人外周血TIM3基因敲除T淋巴细胞的制备及其抗肿瘤作用
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摘要
目的:通过CRISPR-Cas9基因编辑技术及质粒电转染技术制备T细胞免疫球蛋白黏液素3(TIM3)基因敲除的人外周血T淋巴细胞,探讨敲除TIM3基因后能否增强T细胞免疫功能及其抗肿瘤作用。方法:通过电转染法将hTIM3 sgRNA/Cas9双质粒共转染EBV阳性胃癌患者外周血T淋巴细胞,电转24 h后流式细胞术检测转染效率并利用荧光显微镜观察;体外培养过程中观察基因敲除后T细胞增殖活性,流式细胞术验证TIM3基因敲除效率以及细胞表型的变化。用肿瘤抗原肽活化T细胞检测TIM3基因敲除后T细胞分泌细胞因子水平及体外杀伤胃癌AGS-EBV细胞的能力。结果:电转染法能够有效地将hTIM3 sgRNA/Cas9双质粒敲除体系转入人外周血T淋巴细胞,其转染效率平均达(41.5±3.6)%,基因敲除效率波动在40.0%～50.0%(均P<0.01);经抗原活化后的基因敲除组T细胞的增殖活性和免疫表型未见明显变化,表面活化分子中仅见HLA-DR较对照组明显提高(P<0.05);TIM3基因敲除T细胞分泌TNF-α、IFN-γ的水平显著增高(P<0.01或P<0.05),体外杀伤胃癌AGS-EBV细胞的能力也明显增强(均P<0.05)。结论:用CRISPR-Cas9基因编辑及质粒电转染技术制备人外周血TIM3基因敲除T淋巴细胞的方法简易可行,在体外的扩增活化中保持TIM3基因水平下调并具有更高的免疫应答水平以及更强的抗肿瘤作用。这一新技术的研发为基因工程化细胞免疫治疗提供了新的思路。
Objective: To prepare human peripheral blood T lymphocytes with TIM3(T cell immunologlobulin and mucin-3) gene knockout by using CRISPR-Cas9 gene editing technique and plasmid electrotransfection system, and to discuss whether the knockout of TIM3 gene could enhance the immune response and anti-tumor efficacy of T cells. Methods: Double plasmids hTIM3 sgRNA/Cas9 were transfected into human peripheral blood T lymphocytes of EBV positive gastric cancer patients by using electrotransfection system. The transfection efficiency was examined 24h later by flow cytometry and fluorescence microscope. The proliferation activity of the T cells after gene knockout was observed during in vitro culture, and the knockout efficiency and phenotypes of the modified T cells were evaluated by flow cytometry. Furthermore, tumor antigen peptide was used to activate T cells, and the level of modified T cells secreting cytokines and its cytotoxicity against gastric cancer AGS-EBV cells were evaluated. Results: Electrotransfection system could successfully transfect h TIM3 sgRNA/Cas9 double plasmids into human peripheral blood T lymphocytes with an average transfection efficiency of(41.5±3.6)%, and the gene knockout efficiency fluctuated between 40.0% and 50.0%(all P<0.01). The proliferation of the modified T cells was not significantly changed in the TIM3 gene knockout group even after the prolonged co-culturing with tumor antigenic peptide; and for the activated molecules, only HLA-DR exhibited significant elevation as compared with control group(P<0.05).Remarkably, T cells with TIM3 gene knockout showed significantly elevated secretion of TNF-α and IFN-γ(P<0.01 or P<0.05), as well as obviously enhanced in vitro cytotoxicity against gastric cancer AGS-EBV cells(P<0.05). Conclusion: It's simple and feasible of CRISPR-Cas9 gene editing technique and plasmid electrotransfection system to prepare T lymphocytes with engineered TIM3 gene knockout. The expression level of TIM3 was down-regulated in in vitro culture. More importantly, the modified T cells performed superior immune response and cytotoxicity, which may provide a new idea for gene engineering cell immunotherapy.
