【作者】 刘艳；

【导师】 杨寒朔；

【作者基本信息】 四川大学 ， 人类重大疾病生物治疗， 2023， 硕士

【摘要】 研究背景及目的嵌合抗原受体T(Chimeric antigen receptor T cell,CAR-T)细胞疗法在B细胞淋巴瘤等血液瘤中展现出良好的治疗效果,但其对实体瘤的疗效有限。实体瘤具有与血液瘤完全不同的物理和生理特征,其外部由大量结缔组织、纤维基质和肿瘤相关成纤维细胞(Cancer associated fibroblasts,CAFs)组成的致密物理屏障阻碍了CAR-T细胞的迁移和浸润。然而,足量的CAR-T细胞浸润至肿瘤组织内部是其发挥抗肿瘤作用的先决条件。CAR-T细胞成功浸润至实体瘤内是引起肿瘤抗原特异性细胞毒性的第一步。因此,构建具有高浸润能力的新型CAR-T细胞,增强CAR-T细胞在实体瘤内的迁移和浸润,有望实现CAR-T细胞在实体瘤治疗领域的突破。肿瘤浸润淋巴细胞(Tumor infiltrating lymphocytes,TILs)是一类浸润至实体瘤内部的异质性细胞群,由效应性细胞、调节性细胞和炎性细胞等不同类型的细胞组成,包括T细胞、B细胞和NK细胞等。TILs的存在表明部分淋巴细胞具有更强的迁移和浸润能力,其基因表达和蛋白分泌与普通淋巴细胞或许存在差异。若筛选到影响淋巴细胞浸润能力的关键基因,并将其用于升级改造CAR-T细胞,有望提高CAR-T细胞对实体瘤的浸润,进而改善CAR-T细胞疗法在实体瘤中的治疗效果。细胞因子是一类兼具免疫调节和效应功能的分泌蛋白,其作用具多效性、丰裕性、协同性、网络性等特点。淋巴细胞的迁移和浸润能力与其细胞因子、趋化因子的表达水平密切相关。理论上存在既能促进T细胞浸润又能增强其杀伤活性的双功能性细胞因子。为筛选出具备高浸润能力及强杀伤活性的双功能性细胞因子,本研究分析了外周血和肿瘤组织的淋巴细胞中与细胞迁移相关的细胞因子的RNA表达水平,成功筛选出具有高浸润能力的T细胞的关键细胞因子GM-CSF。为了进一步探究GM-CSF对CAR-T细胞浸润能力和杀伤活性的影响,我们在二代CAR结构的基础上构建了过表达GM-CSF的CAR-T细胞(CAR-GM-T)。实验结果表明,过表达GM-CSF不仅能增强CAR-T细胞在体外的迁移、浸润能力和杀伤活性,还能有效地抑制小鼠体内皮下移植瘤的生长。当CAR-GM-T细胞被激活后会表达GM-CSF受体,过表达的GM-CSF与其受体结合后,一方面进一步促进GM-CSF的转录表达,另一方面促进STAT3分子的磷酸化。p STAT3促进其下游基因MMP1转录表达。MMP1分泌到胞外,降解细胞外基质,提高CAR-T细胞的迁移和浸润能力。综上,本研究构建了一种具有高浸润能力的新型CAR-T细胞,为增强CAR-T细胞的浸润能力提供了一种新策略,有望改善CAR-T细胞在实体瘤治疗中的临床效果。研究方法1.筛选具有高浸润能力的T细胞的关键基因从肝癌患者的单细胞测序数据及小鼠肿瘤浸润淋巴细胞中筛选具有高浸润能力的T细胞的关键基因,构建Transwell模型进一步验证该特征基因在迁移能力强的T细胞中的表达情况。2.构建过表达GM-CSF的T细胞及其迁移能力验证使用pWPXLd-GM-CSF重组质粒转导293T细胞后收集病毒,感染激活的CD3~+T细胞制备过表达GM-CSF的T细胞(GM-CSF~+T),检测GM-CSF~+T细胞培养液中的GM-CSF浓度以评估其是否成功过表达,在Transwell模型中检测GM-CSF~+T细胞的迁移能力。3.过表达GM-CSF的人CAR-T细胞制备将含有目的基因的表达载体质粒同包装质粒pMD2.G、pSPAX2共同转染293T细胞进行慢病毒包装,超速离心浓缩病毒,利用Jurkat细胞验证病毒感染效率。从人外周血中分离单核淋巴细胞(Peripheral blood mononuclear cell,PBMC)激活后用慢病毒感染制备CAR-T,流式细胞术检测CAR分子的表达情况。4.过表达GM-CSF的CAR-T细胞迁移及浸润能力研究基于Transwell迁移能力检测模型和3D肿瘤球浸润能力检测模型,综合评估过表达GM-CSF的CAR-T细胞体外的迁移与浸润能力。5.过表达GM-CSF的CAR-T细胞体外杀伤活性研究使用天然表达Her2蛋白及过表达红色荧光蛋白的人卵巢癌细胞系(SK-OV3-mcherry)细胞作为CAR-T细胞杀伤的靶细胞,采用活细胞成像系统监测各组CAR-T细胞杀伤肿瘤细胞的过程。检测CAR-T细胞和肿瘤细胞共培养24h时乳酸脱氢酶(Lactate dehydrogenase,LDH)和IFN-γ的释放情况,以评估CAR-T细胞的杀伤活性。6.过表达GM-CSF的CAR-T细胞的GM-CSF受体表达分析流式细胞术检测T细胞静息状态和激活状态下的GM-CSF受体表达情况及CAR-T细胞受到Her2蛋白刺激前后的GM-CSF受体表达情况。7.过表达GM-CSF的CAR-T细胞转录组测序分析CAR-T细胞与肿瘤细胞共培养后,流式细胞术分选出CAR-T细胞进行转录组测序分析,GO-Pathway以及KEGG-Pathway富集分析转录组测序的差异基因。8.检测JAK-STAT通路下游基因的表达水平使用Western Blot检测磷酸化的STAT3分子表达水平,q-PCR检测STAT3下游基因MMP1的表达水平。探究STAT3抑制剂BP-1-102、MMP1抑制剂Ilomastat对过表达GM-CSF的CAR-T细胞迁移和浸润能力的影响。9.GM-CSF蛋白对CAR-T细胞迁移能力的影响在Transwell模型和3D肿瘤球模型中添加外源GM-CSF蛋白,观察GM-CSF蛋白是否影响普通CAR-T细胞的迁移能力和浸润能力。10.过表达GM-CSF的CAR-T细胞在体内的抗肿瘤活性研究使用过表达Her2蛋白的小鼠结直肠癌细胞MC38-Her2构建皮下移植瘤模型,分离小鼠淋巴结细胞制备小鼠CAR-T和过表达GM-CSF的CAR-GM-T细胞,尾静脉回输两种CAR-T细胞进行治疗,监测肿瘤生长情况、小鼠体重状态。治疗后28天,剥离小鼠皮下移植瘤,免疫荧光和流式细胞术检测皮下移植瘤内CAR-T的浸润情况。实验结果1.成功筛选出具有高浸润能力的T细胞的关键基因CSF2,Transwell模型筛选出的迁移能力强的T细胞的GM-CSF表达水平高于普通T细胞。2.成功构建过表达GM-CSF的T细胞(GM-CSF~+T),过表达GM-CSF的T细胞在体外的迁移能力增强。3.对照病毒、Her2-CAR、Her2-CAR-h GM-CSF病毒对Jurkat细胞的感染效率分别是99.77%、95.73%和95.06%。T细胞被激活48h后,CD3阳性率为98.6%。