Metabolic Mechanisms Of Hyperactivation Of T Cell Caused By Gain-of-Function Mutation In PIK3CD Gene

PART ONE ALTERED HOMEOSTASIS OF T CELL COMPARTMENT IN PATIENTS WITH GAIN OF FUNCTION MUTATION IN PIK3CD GENEObjective:To determine the effects of PIK3CD GOF mutations on the differentiation and homeostasis of T cells,the T cell compartment in peripheral blood of patients with APDS were systemically evaluated.Methods:From December 2013 to September 2018,a total of 25 patients with APDS from 22 unrelated Chinese families were enrolled in the present study.Age and gender-matched healthy controls(n=50)were recruited from subjects who took routine medical check-ups.The basal levels of phosphorylated AKT(Ser473)and ribosomal S6 protein,a major downstream mediator of mTOR signaling,in T cells from patients with APDS were determined.In addition,the T cell subsets in peripheral blood of patients with APDS were systemically evaluated with flow cytometry.Furthermore,the activation,exhaustion and senescence of T cells,especially for CD8+T cells,were also evaluated.Results:T cells from patients with APDS had increased basal levels of phosphorylated AKT(Ser473)and ribosomal S6 protein,and they were constantly higher than that in controls after stimulation with anti-CD3 and anti-CD28,suggesting hyperactive PI3K-mTOR signaling.Despite the similar percentages of total T cells in APDS patients and age-matched healthy controls,all patients exhibited obvious peripheral lymphopenia and reduced T cell numbers.APDS patients displayed decreased frequencies of CD4+T cells and expanded CD8+T cells,thereby yielding reversed CD4/CD8 ratio.In addition,in both CD4+and CD8+T lymphocyte lineages from patients,the naive T cells(TNaive)were severely decreased with a corresponding increase in central memory T cells(TCM),effector memory T cells(TEM)and CD45RA+effector memory(TEMRA)T cells.The circulating follicular helper T cells(cTFH)cells and Th1 were increased in abundance while the Th17 subsets were markedly reduced in APDS patients compared to healthy controls.Moreover,the expression of HLA-DR,CD38,NKG2D and PD-1 which expressed in activated T cell,was higher in T cells from APDS patients than that in health controls.However,patients with APDS in our cohorts exhibited a lower expression of CCR7 and CD62L in T cells than that of controls.Further,the frequency of CD57+in T cells of APDS was also higher than that of controls.Conclusion:T cells from patients with PIK3CD GOF displayed an activeted phenotype and CD4+T cells in patients were biased to differentiation into Th1 and cTfh subsets.PART TWO EFFECTS OF PIK3CD GOF ON THE DEVELOPMENT,DIFFERENTIATION AND FUNCTION OF T CELLSObjective:To further explore how activated mutations in the PIK3CD impact the differentiation and functions of T cells,the development,differentiation,proliferation,and survival of T cells in mice were determined.Methods:A mouse model with CRISPR/Cas9 gene targeting strategy in C57BL/6J mouse embryos to introduce a heterozygous E1024K mutation in PIK3CD gene,which equivalents to the hot spot substitution in PI3Kδ(E102IK)in patients with APDS were generated.The signaling pathways downstream of PI3Kδ in T cells from WT or PIK3CD GOF mice were determined by western blot.The T cells development in the thymus and the distribution of various T cell subsets in the periphery of PIK3CD GOF mice were also evaluated.In addition,the proliferation,activation,survival and functional status of T cells were also evaluated in mice.Results:The mouse model were successfully developed,and all mice were genotyped by PCR for genomic DNA and confirmed by sanger sequencing.T cells from PIK3CD GOF mice had higher basal and TCR-induced activity of PI3K-AKT-FoxO1 and mTOR than did wild-type.Before and following TCR stimulation with anti-CD3/CD28 antibodies,the expressions of p110δ and p85α in T cells from mutant mice(PIK3CD GOF)were comparable to that of the wild-type cells,as was the levels of the other class IA PI3Ks members(p110α,p100β and p110γ)Moreover,the interaction between mutant p110δ and p85α was not impaired.Among the T cell compartments in spleen and mesenteric lymph nodes,PIK3CD GOF mice had a significant decrease in the percentage of CD62LhiCD441o naive T cells compared with control mice,and a correspondingly expanded proportion of CD44hi subset with an activated or memory phenotype.This alteration was further aggravated in aged mice.In addition,both the CD4+and CD8+ T cell populations from PIK3CD GOF mice displayed a much higher expressions of activation markers CD25,CD44,and CD69 in both young and aged mice than that of their wild-type littermates.Interestingly,the expression of CD44 was obviously elevated in the naive T cells of PIK3CD mice.The altered peripheral T cell populations in PIK3CD GOF mice might not be fully cell intrinsic evaluated by bone marrow chimeria model.PIK3CD GOF mice