The Role Of Glycolysis On Selection Of T Lymphocytes In Thymus

BackgroundT lymphocytes act as a central role in the adaptive immune system,which protects us from various pathogens.T lymphocytes,abbreviated as T cells,derive their names from this organ thymus gland where they develop(or mature).The molecular mechanisms for the development and differentiation of T cells that go through multiple stages are quite complicated.T cells originate from early T lineage precursors and then migrate to thymus gland for the further differentiation and clone deletion.Mature T cells left the thymus to the periphery when the selection of T cells is done.Previous studies have documented well in the behavioral mechanisms of thymic T cells selection.The positive selection and negative selection in the thymus mediated by the interplay of TCR and MHC are two critical events to ensure the recognization of foreign epitopes but not against self-antigens.The both selections delete abnormal T cells by means of their apoptosis.However,the underlying mechanism is still poorly understood.It’s already known that the activation of T cells accompanies with negative regulations like re-stimulated induced cell death(RICD),activation induce cell death(AICD)and checkpoints which can help to avoid sustained activation and proliferation of T cells,and thus to avoid immune related injury.We human stay in immune homeostasis under the balance between cellular metabolisms and functions.The dynamic changes of T cells metabolism significantly regulate their sensitivity to apoptosis signals.The dysregulation of metabolisms is one of the causes of autoimmune diseases.Previous investigations have shown that metabolic pattern for energies of effector T cells needs to switch from oxidative phosphorylation to glycolysis with upregulation of glycolysis related genes following the interaction of TCR and MHC.What’s more,their interplay can stimulate the Fas/Fas L signaling.T cells relying on glycolysis are more sensitive to AICD/RICD.However,whether the transition of metabolism pattern and glycolysis also paly a critical role in thymus to involve in T cell selection has been still unknown.In the present study,based on the data of m RNA sequencing,laboratory results with clinical samples and vitro cell experiments,we made a sketch as follow:1.Based on bioinformatics analysis,to identify the expression of glycolysis related genes in thymic T cells in contrast with peripheral T cells,and to determine the role of glycolysis during T cell selection and if PDK kinase family are critical;2.To detect the expression levels of key genes in bioinformatics analysis in thymus samples by q PCR,western blotting and immunostaining;3.Isolating and culturing the peripheral human T cells;Examining the frequency of T cell apoptosis and expansion following co-stimulation with CD3/CD28 antibody after over-expression of the key gene in glycolysis.These studies were performed to reveal the physiological mechanisms by which glycolysis involves in T cell selection in thymus,and to provide a new perspective on the molecular biological mechanisms of T cell selection.MethodsChapter 1 Determination of glycolysis related genes in thymic T cells through bioinformatics analysis1.We downloaded the m RNA sequencing data of T cells in thymus and peripheral PBMC from GEO and DICE database to quantify the activating levels of main energy related metabolic pathways such as glycolysis,oxidative phosphorylation,fatty acid oxidation,amino acid catabolic process in immune cells by enrichment analysis.We observed the differences of activating level in metabolic pathways mentioned above between thymic CD4~+/CD8~+T cells and peripheral CD4~+/CD8~+T cells.2.We sorted the glycolysis specific enzyme genes and PDK family genes,important on mediating glycolysis,and then compared their expressions between thymic CD4~+/CD8~+T cells and peripheral CD4~+/CD8~+T to confirm the role of glycolysis in the negative and positive selection of thymic T cells and to found the critical genes in these processes.In addition,using 10×Genomics single-cell sequencing of PBMC,we aimed to confirmed the expression frofiles of selected possible critical genes in peripheral CD4~+/CD8~+T cells.Chapter 2 Confirmation of the critical genes mediating glycolysis in normal thymus1.Collecting normal thymus samples of patients undergoing cardiac surgery with various ages.The expression levels of key genes mediating glycolysis and PD-1/PD-L1 were detected by q PCR in thymic T cells in contrast with peripheral T cells.2.Western bolt and immunostaining were performed for identification of those possile key genes and checkpoint protein PD-1/PD-L1 and CD31 in contrast with peripheral T cells.Chapter 3 Vitro experimental study of the critical genes