慢病毒感染T细胞制备靶向Her2的普通CAR-T细胞和过表达GM-CSF的CAR--T细胞(CAR-GM-T),CAR-T细胞的CAR表达水平为86.26%,CAR-GM-T细胞的CAR表达水平为88.16%。4.在Transwell迁移模型中,CAR-GM-T细胞的迁移到下层小室的细胞数量显著高于CAR-T细胞;在3D肿瘤球模型中,CAR-GM-T细胞浸润到肿瘤球内的细胞数量显著多于CAR-T细胞,且CAR-GM-T细胞在肿瘤球内的浸润距离比CAR-T细胞更远。与CAR-T细胞相比,CAR-GM-T细胞在体外具有更强的迁移能力和浸润能力。5.在不同效靶比下,CAR-GM-T细胞对SK-OV3的杀伤效率均显著高于CAR-T细胞。CAR-GM-T细胞与肿瘤细胞共培养后IFN-γ分泌量约为CAR-T细胞的5倍。与CAR-T细胞相比,CAR-GM-T在Transwell迁移模型中对SK-OV3的杀伤效率显著升高,IFN-γ释放量增加。6.静息状态的T细胞不表达GM-CSF受体,当T细胞被激活24h后,GM-CSFRα阳性率约为26.3%,GM-CSFRβc阳性率约为20.14%;激活48后,GM-CSFRα阳性率下降至20.1%,GM-CSFRβc阳性率升高至60.24%。CAR-T细胞在受到抗原蛋白Her2刺激前检测不到GM-CSF受体的表达,被Her2蛋白刺激24h后,GM-CSFRα阳性率约为24.34%,刺激48h后,GM-CSFRβc阳性率大于75%。7.Heat-map显示CAR-T细胞和CAR-GM-T细胞在杀伤肿瘤细胞时基因表达水平存在较大差异。基因富集分析显示CAR-GM-T细胞杀伤肿瘤细胞时上调的基因显著富集到JAK-STAT信号通路(NES1.36,FDR0.123,p-value0.019)。8.CAR-T细胞在受到Her2蛋白刺激后STAT3分子的磷酸化水平升高,且CAR-GM-T细胞p STAT3分子的表达水平高于CAR-T细胞。q-PCR结果显示,与CAR-T细胞相比,CAR-GM-T细胞中JAK-STAT的下游基因MMP1的表达水平更高。外源添加BP-1-102、Ilomastat抑制剂,均会抑制CAR-GM-T细胞的迁移和浸润能力。9.外源添加GM-CSF蛋白增强了普通CAR-T细胞在体外的迁移和浸润能力。10.Anti-mouse CD3/CD28抗体激活小鼠淋巴结细胞后,检测CD3阳性率为93.66%。慢病毒感染小鼠T细胞制备小鼠CAR-T细胞和CAR-GM-T细胞,检测其CAR表达水平分别为76.74%和81.08%。两种小鼠CAR-T细胞均可抑制皮下抑制瘤的生长,但CAR-GM-T细胞的效果更佳,且CAR-GM-T细胞可以更多地浸润至小鼠皮下瘤内部。研究结论过表达GM-CSF的CAR-T细胞(CAR-GM-T)在体外具有良好的迁移能力、浸润能力和肿瘤杀伤活性,在小鼠体内能够有效地抑制皮下移植瘤的生长,更多地浸润至实体瘤内部。当CAR-GM-T细胞被激活后会表达GM-CSF受体,过表达的GM-CSF与受体结合,一方面进一步促进GM-CSF的转录表达,另一方面促进STAT3分子的磷酸化,p STAT3促进其下游基因MMP1转录表达。MMP1分泌到细胞外,降解细胞外基质,增强CAR-T细胞的迁移能力和浸润能力。综上所述,本研究构建了一种具有高浸润能力的新型CAR-T细胞,同时探究了其浸润能力和杀伤活性增强的作用机制,为增强CAR-T细胞对实体瘤的治疗效果提供了一种新策略。更多还原

【Abstract】 BackgroundChimeric antigen receptor T(CAR-T)cell therapy has shown promising therapeutic effects in hematological tumors such as B-cell lymphoma,but its benefit in solid tumors is limited.The infiltration of many CAR-T cells into tumor tissue is a prerequisite for their anti-tumor effects.Tumor cells in the hematological tumor are generally found in peripheral blood and are easily recognized and killed by CAR-T cells.Solid tumors have completely different physical and physiological characteristics from hematological tumors,with a dense physical barrier consisting of large amounts of connective tissue,fibrous stroma,and cancer associated fibroblasts(CAFs)that prevent the migration and infiltration of CAR-T cells.The successful infiltration of CAR-T cells into solid tumors is the first step in inducing tumor antigen-specific cytotoxicity.Therefore,the development of a new type of CAR-T cell with high infiltration capacity is expected to achieve a breakthrough in CAR-T cell therapy for solid tumors.Tumor infiltrating lymphocytes(TILs)are a heterogeneous group of cells that infiltrate the interior of solid tumors and consist of different types of cells,including T cells,B cells and NK cells,such as effector,regulatory and inflammatory cells.The existence of TILs indicates that some lymphocytes have stronger migration and infiltration capabilities,with differences in gene expression and protein secretion compared to ordinary