【作者】 金戈；

【导师】 徐小燕；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2023， 博士

【摘要】 目的：当肿瘤细胞在机体恶性增殖时,免疫系统可以识别肿瘤细胞表面的肿瘤相关抗原(Tumour-associated antigens,TAAs),并激活特定的体液和细胞免疫反应,将表达TAAs的肿瘤细胞从体内清除。同时,肿瘤细胞可以通过各种途径对机体的免疫反应进行负向反馈,并试图逃避机体的免疫监控和杀伤。免疫细胞浸润在肿瘤的发生和发展中起着重要作用,并影响着肿瘤患者的临床预后水平。恶性黑色素瘤是由黑色素细胞恶变而来的肿瘤。皮肤黑色素瘤主要表现为在较短时间内(一般几个月到几年)皮肤色素明显的病变,某些黑色素痣异常增大或变得颜色不均匀也应引起注意。皮肤黑色素瘤在亚洲人群中发病率较低,但是其恶性程度较高,而且易发生转移,因此该疾病具有较高的致死率。相比于其他实体瘤,黑色素瘤对免疫治疗应答率相对较高,因此黑色素瘤是肿瘤免疫领域较为理想的研究对象。干扰素γ诱导的硫醇蛋白还原酶(Gamma-Interferon-Inducible Lysosomal Thiol Reductase,GILT),又叫干扰素γ诱导的蛋白30(Interferon GammaInducible Protein 30,IFI30)由IFI30基因编码,其前体由250个氨基酸构成,分子量约35k Da。细胞内IFI30前体可以被甘露糖-6-磷酸受体(mannose-6-phosphate receptors,M6PR)转运到内吞小室并最终定位于溶酶体,在上述转运过程中,N端和C端前体肽逐渐解离形成IFI30成熟体(分子量约为30k Da),酶活性中心得以暴露。IFI30能还原二硫键,促使含有二硫键的蛋白质展开,其硫醇还原酶活性需要溶酶体内的酸性环境。课题组前期研究揭示了IFI30可在RNA腺苷脱氨酶(Adenosine Deaminases Acting on RNA,ADARs)家族成员ADAR1和ADAR2的介导下发生RNA编辑,具体表现为A到I的RNA编辑导致第223位的苏氨酸(Threonine,T)替换为丙氨酸(Alanine,A)(T223A)。然而,IFI30在肿瘤微环境的角色以及RNA编辑是否使得IFI30发生功能变化尚不明确。抗原提呈细胞加工处理抗原肽使之形成合适的结构和长度,加工后的抗原肽与主要组织相容复合体分子(Major Histocompatibility Complex,MHC)即人白细胞抗原(Human Leukocyte Antigen,HLA)I和II结合,并最终引起CD4+T和CD8+T细胞的活化,这一过程对于机体识别、清除病原体,乃至杀伤肿瘤细胞有着至关重要的作用。黑色素瘤细胞过表达IFI30,可以增强CD4+T细胞应答,但在黑色素瘤中激活CD8+T细胞机制以及ADARs介导的IFI30编辑情况及功能改变尚不明确。需要注意的是,IFN-γ诱导黑色素瘤细胞表达IFI30的过程不受经典的II型反式激活蛋白(MHC Class II Transactivator,CIITA)调节,而受信号传导子和转录活化子1(signal transducer and activator of transcription 1,STAT1)调控。此外,有证据表明IFI30抑制晚期人转移性黑色素瘤配对盒基因3(Paired Box 3,PAX3)表达,增加对放化疗的敏感性。CD8+T细胞因其特有的细胞毒性作用,而被广泛地应用于肿瘤免疫治疗中,IFI30对包含二硫键的I类抗原的处理也有助于CD8+T细胞的有效激活。但在肿瘤细胞中IFI30如何介导MHC-I类抗原提呈过程尚不明确,研究IFI30受调控的机制对于我们理解抗原提呈过程,黑色素瘤进展以及免疫治疗具有重要意义。组织蛋白酶(cathepsins,CTS)是一组最初发现于各种动物组织细胞内(尤其是溶酶体部分)的蛋白酶。它是半胱氨酸蛋白酶家族中最重要的成员,与许多严重的疾病如人类的肿瘤、骨质疏松症和关节炎密切相关,是近年来倍受关注的一类蛋白酶。组织蛋白酶是由无活性的前体蛋白酶水解形成的,前体蛋白酶由一个信号肽、两个前肽和一个包含成熟蛋白酶活性部位的催化结构域组成。信号肽有10-20个氨基酸残基,可将前体蛋白酶由核糖体运送到内质网,在那里被水解成为只包含前肽和催化结构域的前体蛋白酶。蛋白酶原的氨基酸残基数从36到315不等,占据了酶的活性位点,这种催化作用的酶原接下来被运送到细胞内溶酶体,在其酸性条件下自动水解,产生具有活性的成熟组织蛋白酶。IFI30与组织蛋白酶之间存在相互调节。正如之前文献报道的,组织蛋白酶参与IFI30的成熟过程,不同的组织蛋白酶对于IFI30前体肽的切割效果不同。反之,IFI30也可以影响组织蛋白酶的表达,有证据表明IFI30与组织蛋白酶S共定位在B细胞溶酶体中,并且IFI30可以降解组织蛋白酶S。类似地,在黑色素瘤细胞中IFI30与组织蛋白酶B和D存在共定位,但IFI30与组织蛋白酶B和D之间具体的作用机制尚不明确。组织蛋白酶介导恒定链(Invariant Chain,Ii)降解,使得MHC分子抗原结合沟充分暴露,从而决定了抗原的提呈效率。探究IFI30对组织蛋白酶的调节以及IFI30本身处理多肽的能力有助于我们理解IFI30在MHC信号通路中扮演的角色。此外,肿瘤免疫微环境中的肿瘤相关巨噬细胞(Tumor-Associated Macrophages,TAM)可通过刺激肿瘤细胞增殖、促进肿瘤血管生成并协同肿瘤逃避免疫监控来帮助肿瘤生长。靶向TAM的手段一般包括去除TAM和重塑TAM两类,即减少免疫抑制性巨噬细胞浸润肿瘤或改变其极化状态。通常来说,肿瘤细胞可以分泌趋化因子来招募循环系统中的巨噬细胞,巨噬细胞也可以通过分泌细胞因子来调控肿瘤细胞的增殖、迁移和侵袭。研究肿瘤细胞与巨噬细胞之间的交互作用可以帮助我们理解肿瘤免疫耐受机制,为免疫治疗提供理论支持。综上,我们想探究以下几个关键问题:1.探究野生型和RNA编辑型IFI30能否改变肿瘤浸润的CD8+T细胞和巨噬细胞数量。2.阐明野生型和RNA编辑型IFI30能否影响巨噬细胞M1/M2极化,以及肿瘤细胞IFI30表达变化是否影响其对巨噬细胞的招募能力。3.揭示野生型和RNA编辑型IFI30对组织蛋白酶B和HLA I稳定性的影响。研究方法：1.分别使用人IFN-γ和组织蛋白酶B特异性抑制剂CA-074刺激人黑色素瘤细胞A375、A2058,检测IFI30、组织蛋白酶B和HLA I蛋白水平。2.使用慢病毒载体,在A375、A2058和小鼠黑色素瘤细胞B16F10过表达对照、野生型IFI30和编辑型IFI30质粒,构建稳转细胞系。