had greater frequency of Ki67+ than wild-type T cells.Likewise,an elevated proportion of splenic T cells from mutant mice incorporated more BrdU,suggesting that PIK3CD GOF promoted T cells to exit from cellular quiescent state.CD4+TNaive cells from PIK3CD GOF mice had an enhanced differentiation towards Th1 lineages accompanied with increased expression of transcriptional factor T-bet compared with littermate controls.However,PIK3CD GOF T cells were less effective in towards Th17 cells,compared to WT cells.Either CD4+or CD8+ T cells from PIK3CD GOF mice were enriched for IFN-y-producing cells following stimulation wth PMA and ionomycin.After TCR stimulation,PIK3CD GOF T cells displayed elevated IFN-y production relative to control T cells Likewise,greater proportion of IFN-y upon TCR activation were detected in TNaive from mutant mice relative to littermate controls.After TCR stimulation,the expression of active markers,including CD25,CD44 and CD69,were also markedly increased compared to wide-type littermates.Conclusion:PIK3CD GOF in mice also leads to overactivated PI3K/AKT as well as mTOR signaling pathway.PIK3CD GOF promots the naive T to acquire the activated functional status and predisposes them to overactivation in response to TCR stimuli.PIK3CD GOF drives CD4+TNaive cells biased differentiation into Th1 subsets.PART THREE PIK3CD GOF PROMOTES T CELL PROLIFERATION,OVERACTIVATION AND CYTOKINES PRODUCTION VIA MTOR KINASEObjective:To determine whether PIK3CD GOF promotes activation and proliferation of naive T cell is via mTOR pathway.Methods:Pan-naive T cells in spleen from mice were isolated,and were activated with plate-coated anti-CD3 and anti-CD28 for the indicated time.For specifically chemical inhibitor treatment,cells were incubated with IC87114,rapamycin or DMSO as vehicle before being stimulated.After stimulation,the activation,proliferation and functional status of T cells were determined.For in vivo study,6 weeks old WT and PIK3CD GOF mice were daily delivered vehicle or rapamycin by intraperitoneal injection for 15 days.After treatment,the activation,proliferation and differentiation of T cells were also determined.Results:Similar to the selective PI3Kδ inhibitor IC-87114,mTOR inhibitor rapamycin completely abolished the elevated activation,proliferation and production of IFN-γ induced by TCR ligation in PIK3CD GOF T cells as well as wide type.These results not only confirmed that mTOR and PI3Kδ play important role in T cells activation,but further demonstrated that PIK3CD GOF promoted T cell activation via mTOR pathway in vitro.Compared with the vehicle-treated group,the enlarged spleen and increased numbers of total splenocytes in PIK3CD GOF mice were obviously decreased and even restored to the same levels as wide-type mice after treatment with rapamycin.Rapamycin blocked the enhanced proliferation of PIK3CD GOF T cells in vivo.Moreover,the expanded activated/memory T cells in the peripheral spleen were significantly reduced,while the proportion of naive T cells was correspondingly increased after rapamycin treatment in PIK3CD GOF mice.Consistently,rapamycin treatment lowered levels of activation markers CD25,CD44 and CD69 in T cells of mutant mice,and also the elevated expression of CD44 in Tnaive from PIK3CD GOF mice was also decreased to control levels.Furthermore,the enriched percentage of IFN-y-producing cells in CD4+or CD8+T cells in mutant mice were also substantially decreased following treatment with rapamycin in vivo.Conclusions:These results illustrate that inhibition of mTOR fully reverse the abnormal T cell homeostasis in PIK3CD GOF mice,and that PIK3CD GOF promotes T cell proliferation,overactivation and cytokines production via mTOR kinase.PART FOUR EFFECTS OF PIK3CD GOF ON THE PROFILES OF MRNA EXPRESSIONS IN NAIVE T CELLObjectives:To further explore the molecular mechanisms by which PIK3CD GOF resulted in exiting of quiescence and also promoting the hyperactivation of peripheral T cells,the RNA-Seq for CD4+ TNaive were detected and analyzed.Methods:Total RNA from isolated naive CD4+T cells from spleen of PIK3CD GOF mice and littermate wide-type mice was extracted.The library construction and sequencing were performed at Shanghai Biotechnology Corporation.Sequenced raw reads were preprocessed by removing rRNA reads,sequencing adapters,short-fragment reads and other low-quality reads.Obtained clean reads were mapped to the mouse mm10 reference genome with two mismatches.Differentially expressed genes were identified with the following filter criteria:FDR≤0.05 and fold-change≥2.Functional analysis was conducted with DAVID Bioinformatics Resources as described.In addition,the data were analyzed using Ingenuity Pathway Analysis to further identify biological processes enriched for differentially expressed genes.Results:Compared to expression profiles from WT,there were 2307 genes differentially