lymphocytes.If the key genes affecting the infiltration ability of lymphocytes are screened and used to upgrade CAR-T cells,it is expected that the infiltration of CAR-T cells into solid tumors will be improved,thus improving the therapeutic efficacy of CAR-T cell therapy in solid tumors.Cytokines are a class of secreted proteins with both immunomodulatory and effector functions,and their actions are characterized by pleiotropy,abundance,synergy and network.The ability of lymphocytes to infiltrate is closely related to the expression levels of cytokines and chemokines.Theoretically,there are bifunctional cytokines that can both promote T cell infiltration and enhance their killing activityTo screen for bifunctional cytokines with high infiltration and strong killing activity,this study analyzed the RNA expression levels of cytokines associated with cell migration in lymphocytes from peripheral blood and tumor tissues,and successfully screened for GM-CSF,a gene characteristic of T cells with high infiltration ability.To further investigate the effect of GM-CSF on the infiltration ability and killing activity of CAR-T cells,we constructed CAR-T cells overexpressing GM-CSF(CAR-GM-T)basis on a second-generation CAR structure.The experimental results showed that overexpression of GM-CSF not only enhanced the migratory infiltration ability and killing activity of CAR-T cells in vitro but also effectively inhibited the growth of subcutaneous transplanted tumors in mice in vivo.When activated,CAR-GM-T cells express the GM-CSF receptor,and overexpressed GM-CSF binds to the receptor and transmits signals into the cell to further promote the transcriptional expression of GM-CSF on the one hand,and the phosphorylation level of STAT3 molecule on the other.p STAT3 further promotes the transcriptional expression of the downstream gene MMP1.The extracellular matrix was secreted by MMP1 to degrade the extracellular matrix and improve the infiltration ability of CAR-T cells.In conclusion,this study constructs a new type of CAR-T cells with high infiltration ability,which provides a new strategy to enhance the infiltration ability of CAR-T cells and is expected to improve the clinical effect of CAR-T cells in solid tumor treatment.Methods:1.Screening characteristic genes of T cells with high infiltration abilityCharacteristic genes of T cells with high infiltration ability were screened from single-cell RNA sequencing data of liver cancer patients and lymphocytes infiltrating mouse tumors.The expression of these characteristic genes in T cells with strong migratory ability was further validated using the Transwell model.2.Construction and migration ability verification of T cells overexpressing GM-CSFThe pWPXLd-GM-CSF recombinant plasmid was used to transfect 293T cells,and activated CD3~+T lymphocytes infected with the virus were collected to prepare T