| Background and objective:Malignant tumors are one of the major threats to human health.After more than a century of development,immunotherapy has become an important tumor treatment method after surgery,chemotherapy and radiotherapy.Immunotherapy is to restore the anti-tumor immune response by activating the "tumor-immune" cycle.CD8+T cells,as one of the important members of the human adaptive immune system,play a maj or role in antitumor.In recent years’ research has found that tumor cells can activate immunosuppressive receptors in CD8+T cells to inhibit the function of the T cells,thereby escape immune surveillance and immune clearance.In response to this problem,antibodies or small molecule inhibitors that target T cell immunosuppressive molecules and release immunosuppressive agents have been extensively developed.Although these immunosuppressive blockers can effectively activate CD8+T cells and achieve certain results in the clinic,there are still a large number of patients who cannot benefit from such treatments.Therefore,in-depth research on the regulation of the expression of immunosuppressive receptors in CDS+ T cells is needed to provide a direction for improving the effect of tumor immunotherapy.Currently,dozens of T-cell immunosuppressive receptors have been discovered.Among them,there are many studies on programmed cell death-1(PD-1),T cell immunoglobulin and mucin domain-3(TIM-3)and cytotoxic T lymph cytotoxic T-lymphocyte-associated protein-4(CTLA-4)and so on.Blocking against a single target or combined blocking of multiple targets can release T cell immunosuppression to a certain extent.However,there is a compensating effect between different immunosuppressive receptors,that is,blocking one or several receptors cannot completely release T cell immunosuppression.In view of this dilemma,we hope to explore the regulatory mechanisms that exist in the expression of different immunosuppressive receptors,and provide directions for the development of broad-spectrum immunosuppressive blocking drugs.As a way of post-transcriptional modification of proteins,glycosylation exists widely in proteins,which is particularly important for the correct positioning,conformational stability,protein stability,and activity of membrane proteins.Therefore,exploring the effect of protein glycosylation on T cell function and expression of immunosuppressive receptors will help the discovery of immunotherapeutic drugs.However,the status of protein glycosylation in CD8+T cells is unknown.Therefore,this topic mainly analyzes the changes in the expression of glycosylation modifying enzymes during the activation of CD8+T cells and the effects of such changes on T cell function and the expression of key immunosuppressive receptor PD-1.For genome-wide changes in gene expression,research using high-throughput sequencing,especially Assay for Transposase-Accessible Chromatin(ATAC)sequencing,has great advantages.Sequencing of chromatin accessibility is through transposase binding to the open chromatin segment.By building and sequencing the processed chromatin fragments,the chromatin status of the sample can be studied and chromatin can be detected across the genome.ATAC sequencing plays an important structural indicator role in studying glycosylation modification.At the same time,due to the high throughput and mature technology of the second-generation(RNA)sequencing technology,it can quickly,accurately and comprehensively detect the expression of genes,which has a great advantage for understanding the gene expression of the entire transcriptome.The combination analysis of the two sequencing could support for studying the effect of glycosylation modification on CD8+T cell function.In order to study the effects of glycosylation modifying enzymes on the function of CD8+T cells and immunosuppressive receptors,this project is mainly divided into three parts:The first part is to construct a model of activated CD8+T cells in vitro through the TCR signaling pathway.Using ATAC sequencing and RNA sequencing technology,a comprehensive analysis of the chromatin and transcriptome of the glycosylation-enzyme-encoding genes during the activation process was performed to find key proteins in the glycosylation pathway during cell activation.The second part is to study the mechanisms on primary CD8+T cells and Jurkat model cells,and use bioinformatics methods and gene editing methods to screen out molecules that have important regulatory significance for the glycosylation pathway in the activation model and finally to verify its regulatory effect on the immunosuppressive molecule PD-1 glycosylation,and explore the impact on T cell function.The third part is to analyze and verify the regulation and mechanism of the selected molecules on the glycosylation of CD8+T cells through the use of xenograft tumor animal models,clinical tumor patient samples and the GEO database to provide improved immunotherapy effects in the future.Part 1 Panoramic analysis of chromatin access and transcription of glycosylation-enzyme-encoding genes during CD8+T Cell ActivationIn order to observe the changes of glycosylation modifying enzymes at different stages of T cell activation,we used anti-CD3 and anti-CD28 antibodies to stimulate naive CD 8+T cells from healthy people on days 0,3 and 7 respectively.We performed ATAC sequencing on T cells that were activated at different times.After confirming that the quality of the submitted samples and sequencing data met