Effects And Mechanism Of PIK3CD Gain Of Function Mutation On ?? T Cell

PART I EFFECTS AND MECHANISM OF PIK3CD GAIN OF FUNCTION MUTATION ON??T CELLBackground:Activated phosphoinositide 3 kinase?syndrome(APDS)is a primary immunodeficiency disease caused by PI3K?overactivation due to the PIK3CD gain of function mutation.Recurrent respiratory infections,lymphoid tissue hyperplasia,autoimmune diseases and tumor susceptibility are the main manifestations.Since its importance in anti-infection and anti-tumor response,whether??T cells are involved in the APDS pathogenesis or not and its mechanism are unknown.Objective:To analyze the effects of PIK3CD gain of function mutation(PIK3CD GOF)on total??T cells and subsets in APDS patients and PIK3CD GOF mice,including the proportion,number,proliferation,activation,apoptosis and tumor killing ability of??T cells,and explore the possible molecular mechanism.Methods:The peripheral blood samples of 16 newly diagnosed APDS patients(c.3063G>A,p.E1021K;heterozygous mutation)who had not been treated with immunosuppressants and steroid were collected.Peripheral blood mononuclear cells were extracted and analyzed by flow cytometry,the proportion and number of??T cells were compared with the reference value of age-matched healthy controls.Meanwhile,the PIK3CD GOF mouse model with homologous mutation site E1021K was constructed,and the number,proportion,subsets(CD27+,CD27-,V?1,V?4),secreted cytokines(IFN-?,IL-17,granzyme B,perforin)and activation markers(CD38,CD69,CD44,CD62L)of??T cells were detected by flow cytometry.We further analyzed the effects of PIK3CD GOF on proliferation and apoptosis of??T cells stimulated by TCR??,and sorted the expanded??T cells to verify the influence on tumor killing ability in vivo and in vitro.Moreover,CD45.1-WT and CD45.1-PIK3CD GOF bone marrow chimera mice models were constructed to verify whether the effects of PIK3CD GOF on??T cells were intrinsic or caused by environmental factors.Combined with the single cell sequencing and bulk RNA sequencing,bioinformatic analysis was used to explore the effects of PIK3CD GOF on??T cells.In addition,the PI3K?inhibitor IC-87114 and m TOR inhibitor rapamycin were applied in vivo and in vitro to detect the effects on??T cells.Results:This study found that although all APDS patients enrolled had recurrent respiratory tract infection,there were still 5 cases and 6 cases who had lower??T cell proportion and number than the normal value in age-matched healthy controls.Compared with WT mice,the PI3K-AKT-m TOR pathway phosphorylation level of PIK3CD GOF??T cells was significantly up-regulated.The proportion of??T cells decreased in the basic state,and because the spleen was significantly larger than that of WT mice,the total number of??T cells was increased,the V?1 and CD27~-??T cells increased,while V?4 and CD27~+??T cells decreased significantly.And the expression of activation markers CD69 and CD38 were increased,and na?ve??T cells were significantly reduced,effector memory??T cells were obviously increased.In addition,the expression of NKG2D and secreted IL-17 in??T cells were significantly reduced,but the perforin and granzyme B were significantly increased.After being stimulated by TCR??,the proliferation of??T cell was significantly inhibited,while apoptosis was significantly increased,and the killing melanoma abilities of??T cells in vivo and in vitro were significantly weakened,the expression of killing-related molecules was abnormal,including decreased expression of NKG2D,and the IFN-?secreting CD27~+??T cell subsets were significantly reduced,although the granzyme B and perforin secreted by the??T cells of PIK3CD GOF mice were also significantly reduced,neither IC-87114 nor rapamycin could increase the levels in vitro.In addition,in the bone marrow chimera mouse model,compared with CD45.1-WT mice,??T cells from CD45.1-PIK3CD GOF mice were still significantly reduced,the expression of CD38 and CD69 were increased,na?ve??T cells were reduced,effector memory??T cells were increased significantly,and the proliferation of stimulated??T cells was still obviously inhibited,the NKG2D expression was decreased significantly after stimulation.Through bioinformatics analysis of single-cell sequencing and general RNA sequencing,we found that differential expressed genes between WT and PIK3CD GOF??T cells were mainly enriched in cell activation,differentiation,tumor,bacterial and viral infection,immune response,rapamycin and telomeric DNA binding pathway.The mice with the injection of rapamycin in vivo obviously reduced the volume of spleen,and down-regulated the S6(Ser235,236)phosphorylation level of PIK3CD GOF??T cells,changed the proportion,total number,na?ve and effector memory??T cells,and there was no significant difference from WT mice injected with rapamycin or saline.The addition of rapamycin or IC-87114 in vitro increased the proportion of??T and CD27~+cells,and decreased CD27~-cells,IC-87114 also reversed the NKG2D expression level.Conclusions:Compared with age-matched healthy controls and WT mice,PIK3CD GOF changed the proportion and number of??T cells in APDS patients and PIK3CD GOF mice.The PI3K-AKT-m