anti-CD154 Monoclonal Antibody Treatment Research Articles

Thrombophilia Associated with Anti-CD154 Monoclonal Antibody Treatment and its Prophylaxis in Nonhuman Primates

We agree with the findings of Koyama et al. (1) published in a recent issue of Transplantation, and would like to share published insight pertaining to FcγRIIA (CD32) mediated platelet aggregation in relation to CD40L/CD154 mABs. The authors describe the efficaciousness of ketorolac in reducing thromboembolism in cynomolgus monkey renal allograft recipients treated with anti-CD154 monoclonal antibody (mAb). Blockade of the CD40L/CD154 pathway has been hailed as a promising strategy in prolonging allograft survival in murine allograft models (2). This leads to the initiation of trials including anti-CD40L/CD154 mAb in the immunosuppressive regimen in human allograft recipients. These trials were, however, precociously terminated because of an unusually high incidence of thromboembolic complications in these allograft recipients. This clinical observation ignited the study presented in Transplantation. From unpublished data in their discussion section, Koyama et al. (1) speculate that the underlying mechanism of platelet activation/aggregation might be Fc-receptor dependent. Two pathologic steps are required for immune mediated thrombocytopenia and platelet activation/aggregation: 1) presence of Ab to a self or neo-Ag; and 2) triggering of Ab effector mechanisms that leads to accelerated platelet clearance and/or platelet activation/aggregation, representing the low affinity receptor FcγRIIA (CD32) on platelets. FcγRIIA (CD32) is a single-chain sialoglycoprotein, which interacts with the Fc-domain of IgG antibodies. When cross-linked, FcγRIIA (CD32) fully activates platelets for secretion and activation (3). The large majority of antibodies have no effect on platelets. By contrast, only a subset of antibodies are able to directly promote platelet activation, although it has been reported that binding of these antibodies, via their antigen-binding domain alone, suffices to induce platelet activation (4). It is of interest to acknowledge, in respect to the murine allograft model, that mice lack the genetic equivalent of human and simian FcγRIIA (CD32) and therefore have not demonstrated thrombosis after being confronted with CD40L/CD154 mABs. In our previous work (5), we were able to define a mechanism by which polyclonal antithymocyte-globulin (ATG)—a purified pasteurized gamma globulin suspension and a drug utilized as induction therapy in allograft recipients—is causing platelet depletion in allograft recipients. ATG (thymoglobulin) induces surface platelet activation markers CD62P and CD63, platelet aggregation, and secretion of platelet-bound sCD40L (CD154) in in vitro assays. AT10, a FcγRIIA blocking antibody, is able to abrogate these described effects. Our results find that ATG, containing activating Fc domains, was able to bind the FcγRIIA (CD32) receptor on platelets as well as autologous Fab-fragments (e.g. CD40L/CD154, MHC-class I, CD31) and thus was responsible for the activation and aggregation of platelets in these in vitro assays (5). We are tempted to believe that CD40L/CD154 related thrombophilia/thrombosis is mediated by platelet activation via CD40L/CD154, as well as the presence of functional human FcγRIIA (CD32) receptor. Georg Alexander Roth Andreas Zuckermann Walter Klepetko Ernst Wolner Hendrik Jan Ankersmit Bernhard Moser Ivo Volf Department of Surgery, General Hospital, Vienna Medical School, Vienna, Austria Department of Surgery, Columbia University, New York, New York Institute of Pathophysiology, Vienna Medical School, Vienna, Austria

Response: Thrombophilia Associated with Anti-CD154 Monoclonal Antibody Treatment and its Prophylaxis in Nonhuman Primates

The observations made by Roth and colleagues on FcγRIIA (CD32)-mediated platelet aggregation are interesting and may be relevant to thromboembolic complications caused by CD154 monoclonal antibody (mAb) (1,2). The mechanism they postulate for platelet activation/aggregation resulting from cross linking of the Fc domain of the administrated mAb with FcyRIIA on platelets has provided the impetus for ongoing efforts to develop a truncated IgG molecule, which retains the remarkable efficacy of the anti-CD154 reagent while avoiding thrombophilia. We are also intrigued by a recent report by Henn et al. on coexpression of CD40 and CD154 on platelets (3). They found that CD40 is constitutively expressed on resting platelets, and interaction with CD154 on activated platelets induces rapid downregulation of membrane-bound CD154 and cleaves it to biologically inactive soluble CD154 (sCD154). The authors concluded that cleavage of CD154 from the surface of activated platelets is a central mechanism limiting the inflammatory action of CD154 in the vascular system. Interestingly, the release of sCD154 was abrogated in the presence of anti-CD40 mAb. It is possible that anti-CD154 mAb also can abrogate downregulation of CD154 on platelets leading to unlimited platelet activation in vivo. We anticipate that further such findings and those reported by Roth et al. (2) will lead a better understanding of the CD40-CD154 platelet interactions and delineate effective means to prevent the thromboembolic complications that have been encountered in the initial anti-CD154 mAb therapy trials. Tatsuo Kawai A. Benedict Cosimi Harvard Medical School, Massachusetts General Hospital, Boston, MA

Thrombophilia associated with anti-CD154 monoclonal antibody treatment and its prophylaxis in nonhuman primates.

