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HLA-Universal Platelet Transfusions Prevent Platelet Refractoriness in a Mouse Model

机译：HLA通用血小板输注可防止小鼠模型中的血小板难治性

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Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA class I antigens constitutes a significant clinical problem. Thus, it would be desirable to have PLT units lacking HLA antigens on the cell surface. Previously, we showed that the generation of functional HLA class I-silenced (HLA-universal) PLTs from CD34~+ cells, using a short hairpin RNA (shRNA) to target (beta)2-microglobulin ((beta)2m) transcripts, is feasible. Here, we assessed the capacity of HLA-silenced PLTs to escape HLA antibody-mediated cytotoxicity in vitro and in vivo. Generation of megakaryocytes (MKs) and PLTs was performed by thrombopoietin-mediated differentiation of HLA-silenced CD34~+ cells within 10 days. Lymphocytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) reporter assays using anti-HLA antibodies and a mouse model for PLT refractoriness were used to assess the immune-evasion capability of HLA-universal MKs and PLTs. To mimic PLT refractoriness in vivo, NOD/SCID/IL-2R(gamma)c~(-/-) mice were injected with specific anti-HLA antibodies followed by the infusion of 1 x 10~6 HLA-universal MKs. In vivo PLT generation was evaluated by flow cytometry using anti-CD42a and CD61 antibodies. Cells expressing a nonspecific shRNA were used as control. Lymphocytotoxicity and ADCC reporter assays showed that HLA silencing protects MKs against HLA antibody-mediated complement-dependent and cell-mediated cytotoxicity. In lymphocytotoxicity assays, 80-90% of HLA-expressing MKs but only 3% of HLA-silenced MKs were lysed. In the circulation of mice, HLA-expressing and HLA-silenced MKs showed PLT production in the absence of anti-HLA antibodies, with human PLT frequencies of up to 0.5% within the PLT population. However, in presence of anti-HLA antibodies HLA-expressing MKs were rapidly cleared from the circulation of mice, whereas HLA-silenced MKs escaped HLA antibody-mediated cytotoxicity and produced PLTs that were detectable up to 11 days. Our data show that HLA-silenced PLTs are efficiently protected against HLA antibody-mediated cytotoxicity and prevent PLT refractoriness in vivo. Provision of HLA-silenced PLTs may become an important component in the management of patients refractory to PLT transfusion.
机译：由针对HLA I类抗原的同种免疫导致的血小板输注（PLT）难治性构成了重要的临床问题。因此，希望在细胞表面具有缺乏HLA抗原的PLT单元。先前，我们证明了使用短发夹RNA（shRNA）靶向β2-微球蛋白（β2m）转录本，从CD34〜+细胞中产生了功能性HLA I类沉默（HLA通用）PLT，是可行的。在这里，我们评估了HLA沉默的PLT在体外和体内逃避HLA抗体介导的细胞毒性的能力。通过血小板生成素介导的HLA沉默的CD34〜+细胞分化，在10天内完成了巨核细胞（MKs）和PLT的生成。使用抗HLA抗体和PLT难治性小鼠模型进行的淋巴细胞毒性和抗体依赖性细胞毒性（ADCC）报告基因分析用于评估HLA通用MK和PLT的免疫逃逸能力。为了模拟体内的PLT难治性，向NOD / SCID /IL-2Rγc-（-/-）小鼠注射特定的抗HLA抗体，然后输注1 x 10〜6个HLA通用MK。使用抗CD42a和CD61抗体通过流式细胞术评估了体内PLT的产生。表达非特异性shRNA的细胞用作对照。淋巴细胞毒性和ADCC报告基因检测表明，HLA沉默可保护MK免受HLA抗体介导的补体依赖性和细胞介导的细胞毒性。在淋巴细胞毒性试验中，裂解了80-90％的表达HLA的MK，但仅裂解了3％的HLA沉默的MK。在小鼠体内，表达HLA和沉默HLA的MK在没有抗HLA抗体的情况下显示出PLT的产生，在PLT人群中人PLT的频率高达0.5％。但是，在存在抗HLA抗体的情况下，表达HLA的MKs从小鼠的循环中迅速清除，而沉默了HLA的MKs逃脱了HLA抗体介导的细胞毒性，并产生了可检测到11天的PLT。我们的数据表明，HLA沉默的PLT可有效保护免受HLA抗体介导的细胞毒性，并防止体内PLT难治性。提供HLA沉默的PLT可能成为难治PLT输血患者的重要组成部分。

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