Studyon T Cell Senescence Caused By Gain-of-function In PIK3CD Gene And Clinical And Immunological Phenotypes Of Five TYK2-Deficiency Patients

PART ? STUDY ON T CELL SENESCENCECAUSED BY GAIN-OF-FUNCTION IN PIK3CD GENEBackground and purpose:Activated phosphoinositide 3-kinase?syndrome,APDS is a primary immune deficiency disease caused by the increased function of PI3K? catalytic subunit P110?(PIK3CD)or regulatory subunit p85?(PIK3R1).The main clinical manifestations are:repeated respiratory tract infection,hepatosplenomegaly,autoimmunity,increased tumor susceptibility,etc.The disease was first reported in 2013.So far,more than 200 cases have been found,involving dozens of mutation genes.Among them,the E1021K mutation of PIK3CD gene is a spot mutation.Cell senescence refers to cell cycle arrest and increased apoptosis resistance while maintaining cell viability and metabolic activity.It is characterized by telomere shortening,decreased telomerase activity,secretion of ?galactosidase,increased expression of p16^INK4a,p21 and p53,and senescence related secretory phenotypes.It is mainly divided into replicative senescence with telomere shortening and stress senescence without telomere shortening.In recent years,studies have found that T cells,especially CD8⁺T cells,in patients with APDS present a state of premature senescence.Senescence T cells may lead to immune dysfunction and autoimmunity.Although some studies believe that this phenomenon may be related to the increased glycolysis process induced by excessive activation of PI3K-Akt-mTOR pathway in patients with APDS,the specific mechanism is still unclear.In this study,we used the APDS patients in our hospital and the previously established APDS mouse mutation model(PIK3CD GOF mice)to explore the phenotype of premature senescence of T cells in APDS patients,the effect on immune system function and the possible mechanism involved.Methods:the changes of different subsets of CD4⁺CD8⁺T cells,the expression of CD57/KLRG1,the activation status of T cells,the cell cycle,the proliferation,apoptosis and cytokine secretion of senescence T cells were detected by flow cytometry in APDS patients and corresponding mouse models.The expression of cell cycle related molecules was detected by Western blotting and QPCR.Finally,single cell sequencing was performed to explore the key molecules involved in T cell premature senescence in APDS patients.Results:the percentage of CD4⁺T cells was declined,while the percentage of CD8⁺T cells was elevated,the ratio of CD4/CD8 was inverted,the proportion of T_na(?)ve cell decreased,the proportion of T_EM cells increased significantly,the proportion of T_EMRA cells increased significantly,the expression of CD57 and related transcription factor T-bet increased significantly,the expression of CD38 and HLA-DR on the surface of T cells increased,and the telomere length decreased significantly.Compared with WT mice of with the same age and sex,PIK3CD GOF mice had decreased T_na(?)ve cell,the proportion of T_EM cells was increased significantly,the expression of KLGR1 and T-bet was significantly increased.The proliferation of senescence T cells was decreased,apoptosis was increased,and the secretion of IFN-?,IL-2,TNF-? and perforin was decreased.The telomere length of CD8⁺T cells was lower than that of WT mice,the proportion of cells in the division phase increased,the expression of cell cycle related positive regulatory molecules increased,and the expression of negative regulatory molecules decreased.After rapamycin treatment,the proportion of T_na(?)ve cell increased,the proportion of T_EM cells decreased,and the proportion of CD4⁺CD69⁺and CD8⁺CD69⁺decreased the proportion of CD4⁺CD27^-CD28^-KLRG1⁺and CD8⁺CD27^-CD28^-KLRG1⁺decreased.Conclusion:T lymphocytes from APDS patients and corresponding mouse models show replicative senescence phenotype,especially CD8⁺ T cells.The function of secreting cytokines and proliferating ability of senescent cells are decreased,and the mutation leads to the over activation of PI3K-Akt-mTOR pathway,which further causes the disorder of T cell cycle,which may be the primary cause of replicative senescence with telomere shortening in APDS patients.PART ? CLINICAL AND IMMUNOLOGICAL PHENOTYPES OF FIVE TYK2-DEFICIENCY PATIENTSBackground and purpose:Tyrosine kinase 2(Tyk2)is a member of the non-receptor tyrosine kinase(JAK)family,which also includes JAK1,JAK2 and JAK3.JAKs are widely involved in cell proliferation,differentiation,apoptosis and inflammation through JAK-STAT pathway.TYK2-deficiency is a rare primary immunodeficiency disease caused by the deletion mutation of TYK2 gene.It was first reported in 2006.The patients mainly manifested as repeated bacterial,fungal,viral infections,and high IgE syndrome(HIES).Further study found that the lymphocyte response to IFN-?/?,IL-6,IL-10,IL-12 and IL-23 pathway was weakened,which may be one of the reasons for the occurrence of intracellular bacteria such as Mycobacterium tuberculosis,Salmonella infection and virus infection,and the damage of IL-6 pathway may be the reason for the occurrence of HIES.At first,TYK2-deficiency was considered as a subtype of HIES.However,with the increase of the number of cases,only 2 of the 13 patients reported in the world had HIES.Therefore,it is still unclear whether TYK2deficiency can lead to HIES and the mechanism of high level of IgE.As a rare disease,we lack sufficient understanding of the exact role of TYK2 in the human immune system and the clinical symptoms.Five patients with TYK2-deficiency were diagnosed and treated in our center.The main symptoms were recurrent sinus and lung infection,eczema,eosinophilia,with or without HIES.We evaluated and studied the clinical phenotype and immune cell function of the patients.Methods:to extract the peripheral blood mononuclear cells,DNA and RNA,to verify the mutation by Sanger sequencing,to detect the expression of TYK2 protein by Western blot,to detect the cellular response of IFN-a/?/?,IL-6,IL-10,IL-12 and IL-23 cytokines;to detect the expression of mRNA and the expression of transcription factors downstream of type I IFN by QPCR.By flow cytometry,we detect the expression of T cells and B cells subsets,lymphocyte proliferation,T-lymphocyte cytokines secretion and NK cell function.The TCR receptor diversity was detected by PCR.Results:There were 8 mutations of TYK2 gene in 5 patients,none of them were reported;TYK2 protein expression in peripheral blood mononuclear cells decreased or disappeared in 4 of 5 patients;TYK2 protein expression in 3 patients was significantly reduced or disappeared.The expression of mRNA was decreased in 3 patients;2 patients had impaired response to IFN-I pathway,1 patient had impaired response to IL-6 pathway,2 patients had impaired IL-10 pathway response,2 patients had impaired IL12 and IL-23 pathway response;T-lymphocyte secretion of IL-17 decreased in 2 patients was decreased;NK cell function in 3 patients was not significantly changed;B cell subsets in 1 patient were abnormal;lymphocyte proliferation in 2 patients was normal and the diversity of TCR receptor was normal.Conclusion:Five cases of TYK2 deficiency were described in this study,all of them showed recurrent bacterial or viral infection,with or without HIES.Five patients carried 8 new mutation sites,which were confirmed by Sanger sequencing.The pathogenicity of TYK2 was confirmed by QPCR and Western blot.The patients' peripheral blood mononuclear cells showed heterogeneous response to IFN-?/?/?,IL-6,IL10,IL-12 and IL-23,and the homeostasis of patients' lymphocytes was destroyed.