3.利用实时荧光定量聚合酶链反应(Real Time-quantitative Polymerase Chain Reaction,RT-q PCR)检测A375、A2058稳转细胞系IFI30 m RNA表达,使用蛋白质免疫印迹法(Western Blot,WB)检测A375、A2058稳转细胞系IFI30和V5标签蛋白水平,检测B16F10稳转细胞系标签6-his蛋白水平。4.利用集落形成实验和CCK-8计数法(Cell Counting Kit-8)检测各组细胞增殖能力。5.用裸鼠成瘤实验和C57BL/6小鼠的皮下肿瘤形成实验比较各肿瘤细胞组在体内的生长速度。6.手术摘取各组荷瘤小鼠脾脏,拍照称重,磁珠分离(Magnetic-Activated cell sorting,MACS)小鼠脾脏CD8+T细胞。7.流式细胞术(Flow Cyto Metry,FCM)检测各组荷瘤小鼠脾脏CD8+T细胞数量。8.利用免疫荧光法(Immunofluorescence,IF)分析各组荷瘤小鼠肿瘤浸润CD8+T细胞数量。9.流式细胞术鉴定鼠IFN-γ刺激各组小鼠黑色素瘤细胞后,膜表面MHC I类分子蛋白表达情况。10.蛋白质膜浆分离法探究黑色素瘤细胞内不同组分HLA I类分子的表达水平。11.使用蛋白酶体抑制剂MG132和蛋白合成抑制剂CHX处理人黑色素瘤细胞A375,检测IFI30、组织蛋白酶B和HLA I蛋白降解趋势。12.使用组织蛋白酶B特异性抑制剂CA-074刺激各组A375细胞,检测IFI30、组织蛋白酶B和HLA I蛋白水平。13.使用PMA刺激THP-1贴壁分化成M0巨噬细胞后,提取A375肿瘤细胞条件性培养基与M0巨噬细胞共培养,RT-q PCR检测巨噬细胞M1/M2极化标志物,WB检测巨噬细胞IFI30、组织蛋白酶B蛋白水平。14.用免疫荧光法分析各组荷瘤小鼠肿瘤组织中肿瘤相关巨噬细胞M1/M2极化标志物。15.手术摘取各组荷瘤小鼠股骨和胫骨,分离小鼠骨髓来源巨噬细胞。16.分离原代小鼠骨髓来源巨噬细胞,与脾来源的CD8+T细胞共培养,流式细胞术检测CD8+T细胞的活化比例(选取颗粒霉素B和CD8双阳性细胞)。17.采用细胞因子体外分别诱导巨噬细胞M1和M2极化,收集肿瘤细胞条件性培养基,与极化巨噬细胞共培养,通过Transwell实验检测不同肿瘤细胞条件性培养基对不同极化的巨噬细胞迁移能力的影响。18.使用PMA刺激THP-1贴壁分化成M0巨噬细胞后,组织蛋白酶B特异性抑制剂CA-074刺激M0巨噬细胞,检测IFI30蛋白水平。研究结果：1.野生型IFI30和RNA编辑型IFI30均抑制黑色素瘤细胞体外生长。裸鼠皮下成瘤实验中,与对照组相比,野生型IFI30抑制黑色素瘤细胞在体内生长,与野生组相比,RNA编辑型IFI30抑制黑色素瘤细胞在体内生长;C57BL/6小鼠皮下成瘤实验中,与对照组相比,野生型IFI30促进黑色素瘤细胞在体内生长,与野生组相比,RNA编辑型IFI30抑制黑色素瘤细胞在体内生长。2.与对照组相比,野生型IFI30荷瘤小鼠CD8+T细胞向肿瘤浸润减少,而编辑型IFI30荷瘤小鼠比野生组CD8+T细胞浸润多;3.野生型IFI30荷瘤小鼠的肿瘤相关巨噬细胞倾向于M2极化,而编辑型IFI30荷瘤小鼠的肿瘤相关巨噬细胞倾向于M1极化;4.野生型IFI30抑制黑色素瘤细胞膜表面HLA I类分子的表达,促进黑色素瘤细胞胞浆HLA I类分子表达,而编辑型IFI30作用相反;5.野生型IFI30通过非蛋白酶体途径促进黑色素瘤细胞HLA I总蛋白降解,而编辑型IFI30通过抑制HLA I蛋白酶体途径来抑制其降解,从而维持其蛋白稳定性;更重要的是野生型IFI30促进黑色素瘤细胞膜表面HLA I蛋白降解,而编辑型IFI30延缓黑色素瘤细胞膜表面HLA I蛋白降解。6.野生型和编辑型IFI30通过蛋白酶体途径促进CTSB成熟体降解,从而干扰其蛋白稳定性;7.IFI30通过CTSB调节HLA I总蛋白表达;8.野生型IFI30肿瘤细胞条件性培养基通过抑制巨噬细胞CTSB成熟来抑制巨噬细胞的M1极化状态;9.野生型IFI30肿瘤细胞条件性培养基由CTSB介导抑制M1型巨噬细胞迁移,促进M2型巨噬细胞迁移,而编辑型IFI30作用相反。结论：1.在鼠黑色素瘤细胞中,IFI30抑制CD8+T细胞浸润肿瘤,并使得肿瘤相关巨噬细胞倾向于M2极化,而IFI30发生RNA编辑后,CD8+T细胞浸润肿瘤增多,肿瘤相关巨噬细胞倾向于M1极化。2.IFI30抑制黑色素瘤细胞膜HLA I蛋白表达,促进黑色素瘤细胞浆HLA I蛋白表达,从而抑制CD8+T细胞浸润肿瘤,而编辑型IFI30作用相反;野生型IFI30通过非蛋白酶体途径促进黑色素瘤细胞HLA I降解,而编辑型IFI30抑制HLA I蛋白酶体途径降解。3.IFI30及其编辑型均促进CTSB成熟体经蛋白酶体途径降解,并且两者均通过CTSB调节HLA I总蛋白表达。4.IFI30抑制巨噬细胞CTSB成熟过程,从而抑制巨噬细胞的M1极化状态;过表达野生型IFI30的人源肿瘤细胞条件性培养基通过CTSB抑制M1型巨噬细胞迁移,促进M2型巨噬细胞迁移,而编辑型IFI30作用相反。更多还原

【Abstract】 Objective:When tumor cells invade the body,the immune system recognizes tumor-associated antigens(TAAs)on the surface of the tumor cells and activates specific humoral and cellular immune responses to clear the invading tumor cells;at the same time,tumor cells can negatively regulate the body’s immune responses through various channels to avoid immune supervision.At the same time,tumor cells can negatively regulate the body’s immune response and escape immune supervision through a variety of mechanisms.Immune infiltration is important in tumorigenesis and development,as well as in the