利用MHC-I/肽结合的预测与免疫学实验相结合的方法鉴定肿瘤抗原表位
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摘要
目的:建立中国人群常见的人类白细胞抗原(HLA)分子限定的肿瘤抗原免疫表位的鉴定技术,为分离及鉴定肿瘤特异性T细胞以及克隆肿瘤抗原特异性T细胞受体基因提供实验基础。方法:以肿瘤/睾丸抗原NY-ESO、MAGE-A1和KK-LC-1作为模型,选用中国人群常见的HLA-I分型,利用主要组织相容性复合体(MHC)/肽预测软件对这3个抗原的MHC-I类表位进行预测。用PCR技术测定健康志愿者的HLA分型,以志愿者含有的中国人群常见HLA-A*11:01和HLA-B*46:01分型选择预测的抗原表位,设计合成KK-LC-1长肽,进行T细胞刺激实验,用ELISPOT和流式细胞仪分析产生干扰素γ(IFN-γ)的T细胞数量和CD137上调反应,以验证肽对T细胞特异性刺激的有效性。对其中一个反应最好的KK-LC-1长肽,开展短肽的验证。结果:(1)3个肿瘤/睾丸抗原在我国人群35种常见HLA分型中存在众多的强结合表位,在不同蛋白质的序列中分布不均匀;(2)KK-LC-1长肽可刺激T细胞产生T细胞活化反应,即释放IFN-γ和T细胞上调CD137分子,与预测的HLA分型基本吻合;(3)KK-LC-1短肽比长肽刺激效果更好,有更精确的抗原表位。结论:利用MHC/肽预测法并结合抗原特异性刺激实验可快速鉴定肿瘤抗原的T细胞表位;根据长肽的结果缩短肽段,可确定抗原表位。本研究为快速确定肿瘤抗原表位提供了新的途径。
AIM: To establish the methods to identify immune epitopes of tumor antigens restricted by human leukocyte antigen(HLA) alleles frequent in the Chinese population for the isolation and identification of tumor-specific T cells and the cloning of tumor antigen-specific T-cell receptor gene. METHODS: Three cancer/testis antigens NY-ESO,MAGE-A1 and KK-LC-1 were selected as the model antigens to predict the MHC-I binding peptides by major histocompatibility complex(MHC)-peptide binding prediction software. The HLA alleles of healthy volunteers were determined by PCR. The predicted epitopes restricted by HLA-A*11:01 and HLA-B*46:01 of the volunteers were selected for the long peptide design. ELISPOT and flow cytometry were used to analyze the interferon-γ(IFN-γ) spots of the activated T-cells and the up-regulation of CD137 to verify the effectiveness of peptides for T-cell-specific stimulation. We selected long peptides with the best response to verify short peptides. RESULTS: Three cancer/testis antigens containing many strong binding epitopes restricted by 35 HLA alleles common in Chinese population were found. It was found that the predicted epitopes were not equally distributed in the protein sequences. KK-LC-1 antigen was selected for further study. The 2 long peptides stimulated T-cell activation: the release of IFN-γ and up-regulation of CD137 molecules by T cells of the volunteers with the matched HLA alleles. The short peptides worked better than the long peptides.CONCLUSION: Tumor antigen epitopes are quickly identified by MHC/peptide binding prediction and T-cell stimulation analysis. According to the reaction of the long peptide, the short peptide is synthesized to verify the precise epitope. This study provides a new way to determine tumor antigen epitopes quickly.
