Anti-HLA Antibodies Bound to Monocytes Altered Antibody-mediated Platelet Phagocytosis and Led to Mild Thrombocytopenia

Xiuzhang Xu¹Nelli Baal²Martin Rick²Dawei Chen¹Huaqin Liang¹Xin Ye¹Wenjie Xia¹Hui Ren¹Yaori Xu¹Yongshui Fu³Gregor Bein²Sentot Santoso^2*

• ¹Guangzhou Blood Center, Guangzhou, China
• ²Justus-Liebig-Universitat Giessen, Giessen, Germany
• ³Southern Medical University, Guangzhou, China

In fetal and neonatal alloimmune thrombocytopenia (FNAIT), maternal antibodies cross the placenta and react with alloantigen expressed on fetal platelets, leading to their clearance via antibody-dependent phagocytosis. In Caucasians, most FNAIT cases are caused by anti-HPA-1a antibodies. In contrast, antibodies against HLA class I antigens are rarely found in FNAIT but are frequently implicated in cases of platelet transfusion refractoriness (PTR). The reason for this phenomenon is unknown, which leads to ongoing debate regarding the role of anti-HLA class I antibodies in FNAIT. In this study, we investigated the platelet clearance mediated by anti-HLA class I antibodies in whole blood both in vitro and in vivo. To mimic FNAIT conditions, whole blood was pretreated with anti-HLA antibodies before the phagocytosis of anti-HPA-1a antibody-opsonized platelets. Compared to untreated whole blood, anti-HLA-ABC and anti-HLA-DR IgG antibodies inhibited the phagocytosis of anti-HPA-1a-antibody-opsonized platelets. Similar results were obtained with purified monocytes, indicating that anti-HLA-ABC antibodies bound to monocytes can interfere with antibody-mediated platelet phagocytosis. Compared to isotype controls, the administration of anti-MHC-I antibodies to Balb/c female mice led to a significant decrease in the platelet count within 24 h. However, compared to anti-MHC-I antibodies, anti-αIIbβ3 antibody administration resulted in significantly higher platelet clearance over different time points. Analysis of antibody-bound platelets showed the presence of anti-αIIbβ3 antibodies on the platelet surface, but not on monocytes. In contrast, anti-MHC-I antibodies were found on both platelets and monocytes. Interestingly, monocytes exhibited higher levels of anti-MHC-I binding than platelets (87.0% vs. 25.5% after 30 min), most likely because platelets express significantly fewer HLA class I antigens than monocytes, as indicated by our flow cytometric analysis of whole blood. These results indicated that anti-MHC-I antibodies preferentially bind to monocytes rather than platelets in whole blood and can be cleared by monocytes via endocytosis. Furthermore, we found that the presence of anti-HLA class I antibodies did not significantly influence platelet clearance induced by anti-αIIbβ3 antibodies. The question of whether these observations can explain the controversial opinions regarding the relative roles of anti-HLA class I and anti-αIIbβ3 antibodies in FNAIT requires further assessment in a murine model of FNAIT.