CD8+ T cells mediate antibody-independent platelet clearance in mice

Abstract

Platelet transfusion provides an important therapeutic intervention in the treatment and prevention of bleeding. However, some patients rapidly clear transfused platelets, preventing the desired therapeutic outcome. Although platelet clearance can occur through a variety of mechanisms, immune-mediated platelet removal often plays a significant role. Numerous studies demonstrate that anti-platelet alloantibodies can induce significant platelet clearance following transfusion. In fact, for nearly 50 years, anti-platelet alloantibodies were considered to be the sole mediator of immune-mediated platelet clearance in platelet-refractory individuals. Although nonimmune mechanisms of platelet clearance can often explain platelet removal in the absence of anti-platelet alloantibodies, many patients experience platelet clearance following transfusion in the absence of a clear mechanism. These results suggest that other processes of antibody-independent platelet clearance may occur. Our studies demonstrate that CD8(+)T cells possess the unique ability to induce platelet clearance in the complete absence of anti-platelet alloantibodies. These results suggest a previously unrecognized form of immune-mediated platelet clearance with significant implications in the appropriate management of platelet-refractory individuals.

Figure 1

MHC-immunized recipients rapidly clear MHC-mismatched platelets. (A-B) Serum from nonimmunized C57BL/6 (H-2^b) recipients (NI) or FVB (H-2^q)-immunized C57BL/6 recipients (I) was incubated with FVB platelets (A) or C57BL/6 (B6) platelets (B) followed by detection of bound antibody by incubation with anti–immunoglobulin G (IgG) and flow cytometric examination (n = 5). (C) Nonimmunized or FVB-immunized C57BL/6 recipients were transfused with C57BL/6.GFP × FVB (FVB^GFP) or C57BL/6.GFP (B6^GFP) platelets followed by flow cytometric examination 24 hours later (gate = percentage of total platelets). (D-E) Percentage of FVB^GFP (D) or B6^GFP (E) platelets remaining, normalized to nonimmunized recipients, as indicated at various time points posttransfusion into nonimmunized (NI) or FVB-immunized (I) C57BL/6 recipients (n = 5). Significance was determined in panels A, B, D, and E by Student t test (**P ≤ .01, ****P ≤ .0001). MFI, mean fluorescence intensity; ns, no significance; plts, platelets; SSC, side scatter.

Figure 2

CD8⁺ T-cell–mediated platelet clearance in immunized B-cell–deficient μMT recipients. (A-B) Serum from nonimmunized μMT (H-2^b) recipients (NI) or FVB (H-2^q)-immunized (I) μMT recipients was incubated with FVB platelets (A) or C57BL/6 (B6) platelets (B) followed by detection of bound antibody by incubation with anti-IgG and flow cytometric examination (n = 5). (C) Serum from nonimmunized or FVB-immunized μMT or C57BL/6 recipients was separated by gel electrophoresis under nonreducing conditions and analyzed by western blot analysis for immunoglobulin as indicated. (D) Nonimmunized or FVB-immunized μMT recipients were transfused with C57BL/6.GFP × FVB (FVB^GFP) or C57BL/6.GFP (B6^GFP) platelets followed by flow cytometric examination 24 hours later (n = 5) (gate = percentage of total platelets). (E-F) Percentage of FVB^GFP (E) or B6^GFP (F) platelets remaining, normalized to nonimmunized recipients as indicated at various time points posttransfusion into nonimmunized (NI) or FVB-immunized (I) μMT recipients (n = 5). (G) Percentage of FVB^GFP platelets remaining, normalized to nonimmunized recipients, at 24 hours following transfusion, as indicated into nonimmunized (NI), FVB-immunized (I), CD8⁺ T-cell (CD8) depleted immunized or NK cell (NK) depleted immunized μMT recipients (n = 4-5). Significance was determined in panels A, B, E, and F by Student t test or by 1-way analysis of variance with the Tukey posttest in panel G (****P ≤ .0001; ns, no significance).