AIM: To establish the methods to identify immune epitopes of tumor antigens restricted by human leukocyte antigen(HLA) alleles frequent in the Chinese population for the isolation and identification of tumor-specific T cells and the cloning of tumor antigen-specific T-cell receptor gene. METHODS: Three cancer/testis antigens NY-ESO,MAGE-A1 and KK-LC-1 were selected as the model antigens to predict the MHC-I binding peptides by major histocompatibility complex(MHC)-peptide binding prediction software. The HLA alleles of healthy volunteers were determined by PCR. The predicted epitopes restricted by HLA-A*11:01 and HLA-B*46:01 of the volunteers were selected for the long peptide design. ELISPOT and flow cytometry were used to analyze the interferon-γ(IFN-γ) spots of the activated T-cells and the up-regulation of CD137 to verify the effectiveness of peptides for T-cell-specific stimulation. We selected long peptides with the best response to verify short peptides. RESULTS: Three cancer/testis antigens containing many strong binding epitopes restricted by 35 HLA alleles common in Chinese population were found. It was found that the predicted epitopes were not equally distributed in the protein sequences. KK-LC-1 antigen was selected for further study. The 2 long peptides stimulated T-cell activation: the release of IFN-γ and up-regulation of CD137 molecules by T cells of the volunteers with the matched HLA alleles. The short peptides worked better than the long peptides.CONCLUSION: Tumor antigen epitopes are quickly identified by MHC/peptide binding prediction and T-cell stimulation analysis. According to the reaction of the long peptide, the short peptide is synthesized to verify the precise epitope. This study provides a new way to determine tumor antigen epitopes quickly.
引文
[1] Yang JC, Rosenberg SA. Adoptive T-cell therapy for cancer[J]. Adv Immunol, 2016, 130:279-294.
[2] Baruch EN, Berg AL, Besser MJ, et al. Adoptive T cell therapy: an overview of obstacles and opportunities[J]. Cancer, 2017, 123(S11):2154-2162.
[3] Rosenberg SA, Yang JC, Sherry RM, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy[J]. Clin Cancer Res, 2011, 17(13):4550-4557.
[4] Dunn GP, Koebel CM, Schreiber RD. Interferons, immunity and cancer immunoediting[J]. Nat Rev Immunology, 2006, 6(11):836-848.
[5] 中华骨髓库CWD表编撰组.中国常见及确认的HLA等位基因表(CWD)[C]//中华造血干细胞捐献者资料库管理中心. 中华骨髓库第四届年会论文集.2013:75-96.
[6] Simpson AJ, Caballero OL, Jungbluth A, et al. Cancer/testis antigens, gametogenesis and cancer[J]. Nat Rev Cancer, 2005, 5(8):615-625.
[7] Li LP, Lampert JC, Chen X, et al. Transgenic mice with a diverse human T cell antigen receptor repertoire[J]. Nat Med, 2010, 16(9):1029-1034.
[8] Li LP, Blankenstein T. Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast-embryonic stem cell fusion[J]. Nat Protoc, 2013, 8(8):1567-1582.
[9] Stevanovic S, Pasetto A, Helman SR. Landscape of immunogenic tumor antigens in successful immunotherapy of virally induced epithelial cancer[J]. Science, 2017, 356(6334):200-205.
[10] Allen PM, Strydom DJ, Unanue ER. Processing of lysozyme by macrophages: identification of the determinant recognized by two T-cell hybridomas[J]. Proc Natl Acad Sci U S A, 1984, 81(8):2489-2493.
[11] Babbitt BP, Allen PM, Matsueda G, et al. Binding of immunogenic peptides to Ia histocompatibility molecules[J]. Nature, 1985, 317(6035):359-361.
[12] Falk K, Rotzschke O, Stevanovic S, et al. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules[J]. Nature, 1991, 351(6324):290-296.
[13] Nielsen M, Lundegaard C, Worning P, et al. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations[J]. Protein Sci, 2003, 12(5):1007-1017.
[14] Fukuyama T, HanagirI T, Takenoyama M, et al. Identification of a new cancer/germline gene, KK-LC-1, encoding an antigen recognized by autologous CTL induced on human lung adenocarcinoma[J]. Cancer Res, 2006, 66(9):4922-4928.
[15] Shida A, Futawatari N, Fukuyama T, et al. Frequent high expression of Kita-Kyushu lung cancer antigen-1 (KK-LC-1) in gastric cancer[J]. Anticancer Res, 2015, 35(6):3575-3579.
