CD40-CD154 Costimulatory Pathway Research Articles

412.10: Blocking of the CD40-CD154 Pathway Inhibits Macrophage Infiltration and Reduces Fibrosis After Renal Transplantation

Introduction: Renal transplantation improves the life expectancy and quality of life of end-stage renal patients. Due to the development of immunosuppressive drugs, the results of short-term renal transplantation have improved significantly, but the long-term survival rate of renal transplantation has hardly changed in the past few decades. Interstitial fibrosis is an important cause of graft loss in chronic allograft renal injury. CD40-CD154 costimulatory pathway block can inhibit T cell activation and prolong the survival of allografts in a variety of transplantation models. In this study, we found that the single use of MR1 (CD154 antibody) can significantly reduce the fibrosis of transplanted kidney. Method: The kidney of BALB/c mice was transplanted into C57BL6/J recipient by orthotopic transplantation. The contralateral kidney of the recipient was removed 1-3 days after transplantation. The MR1 treatment group was intraperitoneally injected with 500ug of MR1 antibody on the day of operation, and the control group was given normal saline at the same time. After 30 days, serum was collected to measure the blood creatinine. The kidney was obtained after cardiac perfusion. PAS staining was used to detect renal injury. The number of T lymphocytes and macrophages and the expression of a-SMA in the transplanted kidney were analyzed by flow cytometry. Sirius red and gomori trichrome staining were used to detect fibrosis. The colocalization of macrophages and a-SMA was detected by multiple immunofluorescence. Results: It was found that serum creatinine from MR1 treatment group was significantly lower than control group. Compared with control group, MR1 treated mice also showed milder renal histological lesions that the loss of brush border of proximal tubules, tubular necrosis, tubular atrophy, protein casting and cell infiltration at the cortical medullary junction were much lower. Sirius red and gomori trichrome staining showed that MR1 treatment group significantly reduced the fibrosis of transplanted kidney. RT-PCR showed that the expressions of collagen deposition related proteins such as ColI and FN mRNA were up-regulated in the control group. Further immunohistochemistry analysis showed that the number of infiltrated macrophages in the MR1 treatment group decreased significantly compared to control group. Through flow cytometry analysis, it was found that the number of macrophages secreting a-SMA in control group were significantly higher than those in MR1 treatment group. The FACS results showed that 92.7%± 2.8% of the infiltrating macrophages were M2 type, and they were all derived from the recipients. Immunofluorescence experiment also confirmed this result. Conclusion: MR1 significantly reduced the fibrosis in kidney graft which may due to less infiltration of M2 macrophages and less a-SMA+ macrophages. National key research and development program 2019YFA0110703. Natural Science Foundation of Hunan Province, China (Grant No.:2021JJ31018).

Functional role of CD40 and CD154 costimulatory signals in IgZ-mediated immunity against bacterial infection

CD40 and CD154 are one of the best-characterized costimulatory molecules essential for adaptive immunity, which extensively involved in T and B cell activation, IgM Ab production, isotype class switching, germinal center formation and affinity maturation. However, the functionality of CD40 and CD154 in IgZ-mediated immunity remains limited. In this study, we explored the regulatory role of Cd40-Cd154 interaction in IgZ-mediated antibacterial immunity in zebrafish. The results showed that the IgZ-mediated antibacterial response can be significantly induced in response to A. hydrophila infection. The percentage of Cd40+IgZ+ B cells and the production of IgZ Ab were substantially increased upon A. hydrophila stimulation, but these reactions were markedly declined in Cd154 blockade fish by administering anti-Cd154 Ab or recombinant sCd40-Ig protein, accompanied with the impairment of the vaccine-initiated IgZ-mediated immunoprotection of fish against A. hydrophila infection. These observations suggested the essential role of Cd40-Cd154 interaction in IgZ-mediated bacterial immunity. Notably, the Cd40 and Cd154 costimulatory signals are required for a TD antigen-induced IgZ immunity, but are not indispensable for a TI antigen-induced IgZ immune response. These findings indicated the differential role of Cd40-Cd154 interaction in bacterial TD and TI antigen-induced IgZ immunity, which suggested the existence of diverse regulatory mechanisms underlying IgZ-mediated antibacterial immune reactions. To our knowledge, this is the first report to show the functional role of Cd40-Cd154 costimulatory signaling pathway in IgZ-mediated immune defense against bacterial infection. We hope this study will improve the current understanding of the coevolution between the IgZ/IgT immunoglobins and CD40/CD154 costimulatory molecules.

