anti-CD40L Monoclonal Antibody Research Articles

Inhibition of CD40L with Frexalimab in Multiple Sclerosis

BackgroundThe CD40–CD40L costimulatory pathway regulates adaptive and innate immune responses and has been implicated in the pathogenesis of multiple sclerosis. Frexalimab is a second-generation anti-CD40L monoclonal antibody being evaluated for the treatment of multiple sclerosis.MethodsIn this phase 2, double-blind, randomized trial, we assigned, in a 4:4:1:1 ratio, participants with relapsing multiple sclerosis to receive 1200 mg of frexalimab administered intravenously every 4 weeks (with an 1800-mg loading dose), 300 mg of frexalimab administered subcutaneously every 2 weeks (with a 600-mg loading dose), or the matching placebos for each active treatment. The primary end point was the number of new gadolinium-enhancing T1-weighted lesions seen on magnetic resonance imaging at week 12 relative to week 8. Secondary end points included the number of new or enlarging T2-weighted lesions at week 12 relative to week 8, the total number of gadolinium-enhancing T1-weighted lesions at week 12, and safety. After 12 weeks, all the participants could receive open-label frexalimab.ResultsOf 166 participants screened, 129 were assigned to a trial group; 125 participants (97%) completed the 12-week double-blind period. The mean age of the participants was 36.6 years, 66% were women, and 30% had gadolinium-enhancing lesions at baseline. At week 12, the adjusted mean number of new gadolinium-enhancing T1-weighted lesions was 0.2 (95% confidence interval [CI], 0.1 to 0.4) in the group that received 1200 mg of frexalimab intravenously and 0.3 (95% CI, 0.1 to 0.6) in the group that received 300 mg of frexalimab subcutaneously, as compared with 1.4 (95% CI, 0.6 to 3.0) in the pooled placebo group. The rate ratios as compared with placebo were 0.11 (95% CI, 0.03 to 0.38) in the 1200-mg group and 0.21 (95% CI, 0.08 to 0.56) in the 300-mg group. Results for the secondary imaging end points were generally in the same direction as those for the primary analysis. The most common adverse events were coronavirus disease 2019 and headaches.ConclusionsIn a phase 2 trial involving participants with multiple sclerosis, inhibition of CD40L with frexalimab had an effect that generally favored a greater reduction in the number of new gadolinium-enhancing T1-weighted lesions at week 12 as compared with placebo. Larger and longer trials are needed to determine the long-term efficacy and safety of frexalimab in persons with multiple sclerosis. (Funded by Sanofi; ClinicalTrials.gov number, NCT04879628.)

The anti-CD40L monoclonal antibody AT-1501 promotes islet and kidney allograft survival and function in nonhuman primates.

Prior studies of anti-CD40 ligand (CD40L)-based immunosuppression demonstrated effective prevention of islet and kidney allograft rejection in nonhuman primate models; however, clinical development was halted because of thromboembolic complications. An anti-CD40L-specific monoclonal antibody, AT-1501 (Tegoprubart), was engineered to minimize risk of thromboembolic complications by reducing binding to Fcγ receptors expressed on platelets while preserving binding to CD40L. AT-1501 was tested in both a cynomolgus macaque model of intrahepatic isletallotransplantation and a rhesus macaque model of kidney allotransplantation. AT-1501 monotherapy led to long-term graft survival in both islet and kidney transplant models, confirming its immunosuppressive potential. Furthermore, AT-1501-based regimens after islet transplant resulted in higher C-peptide, greater appetite leading to weight gain, and reduced occurrence of cytomegalovirus reactivation compared with conventional immunosuppression. These data support AT-1501 as a safe and effective agent to promote both islet and kidney allograft survival and function in nonhuman primate models, warranting further testing in clinical trials.

Fibroblastic reticular cells orchestrate long-term graft survival following recipient treatment with CD40 ligand–targeted costimulatory blockade

Fibroblastic reticular cells (FRCs) maintain the architecture of secondary lymphoid organs, which optimize interactions between antigen-presenting dendritic cells and reactive naive T cells. In this issue of the JCI, Zhao, Jung, and colleagues investigated CD4+FoxP3+ regulatory T cell development and long-term heart allograft survival in recipients treated with peritransplant costimulatory blockade to inhibit CD40/CD40 ligand (CD40L) signaling. Treatment with an anti-CD40L monoclonal antibody (mAb) increased the lymph node (LN) population of Madcam1+ FRCs and altered their transcription profile to express immunoregulatory mediators. Administration of nanoparticles, containing the anti-CD40L mAb and a targeting antibody against high endothelial venules, delivered the treatment into LNs of allograft recipients. Direct LN delivery of the costimulatory blockade allowed decreased dosing and increased the efficacy in extending graft survival. The results provide insights into mechanisms by which FRCs can promote donor-reactive tolerance, and establish a strategy for administering costimulation-blocking reagents that circumvent systemic effects and improve allograft outcomes.

