Co-Stimulatory Blockade Abrogates Alloimmunization to Transfused Platelets in a Mouse Model

Platelet transfusion therapy can be a life-sustaining treatment for patients with severe thrombocytopenia. However, alloimmunization is a potential sequelae of platelet transfusion with serious consequences for chronically transfused patients. Induction of alloantibodies, typically against HLA and/or human platelet antigens, can lead to poor survival of transfused platelets expressing the offending antigens. Patients with alloantibodies against multiple HLAs can become refractory to subsequent platelet therapy. Although leukoreduction of platelets has significantly decreased humoral alloimmunization rates, anti-HLA antibodies still form in a significant percentage of transfused patients. In addition to alloantibody formation, animal models have shown that platelet-induced T cell alloimmunity can contribute to the rejection of MHC identical bone marrow transplants across minor antigen barriers. Currently, there are no approved therapeutic interventions in humans to mitigate the risk of alloimmunization other than leukoreduction. Targeted blockade of T cell costimulation has shown great promise in inhibiting alloimmunity in the setting of solid organ transplantation; however, this strategy has not yet been explored in the context of platelet transfusion. Therefore, we tested the hypothesis that the costimulatory blockade reagent CTLA4-Ig, which interrupts the CD28-B7.1/2 T cell signaling pathway, would prevent alloreactivity against major and minor alloantigens on transfused platelets.

C57BL/6 (H-2^b) mice were transfused with filter leukoreduced platelets from BALB/c (H-2^d) donors. Humoral alloimmunization was measured by indirect immunofluoresence using BALB/c splenocytes or platelets as targets, followed by flow cytometry. Platelet specific CD4⁺ T cell responses were analyzed by adoptive transfer of TCR75 CD4⁺ T cells (specific for a peptide from donor MHC I (K^d) presented by recipient MHCII (I-A^b)). Some transfusion recipients were subjected to a bone marrow transplant under reduced intensity conditioning (700 rads) using BALB.B mice as donors (MHC identical, minor antigen mismatched). Engraftment was studied by flow cytometry. Experimental animals received a 500 ug dose of CTLA-4 Ig or 500 ug of a control antibody.

The combined data from three separate experiments demonstrated that whereas 14/15 (93%) of control animals had anti-H2^d antibodies after 4 weekly platelet transfusions, alloantibody formation was abrogated in all animals (n=15) given a single dose of CTLA4-Ig concurrent with the first platelet transfusion. This effect of CTLA4-Ig lasted at least 49 days after treatment. The proliferation of donor-specific CD4⁺ T cells was completely blocked by CTLA4-Ig, indicating an effect at the CD4⁺ T cell level. In addition, administration of a single dose of CTLA4-Ig prevented platelet transfusion induced BMT rejection. The combined results of three independent experiments demonstrated that while 14/15 (93%) of recipients transfused with BALB/c leukoreduced platelets rejected a bone marrow transplant in the presence of isotype control antibody, only 1/15 (7%) of CTLA4-Ig treated recipients rejected donor bone marrow. However, the timing of CTLA4-Ig administration was found to be critical. Delaying treatment until after platelet transfusion failed to prevent bone marrow transplant rejection.

These findings demonstrate that costimulatory blockade in general, and CTLA4-Ig in particular, have the capacity to prevent both humoral and cellular alloimmunization to transfused platelets. These findings suggest costimulatory blockade may be a viable approach to decrease alloimmunization rates in humans receiving platelet transfusions.

Zimring:Immucor Inc,: Research Funding. Off Label Use: We will describe use of CLTA4-Ig in an animal model for what would be an off label indication in humans. However, there are no human studies in this abstract.