T cells were primed with allogeneic dendritic cells (DC). We found that the cytokine profile of primed cells was dependent upon 1) the ratio between T cells and allogeneic DC and 2) the endogenous production of IL-4 and IL-12. Neutralization of IL-4 during primary MLR increased IFN-gamma production at priming and shifted the phenotype of primed cells from Th0 to Th1. These effects were IL-12 dependent, in that they were suppressed by anti-IL-12 Abs. The production of IL-12 in primary MLR was further evidenced by the presence of IL-12 p40 in the culture supernatant fluids. IL-12 production was suppressed by exogenous IL-4 and increased by anti-IL-4 blocking mAbs, indicating that endogenous IL-4 down-regulated IL-12 production by DC. Finally, IL-12 was produced as a result of T cell/DC interaction involving the CD40/CD40 ligand and CD28/B7 costimulation pathways, as revealed by the inhibitory effect of anti-CD40 ligand mAb and CTLA-4Ig. These observations suggest that in neutral conditions, Ag presentation by DC results in the coordinate production of naive T cell-derived IL-4 and DC-derived IL-12 that in concert shape the cytokine profile of Th cells.

B cells regulate CD40 ligand-induced IL-12 production in antigen-presenting cells (APC) during T cell/APC interactions.

Although stimulation of freshly isolated murine spleen cells with anti-CD3 mAb or Con A failed to generate IL-12 production, the same cell preparations depleted of B cells produced IL-12. Addition of normal B cells inhibited IL-12 production in a cell number-dependent manner. IL-12 production was dependent on the presence of CD4+, but not of CD8+, T cells, and inhibited by addition of anti-CD40 ligand (CD40L) mAb. Anti-CD3 or Con A stimulation induced CD40L expression only on CD4+ T cells, which was inhibited in the presence of B cells. IL-12 production was also induced by interactions between CD40L-transfected Chinese hamster ovary cells and splenocytes depleted of T and B cells, but not of APC, indicating CD40L-induced IL-12 production by APC. The involvement of CD40 molecules was examined by comparing the ability of cells from CD40-deficient (CD40 -/-) and wild-type mice (CD40 +/+) to produce IL-12. Spleen cells from CD40 -/- and CD40 +/+ mice produced comparable amounts of IL-12 in response to bacterial stimuli. However, the B cell-depleted fraction from CD40 -/- mice failed to produce IL-12 when stimulated with anti-CD3 or Con A or when cocultured with CD40L-expressing Chinese hamster ovary cells. These results indicate that CD40L expressed on activated T cells induces APC to produce IL-12 through CD40/CD40L interaction, but this pathway is competitively inhibited by CD40+ B cells incapable of producing IL-12 upon stimulation with CD40L. Thus, this might represent a novel mechanism underlying the regulation of cell-mediated and humoral immunity.

Anti-CD40 ligand antibody treatment prevents the development of lupus-like nephritis in a subset of New Zealand black x New Zealand white mice. Response correlates with the absence of an anti-antibody response.

Systemic lupus erythematosus is characterized by B cell production of pathogenic autoantibodies dependent upon cooperation from CD4+ Th cells. The interaction between CD40 on B cells and CD40 ligand (CD40L) on Th cells is necessary for normal thymus-dependent Ab production. An anti-murine CD40L mAb blocks binding of CD40L to CD40 and prevents primary and secondary immune responses to thymus-dependent Ags. In this study, New Zealand Black x New Zealand White lupus-prone mice treated with this anti-CD40L Ab from ages 4 to 10 mo had reduced anti-DNA autoantibody production and renal disease and significantly prolonged survival compared with control mice. Pathologic examination verified the absence of significant renal damage or immune deposition in responding mice. Mice that responded to treatment did not develop an Ab response to the administered Ab. Long-term survivors mounted a substantial Ab response to keyhole limpet hemocyanin after completion of anti-CD40L Ab treatment, suggesting that some of the immunosuppressive effects of the Ab may be reversible. These results suggest a human form of this Ab may have therapeutic utility in human systemic lupus erythematosus.

High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10.

We have shown previously that dendritic cells (DC) produce IL-12 upon interaction with CD4+ T cells. Here we ask how this IL-12 production is induced and regulated. Quantitative PCR and in situ hybridization for IL-12 p40 and an ELISA specific for the p70 heterodimer were used to determine IL-12 production. We demonstrate that ligation of either CD40 or MHC class II molecules independently trigger IL-12 production in DC, and that IL-12 production is downregulated by IL-4 and IL-10. The levels of bioactive IL-12 that can be released by triggering with an anti-CD40 mAb or with a T cell hybridoma are high (range 260-4700 pg/ml from 1 X 10(6) DC in 72 h). The CD40-mediated pathway indicates that IL-12 production is induced in DC upon interaction with activated, CD40 ligand-expressing helper T cells, even in the absence of cognate antigen recognition. Side-by-side comparison of IL-12 production, and blocking experiments employing an anti-CD40 ligand mAb, suggest that the CD40-mediated pathway is quantitatively more significant than induction via the MHC class II molecule. The importance of the CD40/CD40 ligand interaction for IL-12 induction in DC likely contributes to the recent finding that mice lacking the CD40 ligand are impaired in mounting Th1 type cell-mediated immune responses.