背景：

RhD 阴性孕妇注射抗-D 免疫球蛋白（anti-D immune globulin，RhIg），能够抑制母体产生相应抗体，从而预防或减轻因母婴 RhD 血型不一致引起的胎儿新生儿溶血病（hemolytic disease of the fetus and newborn，HDFN）。目前 RhIg 主要通过主动免疫 RhD 阴性志愿者制备，但是在免疫过程中志愿者可能会发生血管外溶血反应，危害其健康。本课题组前期已经制定了有效的免疫程序，但尚未评价该免疫程序能否保障志愿者的安全，因此，本文进一步对免疫程序的安全性进行分析。其次，相关研究已经报道存在 RhD 阳性红细胞免疫后的 RhD 阴性志愿者不产生抗D抗体现象，即“不应答者”现象，但是其不应答机制尚不清楚。本课题组已经提出了 Toll样受体 3（toll like receptor 3，TLR3）及 I 型干扰素（type Iinterferon，IFN-α/β）可能与 RhD 同种免疫发生相关，RhD 同种免疫发生过程极其复杂，单一的细胞或者通路研究无法对其进行完整解释。目前，蛋白质组学在自身免疫及与炎症有关疾病的研究中得到广泛运用，基于定量蛋白质组学技术筛选出的差异蛋白可能找到免疫调节的新途径。因此我们通过质谱技术定量检测 RhD 主动免疫后的应答者与不应答者外周血单个核细胞的（peripheral blood mononuclear cell，PBMC）蛋白表达量并进行生物信息学分析，以找到可能调节 RhD 同种免疫过程的差异蛋白及其可能参与的信号通路。

目的：本研究旨在探讨目前免疫程序的安全性，以在 RhD 主动免疫中保障志愿者的安全，为我国生产 RhIg 提供数据支持。其次以 RhD 阴性志愿者主动免疫后产生的应答者与不应答者 PBMC 为研究对象，采用质谱技术对 RhD 主动免疫应答者以及不应答者的 PBMC 进行检测。定量并筛选出可能与 RhD 同种免疫过程相关的差异蛋白，分析其可能参与的信号通路，为进一步研究 RhD 同种免疫机制奠定理论基础。

方法：（1）使用 RhD 阳性红细胞免疫本研究纳入的 23 名 RhD 阴性志愿者，免疫程序由初始免疫及加强免疫组成，初始免疫包括首次初免，二次初免以及三次初免。志愿者初始免疫后经检测出现抗 D 抗体即可加强免疫。在主动免疫后，抽取志愿者全血检测相关实验室指标（抗 D 抗体滴度、血细胞分析、肝肾及凝血功能），进而评价本研究主动免疫剂量的安全性。（2）取志愿者中多次免疫后确定为应答者以及不应答者样本共 6 例，分为应答组与不应答组，提取 PBMC。采用数据非依赖采集（data independent acquision，DIA）质谱技术，筛选出差异蛋白并通过生物信息学方法对其进行分析，如基因本体（gene ontology, GO）注释及 KEGG（kyoto encyclopedia of genes and genomes）通路富集。结合 GO 分析与文献报道，找到可能与调节 RhD 同种免疫过程相关的差异蛋白，并分析这些蛋白可能参与的信号通路。