the requirements of subsequent research,we performed a bioinformatics analysis of the relevant data.The analysis methods mainly include principal component analysis,distribution and cluster analysis of gene functions in open segments,functional enrichment analysis of differential genes related to open segments,cluster analysis of genes related to open segments,and post-transcriptional modification of proteins related pathway activation analysis.The results showed that the sequencing quality was good and the samples clustered significantly,which met the requirements of subsequent analysis.During the activation of T cells,the chromatin structure appeared in a wide range of open states,especially a large number of open fragments attached to the transcription start site,suggesting that intense transcriptional regulation of genes occurred during the activation of T cells;functional enrichment analysis found that genes related to the cell activation pathway were significantly enriched,further evidence that T cells were fully activated;cluster analysis of sequencing data at different times of activation revealed cell activation indicators that molecules have a good time-dependent enrichment;combined with T cell in vivo activation sequencing data(public data)analysis,it is found that the T cell activation model constructed in this study is highly consistent with the gene changes in the in vivo activation model,suggesting that the results of this study have broad biological significance.In addition to glycosylation,proteins also undergo modifications such as ubiquitination,phosphorylation,and methylation.By analyzing and comparing the transcriptional activities of these post-transcriptional modification related enzyme encoding genes,we found that the transcriptional activity of glycosylation-modified genes changed most significantly.By comparing and analyzing 15 glycosylation modification pathways,the results show that the chromatin state of N-linked glycosylation modification has obvious accessibility regulation.Therefore,we next focus on N-linked glycosylation modifications.In order to understand the changes in the whole gene transcriptome of glycosylation modifying enzymes at different stages of activation,we performed RNA sequencing analysis on the same batch of samples.After confirming that the data quality meets the requirements of subsequent analysis,we performed a bioinformatics analysis.The analysis methods include the analysis of the set of differential genes between groups and cluster analysis,and the related results are visualized.The results showed that there were 7 glycosylation-modifying enzymes in the 559 genes that differed between groups,and fucosyltransferase 8(Fut8),which is located in the N-linked glycosylation modification pathway,was the most significantly different gene.In summary,the first part of the results showed that the in vitro T cell activation model we constructed can reproduce the relevant processes in vivo,and found that N-linked glycosylation modification is of great significance in the activation process,and Fut8 is the most representative glycosylation modification enzyme.It provides a basis for further research on the mechanism of glycosylation on T cell activation.Part 2 Effect of glycosylation enzyme Fut8 on CD8+ T cell function and PD-1 expressionIn order to verify the change of Fut8 in different stages of T cell activation and its effect on T cell function,we activated T cells according to the established activation system,and then used PCR,western blot,cell immunofluorescence,flow cytometry and enzyme-linked immunosorbent assay(ELISA)to detect cell activation levels and Fut8 expression at different time points.The results suggest that with the activation of T cells,Fut8 expression gradually increases.Secondly,we used small interfering RNA to knock down the expression of Fut8 in primary CD8+ T cells and Jurkat cells to establish a knockdown cell model.Then the quantitative knockdown efficiency was detected by PCR,western blot,cellular immunofluorescence.The results showed that the designed small interfering RNA significantly reduced Fut8 expression at both gene and protein levels.Finally,we used ELISA and flow cytometry to perform functional tests on CD8+T cells with Fut8 knockdown expression.The results showed that the reduction of Fut8 expression significantly inhibited T cell killing and proliferation functions.So far,we confirmed that CD8+T cell activation promotes Fut8 expression,and Fut8 is indispensable for the normal proliferation and killing function of T cells.Next,we explored the transcription factors that regulate Fut8 expression.First,we combined existing sequencing data and bioinformatics to make predictions,and selected 4 transcription factors such as NFATc1,NFATc2,NF-kB,and JUN.Then these four transcription factors were tested by inhibitors,siRNA suppression,and comparison of ATAC sequencing results,and finally found that NFATc2 is a key transcription factor regulated by Fut8.In order to understand the expression of NFATc2 during cell activation and its effect on T cell function,we used PCR,western blot and cell immunofluorescence to detect NFATc2 expression.The results showed that with the activation of T cells,the expression of NFATc2 increased.After knocking down the expression of NFATc2 in primary CD 8+ T cells and Jurkat cells by siRNA,the knockdown efficiency was detected by PCR and western blot,respectively.The results showed that NFATc2 significantly