Objective: To prepare human peripheral blood T lymphocytes with TIM3(T cell immunologlobulin and mucin-3) gene knockout by using CRISPR-Cas9 gene editing technique and plasmid electrotransfection system, and to discuss whether the knockout of TIM3 gene could enhance the immune response and anti-tumor efficacy of T cells. Methods: Double plasmids hTIM3 sgRNA/Cas9 were transfected into human peripheral blood T lymphocytes of EBV positive gastric cancer patients by using electrotransfection system. The transfection efficiency was examined 24h later by flow cytometry and fluorescence microscope. The proliferation activity of the T cells after gene knockout was observed during in vitro culture, and the knockout efficiency and phenotypes of the modified T cells were evaluated by flow cytometry. Furthermore, tumor antigen peptide was used to activate T cells, and the level of modified T cells secreting cytokines and its cytotoxicity against gastric cancer AGS-EBV cells were evaluated. Results: Electrotransfection system could successfully transfect h TIM3 sgRNA/Cas9 double plasmids into human peripheral blood T lymphocytes with an average transfection efficiency of(41.5±3.6)%, and the gene knockout efficiency fluctuated between 40.0% and 50.0%(all P<0.01). The proliferation of the modified T cells was not significantly changed in the TIM3 gene knockout group even after the prolonged co-culturing with tumor antigenic peptide; and for the activated molecules, only HLA-DR exhibited significant elevation as compared with control group(P<0.05).Remarkably, T cells with TIM3 gene knockout showed significantly elevated secretion of TNF-α and IFN-γ(P<0.01 or P<0.05), as well as obviously enhanced in vitro cytotoxicity against gastric cancer AGS-EBV cells(P<0.05). Conclusion: It's simple and feasible of CRISPR-Cas9 gene editing technique and plasmid electrotransfection system to prepare T lymphocytes with engineered TIM3 gene knockout. The expression level of TIM3 was down-regulated in in vitro culture. More importantly, the modified T cells performed superior immune response and cytotoxicity, which may provide a new idea for gene engineering cell immunotherapy.
引文
[1]SHARPE A H.Introduction to checkpoint inhibitors and cancer immunotherapy[J/OL].Immunol Rev,2017,276(1):5-8[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362112/.DOI:10.1111/imr.12531.
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[7]ZHOU Q,MUNGER M E,VEENSTRA R G,et al.Coexpression of Tim-3 and PD-1 identifies a CD8+T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia[J/OL].Blood,2011,117(17):4501-4510[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3099570/.DOI:10.1182/blood-2010-10-310425.
[8]LI Z,LIU X B,GUO R B,et al.TIM-3 plays a more important role than PD-1 in the functional impairments of cytotoxic T cells of malignant Schwannomas[J].Tumour Biol,2017,39(5):1010428317698352.DOI:10.1177/1010428317698352.
[9]LI J,SHAYAN G,AVERY L,et al.Tumor-infiltrating Tim-3+Tcells proliferate avidly except when PD-1 is co-expressed:evidence for intracellular cross talk[J/OL].Oncoimmunology,2016,5(10):e1200778[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087305/.DOI:10.1080/2162402X.2016.1200778.
[10]SHAYAN G,SRIVASTAVA R,LI J,et al.Adaptive resistance to anti-PD1 therapy by Tim-3 upregulation is mediated by the PI3K-Akt pathway in head and neck cancer[J/OL].Oncoimmunology,2017,6(1):e1261779[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283618/.DOI:10.1080/2162402X.2016.1261779.
[11]SU S,ZOU Z,CHEN F,et al.CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer[J/OL].Oncoimmunology,2017,6(1):e1249558[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/28197365/.DOI:10.1080/2162402X.2016.1249558.
[12]SU S,HU B,SHAO J,et al.CRISPR-Cas9 mediated efficient PD-1disruption on human primary T cells from cancer patients[J/OL].Sci Rep,2016,6:20070[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730182/.DOI:10.1038/srep20070.
[13]LONG G V,ATKINSON V,LO S,et al.Combination nivolumab and ipilimumab or nivolumab alone in melanoma brain metastases:a multicentre randomised phase 2 study[J].Lancet Oncol,2018,19(5):672-681.DOI:10.1016/S1470-2045(18)30139-6.