TOR phosphorylation level of??T cells was significantly increased in PIK3CD GOF mice.And??T cells were overactivated in the basic state,while the proliferation was inhibited and apoptosis was enhanced under TCR??stimulation.In addition,PIK3CD GOF obviously inhibited the B16F0melanoma-killing ability of??T cells in vivo and vitro,by inhibiting the secretion of perforin and granzyme B,down-regulating NKG2D expression,and reducing CD27~+??T cells,while IC-87114 did not reverse the levels of perforin and granzyme B in vitro,which may be caused by the insufficient time and dose.The effects of PIK3CD gain-of-function mutations on??T cells were intrinsic.Moreover,the bioinformatics analysis of transcriptome sequencing indicated that the??T cell activation,differentiation,anti-tumor,anti-infection,response to rapamycin and telomeric DNA bingding were abnormal in??T cells from PIK3CD GOF mice.the application of rapamycin in vivo and in vitro can respectively reverse the high phosphorylation level of S6(Ser235,236),overactivation in the basic state,the proportion of total and subsets of??T cells after stimulation.PART II EFFECTS AND MECHANISM OF WAS PROTEIN DEFICIENCY ON ATYPICAL MEMORY B CELLSBackground: The Wiskott-Aldrich syndrome(WAS)is a rare X-linked primary immunodeficiency disease caused by WAS gene mutation and WAS protein deficiency,characterized by eczema,thrombocytopenia,recurrent infections,and increased susceptibility to autoimmune diseases and tumors,with extremely poor prognosis.Since its importance in chronic infections and autoimmune diseases,whether atypical memory B cells(a MBCs)are affected by WAS protein deficiency or not and the related mechanisms are unknown.Objective: To analyze the phenotypes and functional changes of atypical memory B cells in peripheral blood of patients with WAS,and to explore the possible mechanisms.Methods: We collected clinical data and gene mutation information of 23 WAS patients,and collected peripheral blood samples of WAS and age-matched healthy controls(HCs),we extracted peripheral mononuclear cells,detected the proportion of a MBCs by flow cytometry,and analyzed the relationship between WAS clinical score and the proportion of a MBCs in mature B cells.The expression level of surface CD11 c,Fc RL5,CXCR5,CXCR3,CD35,CD40,Ig G1,and intracellular T-bet of a MBCs and classical memory B cells(c MBCs)were detected.We also applied phosphorylation flow to detect the expression levels of key molecules p-Syk,p-BLNK,and p-PLC?2 downstream of the BCR signaling pathways in a MBCs and c MBCs from WAS and HCs.In addition,magnetic beads were used to sort B cells,and we detect the proliferation and apoptosis of a MBCs and c MBCs by stimulation of IFN-?+anti-kappa+anti-lambda.Enzyme-linked immunosorbent assay was used to detect the levels of IFN-?,BAFF and APRIL in the plasma of WAS and HCs,and T cells were stimulated with PMA and ionomycin to detect the level of IFN-? secretion.Results: Compared with HCs,the proportion of a MBCs in mature B cells of WAS was significantly increased,and c MBCs were decreased.The proportion of a MBCs in patients with a higher WAS score was higher.In WAS patients,compared with c MBCs,a MBCs have high expression of T-bet,CD11 c,Fc RL5,low expression of CXCR5,CD35,CD40 and Ig G1,and The phosphorylation levels of p-Syk,p-BLNK of a MBCs and Syk,p-BLNK,and p-PLC?2 of T-bet+ a MBCs were decreased significantly.Meanwhile,compared with c MBCs,the expression of ki67 in a MBCs from WAS was lower,and a MBCs had a lower proportion of alive cells,and a higher proportion of early and late apoptotic cells.There was no significant difference in the expression of ki67 in a MBCs between HCs and WAS patients without stimulantion.However,under the stimulation of IFN-?+anti-kappa+anti-lambda,the expression of ki67 in a MBCs from WAS patients was obviously increased compared with HCs,and the combination of BAFF+IFN-?+anti-kappa+anti-lambda stimulation further increased the expression of ki67.In addition,compared with HCs,IFN-? secreted by CD4+ T and CD8+ T cells from WAS patients was increased,the plasma IFN-? and BAFF levels were significantly higher,and the phosphorylation level of p-PLC?2 in a MBCs was significantly up-regulated compared with HCs a MBCs.Conclusions: Compared with HCs,the proportion of a MBCs in the mature B cells of WAS patients was significantly higher,and it was positively correlated with the WAS score.In WAS patients,a MBCs expressed high levels of T-bet,CD11 c,Fc RL5 than c MBCs,while CXCR5,CD35,CD40,and Ig G1 were lower than c MBCs.Compared with c MBCs,a MBCs had lower phosphorylation levels of BCR signaling pathway,decreased proliferation and increased apoptosis.Compared with HCs,WAS a MBCs proliferated more obviously under stimulation of IFN-?+anti-kappa+anti-lambda,and the addition of BAFF can further promote proliferation.Therefore,the reasons for the higher proportion of a MBCs in WAS patients than that in HCs can be: IFN-? secreted by T cells from WAS patients was more than that from HCs;the plasma IFN-? and BAFF levels were significantly increased in WAS;and the BCR phosphorylation level of a MBCs was significantly up-regulated.