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.

A Novel Anti-Cd40 Monoclonal Antibody, Iscalimab, for Control of Graves’ Hyperthyroidism – A Proof-Of-Concept Trial

Abstract Context The CD40-CD154 co-stimulatory pathway plays an important role in the pathogenesis of Graves’ disease (GD) by promoting auto-reactive B cell activation. Objective Evaluate efficacy and safety of a human, blocking, non-depleting anti-CD40 monoclonal antibody, iscalimab, in hyperthyroid patients with GD Design Open label, phase II proof-of-concept study Setting Multicenter Patients Fifteen with GD Intervention Patients received five doses of iscalimab at 10 mg/kg intravenously over 12 weeks. Main outcome measures Thyroid-related hormones and autoantibodies, plasma soluble CD40, free CD40 on B cells, soluble CXCL13, pharmacokinetics, and safety were assessed. Results The iscalimab intervention resulted in complete CD40 engagement for up to 20 weeks. A clinical response and biochemical euthyroidism was observed in seven of 15 (47%) patients. Free and total T3 and T4 normalized in seven patients who did not receive any rescue medication with anti-thyroid drugs (ATD), and 2/15 (13.3%) showed normal TSH. Six (40%) patients required ATD. Four of seven responders relapsed after treatment completion. Serum concentrations of thyrotropin receptor autoantibodies (TSH-R-Ab) significantly declined in all patients (mean 15.3 IU/L versus 4.0 IU/L, 66% reduction, P<0.001) and TSH-R-Ab levels normalized in four (27%). Thyroperoxidase and thyroglobulin autoantibodies significantly decreased in responders. Iscalimab rapidly reduced serum CXCL13 concentrations (P<0.001). Twelve (80.0%) patients reported at least one adverse event (AE). All treatment-related AE were mild or moderate and resolved by end of the study. Conclusion Iscalimab was generally safe and clinically effective in a subgroup of hyperthyroid GD patients. The potential therapeutic benefit of Iscalimab should be further tested.

A Novel Anti-Cd40 Monoclonal Antibody, Iscalimab, for Control of Graves’ Hyperthyroidism – A Proof-Of-Concept Trial

Abstract Context The CD40-CD154 co-stimulatory pathway plays an important role in the pathogenesis of Graves’ disease (GD) by promoting auto-reactive B cell activation. Objective Evaluate efficacy and safety of a human, blocking, non-depleting anti-CD40 monoclonal antibody, iscalimab, in hyperthyroid patients with GD Design Open label, phase II proof-of-concept study Setting Multicenter Patients Fifteen with GD Intervention Patients received five doses of iscalimab at 10 mg/kg intravenously over 12 weeks. Main outcome measures Thyroid-related hormones and autoantibodies, plasma soluble CD40, free CD40 on B cells, soluble CXCL13, pharmacokinetics, and safety were assessed. Results The iscalimab intervention resulted in complete CD40 engagement for up to 20 weeks. A clinical response and biochemical euthyroidism was observed in seven of 15 (47%) patients. Free and total T3 and T4 normalized in seven patients who did not receive any rescue medication with anti-thyroid drugs (ATD), and 2/15 (13.3%) showed normal TSH. Six (40%) patients required ATD. Four of seven responders relapsed after treatment completion. Serum concentrations of thyrotropin receptor autoantibodies (TSH-R-Ab) significantly declined in all patients (mean 15.3 IU/L versus 4.0 IU/L, 66% reduction, P<0.001) and TSH-R-Ab levels normalized in four (27%). Thyroperoxidase and thyroglobulin autoantibodies significantly decreased in responders. Iscalimab rapidly reduced serum CXCL13 concentrations (P<0.001). Twelve (80.0%) patients reported at least one adverse event (AE). All treatment-related AE were mild or moderate and resolved by end of the study. Conclusion Iscalimab was generally safe and clinically effective in a subgroup of hyperthyroid GD patients. The potential therapeutic benefit of Iscalimab should be further tested.

