CD8⁺ T Cells Mediate Antibody-Independent Platelet Clearance Following Transfusion

Background: Alloimmunization to major histocompatibility complex (MHC) antigens is the leading immunological barrier to transplantation and platelet transfusion therapy. In the context of platelet transfusion medicine, alloimmunization to MHC antigens can substantially diminish the therapeutic efficacy of subsequent blood transfusions. Presently, MHC alloantibodies are recognized as the primary mediators of immune-mediated platelet clearance. However, individuals previously exposed to MHC alloantigens can display significant platelet clearance in the absence of detectable anti-platelet antibodies. Given the ability of cellular immunity to mediate rejection of solid organ allografts across MHC differences, we hypothesized that cellular immunity likewise facilitates the clearance of platelets in MHC alloimmunized recipients.

Methods: FVB (H-2K^q) MHC-immunized or non-immunized C57BL/6 (H-2K^b) recipients were transfused with filter leukoreduced platelet rich plasma (PRP) isolated from C57BL/6 donors expressing green fluorescent protein (GFP) under a H-2K^b promoter (B6 GFP) or PRP obtained from a FVB X B6 GFP F1 cross, which generates GFP⁺ H-2K^q+ platelets. Following transfusion, mice were bled at 10 minutes, 1 hour, 2 hours or 24 hours to calculate the percentage of GFP⁺ transfused platelets remaining using flow cytometric analysis. To test the hypothesis that platelet clearance can occur in an antibody-independent process, FVB H-2K^q MHC-immunized or non-immunized μMT recipients were similarly transfused with B6 GFP or FVB X B6 GFP PRP. CD8⁺ T cells in immunized μMT recipients were depleted by i.p. injection of anti-CD8 (clone 2.43) antibody for 3 consecutive days prior to transfusion. NK cells were similarly depleted by injection of anti-NK1.1 (clone PK-136) antibody, 1 day prior to transfusion. CD4, CD8 T cell and NK cell depletion was evaluated in the peripheral blood prior to platelet transfusion, and compared to non-treated and isotype control treated recipients.

Results: While GFP⁺ platelets were readily detected following transfusion into non-immunized or MHC matched recipients, transfusion of FVB-B6 platelets into H-2K^q MHC-immunized C57BL/6 recipients resulted in rapid clearance within the first hour following transfusion. In contrast, though little platelet clearance was detected in H-2K^q MHC-immunized μMT recipients within an hour following transfusion, very few transfused platelets were observed after 24 hours. Moreover, despite platelet clearance, anti-MHC antibodies were not detectable in μMT recipients. To determine the cellular component responsible for clearance in alloimmunized μMT recipients, CD8⁺ T cells or NK cells were depleted prior to transfusion. NK cell depletion failed to impact platelet clearance in alloimmunized μMT recipients; however, depletion of CD8⁺ T cells prior to platelet transfusion abrogated platelet clearance in immunized μMT recipients.

Conclusion: Immunized recipients rapidly clear platelets in an alloantigen specific fashion following platelet transfusion in a murine model. Though platelet clearance occurred at a faster rate in intact immunized C57BL/6 recipients, B cell deficient MHC-immunized μMT recipients possessed the capacity to induce significant clearance at later time points. Evaluation of the cellular populations responsible for antibody-independent clearance in μMT recipients strongly suggest that CD8⁺ T cells play a key role in antibody-independent immune-mediated platelet clearance. These results suggest that, in addition to antibodies, CD8⁺ T cells can play a role in the development of platelet refractoriness and may contribute to platelet non-responsiveness in patients with little to no detectable anti-platelet alloantibodies.