The Role of Platelets in the Induction of Alloimmunization after Platelet Transfusion

Background Platelet (PLT) transfusion can elicit alloimmune responses leading to alloantibody formation. These alloantibodies can opsonize PLTs which causes rapid clearance of transfused PLTs and thereby render the transfusion ineffective. Contaminating WBCs in PLT concentrates are important for the induction of this alloimmunization which is evident from the fact that leukoreduction of platelet concentrates significantly reduced the frequency of alloimmunization. Despite universal leukoreduction, however, a residual risk remains. Currently, it is unknown if this is due to the residual WBCs (<1*10⁶ / transfusion) in PLT concentrates or if PLTs themselves can also induce primary HLA-alloimmunization. Furthermore, it is not known which factors increase or reduce the likelihood of alloimmunization. Therefore, the aim of our study was, first, to determine the involvement of PLTs in the induction of alloimmunization, and second, to investigate whether storage of PLT concentrates might be a risk factor for alloimmunization.

Methods To determine if PLTs are phagocytosed and presented by antigen presenting cells, monocyte derived dendritic cells (moDCs) were incubated at 37°C with allogeneic PLTs. After incubation, moDCs were either fixed and analysed for phagocytosis of PLTs using z-stacks obtained with confocal microscopy, or moDCs were lysed and HLA-DR was immunoprecipitated from this lysate. Thereafter, HLA-DR presented peptides were eluted and measured using mass spectrometry. Furthermore, HLADRB3*01:01⁺ moDCs were incubated with either HPA1a⁺ or HPA1b⁺ PLTs and later exposed for 24 hours to HPA1a-specific HLADRB3*01:01⁺ CD4 T cells after which production of IFNy by T cells was determined using ELISA. To investigate if storage may be a potential risk factor for alloimmunization, moDCs were incubated with freshly isolated PLTs or PLTs stored 7 days under blood bank conditions. Finally, in some experiments, freshly isolated PLTs were activated using thrombin-receptor agonistic peptide, treated with calcimycin A23187 to induce apoptosis, or incubated with either purified phosphatidylserine (PS) or phosphatidylcholine (PC). Subsequently, phagocytosis of these PLTs by moDCs was quantified using imaging flow cytometry.

Results Confocal microscopy clearly showed that PLTs are phagocytosed by moDCs. Mass spectrometry analyses showed presentation of peptides derived from proteins exclusively expressed in PLT by HLA class II of moDCs. In addition, moDCs induced a significant HPA1a⁺ specific CD4 T cells response (measured as IFNy production) after incubation with HPA1a⁺ PLTs, which was not observed after incubation with HPA1b⁺ PLTs. PLTs obtained from stored PLT concentrates were significantly more phagocytosed compared to fresh PLTs. This might be caused by increased presence of apoptotic platelets in stored PLT concentrate, as phagocytosis of apoptotic platelets was significantly increased compared to activated or unstimulated PLTs. This increased phagocytosis was partially caused by PLT surface expression of PS. Furthermore, apoptotic PLTs also induced a stronger CD4 T cell response.

Discussion Our studies clearly show that peptides of phagocytosed PLTs are presented by DCs, resulting in a platelet-specific CD4 T cell response. This implicates that not only WBCs, but also PLTs themselves can induce alloimmunization after PLT transfusion. As a result, factors that increase phagocytosis of PLTs could also increase the risk of alloimmunization after PLT transfusion. In this respect, PLTs stored for 7 days showed increased phagocytosis as compared to fresh PLTs. This increase is at least partly due surface expressed PS caused by storage-induced apoptosis and resulted in a stronger platelet-specific CD4 T cell responses compared to DCs incubated with untreated fresh PLTs. Although these in vitro experiments only partly represent the complex processes that are involved in an immune response in vivo , these data suggest that storage of PLT concentrates may be associated with increased alloimmunization.