The effect of increasing Body Mass Index on sperm quality of subfertile men

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 3 mg/kg), or subcutaneous iscalimab (3 mg/kg), or placebo. Rheumatoid arthritis patients (n = 20) received single doses of intravenous iscalimab (10 or 30 mg/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 >0.3-0.4 µg/mL. In subjects receiving 3 mg/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.

OR19-6 A Novel Anti-CD40 Monoclonal Antibody, Iscalimab, Successfully Treats Graves’ Hyperthyroidism

Objective: Graves’ disease (GD) is an autoimmune disorder characterized by thyrotropin receptor autoantibody (TSHR-Ab)-mediated pathology, leading to hyperthyroidism. To date there is no pharmacotherapy that addresses the underlying pathogenesis. The CD40-CD154 (CD40L) co-stimulatory pathway plays an important role in the pathogenesis of GD by promoting auto-reactive B cell activation and intrathyroidal ectopic lymphoid structure formation and function. Iscalimab (CFZ533) is a fully human, blocking and non-depleting anti-CD40 monoclonal antibody. The purpose of this study was to investigate the efficacy and safety of Iscalimab in patients with Graves’ hyperthyroidism. Methods: In total, fifteen patients with Graves’ hyperthyroidism were enrolled in an open label Phase I proof-of concept study and all completed the trial. Each patient received five doses of Iscalimab at 10 mg/kg IV over 12 weeks. We assessed the effects of Iscalimab on thyroid-related hormones (baseline TSH, free and total T3/T4) and autoantibodies (TSHR-Ab) over 36 weeks following the first dose of Iscalimab. Safety, pharmacokinetics, soluble CD40 levels in plasma, free CD40 on whole blood CD19-positive B cells, and CXCL13 were also assessed. Results: Based on the CD40 occupancy and plasma levels of soluble CD40, the intervention resulted in full CD40 engagement for up to 20 weeks (12-week treatment + 8 week follow up). A clinical response and biochemical euthyroidism was observed in seven out of 15 (46.7%) by Day 211. Free T3/T4 normalized in seven (46.7%) patients who did not received any rescue medication with anti-thyroid drugs. Two further patients (13.3%) did not normalize but did not require rescue medication. In contrast, six (40%) patients required rescue medication with anti-thyroid drugs by Day 141. No patients achieved normalization of TSH by Day 85, however, two out of 15 patients (13.3%) showed normal TSH by Day 169. The levels of free thyroid hormones and TSHR-Ab levels declined over time up to Day 211. The mean levels of TSHR-Ab declined from 15.3 IU/L (baseline) to 9.4 IU/L at Day 85 (35% reduction, P<0.001), then further decreased to 4.0 IU/L at day 141 (66% reduction, P<0.001). The TSHR-Ab levels fell in normal range in four out of 15 patients (26.7%). Treatment with Iscalimab was associated with a rapid and significant reduction in the serum levels of CXCL13 (94.1 pg/mL at baseline to 68.5 pg/mL at Day 15) and remained at similar levels during the 12 week treatment period. Twelve patients reported at least one adverse event. Most events were mild in nature (34 in 8 patients, 53.3%) and few were moderate (5 in 4 patients, 26.7%). Conclusions: Iscalimab was generally safe and clinically effective in a subgroup of patients with Graves’ hyperthyroidism. These encouraging results suggest that Iscalimab may be an attractive strategy as the immunomodulation treatment for GD.

Characterization of the in vitro and in vivo properties of CFZ533, a blocking and non-depleting anti-CD40 monoclonal antibody.

The CD40-CD154 costimulatory pathway is essential for T cell-dependent immune responses, development of humoral memory, and antigen presenting cell function. These immune functions have been implicated in the pathology of multiple autoimmune diseases as well as allograft rejection. We have generated CFZ533, a fully human, pathway blocking anti-CD40 monoclonal antibody that has been modified with a N297A mutation to render it unable to mediate Fcγ-dependent effector functions. CFZ533 inhibited CD154-induced activation of human leukocytes in vitro, but failed to induce human leukocyte activation. Additionally, CFZ533 was unable to mediate depletion of human CD40 expressing B cells. In vivo, CFZ533 blocked primary and recall T cell-dependent antibody responses in nonhuman primates and abrogated germinal formation without depleting peripheral blood B cells. We also established a relationship between plasma concentrations of CFZ533 and CD40 pathway-relevant pharmacodynamic effects in tissue. Collectively these data support the scientific rationale and posology for clinical utility of this antibody in select autoimmune diseases and solid organ transplantation.