结果：（1）在本研究中，23 名 RhD 阴性志愿者初次免疫及加强免疫后其各项生命体征均无异常。首次初免 20 mL、30 mL、40 mL、50 mL 后，统计分析 24 小时及 1周血常规、肝肾及凝血功能中与溶血性输血不良反应相关的安全性指标，结果显示均无统计学差异（P>0.05）；分析加强免疫 0.5 mL、1 mL、2 mL 后 24 小时以及 1周的上述指标与每次免疫前最近一次检测值差值，也均无明显差异（P>0.05）；进行 1 mL 及 2 mL 加强免疫时，24 小时与 1 周后分别有 1 名志愿者出现间接胆红素升高临近医学参考值上限及超过医学参考值范围，但均无输血不良反应发生；RhD应答者不同剂量加强免疫后安全性指标变化异常及超出医学参考值的情况无差别（P>0.05）；加强免疫 2 mL 的抗体效价增高倍数高于 0.5 mL，免疫效果更优（P=0.024）；加强免疫 1 周后，达到满足不同采浆要求的抗 D 抗体效价时，相应安全性指标差值变化无统计学差异（P>0.05）。（2）本研究共筛选出 178 个差异蛋白（P＜0.05，定量差异倍数绝对值( |Foldchange，FC| )＞1.50)，其中包括 76 个上调蛋白，102 个下调蛋白。上述差异蛋白主要涉及趋化因子通路及钙离子（Ca2+）信号通路，上调蛋白主要涉及 RNA 剪接通路，而下调蛋白主要涉及 GTP 酶活性生物过程。进一步筛选差异蛋白中|FC|＞2且可能与调节 RhD 同种免疫过程有关的蛋白，其中 PYHIN1、CTNNBL1、DCAF1、ISG20、LYST 在 RhD 应答者中显著上调，TNFAIP8L2 在 RhD 应答者中显著下调，这些蛋白参与 Toll 样受体（toll like receptors, TLRs）信号通路，T、B 细胞活化相关信号以及趋化因子通路等。

结论：（1）本研究制定的免疫程序中未发现影响志愿者安全的现象发生，初步认为能够保障志愿者的安全。在满足安全且有效的前提下，建议优化免疫剂量为首次初免 50 mL，加强免疫 0.5mL，为我国研发 RhIg 提供了数据支持。（2）上调蛋白 PYHIN1、CTNNBL1、DCAF1、ISG20、LYST 以及下调蛋白TNFAIP8L2 共 6 个差异蛋白可能参与调节 RhD 同种免疫应答过程。其中 TLRs 信号通路在 RhD 同种免疫中可能受到 PYHIN1、LYST 蛋白正调节，受到 TNFAIP8L2蛋白负调节，趋化因子通路在 RhD 同种免疫中可能受到 ISG20 蛋白正调节，T 细胞活化信号在 RhD 同种免疫中可能受到 DCAF1 蛋白正调节，B 细胞活化信号在 RhD同种免疫中可能受到 CTNNBL1 蛋白正调节，为进一步研究 RhD 同种免疫机制提供科学支撑。

Background: RhD blood group incompatibility between mother and infant can causesevere hemolytic disease of the fetus and newborn (HDFN)，and anti-D immune  globulin(RhIg)  injection of RhD-negative pregnant women can prevent or reduce this disease bykeeping her from producing the corresponding antibodies. At present, RhIg is mainly prepared by immunizing RhD-negative volunteers, but the volunteers may have extravascular hemolysis during the immunization processes, which will endanger their health. An effective immunization program had been established by our research team, but the safety of the volunteers has not yet been evaluated. Therefore, further analysis on the safety of the immunization program is conducted in this article. In addition, it has been reported in related studies that some RhD-negative individuals do not produce anti-D antibodies to RhD-positive red blood cells, called "non-responders". The mechanism of non-response, however, is still unclear. The Toll like receptor 3 (TLR3) and type I interferon (type I interferon, IFN-α/β) has been found in our previous study that they may play essential roles in the occurrence of RhD alloimmunization. The process of RhD alloimmunization is extremely complicated, and single cell or pathway study cannot fully explain it. As a popular technology, proteomics has been widely used in the research fields of autoimmunity and inflammatory diseases. Differential proteins which may be associated with the research purposes can be obtained through proteomics technology, which provide new directions for the investigation on the immune regulation mechanisms. Therefore, mass spectrometry technology was used in this study to quantitatively detect and analyze the protein expression profiles of peripheral blood mononuclear cells (PBMC) between responders and non-responders after RhD immunization. Differential proteins that may be related to the RhD alloimmunization were screened and a following bioinformatics analysis was conducted to predict the potential functions, including the signaling pathways that may be involved.

Objective: To ensure the safety of volunteers in RhD immunization and further provide data support for the production of RhIg in China, the safety of current immunization procedures was explored and analyzed in this study. In addition, the PBMCs of  responders and non-responders were collected after immunization of RhD-negative volunteers for mass spectrometry test. Differential proteins that may regulate the immune process of RhD were quantified and screened, and the signaling pathways that they may participate in were predicted, which may lay a theoretical foundation for further research on the RhD alloimmunization mechanisms.