Antibody based conditioning for allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic option for many patients with hematological malignancies and nonmalignant hematopoietic disorders. To achieve stable engraftment of donor hematopoietic stem cells (HSCs), recipient HSC deletion is needed to create space for incoming donor HSCs and donor HSCs must escape immune rejection by the recipient. Conventional allo-HSCT requires high dose of irradiation and/or chemotherapy to produce sufficient host stem cell and immune system ablation to permit donor HSC engraftment. However, these procedures also result in nonspecific tissue injury that can cause short- and long-term adverse effects as well as incite and amplify graft-versus-host-disease (GVHD). The delivery of targeted radiotherapy to hematopoietic tissues with the use of a radioimmunoconjugate (ROIC) as a part of transplant preparative regimen has shown clinical benefits. ROIC clinical data provide evidence for decreased relapse without increased transplant-related mortality by delivering higher targeted radiation to sites of malignancy than when given in a nontargeted fashion. An alternative approach to allo-HSCT has been developed and tested in preclinical mouse models in which nonmyeloablative preconditioning with low dose of the alkylating agent (busulfan) or lower systemic dose of irradiation combined with co-stimulatory pathway blockade (CTLA4-Ig, anti-CD40L monoclonal antibody) and/or immunosuppressive drugs have been used. Under these conditions, mixed chimerism and transplantation tolerance to fully MHC mismatched donor marrow was observed. Recently, several novel proof-of-concept antibody-mediated preconditioning methods have been developed that can selectively target hematopoietic stem and immune cells with minimal overall toxicity. Antibody-drug-conjugate (ADC) combined with reduced intensity conditioning or high dose ADC as single dose monotherapy have shown promise for allo-HSCT in preclinical models. The purpose of the current review is to discuss the literature exploring antibody-based conditioning that includes native antibody, radiolabeled antibody conjugates, and ADC for allo-HSCT.

Using mTOR Inhibitor Nanoimmunotherapy to Induce Cardiac Allograft Tolerance in Non-Human Primates

<h3>Purpose</h3> Though tolerance to kidney allografts has been consistently achieved using a mixed chimerism model, cardiac allografts remain resistant to tolerance induction. Lipoprotein-based nanobiologics loaded with a mammalian target of rapamycin inhibiting prodrug (mTOR inhibiting nanobiologics, mTORi-NBs) inhibit trained immunity and promote graft infiltrating M2-macrophages that provide a favorable local immunologic milieu for tolerance induction. In this study, we evaluate the ability of mTORi-NBs to induce tolerance in a cardiac allograft model in NHPs. <h3>Methods</h3> Six NHPs underwent simultaneous heterotopic heart and bone marrow transplantation for mixed chimerism induction. Group A (n=3) was conditioned with 3 Gy total body irradiation (TBI), 7 Gy thymic irradiation (TI), ATGAM, anti-CD40L monoclonal antibody on days 0, 2, 5,7, 9, and 12 post-bone marrow transplant (pBMTx), and cyclosporine until day 28 pBMTx. Group B recipients (n=3) underwent the same conditioning regimen with the addition of mTORi-NBs at a dose of 0.15 mg/kg on days 2, 5, 12, 19, and 26 pBMTx. Group B donors were also treated with a single dose of mTORi-NBs two days before transplant. <h3>Results</h3> In group A, all allografts had end stage rejection by day 175 pBMTx with a mean duration of lymphocyte chimerism of 74 days. The first recipient in group B developed durable full chimerism and mild graft versus host disease (GVHD) which resolved after a course of steroids and cyclosporine. After resolution of his GVHD, his graft continued beating strongly with no evidence of rejection through day 239 pBMTx. The second and third recipients received half dose (1.5 Gy) TBI and both allografts are contracting strongly at days 271 and 26 pBMTx, though the second recipient had some evidence of rejection at day 212 pBMTx. Currently, the mean duration of lymphocyte chimerism in Group B is 149 days. <h3>Conclusion</h3> Preliminary studies suggest that adding mTORi-NB nanoimmunotherapy to a mixed chimerism protocol prolongs both donor lymphocyte chimerism and heart graft survival in NHPs even when reducing pre-operative TBI.

