anti-CD40 Ligand Antibody Research Articles

A CD45-targeted antibody-drug conjugate successfully conditions for allogeneic hematopoietic stem cell transplantation in mice

AbstractAllogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment of patients with nonmalignant or malignant blood disorders. Its success has been limited by graft-versus-host disease (GVHD). Current systemic nontargeted conditioning regimens mediate tissue injury and potentially incite and amplify GVHD, limiting the use of this potentially curative treatment beyond malignant disorders. Minimizing systemic nontargeted conditioning while achieving alloengraftment without global immune suppression is highly desirable. Antibody-drug-conjugates (ADCs) targeting hematopoietic cells can specifically deplete host stem and immune cells and enable alloengraftment. We report an anti-mouse CD45-targeted-ADC (CD45-ADC) that facilitates stable murine multilineage donor cell engraftment. Conditioning with CD45-ADC (3 mg/kg) was effective as a single agent in both congenic and minor-mismatch transplant models resulting in full donor chimerism comparable to lethal total body irradiation (TBI). In an MHC-disparate allo-HSCT model, pretransplant CD45-ADC (3 mg/kg) combined with low-dose TBI (150 cGy) and a short course of costimulatory blockade with anti-CD40 ligand antibody enabled 89% of recipients to achieve stable alloengraftment (mean value: 72%). When CD45-ADC was combined with pretransplant TBI (50 cGy) and posttransplant rapamycin, cyclophosphamide (Cytoxan), or a JAK inhibitor, 90% to 100% of recipients achieved stable chimerism (mean: 77%, 59%, 78%, respectively). At a higher dose (5 mg/kg), CD45-ADC as a single agent was sufficient for rapid, high-level multilineage chimerism sustained through the 22 weeks observation period. Therefore, CD45-ADC has the potential utility to confer the benefit of fully myeloablative conditioning but with substantially reduced toxicity when given as a single agent or at lower doses in conjunction with reduced-intensity conditioning.

First-in-human clinical trial to assess pharmacokinetics, pharmacodynamics, safety, and tolerability of iscalimab, an anti-CD40 monoclonal antibody.

Iscalimab is a fully human, CD40 pathway blocking, nondepleting monoclonal antibody being developed as an immunosuppressive agent. We describe a first-in-human, randomized, double-blind, placebo-controlled study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of iscalimab in healthy subjects and rheumatoid arthritis patients. Healthy subjects (n=56) received single doses of intravenous iscalimab (0.03, 0.1, 0.3, 1, or 3mg/kg), or subcutaneous iscalimab (3mg/kg), or placebo. Rheumatoid arthritis patients (n=20) received single doses of intravenous iscalimab (10 or 30mg/kg) or placebo. Iscalimab exhibited target-mediated drug disposition resulting in dose-dependent and nonlinear pharmacokinetics. Complete (≥90%) CD40 receptor occupancy on whole blood B cells was observed at plasma concentrations >0.3-0.4µg/mL. In subjects receiving 3mg/kg iscalimab, antibody responses to keyhole limpet hemocyanin were transiently suppressed. CD40 occupancy by iscalimab prevented ex vivo human rCD154-induced expression of CD69 on B cells in whole blood. All doses were generally safe and well tolerated, with no clinically relevant changes in any safety parameters, including no evidence of thromboembolic events. Iscalimab appears to be a promising blocker of the CD40-CD154 costimulatory pathway with potential use in transplantation and other autoimmune diseases.

Costimulation Blockade of CD40 and CD28 Pathways in Limb Transplantation

Costimulation blockade remains a promising experimental regimen for the induction of transplantation tolerance. The simultaneous blockade of both the CD40 and CD28 pathways has been synergistic in prolonging organ allograft survival but has not previously been investigated in a model of limb allotransplantation. This study determined the efficacy of this combination in the murine limb allograft model. C57B1/6 (H-2K b) female mice were recipients of heterotopic vascularized limb allografts from Balb/c (H-2K d) male donors. Experimental groups received treatment with a short course of MR1 (hamster anti-mouse anti-CD40 ligand antibody) alone or in combination with CTLA4-Ig, a fusion protein that blocks the B7/CD28 pathway. Untreated recipients rejected limb allografts at a mean of 9.6 ± 1.1 (standard error of the mean) days postoperatively. Recipients of a prolonged course of MR1 rejected limb allografts at 75 ± 25 days. When both MR1 and CTLA4-Ig were used, limb allograft survival of >120 days was observed despite a much shorter course of therapy. Rejection in both treatment groups was consistent with a chronic antibody-mediated process. Donor antigen rechallenge in these recipients by in vitro assay and skin allograft demonstrated a hyperacute response consistent with presensitization. Long-term limb allograft survival is produced by the synergistic effect of blocking both the CD40 and CD28 costimulatory pathways. However, permanent acceptance was not achieved, and allografts eventually succumbed to what appeared to be antibody-mediated rejection. The additional use of newer agents that block more recently described costimulatory pathways may be essential for the induction of tolerance by costimulation blockade.