[16] Paret C, Simon P, Vormbrock K, et al. CXorf61 is a target for T cell based immunotherapy of triple-negative breast cancer[J]. Oncotarget, 2015, 6(28):25356-25367.
[17] Bonini C, Mondino A. Adoptive T-cell therapy for cancer: The era of engineered T cells[J]. Eur J Immunol, 2015, 45(9):2457-2469.
[18] Fesnak AD, June CH, Levine BL. Engineered T cells: the promise and challenges of cancer immunotherapy[J]. Nat Rev Cancer, 2016, 16(9):566-581.
[2] Baruch EN, Berg AL, Besser MJ, et al. Adoptive T cell therapy: an overview of obstacles and opportunities[J]. Cancer, 2017, 123(S11):2154-2162.
[3] Rosenberg SA, Yang JC, Sherry RM, et al. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy[J]. Clin Cancer Res, 2011, 17(13):4550-4557.
[4] Dunn GP, Koebel CM, Schreiber RD. Interferons, immunity and cancer immunoediting[J]. Nat Rev Immunology, 2006, 6(11):836-848.
[5] 中华骨髓库CWD表编撰组.中国常见及确认的HLA等位基因表(CWD)[C]//中华造血干细胞捐献者资料库管理中心. 中华骨髓库第四届年会论文集.2013:75-96.
[6] Simpson AJ, Caballero OL, Jungbluth A, et al. Cancer/testis antigens, gametogenesis and cancer[J]. Nat Rev Cancer, 2005, 5(8):615-625.
[7] Li LP, Lampert JC, Chen X, et al. Transgenic mice with a diverse human T cell antigen receptor repertoire[J]. Nat Med, 2010, 16(9):1029-1034.
[8] Li LP, Blankenstein T. Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast-embryonic stem cell fusion[J]. Nat Protoc, 2013, 8(8):1567-1582.
[9] Stevanovic S, Pasetto A, Helman SR. Landscape of immunogenic tumor antigens in successful immunotherapy of virally induced epithelial cancer[J]. Science, 2017, 356(6334):200-205.
[10] Allen PM, Strydom DJ, Unanue ER. Processing of lysozyme by macrophages: identification of the determinant recognized by two T-cell hybridomas[J]. Proc Natl Acad Sci U S A, 1984, 81(8):2489-2493.
[11] Babbitt BP, Allen PM, Matsueda G, et al. Binding of immunogenic peptides to Ia histocompatibility molecules[J]. Nature, 1985, 317(6035):359-361.
[12] Falk K, Rotzschke O, Stevanovic S, et al. Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules[J]. Nature, 1991, 351(6324):290-296.
[13] Nielsen M, Lundegaard C, Worning P, et al. Reliable prediction of T-cell epitopes using neural networks with novel sequence representations[J]. Protein Sci, 2003, 12(5):1007-1017.
[14] Fukuyama T, HanagirI T, Takenoyama M, et al. Identification of a new cancer/germline gene, KK-LC-1, encoding an antigen recognized by autologous CTL induced on human lung adenocarcinoma[J]. Cancer Res, 2006, 66(9):4922-4928.
[15] Shida A, Futawatari N, Fukuyama T, et al. Frequent high expression of Kita-Kyushu lung cancer antigen-1 (KK-LC-1) in gastric cancer[J]. Anticancer Res, 2015, 35(6):3575-3579.
[16] Paret C, Simon P, Vormbrock K, et al. CXorf61 is a target for T cell based immunotherapy of triple-negative breast cancer[J]. Oncotarget, 2015, 6(28):25356-25367.
[17] Bonini C, Mondino A. Adoptive T-cell therapy for cancer: The era of engineered T cells[J]. Eur J Immunol, 2015, 45(9):2457-2469.
[18] Fesnak AD, June CH, Levine BL. Engineered T cells: the promise and challenges of cancer immunotherapy[J]. Nat Rev Cancer, 2016, 16(9):566-581.