L’axe CD40-CD40L : implications actuelles et futures en immunologie clinique

The CD40-CD40 ligand (CD40L) pathway is a backbone of communication between cells of the immune system. It makes it possible to generate a proinflammatory signal and thus participates in the pathogenesis of dysimmune diseases, transplant rejection and atherosclerosis. Because of this therapeutic target of choice, several generations of anti-CD40L monoclonal antibodies have emerged since the 1990s. The first generation of antibodies was responsible for thromboembolic toxicity for which the mechanisms are starting to be defined. New generations of antibodies were designed to overcome this toxicity and are still being developed in lupus, rheumatoid arthritis, Sjogren's syndrome or immunologic thrombocytopenia. In addition to these targeted therapies, there are data suggesting the impact of several drugs among molecules used in cardiology and clinical immunology on the level of CD40L. The objective of this review is to recall the clinical issues related to the CD40-CD40L axis and to present current or future treatments that block CD40L which would allow clinicians to diversify their options for managing dysimmune diseases.

Inhibition of the CD40/CD40L complex protects mice against ALI-induced pancreas degradation.

Acute lung injury (ALI) is a severe complication of transfusion. In a previous study, we saw that inhibition of the CD40/CD40L complex allowed restoration of ALI lesions in an experimental mouse model. This study focused on pancreas-associated injury development during experimental ALI pathogenesis and its limitation through CD40/CD40L complex inhibition. An ALI mouse model was established through intraperitoneal lipopolysaccharide and intravenous anti-major histocompatibility complex class I monoclonal antibody injection. Preemption of lesions was achieved with intravenous injection of neutralizing anti-CD40L monoclonal antibody 30 minutes before the trigger, that is, anti-major histocompatibility complex class I monoclonal antibody administration. Histology and immunoassay analyses were used to evaluate pancreatic lesions. ALI development induced significant degradation of the lungs and pancreas and was associated with pancreatic lesions. Different scores were established showing more severe injury to the pancreas in ALI conditions; however, injury was significantly reduced through CD40/CD40L complex inhibition. This study supports the idea that several organs are exposed during ALI development, and particularly when such experimental ALI aims at mimicking transfusion-associated ALI; nevertheless, preventive treatment inhibiting CD40/CD40L (sCD40L) complex formation provides protection from lung disease as well as disease of other organs.

Anti-CD40 Antibodies do not Cause Thromboembolism as Demonstrated by in Vitro and in Vivo Studies

Clinical trials with non-silenced anti-CD40L (anti-CD154) monoclonal antibodies (mAb) in patients with systemic lupus erythematosus (SLE) and idiopathic thrombocytopenic purpura (ITP) were halted after unexpected fatal thromboembolic events (TE). Previous results have shown that soluble CD40L/anti-CD40L mAb immune complexes (IC) can activate and induce pro-aggregatory effects on platelets in vitro via FcgRIIa. Pulmonary thrombi consisting of platelet aggregates and fibrin, and thrombocytopenia were found in humanized FcgRIIa transgenic mice after injection of pre-formed IC consisting of mouse soluble CD40L and anti-mouse CD40L mAb. To date, no such studies have been performed with IC of mouse sCD40 and anti-mouse CD40 mAbs. We therefore injected either single mouse proteins (soluble CD40L, soluble CD40) or antibodies (isotype controls, anti-mouse CD40L mAb, anti-mouse CD40 mAb), IC of soluble protein with the respective antibody into humanized FcgR transgenic mice. In addition, in vitro platelet aggregation was performed using blood from FcgR transgenic mice and human healthy donors. Whole blood samples from FcgR transgenic mice were treated with the same proteins and antibodies as in the in vivo study. For human whole blood, isotype controls, soluble CD40L, anti-CD40L mAb including IC in different ratios, soluble CD40, aCD40 mAbs (e.g. CFZ533) including IC in different ratios were used. We observed evidence of thromboembolism (thrombi formation in the lung shown by histopathology) and thrombocytopenia with the soluble CD40L/anti-CD40L mAb IC but not with the soluble proteins or antibodies alone, nor with the soluble CD40/anti-CD40 mAb IC. In vitro platelet aggregation assays performed using blood from FcgR transgenic mice and human healthy donors with recombinant proteins, mAbs and IC from respective species confirmed these findings. These data provide in vivo and in vitro evidence that all anti-CD40 mAbs, either alone or IC with soluble CD40 protein do not induce thromboembolism, indicating that the TE associated with anti-CD40L mAbs are target-related but not co-stimulation pathway specific. Combined with clinical evidence, these data further support the notion of minimal risk for patients developing life-threatening thromboembolic events following administration of a pathway blocking anti-CD40 mAb.