Lupus systémique : les traitements du futur

During the last decade, new biotherapies have been developed for the treatment of systemic autoimmune diseases, especially for systemic lupus erythematosus (SLE). These new approaches are based on a better understanding of the auto-immune response. Targets of these new treatments are all the steps of the immune response. These new therapies are: (1) "B lymphocyte (BL)" inhibitors such as anti-CD20 monoclonal antibody, anti-CD22 monoclonal antibody, BlyS antagonists, tolerogens of pathogenic-antibody secreting LB (LJP 394) and edratide; (2) "Inhibitors of the costimulation" between antigen-presenting cells and T lymphocyte (TL) like monoclonal anti-CD40 ligand antibody or CTLA-4-Ig (abatacept); (3) "Cytokine antagonists" inhibiting key cytokines of SLE: interleukin-10, interferon-alpha, interleukin-6 and TNF. These new therapies are currently under development in SLE.

Prolonged Susceptibility to Antibody-mediated Neutralization for Adeno-associated Vectors Targeted to the Liver

Adeno-associated virus (AAV) vectors demonstrate highly efficient gene transfer to hepatocytes in vivo. One of the remaining obstacles to the treatment of hemophilia B patients with AAV vectors is the sensitivity of these vectors to antibody-mediated neutralization following systemic delivery. Testing and implementation of strategies to circumvent pre-existing antibodies requires knowledge of the clearance kinetics of AAV from circulation. In this study, AAV clearance kinetics were established for serotypes 2 and 8 in cell culture and in mice. Administration of pooled neutralizing serum subsequent to administration of the vector was used to define the time period in which the vector is susceptible to antibody-mediated neutralization. These experiments defined the in vivo clearance rates for both AAV2 and AAV8 vectors to be between 2 and 4 hours. In mice, portal vein and tail vein administration of each vector was tested with similar results. Cell culture studies in W162 cells established that cellular attachment and internalization both contribute to the clearance kinetics of AAV vectors. These studies characterize the in vivo clearance rates of AAV vectors for the first time and guide the development of future strategies for the avoidance of antibody-mediated AAV vector neutralization.

Thermodynamic Consequences of Mutations in Vernier Zone Residues of a Humanized Anti-human Epidermal Growth Factor Receptor Murine Antibody, 528

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.

Improvements in Mucopolysaccharidosis I Mice After Adult Retroviral Vector–mediated Gene Therapy with Immunomodulation

Mucopolysaccharidosis I (MPS I) is caused by deficient alpha-L-iduronidase (IDUA) activity and results in the accumulation of glycosaminoglycans and multisystemic disease. Gene therapy could program cells to secrete mannose 6-phosphate-modified IDUA, and enzyme in blood could be taken up by other cells. Neonatal retroviral vector (RV)-mediated gene therapy has been shown to reduce the manifestations of murine MPS I; however, intravenous injection of RV into adults was ineffective owing to a cytotoxic T lymphocyte (CTL) response against transduced cells. In this study, prolonged inhibition of CD28 signaling with CTLA4-Ig, or transient administration of CTLA4-Ig with an anti-CD40 ligand antibody or with an anti-CD4 antibody, resulted in stable expression in most mice that received RV as adults. Mice with stable expression had 81 +/- 41U/ml IDUA activity in serum. This resulted in reductions in bone disease, improvements in hearing and vision, and reductions in biochemical and pathological evidence of lysosomal storage in most organs. Improvements in brain were likely due to diffusion of enzyme from blood. However, aortic disease was refractory to treatment. This demonstrates that most manifestations of MPS I can be prevented using adult gene therapy if an immune response is blocked.