cells overexpressing GM-CSF(GM-CSF~+T).ELISA was used to detect the expression level of GM-CSF in GM-CSF~+T cells to evaluate the success of overexpression.The migration ability of GM-CSF~+T cells was tested using the Transwell model.3.Preparation of human CAR-T cells overexpressing GM-CSFThe expression vector plasmid containing the target gene was co-transfected with packaging plasmids pMD2.G and pSPAX2 into 293T cells for lentivirus packaging,and the virus was concentrated by ultracentrifugation.The virus infection efficiency was verified using Jurkat cells.Mononuclear lymphocytes(PBMCs)were separated from human peripheral blood.T cells were activated with CD3/CD28 Dynabeads and infected with lentivirus concentrate to prepare CAR-T cells.Flow cytometry was used to detect the expression of CAR and to confirm the successful preparation of CAR-T cells.4.Study on migration and infiltration ability of CAR-T cells overexpressing GM-CSFThe infiltration ability of CAR-T cells overexpressing GM-CSF was comprehensively evaluated using the Transwell migration ability detection model and the 3D tumor sphere infiltration ability detection model.5.Study on the in vitro killing activity of CAR-T cells overexpressing GM-CSFSK-OV3-mcherry cells naturally expressing Her2 protein and overexpressing red fluorescent protein(mcherry)were used as target cells for CAR-T cell killing.The process of CAR-T cell killing tumor cells was monitored using a live cell imaging system.The release of lactate dehydrogenase(LDH)and IFN-γwere detected when CAR-T cells and tumor cells were co-cultured for 24 hours to evaluate the killing activity of CAR-T cells.6.Analysis of GM-CSF receptor expression in CAR-T cells overexpressing GM-CSFFlow cytometry was used to detect the expression of GM-CSF receptors in T cells at rest and activation states,as well as the expression of GM-CSF receptors in CAR-T cells before and after stimulation with the Her2 protein.7.Transcriptome sequencing analysis of CAR-T cells overexpressing GM-CSFAfter co-culturing Her2-CAR-T cells and Her2-CAR-GM-T cells with tumor cells for 24 hours,flow cytometry was used to sort out these two CAR-T cells for transcriptome sequencing analysis.GO-Pathway and KEGG-Pathway enrichment analyses were performed to identify differentially expressed genes in Her2-CAR-T cells and Her2-CAR-GM-T cells after killing tumor cells.8.Detection of expression levels of downstream genes in the JAK-STAT pathwayWestern Blot was used to detect the phosphorylation level of the STAT3 molecule,and q-PCR was used to detect the expression level of the MMP1 gene downstream of the JAK-STAT pathway.The effects of exogenous STAT3 inhibitor BP-1-102 and MMP1 inhibitor Ilomastat on the migration and infiltration abilities of CAR-T cells overexpressing GM-CSF were observed.9.The effect of GM-CSF protein on the migration ability of CAR-T cellsExogenous