Costimulation blockade by combining CTLA4Ig with anti-CD40L mAb markedly inhibits the inflammatory response of experimental autoimmune myocarditis

The aim of this study was to investigate the effect of costimulation blockade with cytotoxic T-lymphocyte-associated-antigen 4-immunoglobulin (CTLA4Ig) and anti-CD40L monoclonal antibody (anti-CD40L mAb) on an experimental autoimmune myocarditis (EAM) mouse model. Characteristics of myocardial tissue were observed by hematoxylin and eosin (H&amp;E) staining. The messenger RNA (mRNA) levels of CTLA4, CD40L, IFN-γ, and IL-4 were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Serum concentrations of IFN-γ and IL-4 were determined by ELISA. After immune intervention, the inflammatory score, mRNA levels of CTLA4 and CD40L, and IFN-γ level were decreased. Furthermore, these parameters in the combinational intervention group (blockade by CTLA4Ig and anti-CD40L mAb) were significantly decreased, compared to the single intervention group (blockade by CTLA4Ig or anti-CD40L mAb). However, after costimulation, blockade serum IL-4 levels were increased. Therefore, costimulation blockade by combination CTLA4Ig and anti-CD40L mAb could more effectively inhibit the inflammatory response of EAM than single use of CTLA4Ig or anti-CD40L mAb.

Effect of CD40/CD40L signaling on IL-10-producing regulatory B cells in Chinese children with Henoch-Schönlein purpura nephritis.

The aim of the present study was to examine the role and mechanism of interleukin-10 (IL-10)-producing regulatory B cells (B10 cells) in the pathogenesis of Henoch-Schönlein purpura nephritis (HSPN). We examined the percentage of B10 cells, CD19+CD24hiCD38hi B cells, CD19+CD24hiCD27+ B cells, Th17 cells, and T regulatory (Treg) cells within the peripheral blood mononuclear cell (PBMC) population in healthy subjects and HSP/HSPN patients. The percentage of B10 cells and CD19+CD24hiCD38hi B cells was reduced in HSPN patients and that of CD19+CD24hiCD27+ B cells was decreased only in HSPN patients with hematuria and proteinuria or massive proteinuria. The expression of IL-10 by B10 cells and their subsets was decreased in HSPN patients and returned to normal levels in HSP/HSPN patients in remission. B10 cells and their subsets negatively correlated with the Th17/Treg ratio. There was no difference in B10pro + B10 cells, Th17 cells, Treg cells, and the Th17/Treg ratio between children with HSP/HSPN and healthy controls after CD40L stimulation. On the other hand, the level of IL-10 expressed by CD19+CD40+ B cells was decreased in HSPN, and the percentage of B10pro + B10 cells and Treg cells was reduced and that of Th17 cell was increased in the presence of anti-CD40L monoclonal antibody (mAb). Thus, decreased B10 cells and CD19+CD24hiCD38hi B cells may function as an early marker of renal impairment in HSPN. The dysfunction of B10 cells may play a role in the pathogenesis of HSPN by regulating the Th17/Treg balance. Moreover, the CD40/CD40L signaling pathway may play a role in B10 cell differentiation and functional maturation.

Interleukin-10 From Marginal Zone Precursor B-Cell Subset Is Required for Costimulatory Blockade-Induced Transplantation Tolerance.

Blocking CD40-CD40L costimulatory signals induces transplantation tolerance. Although B-cell depletion prevents alloantibody formation, nonhumoral functions of B cells in tolerance have not been well characterized. We investigated whether specific subsets of B cell or B cell-derived interleukin (IL)-10 contribute to tolerance. Wild type C57BL/6, or B cell-specific interleukin (IL)-10 (CD19-Cre::IL-10) mice, received vascularized BALB/c cardiac allografts. BALB/c donor-specific splenocyte transfusion and anti-CD40L monoclonal antibody were used as tolerogen. B cells were depleted with antimouse CD20 monoclonal antibody. Various B-cell subsets were purified and characterized by flow cytometry, reverse transcription polymerase chain reaction, and adoptive transfer. B-cell depletion prevented costimulatory blockade-induced allogeneic tolerance. Costimulatory blockade increased IL-10 in marginal zone precursor (MZP) B cells, but not other subsets. In particular, costimulatory blockade did not change other previously defined regulatory B-cell subsets (Breg), including CD5CD1d Breg or expression of TIM1 or TIM4 on these Breg or other Breg cell subsets. Costimulatory blockade also induced IL-21R expression in MZP B cells, and IL-21R MZP B cells expressed even more IL-10. B-cell depletion or IL-10 deficiency in B cells prevented tolerance in a cardiac allograft model, resulting in rapid acute cardiac allograft rejection. Adoptive transfer of wild type MZP B cells but not other subsets to B cell-specific IL-10 deficient mice prevented graft rejection. CD40 costimulatory blockade induces MZP B cell IL-10 which is necessary for tolerance. These observations have implications for understanding tolerance induction and how B cell depletion may prevent tolerance.