Anti-CD40 Ligand Antibody Permits Regeneration through Peripheral Nerve Allografts in a Nonhuman Primate Model

Ann Arbor, Mich. Received for publication March 22, 2004. Paul S. Cederna, M.D., University of Michigan Health Systems, Department of Surgery, Section of Plastic and Reconstructive Surgery, 1500 East Medical Center Drive, 2130 Taubman Center, Ann Arbor, Mich. 48109-0340, [email protected]

Anti-CD40 Ligand Antibody Permits Regeneration through Peripheral Nerve Allografts in a Nonhuman Primate Model

Systemic immunosuppression is typically required to prevent allograft rejection. Antibody-based therapies that induce immune unresponsiveness represent an appealing alternative to nonspecific immunosuppression, which is often associated with significant morbidity. In mice, successful prevention of nerve allograft rejection has been demonstrated through interference with the CD40/CD40 ligand interaction. This study investigated the effectiveness of anti-CD40 ligand monoclonal antibody as single-agent therapy in preventing rejection and supporting nerve regeneration across long nerve allografts in nonhuman primates. Twelve outbred cynomolgus macaques were arranged into six genetically mismatched pairs, with each animal receiving a 5-cm ulnar nerve allograft in the right arm and a 5-cm autograft in the left arm. Mixed lymphocyte reaction assays were used to assess resulting immune unresponsiveness. Treated animals (n = 10) received anti-CD40 ligand monoclonal antibody 10 mg/kg one time, locally applied, and 20 mg/kg systemically on postoperative days 0, 1, 3, 10, 18, and 28, and then monthly. Untreated animals (n = 2) served as the untreated controls. At 4 or 6 months after transplantation, nerves were harvested for histological analysis. Four treated animals underwent an additional challenge after cessation of anti-CD40 ligand monoclonal antibody therapy and nerve graft harvests. Autogenous and allogeneic skin and nerve inlay grafting was performed to assess the permanence of immune unresponsiveness induced by anti-CD40 ligand monoclonal antibody. Animals that received anti-CD40 ligand monoclonal antibody demonstrated robust regeneration across nerve allografts, similar to that seen in the autograft control in the contralateral arm. The histomorphometric analysis of allografts in the untreated animals demonstrated significantly worse measurements compared with their matched autograft controls. Animals that received anti-CD40 ligand monoclonal antibody with concomitant skin allografts had virtually no evidence of nerve regeneration through allografts. Allogeneic skin and nerve allografts applied 2 to 12 months after withdrawal of anti-CD40 ligand monoclonal antibody therapy were consistently rejected. This study demonstrates that anti-CD40 ligand monoclonal antibody prevents rejection and allows regeneration of peripheral nerve allografts in nonhuman primates. The effect of anti-CD40 ligand monoclonal antibody appears to be transient, however, with restoration of immunocompetence shortly after withdrawal of therapy.

Anti-CD40 ligand monoclonal antibody induces a permissive state, but not tolerance, for murine peripheral nerve allografts

Anti-CD40 ligand monoclonal antibody prevents the interaction between CD40 and its T-cell-based ligand, thereby resulting in selective inhibition of T cell costimulation without pan-T-cell suppression. This antibody has found application in several animal models of solid organ transplantation. This study investigated use of anti-CD40 ligand antibody to promote acceptance of nerve allografts. In Experiment 1, 40 BALB/cj mice with tibial nerve transplants were administered anti-CD40 ligand antibody, a control antibody, or no treatment. In Experiment 2, 40 BALB/cj mice underwent the same regimen as in Experiment 1, but were later challenged with a second nerve allograft 3 weeks after discontinuation of treatment. Animals treated with anti-CD40 ligand antibody in Experiment 1 exhibited improved functional recovery and greater mean fiber count, fiber density, and percent nerve fiber than animals treated with control antibody or no antibody ( P < 0.05). These permissive effects on nerve regeneration were associated with immune unresponsiveness on Elispot assay. The benefit of anti-CD40 ligand therapy did not persist after withdrawal of treatment (Experiment 2). Active blockade of the CD40 costimulatory pathway with murine anti-CD40 ligand antibody therefore induces a permissive state conducive to nerve regeneration across allografts but does not establish long-term tolerance.

CD40 ligand-dependent maturation of human monocyte-derived dendritic cells by activated platelets.