[14]LARKIN J,CHIARION-SILENI V,GONZALEZ R,et al.Combined nivolumab and ipilimumab or monotherapy in untreated melanoma[J/OL].N Engl J Med,2015,373(1):23-34[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698905/.DOI:10.1056/NEJMoa1504030.
[15]PITT J M,VéTIZOU M,DAILLèRE R,et al.Resistance mechanisms to immune-checkpoint blockade in cancer:tumor-intrinsic and-extrinsic factors[J].Immunity,2016,44(6):1255-1269.DOI:10.1016/j.immuni.2016.06.001.
[16]SHARMA P,HU-LIESKOVAN S,WARGO J A,et al.Primary,adaptive,and acquired resistance to cancer immunotherapy[J/OL].Cell,2017,168(4):707-723[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391692/.DOI:10.1016/j.cell.2017.01.017.
[17]MINN A J,WHERRY E J.Combination cancer therapies with immune checkpoint blockade:convergence on interferon signaling[J].Cell,2016,165(2):272-275.DOI:10.1016/j.cell.2016.03.031.
[18]YANG R,HUNG M C.The role of T-cell immunoglobulin mucin-3and its ligand galectin-9 in antitumor immunity and cancer immunotherapy[J].Sci China Life Sci,2017,60(10):1058-1064.DOI:10.1007/s11427-017-9176-7.
[19]GE R T,ZENG L,MO L H,et al.Interaction of TIM4 and TIM3 induces T helper 1 cell apoptosis[J].Immunol Res,2016,64(2):470-475.DOI:CNKI:SUN:JCXG.0.2017-10-002.
[20]SAKUISHI K,APETOH L,SULLIVAN J M,et al.Targeting Tim-3and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity[J].J Exp Med,2010,207(10):2187-2194.DOI:10.1084/jem.20100643.
[21]ZHOU Q,MUNGER M E,VEENSTRA R G,et al.Coexpression of Tim-3 and PD-1 identifies a CD8 T cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia[J].Blood,2011,117(17):4501-4510.DOI:10.1182/blood-2010-10-310425.
[22]GUO Z,CHENG D,XIA Z,et al.Combined TIM3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer[J/OL].J Transl Med,2013,11:215[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853027/.DOI:10.1186/1479-5876-11-215.
[23]CHIBA S,BAGHDADI M,AKIBA H,et al.Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1[J/OL].Nat Immunol,2012,13(9):832-842[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3622453/.DOI:10.1038/ni.2376.
[24]谢一方,王永明.基因编辑技术的原理及其在癌症研究中的应用[J].中国肿瘤生物治疗杂志,2017,24(8):815-827.DOI:10.3872/j.issn.1007-385x.2017.08.001.
[25]LLOYD A,VICKERY O N,LAUGEL B.Beyond the antigen receptor:editing the genome of T-cells for cancer adoptive cellular therapies[J/OL].Front Immunol,2013,4:221[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733021/.DOI:10.3389/fimmu.2013.00221.
[26]刘宝瑞.实体肿瘤免疫治疗的关键问题与对策[J].中国肿瘤生物治疗杂志,2017,24(6):575-580.DOI:10.3872/j.issn.1007-385X.2017.06.001.
[27]苏舒,邹征云,杜娟,等.EBV阳性胃癌免疫卡控点干预治疗意义及抗肿瘤作用研究[J].肿瘤综合治疗电子杂志,2018,(2)[2018-11-28].http://www.cnki.com.cn/Article/CJFDTotal-ZLZD201802018.htm.DOI:10.3969/j.issn.2095-5324.2018.02.014.
[28]LIU J F,WU L,YANG L L,et al.Blockade of TIM3 relieves immunosuppression through reducing regulatory T cells in head and neck cancer[J].J Exp Clin Cancer Res,2018,37(1):44.DOI:10.1186/s13046-018-0713-7.