Murine Fibroblastic Reticular Cells From Lymph Node Interact With CD4+ T Cells Through CD40-CD40L.

Costimulatory blockade with anti-CD40L monoclonal antibody (mAb) plus donor-specific splenocyte transfusion (DST) induces alloantigen-specific tolerance. We previously showed that lymphotoxin signaling in the fibroblastic reticular cell (FRC) stromal subset was required for proper lymph node structure and function during tolerization in murine cardiac transplantation. Here we focused on FRC functions and hypothesized that DST and anti-CD40L mAb-modulated FRC interactions with CD4(+) T cells in mice. Mice were immunized or tolerized by DST or DST plus anti-CD40L mAb. Fibroblastic reticular cells were flow-sorted at different timepoints for characterization and in vitro proliferation and activation assays. Fibroblastic reticular cells responded rapidly to DST by transcribing inflammatory cytokine and chemokine messenger RNAs, such as CXCL2, CXCL9, CXCL10, and CCL21. Conversely, anti-CD40L mAb inhibited FRC inflammatory responses. CD40 was expressed on FRC and agonistic anti-CD40 mAb activated FRC, which supported CD4(+) T-cell proliferation, whereas unstimulated FRC did not. Anti-CD3 mAb-activated CD4(+) T cells induced inflammatory cytokine and chemokine expressions by FRC, which were inhibited by anti-CD40L mAb. Thus, FRC phenotype was altered by interaction with CD4(+) T cells through CD40-CD40L, and activated FRC interacted directly with CD4(+) T cells to support T cell activation and proliferation in vitro. Taken together, these results demonstrated that CD40 on FRC facilitated bidirectional communication between FRC and CD4(+) T cells via CD40-CD40L, thereby altering FRC gene expression of immune regulatory molecules. Because blockade of CD40-CD40L interactions results in tolerance in mice, identification of FRC-T cell interactions provides a new research target for tolerance induction.

Lymph Node Stromal Fiber ER-TR7 Modulates CD4+ T Cell Lymph Node Trafficking and Transplant Tolerance.

Trafficking and differentiation of naive CD4+ and regulatory T cells (Treg) within the lymph node (LN) are integral for tolerance induction. The LN is comprised of stromal fibers that dictate lymphocyte migration and LN structure, organization, and microanatomic domains. Distribution of the stromal fiber ER-TR7 changes within the LN after antigenic challenge, but the contributions of ER-TR7 to the resulting immune response remain undefined. We hypothesized that these stromal fiber structural changes affect T cell fate and subsequently allograft survival. C57BL/6 mice were left naive (untreated) or made immune or tolerant (donor-specific BALB/c splenocyte transfusion -/+ anti-CD40L monoclonal antibody), or made tolerant and received anti-ER-TR7 monoclonal antibody. Donor-specific T-cell migration was visualized by adoptive transfer of carboxyfluorescein diacetate, succinimidyl ester-labeled TEa T cell receptor transgenic CD4+ cells. Immunohistochemistry was performed on LNs to detect stromal fiber distribution, structure, CCL21 presence, and Treg and donor-specific cell location relative to high endothelial venules (HEV). Naive, tolerant, and tolerant + anti-ER-TR7 mice received BALB/c heterotopic cardiac allografts and graft survival was monitored. The ER-TR7 distribution changed after the induction of tolerance vs. immunity. Treating tolerant mice with anti-ER-TR7 altered HEV basement membrane structure and the distribution of CCL21 within the LN. These differences were mirrored by changes in the migration of naive and Treg cells within and surrounding the HEV. Anti-ER-TR7 prevented tolerance induction and resulted in allograft inflammation and rejection. These results identify ER-TR7 as an important component of LN structure in tolerance and a direct target for immune modulation.

Overexpression of Jagged-1 combined with blockade of CD40 pathway prolongs allograft survival.