clinical prognosis of cancer patients.Malignant melanoma is a tumor that develops from melanocytes in the body’s skin,mucous membranes,and retina.Cutaneous melanoma manifests as pigmented lesions that change dramatically over months or years.Despite its low incidence,it is highly malignant and susceptible to metastasis,with a high lethality rate.Melanoma responds to immunotherapy more favorably than other solid tumors,making it a more desirable candidate for research in the field of tumor immunology.The IFI30(Interferon Gamma-Inducible Protein 30)gene encodes the Gamma-Interferon-Inducible Lysosomal Thiol Reductase(GILT),a 250 amino acid precursor with a molecular weight of approximately 35 k D.The mannose-6-phosphate receptor can activate the intracellular IFI30 precursor(mannose6-phosphate receptor).IFI30could reduce disulfide bonds and promote the unfolding of proteins that contain disulfide bonds.Its thiol reductase activity requires an acidic lysosome environment.Previously,Professor Xu Xiaoyan’s team discovered that IFI30 can undergo RNA editing mediated by ADAR1 and ADAR2,members of the Adenosine Deaminases Acting on RNA(ADARs)family,as evidenced by RNA editing from A to I resulting in the replacement of the threonine at position 223(Threonine,T)to alanine,A.(T223A).However,the role of IFI30 in the tumor microenvironment and whether RNA editing results in altered IFI30 function is unclear.Antigen-presenting cells process antigenic peptides and bind to MHC I and II,resulting in the activation of CD4~+and CD8~+T cells,a process required for the body to recognize,clear pathogens,and even kill tumor cells.Overexpression of IFI30 in melanoma cells enhances CD4~+T cell responses,but the mechanism of activation of CD8~+T cells in melanoma and ADAR-mediated editing of IFI30 and functional alterations are unclear.It is important to note that IFN-γinduced IFI30 expression in melanoma cells is not regulated by classical CIITA,but by STAT1.Furthermore,there is evidence that IFI30 inhibits PAX3 expression in advanced human metastatic melanoma and increases sensitivity to radiotherapy.Because of its unique cytotoxic effects,CD8~+T is widely used in tumor immunotherapy,and IFI30 processing of class I antigens containing disulfide bonds also contributes to effective activation of CD8~+T cells.However,it is unclear how IFI30 mediates MHC class I antigen presentation in tumor cells,and it is critical to investigate the mechanisms by which IFI30 is regulated to understand the process of antigen presentation,melanoma progression,and immunotherapy.Cathepsin are a group of proteases found in the intracellular(especially the lysosomal fraction)of various animal tissues and are the main members of the cysteine protease family,which are closely associated with several major diseases such as human