Novel insights into anti-CD40/CD154 immunotherapy in transplant tolerance.

Since the discovery of the CD40-CD154 costimulatory pathway and its critical role in the adaptive immune response, there has been considerable interest in therapeutically targeting this interaction with monoclonal antibodies in transplantation. Unfortunately, initial promise in animal models gave way to disappointment in clinical trials following a number of thromboembolic complications. However, recent mechanistic studies have identified the mechanism of these adverse events, as well as detailed a myriad of interactions between CD40 and CD154 on a wide variety of immune cell types and the critical role of this pathway in generating both humoral and cell-mediated alloreactive responses. This has led to resurgence in interest and the potential resurrection of anti-CD154 and anti-CD40 antibodies as clinically viable therapeutic options.

Blockade of CD40–CD154 Costimulatory Pathway Promotes Long-Term Survival of Full-Thickness Porcine Corneal Grafts in Nonhuman Primates: Clinically Applicable Xenocorneal Transplantation

The porcine cornea may be a good solution for the shortage of human donor corneas because its size and refractive properties are comparable to those of the human cornea. However, antigenic differences need to be overcome to apply xenocorneal transplantation in actual clinical practice. We aimed to investigate the feasibility of full-thickness porcine corneas as human corneal substitutes using a CD40-CD154 costimulatory pathway blocking strategy in a clinically applicable pig-to-nonhuman primate corneal transplantation model. As a result, the mean survival time of the xenocorneal grafts in recipients who received anti-CD154 antibody-based immunosuppressants (POD318 (n = 4); >933, >243, 318 and >192) was significantly longer than that in controls (POD28 (n = 3); 21, 28 and 29; p = 0.010, log-rank test). Administration of anti-CD154 antibodies markedly reduced inflammatory cellular infiltrations (predominantly CD8 T cells and macrophages) into the xenocorneal grafts and almost completely blocked xenoantigen-triggered increases in Th1-associated cytokines, chemokines and C3a in the aqueous humor. Moreover, systemic expansion of memory T cells was effectively controlled and responses of anti-Gal/donor pig-specific antibodies were considerably diminished by programmed injection of anti-CD154 antibodies. Consequently, porcine corneas might be promising human corneal substitutes when the transplantation is accompanied by potent immunosuppression such as a CD40-CD154 costimulatory pathway blockade.

Foxp3+ Alloantigen-Specific iTreg Highly Express CTLA4 and Preferentially Survive Following CTLA4-Ig Treatment.

The CD40-CD154 costimulatory pathway is one of the most attractive targets for therapeutic intervention in transplantation. However, thromboembolic complications during early clinical trials with anti-CD154 antibodies, due to Fc-mediated platelet activation, prevented their translation. Recent studies by our group have suggested that Fc-silent anti-CD154 domain antibodies may be a safer and equally efficacious means of prolonging allograft survival. Using a transgenic mouse model where OT-I CD8 and OT-II CD4 T cells were adoptively transferred to B6 mice that then received skin grafts that ubiquitously express the OVA protein, we showed that Fc-silent anti-CD154 antibodies prolong graft survival and lead to the conversion of naïve donor-specific CD4 T cells into Foxp3+ iTreg. Given the potential for clinical translation of Fc-silent anti-CD154 therapy and long-standing interest in combination therapy with CD28 costimulation blockade, we sought to determine the effect of CTLA4-Ig on CD154 antagonism-induced iTreg generation. This question was especially pertinent due to the known negative impact of CTLA4-Ig on the frequency of Treg. While the addition of CTLA4-Ig treatment to an Fc-silent anti-CD154 regimen led to a drastic decrease in the percentage of endogenous CD4+CD25+Foxp3+ Treg (7.2% vs. 11.1% with anti-CD154 alone), the percentage of alloantigen-specific iTreg actually increased following the addition of CTLA4-Ig (25.0% vs. 14.4% with anti-CD154 alone), which may be an important mechanism of synergy with these two therapies. To determine the requirements for CD28 signals for survival of these cells, we treated animals with anti-CD28 domain antibodies that specifically block CD28 signaling while leaving CTLA4 signaling intact. Results indicated that in contrast to nTreg, the survival of iTreg is independent of CD28 costimulation. Furthermore, in our model, iTreg express higher amounts of CTLA4 (MFI 5187) than their endogenous counterparts (MFI 1668). Previous studies have shown that proliferation leads to higher expression of CTLA4 on Treg, and we hypothesize that exposure to cognate alloantigen following transplantation may supplant the requirement of CD28 for the survival of these iTreg. Taken together, these results suggest that a novel anti-CD154 therapeutic may synergize with an already-approved agent, opening the door for translation of more effective immunosuppression regimens. DISCLOSURE:Nadler, S.: Employee, Bristol Myers Squibb.

