In Primary Immunodeficiency Disease, WAS Protein Regulates The Formation And Function Maintenance Of Memory T And The Study Of Granulocyte Function In Children With SCN

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WASP REGULATE EFFECTOR-TO-MEMORY CONVERSION AND MAINTENANCE OF CD8+T CELL MEMORYEBackground: Wiskott-Aldrich syndrome(WAS)is a very rare X-linked primary immunodeficiency featured by recurrent infections,eczema,micro thrombocytopenia,and an increased incidence of autoimmunity and malignancies.Compromised cellular adaptive immunity is a hallmark of classical WAS.Intriguingly,the incidence of infection increased dramatically with age.Recurrent infection even become the leading cause of death in elder patients.In this study,using WAS KO mice,we investigated how WASp regulates Ag-driven memory CD8+T cells formation and maintenance following acute LCMV infection in mice.Methods: we measured the percentage and kinetics of gp33 specific T cell by Tetramer staining.we evaluated the ability of antigen-specific CD8+T cells from WT and WAS KO mice to produce cytokine upon Ag stimulation ex vivo.We also use adoptive transfer to compare the recall response and protective capacity of the antigen-specific T cells.By using of bone marrow chimeric mice,we want to explore whether these altered aspects are cell intrinsic.Results: 1)Efficient clonal expansion and effector differentiation of WAS-deficient CD8+T cells.WAS deficiency decreased the percentage and absolute number of the CD8+T cells in uninfected mice,but the percentages and total number of CD8+T cells that are specific to the GP33 epitope were comparable on D8 after LCMV infection.WAS GP33 specific CD8+T cells displayed no significance in the expression of T-bet,Ki67 and BCL-2 compared with WT effector CD8+T cells.The IFN-r,CD107 a,Granzyme B producing cells were much higher in KO mice.2)Partial impaired memory CD8+T cell contraction and early memory cell formation in the absence of WAS.At D15 and D30 PI,the frequencies and total numbers of LCMV-specific memory CD8+T cells in WAS mice were comparable to those in WT mice.However,in GP33 specific T cell,the proportion of TCM decreased while the CD95 expression is increased in KO mice.The percentage of epitope-specific cytokine–producing CD8+T cells in WASp KO mice were significantly higher,as compared with those in WT mice Which infers that the CD8+T cell from WASp KO mice are harder to get a quiescent state.3)WASp deficiency limits the size of the memory CD8+T cell compartment and promotes exhaustion T cell generation.On D60 and D100,the percentage of GP33-specific CD8+T cells decreased in WAS deficiency mice after D60 and both low percentages and numbers of GP33 specific memory CD8+T cells were maintained in WAS deficiency mice until day 200 PI and later time point.WASp deficient mice exhibit a substantial increase in the percentage of IFN-r,TNF-a,CD107 a,and Granzyme B upon stimulation with the cognate antigenic peptide at indicated time.The GP33-specific CD8+T cell in WT and KO exhibit comparable Ki 67 and BCL-2 expression.Meanwhile,the percentage of exhaustion T cells in LCMV GP33 specific CD8+T cells increased dramatically in KO mice.4)Wasp didn't alter the development of CD8+T cell while accelerated antigen specific CD8+ T cells consumption via the extrinsic pathway.WT and KO mice had comparable numbers of total thymocytes as well as CD8+ single-positive subsets and had similar expression of thymocyte-maturation markers CD62 L,CD69.there are consistently more CD95 and CD178(FASL)positive cells in LCMV GP33 specific CD8+T cells in KO mice at different time point.the percentage of live cells in GP33 specific CD8 T cell is significantly higher while the proportion of apoptotic cells is consistently lower in WT than that in KO mice after GP33-41 peptide stimulation.5)Secondary CD8+T cell responses and protective immunity is impaired in WAS mice.Equal numbers of CD8+GP33+T cells were transferred into naive congenic recipients(CD45.1)and challenged the next day with Lm-GP33.Five days post challenge,we found a significant decrease in expansion and weaken protective capacity in KO mice.6)WASp regulates CD8+T cell memory by T cell intrinsic mechanisms.In chimeric mice,60 days post infection,KO mice exhibited a significant decrease in the percentage and number of LCMV-specific CD8+T cells,simultaneously,the cytokine producing CD8+ T cell is consistently higher in KO mice than that of the WT mice.These results clearly demonstrated the intrinsic role of WASp in the formation of the memory CD8.Conclusion: WASp acts as a key regulator of functional CD8+T cell memory.PART ? TWO PATERNAL MOSAICISM OF MUTATION IN ELANE CAUSING SEVERE CONGENITAL NEUTROPENIA EXHIBIT NORMAL NEUTROPHIL MORPHOLOGY AND ROS PRODUCITONBackground: Severe congenital neutropenia(SCN)encompasses a family of neutropenic disorders that can cause life-threatening pyogenic infections,acute gingivostomatitis,and chronic periodontal disease,Mutations in the ELANE gene encoding neutrophil elastase are the most common cause.which occur in about 40%–55% of patients with congenital neutropenia.To date,more than 200 different ELANE mutations have been identified;however,mosaicism of an ELANE mutation has been described in only four families with SCN.no mosaicism of an ELANE mutation has been reported in a Chinese population.The mosaic father is asymptomatic;however,it is not known whether granulocytes harbored by the mosaic father of an affected child are immunocompetent.We described in detail the clinical and laboratory findings of two Chinese patients harboring a novel mutation that extends the phenotype and functional spectrum of ELANE deficiency.Methods: PBMC and granulocytes were separated,Sanger sequencing and TA cloning were performed.NADPH oxidase activity was measured by luminol-enhanced chemiluminescence;elastase expression was acquired under a confocal microscope.NADPH oxidase components were measured by western blot.Results: Two patient harbor a pathogenic mutation,analysis of granulocytes isolated from the fathers revealed no genetic mutations.DNA extracted from fractionated peripheral blood mononuclear cells(PBMCs)and fingernails obtained from both fathers did harbor the mutation,suggesting mosaicism.Granulocytes isolated from the patients displayed significantly weaker ionomycin-induced intracellular reactive oxygen species(ROS)responses than those isolated from the fathers.Both patients showed increased expression of neutrophil elastase,whereas the mosaic fathers showed normal expression.Both the patients and their fathers showed normal expression of P22 phox,P40phox,P47 phox,P67phox,gp91 phox.Conclusion: Besides the number reduction,the function of the granulocytes in ELANE patients were also immunocompromised,whereas those from the mosaic fathers are normal.These findings may provide new insight into disease diagnosis,prognosis,therapy and genetic counseling.