Method: (1) Twenty-three RhD-negative volunteers were immunized with RhD-positive red blood cells (RBCs). The immunization program includes initial immunization and booster immunization. The initial immunization was divided into the first, the second, and the third initial immunization. The volunteers will give booster immunization once they developed anti-D antibody after the initial immunization. Blood samples were collected after each immunization and the anti-D titer, blood cell analysis, liver and kidney function, and routine blood coagulation were detected to evaluate the safety of RhD immunization. (2) PBMCs from Six volunteers who were determined to be responders and non-responders after multiple immunizations were extracted and the samples were divided into respond group and non-respond group. The differential proteins were screened by data independent acquision (DIA) mass spectrometry and analyzed by bioinformatics methods, such as gene ontology (GO) annotation and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment. Combined with GO analysis and literature reports, differential proteins that may be related to the regulation of RhD alloimmunization process were screened, and the signaling pathways that these proteins may be involved in were analyzed.

Result: (1) In the safety study of RhD immunization, the vital signs of twenty-three volunteers were normal after the initial and booster immunization. After first immunization with 20 mL, 30 mL, 40 mL, 50 mL, the safety indexes related to hemolytic blood transfusion adverse reactions in blood routine, liver and kidney function and coagulation routine at 24 hours and 1 week were statistically analyzed, and the results showed no statistical significance (P > 0.05). The differences between the above index values and the last test values before each immunization also showed no significantly difference at 24 hours and 1 week after 0.5 mL、1 mL、2 mL of booster immunizations (P > 0.05).After 1 mL and 2 mL of booster immunization for 24 hours and 1 week respectively, 1 volunteer had an indirect bilirubin elevation near the upper limit of the medical reference value and exceeded the medical reference value range, but no adverse transfusion reactions occurred. No statistically significant differences were observed comparing the conditions of abnormal changes and exceeding the medical reference  value of safety indicators in RhD responders after different doses of booster immunizations (P > 0.05). The immune effect of booster immunization with 2 mL is better than 0.5 mL, with higher antibody titer (P = 0.024). After 1 week of booster immunization, the changes of safety indexes were not statistically different (P > 0.05) in anti-D antibodies titers that meet the requirements of different plasma collection. (2) According to the mass spectrometry, a total of 178 differential proteins were screened (P < 0.05, the absolute value of fold change (|FC|)>1.50), including 76 up-regulated proteins and 102 down-regulated proteins. All the differential proteins mainly involve in chemokine pathways and calcium ion (Ca2+) signaling pathways. The up-regulated proteins mainly involve in RNA splicing pathways, while the down-regulated proteins mainly involve in GTPase activity biological processes. The differential proteins with |FC|>2 and which may be related to the regulation of RhD alloimmunization process were further screened. Among them, PYHIN1, CTNNBL1, DCAF1, ISG20, LYST were significantly up-regulated and TNFAIP8L2 was significantly down-regulated in RhD responders, and these proteins were involved in Toll like receptors (TLRs) signaling pathways, T and B cell activation related signals and chemokine pathways, etc.

Conclusion: (1) The immunization program developed in this study did not find any phenomenon affecting the safety of volunteers, and it is preliminarily believed that the safety of volunteers can be guaranteed. On the premise of safe and effective, it is recommended to optimize the immunization dose to 50 mL for the first immunization and 0.5 mL for the booster immunization, which provides data support for the

development of RhIg in China. (2) Up-regulated proteins of

PYHIN1, CTNNBL1, DCAF1, ISG20, LYST, and down-regulated protein of TNFAIP8L2 may be involved in the regulation of RhD allimmunization response. Among them, TLRs signaling pathway may be positively regulated by PYHIN1 and LYST proteins in the RhD alloimmunitization, and negatively regulated by TNFAIP8L2 protein. Chemokine signaling pathway may be positively regulated by ISG20 protein, T cell activation signals may be positively regulated by DCAF1 protein, and B cell activation signals may be positively regulated by CTNNBL1 protein, which provides scientific supports for further research on RhD alloimmunization mechanisms.