GM-CSF protein was added to the Transwell model and 3D tumor sphere model to observe whether GM-CSF affects the migration ability of normal Her2-CAR-T cells.Flow cytometry was used to detect the expression levels of GM-CSF receptors in T cells at rest and activation states,as well as in CAR-T cells before and after antigen stimulation.10.In vivo anti-tumor activity study of CAR-T cells overexpressing GM-CSFA subcutaneous transplantation tumor model was constructed using MC38-Her2colon cancer cells overexpressing Her2 protein in mice.Mouse CAR-T and CAR-GM-T cells were prepared by isolating mouse lymph node cells.The two types of CAR-T cells were intravenously infused for treatment,and tumor growth and mouse body weight were monitored.After 28 days of treatment,subcutaneous transplant tumors were removed,and flow cytometry was used to detect the number of infiltrating CAR-T cells.Experimental results:1.The characteristic gene CSF2,which has high invasive ability,was successfully screened out in T cells.In the Transwell model,the expression of GM-CSF in T cells with strong migration ability was higher than that in normal T cells.2.T cells overexpressing GM-CSF(GM-CSF~+T)were successfully constructed.Overexpression of GM-CSF helps to enhance the migration ability of T cells.3.The lentivirus packaging system was successfully used in 293T cells.The infection efficiency of the control virus,Her2-CAR,and Her2-CAR-h GM-CSF viruses on Jurkat cells was 99.77%,95.73%,and 95.06%,respectively.Human T-Expander CD3/CD28 Dynabeads were used to activate T cells,and after 48 hours,the CD3-positive rate was 98.6%.CAR-T cells and CAR-GM-T cells targeting Her2 were prepared by infecting T cells with lentivirus.The positive rate of CAR-T cells was86.26%,and the positive rate of CAR-GM-T cells was 88.16%.4.In the Transwell migration model,the number of cells that migrated to the lower chamber by CAR-GM-T cells was significantly higher than that by CAR-T cells.In the 3D tumor sphere model,the number of cells infiltrated by CAR-GM-T cells was significantly higher than that by CAR-T cells,and the infiltration distance of CAR-GM-T cells in the tumor sphere was farther than that of CAR-T cells.5.The killing efficiency of CAR-GM-T cells against SK-OV3 was significantly higher than that of CAR-T cells at different effector-to-target ratios.After co-culture with tumor cells,the IFN-γsecretion level of CAR-GM-T cells was approximately 5 times that of CAR-T cells.Compared with CAR-T cells,CAR-GM-T cells exhibited significantly increased killing efficiency and IFN-γrelease in the Transwell migration model against SK-OV3.6.T cells in the resting state have low levels of GM-CSF receptor expression.After 24hours of activation,the positivity rates of GM-CSFRαand GM-CSFRβc were approximately 26%and 20%,respectively.After 48 hours of activation,the positivity rate of