Dendritic cells (DCs) have the tolerogenic potential to regulate adaptive immunity and induce allografts acceptance. Here we investigated whether blockade of the CD40 pathway could enhance the immune tolerance induced by DC2.4 cells modified to express Jagged-1 (JAG1-DC) in heart transplantation. Results showed that JAG1-DC treatment combined with anti-CD40L monoclonal antibody (mAb) administration significantly prolonged cardiac allograft survival in mice, with long-term survival (>110 days) of 50% of the allografts in the recipients. The therapy specifically inhibited the immune response, induced alloantigen-specific T-cell hyporesponsiveness, upregulated transforming growth factor-β synthesis and increased the population of regulatory T cells (Tregs) driven by Jagged-1-Notch activation. These results highlight the potential application of gene therapy to induce alloantigen-specific Tregs effectively by providing the Jagged-1 stimulation.

Identification of Novel Mechanisms of Tolerance Resistance Induced By OX40.

Induction of donor specific tolerance remains an important goal in transplantation and to identify and overcome barriers to tolerance will significantly improve transplant outcomes in the clinic. In the present study, we examined the effector programs that evade tolerance induction by costimulatory blockade in a mouse heart transplant model. Blocking CD40-CD40L costimulation using an anti-CD40L monoclonal antibody (MR1) induced prolonged survival of C57BL/6 heart allografts in BALB/c recipients (MST>60 days). However, we found that MR1 completely failed to prolong survival of heart allografts that express the OX40L transgene (OX40Ltg grafts) (MST=9 days), indicating that OX40 signaling evades tolerance induction by costimulatory blockade. Histologically, rejection of OX40Ltg heart allografts was associated with extensive lymphocyte infiltration, and a significant proportion of graft infiltrating T cells produced IL-9 (40%), while few of them produced IL-4 or IFN-γ, suggesting that OX40 may drive a new mechanism of tolerance resistance. In an in vitro T cell polarization assay, we showed that OX40 engagement inhibited the induction of TH17 cells by TGF-β/IL-6 and that of inducible Treg cells by TGF- β/IL-2. Instead, OX40 diverted CD4+ T cells to IL-9 producing TH9 cells. Mechanistically, OX40 signaling mediated the induction of TH9 cells by triggering noncanonical NF-κB activation in activated T cells. In an in vivo CFSE model in which CD4+ T cells were stimulated by alloantigens, blocking CD40-CD40L costimulation induced TH9 cells only in the presence of OX40 signaling. These data suggest that induction of TH9 cells may constitute a novel mechanism of tolerance resistance. Interestingly, blocking OX40 costimulation induced long-term heart allograft survival (MST>100 days) and markedly reduced transplant vasculopathy (chronic allograft rejection) in CD40L-/- recipient mice. In conclusion, we identified that OX40 dramatically induces the generation of TH9 cells both in vitro and in vivo, and TH9 cells are closely involved in tolerance resistance. These findings may lead to the development of new therapeutics to promote graft tolerance in transplantation.

In Vivo and In Vitro Characterization of Domain Antibodies (dAbs) Targeting CD40 Ligand (CD40L)

CD40:CD40L interactions play a critical role in regulating immune responses and offer a potential for targeted prevention of transplant rejection. Herein we describe the identification and biological properties of potent domain antibodies (dAbs) targeting human and murine CD40L molecules. Initial screens identified monomeric dAbs against murine and human CD40L, which were then formatted as bivalent Fc proteins. In vitro primary immune cell-based assays identified potent leads which were further optimized to generate molecules that exhibited favorable safety profile and biophysical properties coupled with potent biological activity. The murine CD40L dAb exhibited notable efficacy in various pre-clinical models including anti-KLH Ab responses, TNBS-colitis, a heart-to-ear transplant model and in the NZB/W lupus model. The human anti-CD40L dAb, BMS-X, was extensively characterized in various primary cell assays including B cell activation, dendritic cell activation and cytokine production, and in a T cell-B cell mixed lymphocyte reaction (MLR) assay. Furthermore, extensive investigative studies were conducted with BMS-X to address the potential for thromboembolism (TE), observed with other anti-CD40L monoclonal antibodies (such as BG9588 [hu5c8, Biogen] and IDEC-131). In vitro assays demonstrated that complexes of soluble CD40L and anti-CD40L mAb induced robust activation of platelets. Platelet activation was not evident when F(ab)2 fragment were used or when the assay was performed in the presence of an anti-FcgRIIa mAb. These results supported the conclusion that Fc-FcgR interactions contributed towards activation of platelets. Next, we formatted an anti-CD40L mAb with different Fcs to identify one that was inert in terms of platelet activation. These studies identified a modified IgG1 that demonstrated no activity in the in vitro platelet assays. Subsequently, BMS-X was formatted using this inert Fc molecule and tested for its potential to activate human platelets. No platelet activation was evident with BMS-X across a wide diversity of donors, which indicated that BMS-X has a reduced risk for causing TE, allowing it to advance to clinical studies. This risk will be monitored in future toxicology and human studies.