tumors,osteoporosis,and arthritis,and are a group of target proteases that have received much attention in recent years.Cathepsin are all made by the hydrolysis of inactive procathepsin,which consists of a signal-peptide,a propeptide and a catalytic domain containing the active center of the mature protease.The signal-peptide is between 10 and 20 amino acid residues in length and is responsible for transporting the ribosomally expressed procathepsin to the endoplasmic reticulum,where it is hydrolysed to form a procathepsin containing only the precursor peptide and the catalytic domain.The IFI30 and Cathepsin are regulated by each other.As mentioned earlier,cathepsin is involved in the maturation of IFI30 and different histases have different effects on the cleavage of IFI30 precursor peptides.Similarly,in melanoma cells IFI30 co-localized with Cathepsin B and D,but the underlying mechanism of regulation between IFI30 and Cathepsin B and D remains unclear.Cathepsin mediate the degradation of invariant chain li,allowing appropriate exposure of the antigen-binding furrow of the MHC molecule and thus determining the efficiency of antigen presentation.Exploring the regulation of cathepsin by IFI30 and the ability of IFI30itself to process peptides has helped us to understand the role of IFI30 in the MHC signalling pathway.In addition,Tumor-Associated Macrophages(TAM)are an important component of the tumor immune microenvironment,infiltrating most solid tumors in large numbers and promoting tumor progression by stimulating tumor cell proliferation,angiogenesis,metastasis and protecting tumor cells from immune system a.Therapeutic approaches to TAM can be broadly classified into two categories:TAM removal and TAM reprogramming,that is,inhibiting infiltrated macrophages or change its polarization.In general,tumor cells can secrete chemokines to recruit circulating macrophages,and macrophages can also secrete cytokines to regulate the biological functions of tumor cells.Studying the interaction between tumor cells and macrophages can help us understand the mechanism of tumor immune tolerance and provide theoretical support for immunotherapy.In summary,we would like to explore the following key questions:1.To investigate whether IFI30 and RNA-edited IFI30 can alter the HLA-I-specific antigen presentation process.2.To elucidate whether IFI30 and RNA-edited IFI30 can affect the M1/M2polarization of macrophages and whether changes in IFI30 expression in tumor cells affect their recruitment ability to macrophages.3.To reveal whether RNA-edited IFI30on the stability of cathepsin B.Methods:1.A375 and A2058 were stimulated with human IFN-γand Cathepsin B-specific inhibitor CA-074,respectively,to detect IFI30,Cathepsin B and HLA I protein levels.2.Lentiviral vectors were used to construct stable