| Background:RhD blood group incompatibility between mother and infant can cause severe 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 by keeping 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. |
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