The Role Of CD4~+CD25~+ Regulatory T Cells In Immune Tolerance Induced By Avain Leukosis Virus Subgroup J

ALV-J-induced chicken immune tolerance is a prerequisite for tumorigenesis.However,the causes and pathogenesis of the virus-induced immune tolerance are not fully understood.Previous studies have shown that ALV-J can cause severe depletion of lymphocytes in immune organs,suggesting that T and B lymphocytes have been severely damaged and suppressed,and that immunosuppressive effects dominate the body.Moreover,a large number of studies have shown that CD4~+CD25~+Tregs play an important regulatory role in maintaining the body's immune tolerance and participate in the development of a variety of chronic infectious diseases.So,are Tregs involved in ALV-J-induced immune tolerance,and by what means?Therefore,in order to analyze the role of Tregs in ALV-J-induced immune tolerance,this study established an ALV-J immune tolerance model and assessed the impairment of immune response ability from multiple perspectives.Using molecular biology techniques,we systematically studied the dynamic changes in the frequency of CD4~+CD25~+Tregs under ALV-J congenital infection,and its relationship with cytokines and viral load,in order to reveal the role of CD4~+CD25~+Tregs in the immune tolerance induced by ALV-J.In order to simulate ALV-J-induced persistent immune tolerance in the body,an animal model of congenital infection was established by injecting ALV-J into the allantoic cavity of 6embryos.The ELISA and qPCR methods were used to detect that the chickens were continuously toxic and did not produce specific antibodies.Evaluation of the immune organ damage in chickens was performed by gross necropsy observation,immune index detection,and histopathological observation.Gross necropsy results showed that the thymus,bursa,and spleen of the ALV-J-infected chickens were dysplastic,showing a smaller volume and a significantly lower immune index.Histopathological examination showed that there were more interstitial connective tissues in the thymus and bursa of chickens in the infected group,while the number of lymphocytes was less than in the normal group,suggesting that lymphocyte damage was severe after ALV-J infection.In order to further explore the damage of lymphocytes by ALV-J,the changes in the number and function of B cells in the spleen were detected by IF and IHC methods.The results showed that after ALV-J infection,the number of B cells decreased significantly,the number of IgM~+and IgG~+cells decreased significantly,and LPS could not stimulate B cell activation.In order to investigate the changes of immune function of T cells after ALV-J infection,the changes of the proportion of CD4~+,CD8~+T cells subsets and cytokine expression in chickens were detected by FCM and qPCR methods.FCM results showed that the proportion of CD4~+T cell subsets in immune organs and blood decreased significantly after ALV-J infection,especially at 7 days of age.The proportion of CD8~+T cell subpopulations did not show a significant and continuous decrease.The qPCR results showed that after ALV-J infection,the expression of IL-2 and IL-4continued to decrease,IFN-?expression increased and decreased in the early stage,and there was almost no difference with the control group at 45 days of age.IL-10 expression continued to increase.In contrast,IL-10 expression continued to increase.The above results suggest that ALV-J inhibits the proliferation and function of T cells,the CD4~+T cells are more severely inhibited,and the body's immune suppressive function is dominant.In order to analyze the role of CD4~+CD25~+Tregs in ALV-J-induced immune tolerance,FCM was used to continuously monitor the changes of CD4~+CD25~+Tregs frequency in immune organs and blood of 7-60 days old chickens.The results showed that the frequency of Tregs in blood and immune organs increased significantly after ALV-J infection,especially in the young age(7-15 days of age).As the age increases,the frequency of Tregs fluctuates in certain immune organs.CD4~+CD25~+Tregs subgroups were further selected by FCM,and the transcription levels of IL-10,TGF-?,CTLA-4 in cells were detected by qPCR.The results showed that IL-10,TGF-?,and CTLA-4 in the thymus,bursa,and spleen all increased significantly,and the change pattern was consistent with the change trend of the frequency of CD4~+CD25~+Tregs.However,the change trend of Tregs frequency of the cecum tonsil was only consistent with the increase of the transcription level of IL-10,and the transcription levels of TGF-?and CTLA-4 only increased at certain ages.The above results suggest that ALV-J infection leads to continuous proliferation and activation of CD4~+CD25~+Tregs in immune organs and blood,and exerts immunosuppressive functions by secreting factors such as IL-10,TGF-?,CTLA-4.And the ability of Tregs to proliferate is different in different immune organs.In order to further investigate whether the difference in Tregs immune function in different immune organs is related to the virus,dynamic monitoring of ALV-J virus load in immune organs by qPCR.The results showed that the trend of ALV-J viral load change was positively correlated with Tregs frequency and function change.Moreover,the viral load of cecum tonsil with lower immune suppressive function of Tregs was lower than that of other immune organs.The above phenomena suggest that ALV-J can induce the proliferation of Tregs in the body,but the activation of Tregs function needs to reach a certain viral load.Also suggested that ALV-J can be copied in Tregs.To verify the inference,the expression of ALV-J in CD4~+CD25~+Tregs was detected by FCM.The results showed that about 50%of Tregs surface expressed the SU protein of ALV-J.This further proves that ALV-J can be replicated in Tregs,that is,ALV-J can directly act on Tregs.In summary,the continuous proliferation and activation of CD4~+CD25~+Tregs in vivo is an important reason for ALV-J to induce persistent immune tolerance.Tregs of immune organs such as the thymus,spleen,and bursa of bursa play important immunosuppressive functions.ALV-J can directly affect Tregs and induce a large number of Tregs in the body,but the activation of Tregs functions needs to reach a certain viral load.However,the molecular mechanism of the interaction between ALV-J and Tregs needs to be further studied.