Importantly, transfusion of MHC-matched B6GFP platelets into immunized or nonimmunized recipients failed to result in any detectable changes in platelet clearance (Figure 2D,F), strongly suggesting the clearance of FVBGFP platelets reflected an immune-mediated process

Importantly, transfusion of MHC-matched B6GFP platelets into immunized or nonimmunized recipients failed to result in any detectable changes in platelet clearance (Figure 2D,F), strongly suggesting the clearance of FVBGFP platelets reflected an immune-mediated process. alloantibodies, many individuals encounter platelet clearance following transfusion in the absence of a clear mechanism. These results suggest that additional 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. Intro Although over 1.5 million platelet transfusions happen each year,1 a significant portion of individuals who receive platelets fail to achieve the desired therapeutic benefit due to accelerated platelet clearance.2,3 While clearance can occur through nonimmune-related mechanisms,4 many studies demonstrate the importance of immune-mediated clearance.2,3,5-8 Historically, immune-mediated platelet clearance, termed refractoriness, was attributed solely to anti-platelet alloantibodies predominately Enalaprilat dihydrate targeted to major histocompatibility complex (MHC) antigens.5,7 In the absence of detectable anti-platelet alloantibodies, platelet clearance is invariably considered nonimmune in nature.5,6 However, although studies demonstrate that some individuals can fail platelet therapy in KITH_HHV11 antibody the complete absence of detectable anti-platelet alloantibodies,2,3 nonimmune mechanisms often fail to fully explain platelet clearance, suggesting that immune-mediated platelet clearance may occur independent of anti-platelet alloantibodies. Study design Generating a mouse model for immune-mediated platelet clearance C57BL/6 (H-2b) mice were immunized for 3 consecutive weeks by intraperitoneal injections of 10 106 total splenocytes from FVB (H-2q) mice. Generation of anti-platelet alloantibodies was confirmed by circulation cross-match with FVB (H-2q) and C57BL/6 (H-2b) platelets. Immunized mice were transfused, as indicated, with platelets isolated as previously explained9 from H2Kb-eGFP (B6GFP) (GFP+, H-2b) or FVB H2Kb-eGFP (FVBGFP) (GFP+, H-2b, H-2q) mice. Subsequent green fluorescent proteinCpositive (GFP+) platelet clearance was assessed by circulation cytometry at the changing times indicated following transfusion. Assessing antibody-independent platelet refractoriness To evaluate antibody-independent platelet clearance, MT mice (B-cellCdeficient C57BL/6, H-2b) were immunized and transfused with B6GFP or FVBGFP platelets, followed by evaluation of platelet clearance, as defined in the previous paragraph. Absence of antibody was confirmed by western blot analysis of serum from naive and immunized C57BL/6 and MT mice. Specific immune cell subsets were eliminated from immunized MT mice prior to platelet transfusion by injection of monoclonal CD8-depleting antibody (clone 2.43) or NK1.1 monoclonal antibody (clone PK-136), respectively. Depletions were confirmed by circulation cytometry. Please refer to supplemental Materials (available on the web page) for detailed Enalaprilat dihydrate methodology. Results and conversation Although earlier studies provide insight into the development of anti-platelet alloantibodies,2,9-14 few models exist to evaluate mechanisms of platelet refractoriness in transfused recipients. Consequently, we 1st developed a model to evaluate mechanisms whereby platelet clearance may occur following MHC alloimmunization. To accomplish this, C57BL/6 (H-2b) recipients were immunized with FVB (H-2q) splenocytes, which resulted in reproducible MHC alloimmunization monitored by evaluating anti-MHC alloantibody formation. Consistent with earlier results, specific anti-H-2q alloantibodies were produced that identified platelets isolated from FVB donors (Number 1A). Importantly, these interactions appeared to be specific to FVB platelets, as serum from FVB-immunized C57BL/6 recipients failed to cross-react with platelets isolated from MHC-identical C57BL/6 donors (Number 1B). Open in a separate windowpane Number 1 MHC-immunized recipients rapidly obvious MHC-mismatched platelets. (A-B) Serum from nonimmunized C57BL/6 (H-2b) recipients (NI) or FVB (H-2q)-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 antiCimmunoglobulin G (IgG) and circulation cytometric exam (n = 5). (C) Nonimmunized or FVB-immunized C57BL/6 recipients were transfused with C57BL/6.GFP FVB (FVBGFP) or C57BL/6.GFP (B6GFP) platelets Enalaprilat dihydrate followed by circulation cytometric examination 24 hours later (gate = percentage of total platelets). (D-E) Percentage of FVBGFP (D) or B6GFP (E) platelets remaining, normalized to nonimmunized recipients, as indicated at numerous time points posttransfusion into nonimmunized (NI) or FVB-immunized (I) C57BL/6 recipients (n = 5). Significance was identified in panels A, B, D, and E by College student test (** .01, **** .0001). MFI, mean fluorescence intensity; ns, no significance; plts, platelets; SSC, part scatter. To avoid labeling strategies that may change platelet clearance in an immune-independent fashion,15-18 we crossed C57BL/6 transgenics expressing GFP under a H-2Kb promoter19 with FVB, to generate C57BL/6.GFP FVB progeny (FVBGFP) that express GFP and H-2q antigens. To determine whether FVB immunization improved FVBGFP platelet clearance, FVB-immunized C57BL/6 recipients were transfused with FVBGFP platelets and evaluated for platelet clearance at numerous time points posttransfusion. Transfused platelets could be recognized as GFP and CD41-positive events immediately following transfusion (Number 1C; supplemental Number 1). Following.