Atherosclerosis is defined as an inflammatory immunological disease that is triggered by platelet activation, endothelial injury and consequent innate and adaptive immune processes. Dendritic cells are critical for the cell-mediated arm of the immune response as they activate naïve T cells after maturation. Platelets play a crucial role in thrombus formation in the injured vessel walls. We investigated the role of resting and thrombin-activated platelets in dendritic cell maturation in vitro using platelets and monocyte-derived dendritic cells from healthy donors. Resting platelet supernatants did not affect maturation, whereas supernatants from thrombin-activated platelets induced dendritic cell maturation as demonstrated by FACS analysis of HLA-DR expression. This effect was inhibited by anti CD40 ligand antibody, but not by aspirin pretreatment of platelets. Supernatants of platelet-dendritic cell co-cultures induced augmented monocyte migration when platelets were activated by thrombin, again reversible by blocking CD40 ligand. These data show that activated platelets trigger dendritic cell maturation independent of cyclooxygenase-derived arachidonic acid metabolites by mechanisms involving CD40 ligand, which is also involved in monocyte chemotactic mediator release from platelets and dendritic cells. The results of this study suggest a role of CD40 ligand from activated platelets in connecting innate and adaptive immunity.

Tolerance induction by costimulator blockade in 100 cGy treated hosts with varying degrees of genetic disparity.

Long-term multilineage allochimerism can be obtained in H2-mismatched B6.SJL to BALB/c transplants with host irradiation of 100 cGy, donor spleen cell pre-exposure and costimulator blockade with anti-CD40 ligand (CD40L) antibody. We evaluated this allochimerism approach in murine marrow transplants with different degrees of major histocompatibility complexe (MHC) mismatching; these include: (1) H2-mismatched transplant H2Kk to H2Kb, (2) full haplo-identical transplant H2Kbd to H2Kbk, (3) a partial haplo-identical transplant H2Kd to H2Kbd and (4) an MHC class II mismatch. Levels of chimerism increased up to 12 weeks and then stayed relatively stable up to 1 year after transplant. At 18 weeks post-transplant, the H2-mismatched, haplo-identical, partial haplo-identical and class II-mismatch transplants evidenced 17.9+/-4.4, 40.7+/-0.9, 25.1+/-4.19 and 33.7+/-3.5% donor chimerism, respectively. Dropping the anti-CD40 antibody treatment and spleen cells or changing the schedule of antibody to one injection, in haplo-identical or full-mismatched transplants resulted in no donor-derived chimerism. On the other hand, these still resulted in minor chimerism in class II-mismatched transplants. Lineage analysis of peripheral blood at 6 and 12 months post-transplant demonstrated a significant shift toward increased chimeric lymphocytes and decreased chimeric granulocytes in the full H2 as compared with haplo-identical or class II transplants. Transplantation with anti-CD40L antibody eliminated both graft-versus-leukemia and graft-versus-host disease (GVHD) and delayed lymphocyte infusion did not rescue animals from fatal leukemia. In conclusion, under the conditions of our tolerization regimen, a haplo transplant gives higher engraftment levels than a full H2 mismatch, and despite lower engraftment levels, a class II-mismatched transplant can be successfully accomplished with only 100 cGy and no CD40L blockade.

A short course of BG9588 (anti–CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis

CD40-CD40 ligand (CD40L) interactions play a significant role in the production of autoantibodies and tissue injury in lupus nephritis. We performed an open-label, multiple-dose study to evaluate the safety, efficacy, and pharmacokinetics of BG9588, a humanized anti-CD40L antibody, in patients with proliferative lupus nephritis. The primary outcome measure was 50% reduction in proteinuria without worsening of renal function. Twenty-eight patients with active proliferative lupus nephritis were scheduled to receive 20 mg/kg of BG9588 at biweekly intervals for the first 3 doses and at monthly intervals for 4 additional doses. Safety evaluations were performed on all patients. Eighteen patients receiving at least 3 doses were evaluated for efficacy. The study was terminated prematurely because of thromboembolic events occurring in patients in this and other BG9588 protocols (2 myocardial infarctions in this study). Of the 18 patients for whom efficacy could be evaluated, 2 had a 50% reduction in proteinuria without worsening of renal function. Mean reductions of 38.9% (P < 0.005), 50.1% (P < 0.005), and 25.3% (P < 0.05) in anti-double-stranded DNA (anti-dsDNA) antibody titers were observed at 1, 2, and 3 months, respectively, after the last treatment. There was a significant increase in serum C3 concentrations at 1 month after the last dose (P < 0.005), and hematuria disappeared in all 5 patients with significant hematuria at baseline. There were no statistically significant reductions in lymphocyte count or serum immunoglobulin, anticardiolipin antibody, or rubella IgG antibody concentrations after therapy. A short course of BG9588 treatment in patients with proliferative lupus nephritis reduces anti-dsDNA antibodies, increases C3 concentrations, and decreases hematuria, suggesting that the drug has immunomodulatory action. Additional studies will be needed to evaluate its long-term effects.