expressed in mutant naive T cells,of which 1749 were increased and 558 were decreased.Gene-ontology analysis with DAVID bioinformatics tool(version 6.8)showed that naive PIK3CD GOF T cells up-regulated the expression of genes encoding molecules involved in cell cycle and mitosis.In addition,the mRNA expression of genes encoding the T cell activation,cytokines and cytokines receptors,chemokines and chemokine receptors,transcriptional factors,adhesion molecules,and the metabolism regulators were differentially regulated in PIK3CD GOF mice compared to the littermate controls.Kyoto encyclopedia of genes and genomes(KEGG)analysis and showed that altered genes in naive T cells of PIK3CD GOF mice displayed significant enrichment of several sets associated with infection,inflammatory and autoimmune disease.Ingenuity pathway analysis(IPA)also revealed that the signaling pathways regulating cellular growth,proliferation,activation,metabolism and cell-cycle pathways were upregulated in naive PIK3CD GOF T cells.In addition,enriched networks with the highest score among the differentially expressed genes were strongly associated with Thl activation,T helper cell differentiation,T cell exhaustion,interferon signaling and inflammatory.Conclusions:PIK3CD GOF results in a loss of quiescence gene-expression patterns in naive T cells by collectively coupling cell-cycle,nutrients metabolism,cell trafficking and signal transduction.PART FIVE GLUCOSE METABOLIC MECHANISMS OF HYPERACTIVATION OF NAIVE T CELL MEDIATED BY PIK3CD GOFObjectives:To determine the effects of PIK3CD GOF on the glucose metabolism of naive T cell,and to further explore the metabolic regulatory mechanisms of hyperactivation of naive T cell induced by PIK3CD GOF.Methods:The cell size and glucose uptake of T cells from WT and PIK3CD GOF mice were determined.The extracellular acidification rate and oxygen consumption rate for resting or active T cells were detected by using a XF24 Extracellular Flux Analyzers(Seahorse).The expressions of glycolytic genes and key transcription factors to orchestrating the expression of glycolytic enzymes were also determined by QRT-PCR and western blot.In vivo,both wide-type and PIK3CD GOF mice were treated with normal or 2-DG-containing drinking water for 6 weeks.After that,the activation,proliferation and differentiation of T cells were also evaluated.Results:The size of freshly isolated T cells from newly diagnostic patients with APDS were much larger than that of healthy controls.Either CD4+T cells or CD8+ T cells from patients exhibited increased glucose uptake compared with controls.The glucose uptake evaluated by PET-CT was obvious in cervical lymph nodes,bilateral inguinal lymph node,spleen of patient with APDS.Similarly,the sizes and glucose uptake of naive or total T cells from PIK3CD GOF mice were also greater than that of controls.The glycolytic capacity was slightly higher in mutant naive T cells than that of wide-type controls.Upon activation with TCR ligation,either basal ECAR or capacity of glycolysis were enhanced in T cells from wide-type,which was further increased in PIK3CD GOF T cells.In contrast,baseline OCR in both naive and activated T cells were decreased in PIK3CD GOF mice.Consistent with these results mentioned above,compared with controls,lactate production was significantly upregulated in PIK3CD GOF T cells following activation.Furthermore,the expressions of glycolytic genes and the levels of HIF-1α,Myc,and IRF4 protein were also higher in activated T cells isolated from PIK3CD GOF mice than that of controls.Inhibition of glycolysis by chemical inhibitor significantly repressed the enhanced production of IFN-y and elevated proliferation of PIK3CD GOF T cells as well as wide-type.However,inhibition of fatty acid oxidation had no obvious effect on the frequency of IFN-y-producing T cells.2-DG treatment in vivo decreased the enlarged spleen in PIK3CD GOF mice,concomitant with the reduced total splenocytes.In addition,the proportion of naive T cells were increased while the frequency of effector memory T cells was decreased in PIK3CD GOF mice after 2-DG treatment.Importantly,the treatment also decreased the expressions of activation markers(CD69,CD25 and CD44)and the proliferation in PIK3CD T cells,similar to that of age-matched WT mice.Furthermore,the production of IFN-y and IL-2 in T cells derived from treated mutant mice decreased and was comparable to the wide type mice.Conclusions:Naive T cells of PIK3CD GOF mice displayed an increased glycolysis capacity ex vivo and in response to activation mediated by TCR,and PIK3CD GOF TNaive cells rely largely on aerobic glycolysis than mitochondrial respiration.Therefore,enhanced aerobic glycolysis were required for PIK3CD GOF-induced T cell hyperactivation,and targeting aerobic glycolysis could restore the abnormal homeostasis induced by PIK3CD GOF ex vivo and in vivo.