Study Of The Construction Of Immunodeficient Pigs And Maternal-fetal Microchimerism

For a long time,it was generally believed that due to the existence of the placental barrier during pregnancy,only nutrients could be exchanged between mother and fetus,but no cell transmission could be carried out.Studies in recent years have found that maternal cells can enter the fetus during pregnancy and persist into adulthood,called maternal microchimerism.At the same time,fetal cells can also enter the mother,which is considered fetal microchimerism.Maternal microchimerism is more likely to occur in immunodeficient patients and greatly affects the prenatal diagnosis of immunodeficiency.The physiological structure and organ size of pigs are very similar to those of humans.As a large animal model to study human diseases,the pig has attracted more and more attention,and the most valuable animal model is the immunodeficient pig.However,whether the immunodeficient pigs carry the maternal cells at birth and whether the maternal cells can affect the subsequent application of the immunodeficient pigs have not been reported.Therefore,a series of studies on maternal microchimerism in immunodeficient pigs were conducted in this study.It has been found that maternal microchimerism widely exists in immunodeficient pigs,and the cells in which microchimerism occurs are mainly T cells.Through the TCR-seq analysis,we found that most of the maternal T cells can enter the piglets during pregnancy,with no clonal selectivity but limited proliferative capacity.Subsequently,fetal microchimerism was also studied and GFP fetal cells were detected in recipient sows after the GFP pigs were cloned.We found that fetal cells can also pass through the placenta into the mother during pregnancy.The main results are as follows:1.In this study,we established two types of immunodeficient pigs using CRISPR/Cas9 gene editing technology and animal cloning technology.First,we produced IL2RG knockout pigs that exhibited certain symptoms of immunodeficiency.Compared with wild-type pigs,the thymus of IL2RG^-/Y piglets was structurally abnormal,and thymic corpuscles were poorly developed.In addition,the spleens of IL2RG^-/Y piglets developed dysplasia,which was mainly manifested as dysplasia of lymphatic sheaths around the central artery of the spleen and a significant reduction in the number of splenic lymphocytes.The proportion of T cells,B cells and NK cells in the peripheral blood and spleen of the IL2RG^-/Y pig were lower than that of the wild-type pig.The IL2RG^-/Ypigs were able to support the growth of human melanoma cells.At the same time,we also established immunodeficient pigs with more severe symptoms of immunodeficiency,which were deficient in RAG1 and IL2RG.V（D）J rearrangement could not be completed after the knockout of the RAG1 gene.The number of lymphocytes in the thymus of this immunodeficient pig was significantly reduced,and the number of epithelioid cells was increased.The boundary between the cortex and medulla was not clear,and no thymic bodies were observed.The spleen of this pig showed indistinct boundaries of red and white pulp,diffuse lymphocytes,and no obvious periarterial lymphatic sheath structure.Immunohistochemistry also showed the absence of CD3⁺T cells in the thymus and spleen;Flow cytometry analysis showed that T cells,B cells,and NK cells in the peripheral blood of the immunodeficient pig were significantly reduced.2.The present study investigated the presence of maternal microchimerism in RAG1 and IL2RG knockout immunodeficient pigs.By fluorescence quantification,we found maternal microchimerism in 80%of cord blood and 52.94%of the spleen,with chimerism frequencies ranging from 0.05 to 1.85%and 0.04 to 2.21%,respectively.Furthermore,we found that the spleen was the organ most easily detected for maternal microchimerism.We found that the cells entering the piglet through the placental barrier during pregnancy were mainly composed of T cells,which were distributed in multiple organs except for the thymus,and the number of T cells in the spleen was the highest.We found that these maternal T cells had completed the V（D）J rearrangement and further demonstrated that they developed and matured in the thymus of the sow.Through TCR-seq analysis,we found that most types of T cells can cross the placental barrier and enter the fetus during pregnancy,but their proliferation capacity is limited.Under the stimulation of piglet antigen,only a few types of T cells can significantly proliferate.Combined with RNA-seq analysis,we found that the highly expressed genes in the peripheral blood of immunodeficient pigs with maternal microchimerism could be enriched in the pathways related to lymphocyte chemotaxis,lymphocyte migration,myeloid cell migration,and granulocyte migration,suggesting that in addition to T cells,other types of cells such as granulocytes and myeloid cells could also enter the fetus.3.Studies in humans and mice have shown that this transfer from the placenta during pregnancy is bidirectional.Therefore,we investigated fetal microchimerism in pigs during the production of GFP piglets.We detected GFP fetal cells in the blood of recipient sows bearing GFP piglets.By flow cytometry analysis,we found that peripheral blood contained 0.043%GFP fetal cells,of which 88.3%were CD3⁺T cells.Furthermore,we found that fetal-derived GFP cells were mainly distributed in immune organs such as the spleen and lymph nodes,a small amount of GFP cells were observed in the lung,liver,intestine,brain and heart,but no obvious GFP cells were observed in the kidney.In conclusion,we obtained immunodeficient pigs and GFP pigs using CRISPR/Cas9 gene editing technology and animal cloning technology and carried out a systematic study on the maternal-fetal microchimerism of pigs.The phenomenon of maternal microchimerism was discovered in immunodeficient pigs for the first time,which provided an important reference for the application of immunodeficient pigs and the construction of humanized pigs.Piglet GFP cells were found in surrogate recipient sows,indicating that cell migration between mother and fetus is bidirectional during pregnancy,suggesting that pigs can be used as a large animal model to study the problem of maternal-fetal microchimerism.