GM-CSFRαwas 20%,while the positivity rate of GM-CSFRβc increased to60%.CAR-T cells almost did not express the GM-CSF receptor before being stimulated by the Her2 antigen protein.After being stimulated by Her2 for 24 hours,the positivity rate of GM-CSFRαwas approximately 24%,while after 48 hours of stimulation,the positivity rate of GM-CSFRβc was more than 75%.7.Heat-map analysis showed a significant difference in gene expression levels between CAR-T cells and Her2-CAR-GM-T cells in killing tumor cells.GSEA analysis showed that the upregulated genes in CAR-GM-T cells during tumor cell killing were significantly enriched in the JAK-STAT signaling pathway(NES1.36,FDR0.123,p-value0.019).8.Both CAR-T and CAR-GM-T cells showed increased levels of phosphorylated STAT3 molecules after being stimulated by the Her2 protein,with the phosphorylation level of STAT3 in CAR-GM-T cells being higher than that in CAR-T cells.q-PCR results showed that the expression level of MMP1,a downstream gene of JAK-STAT,was higher in CAR-GM-T cells than in CAR-T cells.The exogenous addition of the STAT3 inhibitor BP-1-102 and the MMP1 inhibitor Ilomastat both inhibited the migration and infiltration ability of CAR-GM-T cells.9.Exogenous addition of GM-CSF protein enhances the migration and infiltration capacity of regular CAR-T cells in vitro.10.After activation with anti-mouse CD3/CD28 antibodies,the positivity rate of CD3was 93.66%in mouse lymph node cells.Mouse CAR-T cells and CAR-GM-T cells were prepared by infecting mouse T cells with lentivirus,and the expression levels of CAR were 76.74%and 81.08%,respectively.Both mouse-based CAR-T cells and CAR-GM-T inhibited the growth of subcutaneous tumors in immunized mice,but CAR-GM-T cells were more effective and CAR-GM-T cells were able to infiltrate more into the subcutaneous tumors in mice.Conclusion:CAR-T cells overexpressing GM-CSF(CAR-GM-T)have good migration,infiltration,and tumor-killing activities in vitro,and can effectively inhibit the growth of subcutaneous transplanted tumors and infiltrate more into solid tumors in mice.When CAR-GM-T cells are activated,they express the GM-CSF receptor,and the overexpressed GM-CSF binds to the receptor and transmits signals into the cell to further promote the transcriptional expression of GM-CSF on the one hand,and the phosphorylation level of STAT3 molecule on the other.p STAT3 further promotes the transcriptional expression of the downstream gene MMP1.MMP1 was secreted into the extracellular matrix to degrade the extracellular matrix and improve the migration and infiltration ability of CAR-T cells.In summary,this study constructs a novel CAR-T cell with high infiltration capacity and also explores the mechanism of action of its enhanced infiltration capacity and killing activity,providing a new strategy to enhance the therapeutic effect of CAR-T cells on solid tumors.更多还原