Flt3L-Induced Dendritic Cells Suppress Donor-Specific Immune Reaction but not Prolong the Graft Survival in Pancreatic Islet Transplantation

Introduction: It has recently been reported that Flt3L-induced bone marrow-derived Dendritic Cells (FLDCs) induce donor-specific tolerance and prolong the acceptance of donor skin grafts. In the present study, we examined whether this attractive regimen is applicable to the pancreatic islet transplantation. Methods: BALB/c male mice were used as donors. C57BL/6J male mice were used as recipients. Recipients were divided as follows: with FLDCs (FLDCs group) and without FLDCs (control group). In the FLDCs group, bone marrow cells were harvested, then cultured with 100 ng/mL Flt3L. At day 8, loosely adherent cells were harvested and used as immature FLDCs. FLDCs were injected into a recipient's tail vein twice. To block the costimulatory signals through CD28/B7 and CD40/CD40L, hCTLA4-Ig and anti-CD40L monoclonal antibody (mAb) was injected intraperitoneally three times after FLDCs infusion. Natural killer (NK) depletion was carried out by injecting mouse anti-NK1.1 mAb (PK136) twice before FLDCs were transferred. The exactly same amount of each mAb was administered to the recipients in the control group. Streptozotocin (170 mg/kg) was injected intravenously through a tail vein 3 days before FLDCs infusion. On day 8 after FLDCs infusion, islets were isolated by collagenase digestion. Sufficient amount (1,400 IEQs) or marginal amount (600 IEQs) of the islet grafts were transplanted under the subcapsular space of the left kidney. The blood glucose levels of all transplanted mice were measured twice a week, and the curative rate was evaluated by the blood glucose level at 28 days after islet transplantation. In order to assess whether the infusion of FLDCs could induce donor-specific tolerance, the mixed-lymphocyte reaction (MLR) assays were performed during 14 to 28 days after islet transplantation. In the MLR assay, C3H male mice were used as a third party. Results: The characterization of FLDCs by FACS analysis was as follows; CD11c+B220-: 35.78%±1.29, CD11c+B220+: 38.75%±1.73. In the MLR assay, the stimulation index of the recipient lymphocytes against BALB/c strain was significantly lower in the FLDCs group compared with that of the control group (1,400 IEQs: 1.72±0.10 vs. 2.72±0.19 p=0.0001 (n=23), 600 IEQs: 2.05±0.31 vs. 2.52±0.27 p=0.264 (n=16)). Of particular note, this tendency was more pronounced in the normoglycemic mice (1.52±0.12 vs. 2.79±0.19 p=0.0004 (n=19)). Nevertheless, the curative rate of the transplanted mice was considerably lower in the FLDCs group compared with that of the control group irrespective of the amount of transplanted islet grafts (1,400 IEQs: 25% vs. 50% p=0.26 (n=24), 600 IEQs: 12.5% vs. 62.5% p=0.05 (n=16)). Conclusion: These data show that Flt3L-induced dendritic cells were effectively able to suppress donor-specific immune reaction especially when normoglycemia had been achieved, but not enough to prolong the graft survival in pancreatic islet transplantation.

NK Cells are Required for Costimulatory Blockade Induced Tolerance to Vascularized Allografts