DNA vaccine encoding CD40 targeted to dendritic cells in situ prevents the development of Heymann nephritis in rats

The CD40-CD154 costimulatory pathway has been shown to be critical for both T- and B-cell activation in autoimmune disease. Here, we assessed the effects of blocking this pathway using CD40 DNA vaccine enhanced by dendritic cell targeting on the development of active Heymann nephritis, a rat model of human membranous nephropathy. DNA vaccination delivers plasmid DNA encoding the target antigen, either alone or in combination with enhancing elements, to induce both humoral and cellular immune responses. To determine whether CD40 DNA vaccine targeting the encoded CD40 directly to dendritic cells would improve the efficacy of the vaccination against self-protein CD40, we utilized a plasmid encoding a single-chain Fv antibody specific for the dendritic cell-restricted antigen-uptake receptor DEC205 (scDEC), the target gene CD40, and the adjuvant tetanus sequence p30. This vaccine plasmid was compared to a control plasmid without scDEC. Rats vaccinated with scDEC-CD40 had significantly less proteinuria and renal injury than did rats receiving scControl-CD40 and were protected from developing Heymann nephritis. Thus, CD40 DNA vaccination targeted to dendritic cells limits the development of Heymann nephritis.

Activation of CD40-CD154 co-stimulatory pathway and its correlation with disease activity in patients with inflammatory bowel disease

Objective To investigate the expression of CD40-CD154 co-stimulatory pathway in peripheral circulation and intestinal mucosa in patients with inflammatory bowel disease （IBD）, the difference between the expression of CD40-CD154 in patients with IBD and that in healthy controls, and the correlation between CD40-CD154 levels and disease activity. Methods A total of 62 patients with Crohn＇s disease （CD）, 64 patients with ulcerative colitis （UC） and 56 healthy controls were enrolled. Enzyme-Linked Immuno Sorbent Assay （ELISA）, SYBR-green real time PCR and immunohistochemical assay were respectively applied to evaluate expression of CD40-CD154 in plasma, mononuclear cells of peripheral blood and intestinal mucosa. Results Levels of CD40 （ P = 0. 000） and CD154 （P = 0. 001 ） in plasma, mononuclear cells of peripheral blood and intestinal mucosa were significantly higher in patients with CD and UC than in healthy controls. However, no correlation between disease activity and CD40-CD154 expression in peripheral circula- tion or intestinal mucosa was detected （ P 〉 0. 05 ）. Conclusion CD40-CD154 pathway activation is found in plasma, peripheral blood mononuclear ceils and intestinal mucosa in patients with IBD, but is not correlated with disease activity. Key words: Colitis, ulcerative ; Complement pathway; Crohn＇s disease

Indoleamine 2,3-dioxygenase (IDO) modulates immunological tolerance to cardiac allografts

Objectives: Negative or inhibitory costimulatory pathways regulate T cell activation and play a role in peripheral tolerance. The concept that Indoleamine 2, 3-dioxygenase (IDO) expressing dendritic cells (DCs) are able to suppress T-cell responses and promote tolerance is a relatively new paradigm in immunology. This study aimed to investigate the interplay of IDO and CD28:B7 and/or CD40-CD154 co-stimulatory pathway blockade in a fully mismatched allograft model.