[29]JIE H B,SRIVASTAVA R M,ARGIRIS A,et al.Increased PD-1+and TIM-3+TILs during cetuximab Immunol Res,2017,5(5):408-416[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497750/.
[30]WANG Y,ZHAO E,ZHANG Z,et al.Association between Tim-3and Gal-9 expression and gastric cancer prognosis[J].Oncol Rep,2018,40(4):2115-2126.DOI:10.3892/or.2018.6627.
[2]MAHONEY K M,RENNERT P D,FREEMAN G J.Combination cancer immunotherapy and new immunomodulatory targets[J].Nat Rev Drug Discov,2015,14(8):561-584.DOI:10.1038/nrd4591.
[3]POSTOW M A,CALLAHAN M K,WOLCHOK J D.Immune checkpoint blockade in cancer therapy[J].J Clin Oncol,2015,33(17):1974-1982.DOI:10.1200/JCO.2014.59.4358.
[4]YANG R,HUNG M C.The role of T-cell immunoglobulin mucin-3and its ligand galectin-9 in antitumor immunity and cancer immunotherapy[J].Sci China Life Sci,2017,60(10):1058-1064.DOI:10.1007/s11427-017-9176-7.
[5]DAS M,ZHU C,KUCHROO V K.Tim-3 and its role in regulating antitumor immunity[J/OL].Immunol Rev,2017,276(1):97-111[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512889/.DOI:10.1111/imr.12520.
[6]DEKRUYFF R H,BU X,BALLESTEROS A,et al.T cell/transmembrane,Ig,and mucin-3 allelic variants differentially recognize phosphatidylserine and mediate phagocytosis of apoptotic cells[J/OL].J Immunol,2010,184(4):1918-1930[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128800/.DOI:10.4049/jimmunol.0903059.
[7]ZHOU Q,MUNGER M E,VEENSTRA R G,et al.Coexpression of Tim-3 and PD-1 identifies a CD8+T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia[J/OL].Blood,2011,117(17):4501-4510[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3099570/.DOI:10.1182/blood-2010-10-310425.
[8]LI Z,LIU X B,GUO R B,et al.TIM-3 plays a more important role than PD-1 in the functional impairments of cytotoxic T cells of malignant Schwannomas[J].Tumour Biol,2017,39(5):1010428317698352.DOI:10.1177/1010428317698352.
[9]LI J,SHAYAN G,AVERY L,et al.Tumor-infiltrating Tim-3+Tcells proliferate avidly except when PD-1 is co-expressed:evidence for intracellular cross talk[J/OL].Oncoimmunology,2016,5(10):e1200778[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087305/.DOI:10.1080/2162402X.2016.1200778.
[10]SHAYAN G,SRIVASTAVA R,LI J,et al.Adaptive resistance to anti-PD1 therapy by Tim-3 upregulation is mediated by the PI3K-Akt pathway in head and neck cancer[J/OL].Oncoimmunology,2017,6(1):e1261779[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283618/.DOI:10.1080/2162402X.2016.1261779.
[11]SU S,ZOU Z,CHEN F,et al.CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer[J/OL].Oncoimmunology,2017,6(1):e1249558[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/28197365/.DOI:10.1080/2162402X.2016.1249558.
[12]SU S,HU B,SHAO J,et al.CRISPR-Cas9 mediated efficient PD-1disruption on human primary T cells from cancer patients[J/OL].Sci Rep,2016,6:20070[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730182/.DOI:10.1038/srep20070.
[13]LONG G V,ATKINSON V,LO S,et al.Combination nivolumab and ipilimumab or nivolumab alone in melanoma brain metastases:a multicentre randomised phase 2 study[J].Lancet Oncol,2018,19(5):672-681.DOI:10.1016/S1470-2045(18)30139-6.
[14]LARKIN J,CHIARION-SILENI V,GONZALEZ R,et al.Combined nivolumab and ipilimumab or monotherapy in untreated melanoma[J/OL].N Engl J Med,2015,373(1):23-34[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698905/.DOI:10.1056/NEJMoa1504030.