The role of natural killer (NK) cells in organ transplantation is poorly understood because studies link these cells to both regulatory and inflammatory functions. NK cells exacerbate inflammation and adaptive immunity under conditions of allograft rejection, but little is known regarding their roles in allograft tolerance. We test the hypothesis that NK cells have regulatory function and promote tolerance induction to murine cardiac allografts. Murine hearts were transplanted as fully vascularized heterotopic grafts from BALB/c donors into C57BL/6 recipients. Allograft tolerance was achieved using donor splenocyte transfusion + anti-CD40L monoclonal antibody (mAb) before transplantation. The requirement for NK cells in tolerance induction was tested by administering anti-NK1.1-depleting mAb or anti-NKG2D-blocking mAb. Intragraft and peripheral immune cell populations were determined by flow cytometry and immunohistochemistry. CD4 T-cell alloantigen-specific responses and donor-specific alloantibody were also determined. NK cell-depleted recipients acutely reject allografts despite anti-CD40L blockade, but rejecting recipients lacked alloantibody and alloantigen-specific CD4 T-cell responses. NK cell depletion resulted in elevated numbers of graft-infiltrating macrophages. NKG2D blockade in tolerized recipients did not cause acute rejection but increased macrophage graft infiltration and increased the expression of NKG2D ligand Rae-1γ on these cells. Our data show that NK cells are required for tolerance induction in recipients given donor splenocyte transfusion + anti-CD40L mAb. Our data suggest NK cells regulate monocyte or macrophage activation and infiltration into allografts by a mechanism partially dependent on NKG2D receptor-ligand interactions between NK cells and monocytes/macrophages.

English

Replication-defective adenovirus vector without both E1 and E3 is one of the most popular tools in transgenic therapies. However, more attention should be paid to adenovirus vectors mediated-gene modified study on endothelial cells (ECs). To verify the possible danger in that process, we explored the effect of adenovirus on ECs in this study. By using western blot analysis, we showed that the level of both CD40 and CD40L on human umbilical vein endothelial cells (HUVECs) were upregraduated by adenovirus vector infection at 100 multiplicity of infection (MOI). The activation of ECs induced by adenovirus vector infection at MOI 100 can be partly inhibited by a blockade of CD40/CD40L interactions by using the recombinant adenovirus Ad-sCD40LIg or an anti-CD40L monoclonal antibody (mAb) in vitro. On ECs, blockade of CD40/CD40L decreased the expression of IL (interleukin)-6, IL-8 and intercellular adhesion molecule (ICAM) in adenovirus vector-induced cells. In electrophoretic mobility shift assay (EMSA), both Ad-sCD40LIg and anti-CD40L mAb can attenuate the activity of NF-kappaB (NF-κB) pathway contributing to the activation of ECs, which indicated that CD40-CD40L interactions played significant role in the activation of ECs induced by adenovirus vectors via an NF-κB pathway. Our study provide evidences for a supplementary mechanism of the ECs activation induced by adenovirus vector infection and suggests that CD40-CD40L interactions partly participate in the ECs activation induced by adenovirus vectors in an NF-κB-dependent manner. Key words : Adenovirus vector, CD40, CD40L, endothelial cells, NF-kappaB.

Transplant long-surviving induced by CD40–CD40 ligand costimulation blockade is dependent on IFN-γ through its effect on CD4+CD25+ regulatory T cells

BackgroundIFN-γ was documented to be commonly associated with acute rejection. In the present study, we investigated the role of IFN-γ in the transplant long-surviving induced by blocking CD40–CD40 ligand (CD40–CD40L) costimulation and its mechanisms. MethodsIFN-γ expression in cardiac allografts and spleens from syngeneic and allogeneic recipients with or without anti-CD40L monoclonal antibody (MR-1) treatment was examined by real-time RT-PCR. The grafts survival time in Wild type (IFN-γ+/+) and IFN-γ deficient (IFN-γ−/−) recipients was investigated. Mixed lymphocyte reaction (MLR) of CD4+ T cells and cytotoxic T lymphocyte (CTL) assay of CD8+ T cells were also studied. FoxP3 expression in allografts and spleens from IFN-γ+/+ or IFN-γ−/− recipients with MR-1 treatment was examined. Furthermore, FoxP3, IL-10 and CTLA-4 expressions and the suppressive capability of CD4+CD25+ regulatory T cells were examined. ResultsRejected allografts showed significantly higher IFN-γ expression than long-surviving allografts. Allograft survival was not prolonged in nonimmunosuppressed IFN-γ−/− mice. Administration of MR-1 induced long-term survival in 90.1% of IFN-γ+/+ recipients (98±6.6days) but failed to do so in IFN-γ−/− group (16.2±4.0days). IFN-γ−/− recipients facilitated the proliferation and CTL generation of T cells. The allografts and spleens from IFN-γ+/+ recipients contained higher FoxP3 expression than IFN-γ−/− recipients. Moreover, CD4+CD25+ T cells from IFN-γ+/+ recipients displayed a higher FoxP3 and IL-10 expression and suppressive capability. ConclusionIFN-γ plays an important role in the long-surviving induced by blocking CD40–CD40L through inhibiting the function of activated T cells and increasing suppressive capability of CD4+CD25+ regulatory T cells.