Combined gene therapy with adenovirus vectors containing CTLA4Ig and CD40Ig prolongs survival of composite tissue allografts in rat model.

The blockade of costimulatory signal pathway by anti-CD40 ligand antibody or cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) prolongs allograft survival in various vascularized organ transplantations. Because of the short half life of these agents, repeated administration of proteins is required to achieve significant graft survival. Furthermore, there is limited information regarding the effect of cosimulatory blockade on the survival of composite tissue allografts. Therefore, we examined the effect of adenovirus-mediated gene transfer of CTLA4Ig or CD40Ig gene or both in composite tissue allotransplantation. The hind limbs removed from male ACI rats (RT1 ) were transplanted into female Lewis rats (RT1 ) heterotopically. The recombinant adenovirus carrying CTLA4Ig (AxCTLA4Ig) or CD40Ig (AxCD40Ig) was intravenously administered after limb transplantation. Limb allograft survival was significantly prolonged by either AxCTLA4Ig or AxCD40Ig treatment at 1 x 10 plaque forming unit (mean survival time [MST] of 39.4+/-6.0 and 13.0+/-2.9, respectively) compared with the adenovirus vector containing beta-galactosidase-treated group (MST of 4.8+/-0.8). Combination of AxCTLA4Ig and AxCD40Ig led to significant prolongation of graft survival (MST of 49.2+/-6.6). Serum levels of CD40Ig were higher in rats treated with combination therapy than those treated with AxCD40Ig alone, whereas the serum levels of CTLA4Ig in rats treated with AxCTLA4Ig alone and AxCTLA4Ig and AxCD40Ig combined were very similar. This study indicates that an adenovirus-mediated gene therapy of CTLA4Ig or CD40Ig has a therapeutic potential for preventing rejection in composite tissue transplantation. Furthermore, a combination therapy of AxCTLA4Ig and AxCD40Ig was even more effective in preventing acute rejection and prolonging the survival of allografted limbs without apparent complication.

IFN-gamma production is specifically regulated by IL-10 in mice made tolerant with anti-CD40 ligand antibody and intact active bone.

We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-gamma production that is donor specific, and a reduction in the frequency of IFN-gamma-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-gamma responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-beta, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-gamma response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-gamma production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-gamma levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-gamma. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-gamma production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.

Interaction of monocytes with vascular endothelial cells synergistically induces interferon γ-inducible protein 10 expression through activation of specific cell surface molecules and cytokines

To further understand the regulatory mechanisms involved in the process of angiogenesis, the present study was designed to determine the expression and regulation of interferon γ-inducible protein 10 (IP-10) in peripheral blood monocytes and human umbilical vein endothelial cells (HUVECs). We found that the interaction of monocytes with HUVECs resulted in synergistic increases in IP-10 expression and secretion, which consequently inhibited endothelial tube formation in vitro. Induction of IP-10 was mediated via specific cell surface molecules, as indicated by the finding that IP-10 secretion was significantly inhibited by anti-CD40 ligand antibody, and to a lesser extent by anti-CD40 antibody. Furthermore, we examined the effects of soluble mediators, such as inflammatory and immune cytokines on IP-10 secretion. Addition of interleukin (IL)-1, as well as interferon γ, induced a marked augmentation of IP-10 secretion by unstimulated monocytes, unstimulated HUVECs, and co-cultures of the two cell types. In contrast, IL-10, recognized as an anti-inflammatory cytokine, significantly inhibited IP-10 secretion by co-cultures. Our results suggest that the interaction of monocytes with endothelial cells results in synergistic increases in IP-10 expression and secretion, which contribute to the regulation of angiogenesis and initiation of inflammatory vascular diseases.