[15]PITT J M,VéTIZOU M,DAILLèRE R,et al.Resistance mechanisms to immune-checkpoint blockade in cancer:tumor-intrinsic and-extrinsic factors[J].Immunity,2016,44(6):1255-1269.DOI:10.1016/j.immuni.2016.06.001.
[16]SHARMA P,HU-LIESKOVAN S,WARGO J A,et al.Primary,adaptive,and acquired resistance to cancer immunotherapy[J/OL].Cell,2017,168(4):707-723[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391692/.DOI:10.1016/j.cell.2017.01.017.
[17]MINN A J,WHERRY E J.Combination cancer therapies with immune checkpoint blockade:convergence on interferon signaling[J].Cell,2016,165(2):272-275.DOI:10.1016/j.cell.2016.03.031.
[18]YANG R,HUNG M C.The role of T-cell immunoglobulin mucin-3and its ligand galectin-9 in antitumor immunity and cancer immunotherapy[J].Sci China Life Sci,2017,60(10):1058-1064.DOI:10.1007/s11427-017-9176-7.
[19]GE R T,ZENG L,MO L H,et al.Interaction of TIM4 and TIM3 induces T helper 1 cell apoptosis[J].Immunol Res,2016,64(2):470-475.DOI:CNKI:SUN:JCXG.0.2017-10-002.
[20]SAKUISHI K,APETOH L,SULLIVAN J M,et al.Targeting Tim-3and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity[J].J Exp Med,2010,207(10):2187-2194.DOI:10.1084/jem.20100643.
[21]ZHOU Q,MUNGER M E,VEENSTRA R G,et al.Coexpression of Tim-3 and PD-1 identifies a CD8 T cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia[J].Blood,2011,117(17):4501-4510.DOI:10.1182/blood-2010-10-310425.
[22]GUO Z,CHENG D,XIA Z,et al.Combined TIM3 blockade and CD137 activation affords the long-term protection in a murine model of ovarian cancer[J/OL].J Transl Med,2013,11:215[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853027/.DOI:10.1186/1479-5876-11-215.
[23]CHIBA S,BAGHDADI M,AKIBA H,et al.Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1[J/OL].Nat Immunol,2012,13(9):832-842[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3622453/.DOI:10.1038/ni.2376.
[24]谢一方,王永明.基因编辑技术的原理及其在癌症研究中的应用[J].中国肿瘤生物治疗杂志,2017,24(8):815-827.DOI:10.3872/j.issn.1007-385x.2017.08.001.
[25]LLOYD A,VICKERY O N,LAUGEL B.Beyond the antigen receptor:editing the genome of T-cells for cancer adoptive cellular therapies[J/OL].Front Immunol,2013,4:221[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733021/.DOI:10.3389/fimmu.2013.00221.
[26]刘宝瑞.实体肿瘤免疫治疗的关键问题与对策[J].中国肿瘤生物治疗杂志,2017,24(6):575-580.DOI:10.3872/j.issn.1007-385X.2017.06.001.
[27]苏舒,邹征云,杜娟,等.EBV阳性胃癌免疫卡控点干预治疗意义及抗肿瘤作用研究[J].肿瘤综合治疗电子杂志,2018,(2)[2018-11-28].http://www.cnki.com.cn/Article/CJFDTotal-ZLZD201802018.htm.DOI:10.3969/j.issn.2095-5324.2018.02.014.
[28]LIU J F,WU L,YANG L L,et al.Blockade of TIM3 relieves immunosuppression through reducing regulatory T cells in head and neck cancer[J].J Exp Clin Cancer Res,2018,37(1):44.DOI:10.1186/s13046-018-0713-7.
[29]JIE H B,SRIVASTAVA R M,ARGIRIS A,et al.Increased PD-1+and TIM-3+TILs during cetuximab Immunol Res,2017,5(5):408-416[2018-11-28].https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497750/.
[30]WANG Y,ZHAO E,ZHANG Z,et al.Association between Tim-3and Gal-9 expression and gastric cancer prognosis[J].Oncol Rep,2018,40(4):2115-2126.DOI:10.3892/or.2018.6627.