reduced in the gene and protein levels.At the same time,Fut8 protein expression was also down-regulated in NFATc2 knockout-expressing cells.Functional assays of CD8+T cells that knocked down NFATc2 were performed using ELISA and flow cytometry.The results showed that the killing and proliferation functions of NFATc2 knockdown cells were significantly inhibited.In order to prove the direct regulatory relationship between the two,we used chromatin immunoprecipitation(ChIP)to detect the binding of NFATc2 to Fut8 gene.The results showed that NFATc2 can bind to the regulatory region of Fut8 coding gene and directly promote Fut8 expression.So far,we found that during the activation of CD8+ T cells,the expression of the transcription factor NFATc2 is up-regulated and promotes the enhancement of Fut8 transcription,thereby promoting T cell proliferation and killing activity.Finally,we explored the effect of Fut8 on PD-1 expression.We first analyzed six immunosuppressive and activated receptors using flow cytometry,western blot,and PCR.The results showed that PD-1 was inconsistently expressed at the gene and protein levels.Subsequently,we performed PD-1 detection on primary CD8+T cells that knocked down Fut8 expression,and the results showed that PD-1 expression was significantly reduced in T cells that knocked down Fut8 expression.Therefore,Fut8-mediated glycosylation plays a crucial role in PD-1 expression.After mutating four potential glycosylation modification sites in PD-1 protein,it was found that amino acid positions 49(N49)and 74(N74)play a major regulatory function.In order to explore the effect of fucosylation on PD-1 during the activation of CD8+T cells,we used CD8+T cells as tool cells to design single and simultaneous mutant gene sequences for these two sites The transfection was performed using the cell electroporation test method to change the amino acid at the glycosylation modification site.Then we used western blot and flow cytometry to detect PD-1 expression,and treated the PD-1 protein on the surface of CD8+T cells with glycolytic enzyme(PNGase F)to detect the glycosylation modification.CD8+T cells modified at different sites were also co-incubated with PD-L1,and ELISA and membrane labeling flow cytometry were used to detect cytokine secretion and cell proliferation.The results showed that N49 and N74 site mutations can significantly affect the molecular weight of PD-1 protein and reduce PD-1 expression.The effect of changing both sites at the same time is the most obvious.After PNGase F treatment,the molecular weight of PD-1 protein decreased significantly,which proved that PD-1 has glycosylation modification.After co-incubation with PD-L1,PD-1 point mutated CD8+T cells showed better killing and proliferation functions.In conclusion,the second part clarifies that the transcription factor NFATc2 affects cell function and PD-1 expression through regulating glycosylation pathway during the activation of CD8+T cells.Therefore,after conducting in vitro studies,we verified these results in mouse models as well as in clinical samples.Part 3 Using animal models and clinical samples to verify the effect of Fut8 on CD8+T cellsAfter clarifying the regulation of T cells by the NFATc2-Fut8 pathway,we first performed in vivo validation using chimeric antigen receptor T(CAR-T)cells and xenograft tumor animal models.We subcutaneously inoculated mesothelin-positive tumor cells into SCID-Beige mice.On the fifth day after inoculation,the tail vein was injected with mesothelin-targeted CD8+CAR-T cells.Three days after the injection of CAR-T cells,mice were sacrificed and tumors and spleens were isolated.A single-cell suspension was obtained after processing the dissected tumor and spleen,and CD8+T cells were obtained by magnetic sorting.Then,western blot,cell immunofluorescence,and flow cytometry were used to detect NFATc2,Fut8,and PD-1,and CD69 and IFN-y which represent cell activation and function,were also detected.The results showed that compared with spleen-derived cells,tumor-derived T cells had a higher degree of activation and stronger expression of NFATc2,Fut8,and PD-1.From this we can conclude that the NFATc2-Fut8 pathway is very important in T cell activation.To verify changes in the relevant genes in the human,we tested freshly isolated lung cancer patients with peripheral blood and tumor-infiltrating CD8+T cells.The results showed that NFATc2,Fut8,and PD-1 were more highly expressed in tumor-infiltrated T cells than in peripheral blood,and tumor-infiltrated CD8+T cells had extensive glycosylation modification,which was consistent with previous results.We also screened the sequencing data of CD8+T cells isolated from untreated lung cancer patients and adjacent cancer cells from the GEO database,and compared the expressions of PD-1,Fut8 and NFATc2.The results showed no difference in PD-1 expression between cancer and adjacent cancers,but the expression of Fut8 and NFATc2 was higher in cancers than adjacent cancers,which is consistent with the results of previous studies.Therefore,in this paper,we verified that the fucosylation modification mediated by the NFATc2-Fut8 pathway plays an important role in T cell activation and PD-1 expression from in vivo and in vitro.The clarification of this pathway provides new ideas for clinical immunotherapy for protein glycosylation modification in the future,and helps promote related treatment progress. |
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