Neutralizing Interleukin-4 Prevents Transplant Arteriosclerosis Mediated by Indirect Pathway T Cells Under CD40-CD154 Costimulation Blockade

Blockade of the CD40-CD154 costimulatory pathway can prolong allograft survival, but does not prevent the development of transplant arteriosclerosis in several models. In this study, we investigated the mechanisms of CD40-CD154-independent transplant arteriosclerosis in major histocompatibility complex (MHC)-class I-mismatched aortic allografts. MHC class I-mismatched CBK (H2k+Kb) donor aortas were transplanted into CBA (H2k) recipients who can only recognize the graft through CD8+ T cells and CD4+ T cells responding to the class I MHC mismatch through the indirect pathway of allorecognition. Recipients were treated with anti-CD154 antibody (MR1) alone or in combination with anti-CD8 (YTS169) or anti-interleukin (IL)-4 (11B11) antibodies. Grafts were analyzed by histology on days 30 and 60 and for intragraft mRNA expression on day 14 after transplantation. Repeated treatment with anti-CD154 alone or in combination with anti-CD8 antibody did not prevent intimal proliferation compared with untreated controls (65%+/-6%, 62%+/-9%, and 71%+/-7% luminal occlusion, respectively, 60 days after transplantation). In both treatment groups, the expression of IL-4, IL-5, and eotaxin was increased compared with control grafts, and an eosinophilic infiltration was observed. Neutralizing IL-4 in combination with CD40-CD154 blockade and CD8+ T-cell depletion abrogated transplant arteriosclerosis (9%+/-4% luminal occlusion 60 days after transplantation). Prolonged treatment with anti-CD154 was not able to prevent the development of transplant arteriosclerosis in MHC class I-mismatched aortic allografts, in the presence or absence of CD8+ T cells. This CD40-CD154 pathway resistant transplant arteriosclerosis was mediated by IL-4, because neutralizing IL-4 in addition to CD40-CD154 costimulation blockade and CD8+ T-cell depletion prevented its development.

Inhibition of CD40–CD154 costimulatory pathway by a cyclic peptide targeting CD154

Disruption of the CD40-CD154 interaction was found to be effective in the prevention and treatment of several immune-mediated diseases. The antibody-based strategy of inhibition was in humans limited by platelet activation leading to thrombotic effects. Other strategies different from antibody technology may be useful to create tools to interfere with CD40-CD154 pathway. In the present study, we selected and characterized from a phage display library, cyclic hepta-peptides specific for human CD154 through biopanning against plate-immobilized recombinant hCD154-muCD8. Nine phage clones were selected for the ability to bind CD154 expressed on the surface of J558L cells transfected with human CD154. From the nine selected phage clones, we obtained seven different amino acidic sequences, and the corresponding hepta-peptides rendered cyclic by two cysteines were synthesized. All the peptides specifically bound CD154 expressed on J558L. However, only the peptide 4.10 (CLPTRHMAC) was found to recognize the active binding site of CD154, as it competed with the blocking anti-CD154 antibody. When changes in the amino acid composition were introduced in the sequence of 4.10 peptide, the binding to CD154 was abrogated, suggesting that the amino acid sequence was critical for its specificity. This peptide was found to inhibit the CD40-CD154 interaction, preventing CD40-dependent activation of B lymphocytes in vitro as it was able, as the blocking anti-human CD154 mAb, to prevent the expression of CD80 and CD86 costimulatory molecules and switching of Ig isotype induced by CD154. Moreover, the peptide 4.10 inhibited the in vitro endothelial cell motility and organization into capillary-like structures, and the in vivo angiogenesis of human umbilical cord-derived endothelial cells implanted in Matrigel in severe combined immunodeficiency mice. In vitro studies on platelet activation demonstrated that the 4.10 peptide, at variance of the anti-CD154 mAb, was unable to prime human platelet activation and aggregation. In conclusion, we identify a cyclic hepta-peptide able to displace the binding of human CD154 to CD40 expressed on cell surface and to abrogate some biological effects related to the CD40 stimulation, such as B cell activation and endothelial triggered angiogenesis.

The proportion of CD40+ mucosal macrophages is increased in inflammatory bowel disease whereas CD40 ligand (CD154)+ T cells are relatively decreased, suggesting differential modulation of these costimulatory molecules in human gut lamina propria