transfected cell lines in human melanoma cells A375,A2058 and mouse melanoma cells B16F10 overexpressing control,wild-type IFI30 and edited IFI30 plasmids.3.Real Time-quantitative Polymerase Chain Reaction(RT-q PCR)was used to detect IFI30 m RNA expression in A375 and A2058 stable-transformed cell lines,and Western Blot(WB)was used to detect the levels of IFI30 and V5 tag proteins in A375 and A2058 stable-transformed cell lines,and the levels of 6-his tag protein in B16F10 stable-transformed cell line were detected.4.In vitro proliferation of each group of cells was measured by colony formation assay and CCK8 counting(Cell Counting Kit-8).5.The growth rate of each tumor cell line in vivo was compared using tumorigenesis assay in BALb/c-nu mice and subcutaneous tumor formation assay in C57BL/6 mice.6.Spleens of each group of tumor-bearing mice were surgically removed,photographed,and weighed.Magnetic-activated cell sorting(MACS)was used to isolate the spleen CD8~+T.7.Flow Cyto Metry(FCM)was used to analyze the number of splenic CD8~+T in each group of tumor-bearing mice.8.Immunofluorescence(IF)was used to analyze the spleen CD8~+T in each group of tumor-bearing mice.The tumor-infiltrating CD8~+T cells of each group of tumor-bearing mice were analyzed.9.The expression of MHC class I molecules on the membrane surface of each group of mouse melanoma cells was identified by flow cytometry after stimulation with murine IFN-γ.10.The expression levels of HLA class I molecules at different locations in melanoma cells were probed by cytoplasmaic/membrane protein isolation.11.The human melanoma cells A375were treated with the proteasome inhibitor MG132 and the protein synthesis inhibitor CHX to detect protein degradation of IFI30,Cathepsin B and HLA I.12.Stimulation of groups of A375 cells using Cathepsin B-specific inhibitor CA-074 to detect IFI30,Cathepsin B and HLA I protein levels.13.After stimulation of THP-1 apposition and differentiation into M0 macrophages via PMA,extraction of the A375 cell supernatant was co-cultured with M0 macrophages,and the M1/M2 polarization markers of macrophages were detected by q PCR,and the levels of IFI30 and Cathepsin B were detected by WB.14.The M1/M2 polarization markers of tumor-associated macrophages in each group of tumor-bearing mice were analyzed by immunofluorescence.15.The femurs and tibias of each group of tumor-bearing mice were surgically removed,and the bone marrow-derived macrophages of mice were isolated and cultured ex vivo.16.The primary mouse bone marrow-derived macrophages were isolated and co-cultured with splenic-derived CD8~+T cells,and the activation ratio of CD8~+T cells(selected from granulomycin B and CD8 double-positive cells)was detected by flow cytometry.17.Cytokines were used to induce macrophage M1 and M2 polarization