The authors previously reported thromboembolic complications associated with anti-CD154 monoclonal antibody (mAb) treatment in nonhuman primates. The underlying mechanisms of this complication and its management have not been established. Eighty cynomolgus monkey renal allograft recipients treated with anti-CD154 mAb were studied for the incidence of thrombosis and its prophylaxis. Without anticoagulation prophylaxis, thromboembolic complications were seen in 5 of 11 recipients. With addition of perioperative heparin, the incidence was decreased to 2 of 10. No further improvement was observed by adding intraoperative prostaglandin (PG) E1. However, addition of ketorolac tromethamine to PGE1 and heparin decreased the incidence of thrombosis (one of eight). Most recently, the authors have found that ketorolac administration alone resulted in no thrombosis in 25 consecutive recipients. Ketorolac is remarkably effective in preventing thromboembolism associated with anti-CD154 mAb treatment, suggesting the mechanism underlying this complication may be related to platelet activation leading to enhanced aggregation.

Characterization of the CD154-positive and CD40-positive cellular subsets required for pathogenesis in retrovirus-induced murine immunodeficiency.

Genetically susceptible C57BL/6 (B6) mice that are infected with the LP-BM5 isolate of murine retroviruses develop profound splenomegaly, lymphadenopathy, hypergammaglobulinemia, terminal B-cell lymphomas, and an immunodeficiency state bearing many similarities to the pathologies seen in AIDS. Because of these similarities, this syndrome has been called murine AIDS (MAIDS). We have previously shown that CD154 (CD40 ligand)-CD40 molecular interactions are required both for the initiation and progression of MAIDS. Thus, in vivo anti-CD154 monoclonal antibody (MAb) treatment inhibited MAIDS symptoms in LP-BM5-infected wild-type mice when either a short course of anti-CD154 MAb treatment was started on the day of infection or a course was initiated 3 to 4 weeks after LP-BM5 administration, after disease was established. Here, we further characterize this required CD154-CD40 interaction by a series of adoptive transfer experiments designed to elucidate which cellular subsets must express CD154 or CD40 for LP-BM5 to induce MAIDS. Specifically with regard to CD154 expression, MAIDS-insusceptible B6 nude mice reconstituted with highly purified CD4+ T cells from wild-type, but not from CD154 knockout, B6 donors displayed clear MAIDS after LP-BM5 infection. In contrast, nude B6 recipients that received CD8+ T cells from wild-type B6 donors did not develop MAIDS after LP-BM5 infection. B6 CD40 knockout mice, which are also relatively resistant to LP-BM5-induced MAIDS, became susceptible to LP-BM5-induced disease after reconstitution with highly purified wild-type B cells but not after receiving purified wild-type dendritic cells (DC) or a combined CD40+ population composed of DC and macrophages obtained from B6 SCID mouse donors. Based on these and other experiments, we thus conclude that the cellular basis for the requirement for CD154-CD40 interactions for MAIDS induction and progression can be accounted for by CD154 expression on CD4+ T cells and CD40 expression on B cells.

CTLA4 Signals Are Required to Optimally Induce Allograft Tolerance with Combined Donor-Specific Transfusion and Anti-CD154 Monoclonal Antibody Treatment

Abstract Sensitization to donor Ags is an enormous problem in clinical transplantation. In an islet allograft model, presensitization of recipients through donor-specific transfusion (DST) 4 wk before transplantation results in accelerated rejection. We demonstrate that combined DST with anti-CD154 (CD40L) therapy not only prevents the deleterious presensitization produced by pretransplant DST in the islet allograft model, it also induces broad alloantigen-specific tolerance and permits subsequent engraftment of donor islet or cardiac grafts without further treatment. In addition, our data strongly indicate that CTLA4-negative T cell signals are required to achieve prolonged engraftment of skin allograft or tolerance to islet allograft in recipients treated with a combination of pretransplant DST and anti-CD154 mAb. We provide direct evidence that a CD28-independent CTLA4 signal delivers a strong negative signal to CD4+ T cells that can block alloimmune MLR responses. In this study immune deviation into a Th2 (IL-4) response was associated with, but did not insure, graft tolerance, as the inopportune timing of B7 blockade with CTLA4/Ig therapy prevented uniform tolerance but did not prevent Th2-type immune deviation. While CTLA4-negative signals are necessary for tolerance induction, Th1 to Th2 immune deviation cannot be sufficient for tolerance induction. Combined pretransplant DST with anti-CD154 mAb treatment may be attractive for clinical deployment, and strategies aimed to selectively block CD28 without interrupting CTLA4/B7 interaction might prove highly effective in the induction of tolerance.