Primed Allospecific T Cells Prevent the Effects of Costimulatory Blockade on Prolonged Cardiac Allograft Survival in Mice

Costimulatory blockade can induce long-term allograft survival in naive animals, but may not be as effective in animals with previously primed immune repertoires. We attempted to induce long-term graft survival in B10.D2 recipients of B10.A cardiac allografts using donor-specific transfusion (DST) plus anti-CD40 ligand antibody (alphaCD40L). Recipients were either naive mice, or mice previously primed to B10.A or third party alloantigens through engraftment and rejection of skin transplants. Untreated naïve mice rejected cardiac transplants by day 15 and contained a high frequency of primed, donor-reactive T cells. Donor-specific transfusion/alphaCD4OL treatment of naïve animals induced long-term graft survival associated with low frequencies of donor-reactive T cells. Previous priming of donor-specific T cells through rejection of B10.A, but not third party, skin grafts prevented the effects of DST/alphaCD40L on prolonging survival of B10.A hearts. Moreover, adoptive transfer of CD3+, CD4+ or CD8+ T cells from B10.A skin-graft-primed animals prevented the effects of DST/alphaCD40L. The data demonstrate that animals with immune repertoires containing previously primed, donor-reactive T cells are resistant to the effects of costimulatory blockade. The findings have important implications for ongoing, costimulatory blockade-based trials in humans, whose T-cell repertoires are known to contain memory alloreactive T cells.

T cell-mediated signaling to vascular endothelium: induction of cytokines, chemokines, and tissue factor

Adhesion of leukocytes to the vascular endothelium is an early event in inflammation. Since cell-cell signaling may be an important stimulus for endothelial activation, we focused in this study on the role of contact-mediated activation by T lymphocytes of endothelial cells (EC). T lymphocytes were cultured with anti-CD3 monoclonal antibody or in the presence of a combination of TNF-alpha, interleukin (IL)-6, and IL-2, prior to fixation and coculture with human umbilical vein EC. Fixed, activated (anti-CD3- or cytokine-stimulated), but not unstimulated T cells, induced release of monocyte chemotactic protein-1, IL-8, and IL-6 by EC in a contact-dependent manner. Moreover, expression of tissue-factor antigen and activity was also significantly increased. Addition of anti-CD40 ligand antibody abolished T cell-induced activation of EC. Our data suggest that contact-mediated activation of EC by T cells, involving ligand:counter ligand interactions such as CD40:CD40 ligand, may represent a novel pathogenic mechanism of progression in inflammatory diseases such as atherosclerosis or rheumatoid arthritis.

CD40-CD40 Ligand Disruption Does Not Prevent Hyperoxia-Induced Injury

Recent studies suggest that apoptosis plays a role in oxygen-induced injury, although the activation pathways and the executioner proteases that lead to cleavage of lung cell proteins and DNA, are not yet identified. We explored previously the tumor necrosis factor/tumor necrosis factor receptor and the Fas/FasL, belonging to the intrinsic pathway, and could not demonstrate any protective effect by interfering with these cell receptors. Lately, it has been shown that interacting with the CD40 system, also known to promote cell death, by administering anti-CD40 ligand (L) antibody was beneficial in several diseases and, in particular, in hyperoxia-induced injury. Using CD40- and CD40L-deficient mice (-/-) as well as administering anti-CD40L antibody, we examined the extent of lung injury in oxygen-breathing mice by several ways (lung weight, histology, inflammatory mediators, and DNA ladder) as well as the mortality. The development of lung injury was similar in wild-type, CD40-/-, CD40L-/-, or in wild-type mice treated with anti-CD40L antibody. Apoptosis was present in all conditions at 72 hours of oxygen exposure. These results show that oxygen-induced injury does not require CD40-CD40L interaction and that apoptosis of lung cells does not involve this pathway.

Spontaneous formation of germinal centers in autoimmune mice

The mechanisms of autoantibody production are not well understood. Germinal centers (GC) may be important sites of immune disregulation in autoimmune diseases. In this study, we document the presence of spontaneous GC formation in the spleens of several autoimmune mouse strains that spontaneously develop autoimmune Type I diabetes and a lupus-like disease. In contrast, mouse strains that do not develop lupus did not exhibit spontaneous formation of GC. In all of the autoimmune strains studied, GC were present at 1-2 months of age, a time that closely parallels the appearance of autoantibodies. Like the GC that develop after purposeful immunization, GC in autoimmune mice contained B220(+), PNA(+), and GL-7(+) B cells, and FDC-M1(+) follicular dendritic cells. In addition, spontaneously formed GC in autoimmunity and those caused by immunization were abrogated in a similar way by a short-term treatment with anti-CD40 ligand antibody. These data indicate that spontaneously forming GC in autoimmunity are similar to those appearing after purposeful immunization.