Signal transduction through binding of CD40 on antigen-presenting cells and CD40 ligand (CD154) on T cells appears to be crucial for mutual cellular activation. Antibodies aimed at blocking the CD40-CD154 costimulatory pathway dampen the severity of experimental colitis. To elucidate the microanatomical basis for signaling through this costimulatory pathway in human inflammatory bowel disease, we studied in situ the cellular distribution of these 2 molecules on lamina propria macrophages and T cells, respectively. Colonic specimens from 8 patients with ulcerative colitis and 8 with Crohn's disease, 8 small bowel specimens of Crohn's disease, and histologically normal control samples (6 from colon and 6 from small bowel) were included. Multicolor immunofluorescence in situ staining was performed to determine the percentage of subepithelial macrophages expressing CD40 and that of lamina propria T cells expressing CD154 while avoiding cells in lymphoid aggregates. The proportion of subepithelial CD40CD68 macrophages was significantly increased in normal colon compared with normal small bowel and showed further elevation in both colon and small bowel afflicted with inflammatory bowel disease. In addition, on a per-CD68-cell basis, CD40 expression was significantly increased in severely inflamed compared with moderately inflamed colonic specimens. Conversely, the proportion of CD154 T cells was similar in colon and small bowel, and interestingly, it was significantly reduced in colonic inflammatory bowel disease. Our findings suggested that modulation of CD40 expression by subepithelial macrophages and CD154 by lamina propria T cells is inversely modulated in the human gut.

Inhibition of Pemphigus Vulgaris by Targeting of the CD40–CD154 Co-Stimulatory Pathway: A Step Toward Antigen-Specific Therapy?

The signaling of CD40 by its ligand, CD154, between T and B cells is required for humoral immune responses. Thus, blockade of this ligand-receptor interaction can prevent the development of antibody-mediated autoimmune diseases through different mechanisms.

Dependency of direct pathway CD4+ T cells on CD40-CD154 costimulation is determined by nature and microenvironment of primary contact with alloantigen.

Blockade of the CD40-CD154 costimulatory pathway can inhibit CD4(+) T cell-mediated alloimmune responses. The aim of this study was to define the in vivo requirement for CD40-CD154 costimulation by CD4(+) T cells that respond to alloantigen following direct recognition. We used TCR-transgenic CD4(+) T cells that are reactive to the MHC class II alloantigen, H2A(s). An experimental in vivo model was established that allowed direct comparison of the fate of a trace population of H2A(s)-reactive CD4(+) T cells when challenged with different forms of H2A(s+) alloantigen under conditions of CD40-CD154 costimulation blockade. In this study, we demonstrate that an i.v. infusion of H2A(s+) leukocytes in combination with anti-CD154 therapy rapidly deletes H2A(s)-reactive CD4(+) T cells. In contrast, following transplantation of an H2A(s+) cardiac allograft, H2A(s)-reactive CD4(+) T cell responses were unaffected by blocking CD40-CD154 interactions. Consistent with these findings, combined treatment with donor leukocytes and anti-CD154 therapy was found to be more effective in prolonging the survival of cardiac allografts compared with CD154 mAb treatment alone. The dominant mechanism by which donor leukocyte infusion and anti-CD154 therapy facilitate allograft acceptance is deletion of donor-reactive direct pathway T cells. No evidence for the generation of regulatory cells by this combined therapy was found. Taken together, these results clearly demonstrate that naive alloreactive CD4(+) T cells have distinct requirements for CD40-CD154 costimulation depending on the form and microenvironment of primary alloantigen contact.

Bicistronic adenovirus-mediated gene transfer of CTLA4Ig gene and CD40Ig gene result in indefinite survival of islet xenograft

Blockade of CD40-CD154 costimulatory pathway in mice and primates with anti-CD154 monoclonal antibodies results in prolonged survival of vascularized organs and islet grafts. CD40Ig, a recombinant fusion protein comprised of the extracellular domain of human CD40 molecule in frame fused with the site-mutated human IgG1 Fc region, abrogated the cognate interaction of CD40-CD154 pathway by binding the CD154 molecule. In this study, replication-defective adenovirus containing the CD40Ig gene was prepared by homologous recombination and used to infect freshly isolated islets from LEW rats (RT-1 1) in vitro using a titered dose. The islet transfectants (500 per recipient) were transplanted under the left kidney capsule of streptozocin-rendered diabetic C57BL/6 mouse recipient (H-2 b). The mean survival time of AdCD40Ig-transfected islet grafts was significantly prolonged, while mock-infected grafts and AdEGFP-transfected grafts were rejected in normal fashion. Additionally, dose-dependent prolongation of islet graft survival was observed in mice receiving AdCD40Ig-transfected grafts. In conclusion, local production of Cd40Ig via adenoviral-mediated gene transfer induced dose-dependent prolongation of LEW → Balb-c islet xenografts.