upregulating AICD sensitization in T cells from peripheral blood1.Constructing the overexpression lentiviral vector of human PDK4,and then preparing the lentivirus through transfecting lentiviral vector into HEK-293T.2.After isolation and 5-day culture of peripheral T cells,the lentivirus for PDK4overexpression infected the cultured T cells.After infection of 24 hours,recombinant CD3/CD28 antibody was used to activate TCR signaling,and then clonal expansion of T cells can be observed and T cell apoptosis can be detected by flow cytometry.ResultsChapter 1 Determination of glycolysis related genes in thymic T cells through bioinformatics analysis1.The main metabolic pathways were active to a certain extent including glycolysis,oxidative phosphorylation,fatty acid oxidation,amino acid catabolic process.The last three were more enhanced in thymic CD4~+/CD8~+T cells than in peripheral CD4~+/CD8~+T cells.For glycolysis,there was a significantly higher level in thymic CD4~+T cells than in peripheral CD4~+T but not CD8~+T.2.These genes of key enzymes in glycolysis pathway expressed signicantly differently between thymus CD4~+/CD8~+T cells and blood CD4~+/CD8~+T cells.Specially,in contrast to peripheral CD4~+/CD8~+T cells,the genes ADH1B、ENO2、ADH5、ACDH3B2、PDHA1、ALDH7A1、PFKM、MINPP1、PGAM2、PGM2were all upregulated in thymic CD4~+/CD8~+T cells but HK1、HK3、FBP1、ALDH3B1、ALDOC、HKDC1、ACSS2 were downregulated.3.Our results showed that PDK family,as a critical role regulating glycolysis,PDK1,PDK2,PDK3 of them were downregulated in thymic CD4~+T compared to periheral CD4~+T cells but PDK4 was upregulated.PDK2,PDK3,PDK4 were downregulated in thymic CD8~+T compared to periheral CD8~+T cells but PDK1 was upregulated.Single-cell sequencing using 10×Genomics for PBMC demonstrated that there was minimal m RNA of PDK4 in blood CD4~+/CD8~+T cells,which indicated that PDK4 was a possible key gene activating glycolysis in thymic T cells.Chapter 2 Confirmation of the critical genes mediating glycolysis in normal thymus1.Quantitative PCR showed PDK1,PDK2,PDK3 and PDK4 all expressed in thymus CD4~+/CD8~+T cells of 25-day-old,5-month-old,3-year-old,25-year-old,67-year-old and 70-year-old thymus samples.Additionally,PDK1,PDK2 and PDK3expressed in transcript level in the peripheral CD4~+/CD8~+T cells,but not PDK4.PDK4of CD4~+/CD8~+T cells expressed significantly higher in thymus than in blood.Meanwhile,m RNA of PD-1/PD-L1 also were observed in thymic and peripheral CD4~+/CD8~+T cells.2.Western blotting showed that PDK1,PDK2,PDK3,PD-1,PD-L1 expressed low and PDK4 expressed relatively high in thymus of 25-day-old,5-month-old,3-year-old,25-year-old,67-year-old and 70-year-old thymus samples.In peripheral CD4~+/CD8~+T cells,PDK1,PDK2 and PDK3 expressed low,and PDK4 and PD-L1 did not expressed,PD-1 expressed relatively high.3.Results of immunohistochemistry and immunofluorescence showed that PDK1and PDK3 expressed low in thymus of 25-day-old,5-month-old,3-year-old,25-year-old,67-year-old and 70-year-old thymus samples,but PDK4 expressed high in thymic CD4~+/CD8~+T cells including in thymic cortex and medulla.PDK4~+cells of CD4~+cells were found no signicant difference between cortex and medulla except 5-month sample,the same as PDK4~+cells of CD8~+cells.Additionally,PDK2 and PD-1 showed no expression,PD-L1 expressed low in stromal cells,CD31 staining suggested that the microvessels distributed sparsely in cortex and densely in medulla.Chapter 3 Vitro experimental study of the critical genes upregulating AICD sensitization in T cells from peripheral blood1.The human T cells isolated from peripheral blood were cultured in vitro for 5days.Afterwards,we upregulated PDK4 in T cells through infection using the lentivirus.And then,we found the clonal expansion was impaired in PDK-OE group after activating TCR with recombinant CD3/CD28 antibody(CTL average clone:52.0vs.PDK-OE average clone:37.3,P=0.0029).2.Also,T cells after activation were checked the apoptosis level through flow cytometry and it showed that the frequency of apoptosis was higher in PDK-OE group than in CTL group(5.79%vs.15.17%,P=0.0013).ConclusionBased on the multi-expression levels data,we found that transcript expressions of glycolysis related enzymes were significant difference between thymus T cells and periheral T cells.PDK4 showed upregulated expression in thymic T cells compared to peripheral T cells.In thymus,PDK4 expressed mainly in CD4~+/CD8~+T cells in cortex and medulla.Our preliminary results suggested that PDK4 probably activates glycolysis to mediates positive and negative selection.Bedsides,Vitro functional studies revealed that PDK4 mediate the apoptosis of T cells by sensitizing their AICD,which was probably the mechanism that PDK4 regulated the selection of T cells in thymus.