Molecular And Phenotypic Characterization Of STAT1 Gene Mutations

PART ? MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF PATIENTS WITH STAT1 GENE MUTATIONSBackground:Signal transducer and activator of transcription 1(STAT1)is a transcription factor that mediates cellular responses to interferons(IFNs)and other cytokines and growth factors in diverse cell types.STAT1 gain-of-function(GOF)mutations result in an unexpectedly wide range of clinical features.And STAT1 loss-of-function(LOF)mutations cause Mendelian susceptibility to mycobacterial diseases(MSMD).It remains unclear why STAT1 GOF mutations result in such a broad spectrum of phenotypes.Moreover,some new mutations we have found may extended the phenotype of STAT1 activity and STAT1deficiency.Sometimes there is crossover with AD STAT1 deficiency,which is difficult to identify.These two diseases are extremely rare,especially STAT1 LOF cases.Up to now,no more than 20 cases have been reported.It is very necessary to use precious clinical resources to improve the understanding of clinical phenotypes and study their immune pathogenesis,so as to obtain first-hand information from human diseases.Methods:We analyzed the clinical,molecular,and phenotypic characteristics of eleven patients with STAT1 GOF mutations and five patients with STAT1 LOF mutations.Results:This study enrolled eleven patients with STAT1 GOF mutations and five patients with STAT1 LOF mutations including nine novel mutations.In our study we found that patients with STAT1 GOF mutations had defects in both innate and adaptive immunity,including impaired T cell receptor(TCR)diversity;reduced numbers of na(?)ve and effector memory CD4~+T cells,memory B cells,NK cells;and defects in the production of IL-17A and IFN-?.In addition,experiments with primary immune cells revealed that enhanced STAT1 phosphorylation resulted from not only lower rates of STAT1 dephosphorylation but also increased total STAT1 expression.We also found that AD STAT1 deficiency causes not only mycobacterial infection,but also Nocardia farcinica and congenital multiple malformations.Consistently,we showed that these mutations impaired IFN-?mediated STAT1 phosphorylation,GAS and ISRE transcription activity and IFN induced gene expression,which might contribute to the clinical manifestations in these patients.Conclusion:Our report provides the first comprehensive overview of the molecular genetics,clinical heterogeneity,and underlying immunological abnormalities of patients with STAT1 GOF mutations and STAT1 LOF mutations in China.The diversity of infections and immunological phenotypes in our patients suggests the important roles of STAT1 in host-pathogen interaction and immunity.PART ? THE DEVELOPMENT AND FUNCTION OF REGULATORY T CELLS IN PIK3 CD GOF MICEBackground: The main clinical manifestations of patient with Activated PI3K-? Syndrome are recurrent respiratory tract infections,non-neoplastic lymphocytic hyperplasia,autoimmune autoinflammatory disease,and lymphoma.The immunological phenotype was mainly manifested as thymus output dysfunction,CD4+ T cells decreased,and T cells were in a state of abnormal activation and depletion.Treg cells can inhibit the abnormal activation of immune cells and maintain immune homeostasis of the body.Previous studies have shown that PI3 K overactivation can activated downstream of Akt and mTOR,affecting cell differentiation,proliferation,apoptosis and metabolism.It has also been reported that mTOR overactivation can damage the stability of Treg cells,which is mainly exhibited transient or unstable expression of Foxp3,express an activated-effector T cell phenotype and produced inflammatory cytokines,had a phenotype with down-regulation of CD25 and overexpression of CD127.Therefore,we speculate that the imbalance of immune homeostasis in APDS1 patients is related to the impaired Treg suppression function,but the specific mechanism has not yet been clarified.Methods: In this study,the proportion of Treg cell subsets in eight APDS1 patients was included and analyzed,and PIK3 CD GOF mouse model was constructed according to the hot spot mutation E1021 K which account for approximately 85% of reported cases worldwide in APDS1 patients.The mouse model was used to detect the development of Treg cells in the thymus,differentiation and supression of Treg cells in peripheral lymphatic organs.Finally,mice were intraperitoneally injected with Rapamycin to observe the therapeutic effect on the differentiation and function of Treg cells.Results: The results show that 1)Compared with the normal control,the Resting Treg of APDS1 patients decreased significantly while the Nonsuppressive Treg increased;moreover,compared with the normal control,the proportion of CD25+Foxp3+ Treg cells in APDS1 patients decreased significantly,while the proportion of CD25-Foxp3+ Treg cells increased;2)Like the patients with APDS1,PIK3 CD GOF mice also had lymphocyte proliferation and splenectasis;3)The proportion of CD4 SP cells in thymus of PIK3 CD GOF mice was significantly lower than that of WT mice,and the proportion of CD25+Foxp3-Treg and CD25-Foxp3+Treg precursors was also significantly decreased,as was the proportion of mature CD25+Foxp3+Treg cells.4)Compared with WT mice,the proportion of Treg cells in spleen of PIK3 CD GOF mice was significantly increased,while the proportion of iTreg cells was significantly decreased.The ability of CD4 naive T cells to differentiate into iTreg cells in vitro was significantly impaired.5)PIK3CD GOF mutation can lead to impaired supression function of Treg cells,thus causing Th1-type inflammatory response in mice;6)The proportion of Treg cells expressing Ki67+,CD44+CD62L-and CTLA-4,ICOS,PD-1 and LAG3 were significantly increased in PIK3 CD GOF mice.7)Abnormal differentiation and function of Treg cells were partially reversed after intraperitonal injection of Rapamycin.Conclusion: The Treg subsets of APDS1 patients emerges out of the proportion;PIK3CD GOF mice showed lymphocytic hyperplasia;In PIK3 CD GOF mice,the development,differentiation,suppression function of Treg cells was impaired;and Treg cells exhibited hyperproliferation, hyperactivation and tended to be exhausted;Rapamycin could partially reverse the abnormal differentiation of Treg cells and improve the expression of lymphocyte hyperplasia in PIK3 CD GOF mice.