Background-Storage of RBCs is necessary for an adequate blood supply. However, reports have identified potential negative sequelae of transfusing stored RBCs. An animal model would be useful to investigate the pathophysiology of transfusing stored RBCs. However, it has been reported that storage of rat RBCs in CPDA-1 resulted in an unexpected sudden decline in posttransfusion survival. We developed a mouse model of RBC storage and transfusion to assess survival kinetics of mouse RBCs.
Blockade of the CD40/CD154 signaling pathway using anti-CD154 Abs has shown promise in attenuating the alloimmune response and promoting long-term graft survival in murine model systems, although side effects observed in humans have hampered its progression through clinical trials. Appropriately designed anti-CD40 Abs may provide a suitable alternative. We investigated two isoforms of a novel monoclonal rat anti-mouse CD40 Ab (7E1) for characteristics and effects mirroring those of anti-CD154: 7E1-G1 (an IgG1 isotype); and 7E1-G2b (an IgG2b isotype). In vitro proliferation assays to measure the agonist properties of the two anti-CD40 Abs revealed similar responses when plate bound. However, when present as a soluble stimulus, 7E1-G1 but not 7E1-G2b led to proliferation. 7E1-G2b was as effective as anti-CD154 when administered in vivo in concert with CTLA4-Ig in promoting both allogeneic bone marrow chimerism and skin graft survival, whereas 7E1-G1 was not. The protection observed with 7E1-G2b was not due to depletion of CD40-bearing APCs. These data suggest that an appropriately designed anti-CD40 Ab can promote graft survival as well as anti-CD154, making 7E1-G2b an attractive substitute in mouse models of costimulation blockade-based tolerance regimens.
Background
Alloantibodies are a clinically significant sequelae of platelet transfusion, potentially rendering patients refractory to ongoing platelet transfusion support. These antibodies are often IgG class switched, suggesting the involvement of CD4+ T cell help; however, platelet specific CD4+ T cells have not been visualized in vivo and specifics of their stimulation are not completely understood.
Study Design and Methods
A murine model of alloimmunization to transfused platelets was developed to allow in vivo assessment and characterization of CD4+ T cells specific for platelet MHC alloantigen. Platelets were harvested from BALB/c mice, filter leukoreduced, and transfused into C57BL/6 recipients. Platelet specific CD4+ T cell responses were visualized by using a TCR transgenic mouse that detects peptide from donor MHC I presented on recipient MHC II. Antibody responses were determined by indirect immunofluorescence using BALB/c donor targets.
Results
C57BL/6 recipients of BALB/c leukoreduced platelet transfusions produced anti-BALB/c antibodies, with proliferation of antigen specific CD4+ T cells seen in the spleen but not lymph nodes or liver. Depletion of recipient CD4+ cells or splenectomy independently abrogated the alloantibody response.
Conclusion
We report a novel model to study antigen-specific CD4+ T cells during alloimmunization to platelet transfusion. The presented data support a critical role for CD4+ T cell help in the humoral response to platelet transfusion and establish the spleen as a required microenvironment for effective CD4+ T cell priming against donor platelet derived MHC I.
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