in vitro,respectively.Tumor cell conditioned medium was collected and co-cultured with polarized macrophages,and the effect of different tumor cell conditioned medium on the migration ability of differently polarized macrophages was examined by Transwell assay.18.After stimulation of THP-1 apposition and differentiation into M0 using PMA,M0 was stimulated with the cathepsin B specific inhibitor CA-074 and IFI30 protein levels were measured.Results:1.Both wild-type IFI30 and RNA-edited IFI30 inhibited the growth of melanoma cells in vitro.In the subcutaneous tumorigenesis assay in BALb/c-nu mice,wild-type IFI30 inhibited melanoma cell growth in vivo compared with the control group,and RNA-edited IFI30 inhibited melanoma cell growth in vivo compared with the wild group;in the subcutaneous tumorigenesis assay in C57BL/6 mice,wild-type IFI30 promoted melanoma cell growth in vivo compared with the control group,and RNA-edited IFI30 inhibited melanoma cell growth in vivo.2.Compared to controls,wild-type IFI30 tumor-bearing mice have fewer tumor-infiltrating CD8~+T cells,whereas edited IFI30 tumor-bearing mice have more CD8~+T cells.3.Tumor-associated macrophages in wild-type IFI30 tumor-bearing mice were predominantly M2,whereas tumor-associated macrophages in edited IFI30 tumor-bearing mice were predominantly M1.4.In melanoma cells,wild-type IFI30 inhibited the expression of HLA class I molecules on the membrane surface while promoting the expression of cytoplasmic HLA class I molecules,whereas edited IFI30 had the opposite effect.Wild-type IFI30promoted the degradation of total HLA I protein in melanoma cells by non-proteasome pathway,while the edited IFI30 inhibited the degradation by inhibiting the HLA I proteasome pathway,thus maintaining the protein stability.More importantly,wild-type IFI30 promoted the degradation of HLA I protein on the surface of melanoma cell membrane,while the edited type IFI30 delayed the degradation of HLA I protein on the surface of melanoma cell membrane.6.IFI30,both wild-type and edited type,promotes the degradation of mature CTSB via the proteasome hydrolysis pathway,interfering with its protein stability.IFI30 regulates the expression of total HLA I protein through CTSB.8.wild-type IFI30 tumor supernatant inhibits macrophage M1polarization by inhibiting CTSB maturation in macrophages.9.wild-type IFI30 tumor supernatant inhibits M1 macrophage migration and promotes M2 macrophage migration mediated by CTSB,whereas edited-type IFI30 has the opposite effect.Conclusion:1.IFI30 inhibits tumor-infiltrating CD8~+T cells and reduces the M1polarization state of tumor-associated macrophages in melanoma cells.2.IFI30suppresses HLA I expression by blocking CTSB.3.IFI30 may affect CTSB and HLA I protein intracellular stability by promoting their degradation.更多还原