抗-CD3/CD28磁珠活化T细胞的实验研究
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摘要
目的探究在不同磁珠比例条件下,抗-CD3/CD28磁珠刺激活化T淋巴细胞,对T细胞的增殖和CD8+T细胞亚群中干细胞样中心记忆性T细胞(Stem Central Memory T cells, TSCM)的影响,为T细胞相关奠定实验基础。方法采集健康成年人外周血及脐带血,通过密度梯度离心法分离获得外周血单个核细胞(Peripheral Blood Mononuclear Cell, PBMC)和脐血单个核细胞(Umbilical Cord Blood Mononuclear Cell,UCBMC),采用不同比例(磁珠比细胞:1:2、1:1、2:1、3:1)的磁珠刺激活化PBMC及UCBMC,诱导培养7d,通过多色流式细胞术检测T细胞的增殖和CD8+细胞亚群中TSCM变化情况。结果 T淋巴细胞采用磁珠刺激活化后,实验组T细胞增殖倍数较空白对照组均明显升高,差异具有统计学意义(P<0.05);活化PBMC的实验中,当磁珠与细胞比例为1:2刺激活化7d后,T细胞累积增殖倍数为(9.07±0.04,n=3)均高于1:1、2:1、3:1各组,差异具有统计学意义(P<0.05);TSCM亚群比例为(86.74%±0.79,n=3)也均高于1:1、2:1、3:1各组,差异具有统计学意义(P<0.05)。活化UCBMC的实验中,当磁珠与细胞比例为3:1刺激活化7d后,T细胞累积增殖倍数为(8.75±0.08,n=3)均高于2:1、1:1、1:2各组,差异具有统计学意义(P<0.05);TSCM亚群比例为(95.46±0.16,n=3)亦均高于2:1、1:1、1:2各组,差异具有统计学意义(P<0.05)。结论在PBMC中,采用磁珠与细胞比例为1:2刺激活化时,T细胞增殖状态及细胞亚群中TSCM比例明显优于其他实验组;在UCBMC中,采用磁珠与细胞比例为3:1刺激活化时,T细胞增殖状态及细胞亚群中TSCM比例明显优于其他实验组。实验和临床研究可更根据不同的T淋巴细胞来源,选择最佳的磁珠活化比例。
Objective Investigate the effect of anti-CD3/CD28 magnetic beads stimulation on T lymphocyte activation under differentmagnetic beads proportion conditions, which influence on the proliferation of T cells and the Stem Central Memory T cells(TSCM) inthe subtypes of CD8+T cells, as the experimental basis for the correlation of T cells. Methods Density gradient centrifugation isolatedPBMC and UCBMC from healthy adult peripheral blood and cord blood, adopt different proportions(magnetic beads than cells: 1:2, 1:1,2:1,3:1) magnetic beads stimulate PBMC and UCBMC, after 7 d induction, by multicolor flow cytometry to detect T cell proliferation andTSCM variation. Results After T lymphocytes activated by magnetic beads stimulation, compared with the blank control group T cellproliferation was significantly increased in the experimental group, and the difference was statistically significant(P<0.05).In PBMC,when the ratio of magnetic beads and cells was 1:2 and activated for 7 days, the cumulative proliferation of T cells was(9.07 + 0.04, n=3)higher than that of groups 1:1, 2:1 and 3:1, with statistically significant differences(P<0.05).The TSCM subtype proportion(86.74%+0.79,n=3) was higher than other groups, with statistically significant differences(P<0.05).In UCBMC, when the ratio of magnetic beads andcells was 3:1 and activated for 7 days, the cumulative proliferation factor of T cells was(8.75+0.08, n=3) higher than that of groups 2:1,1:1 and 1:2, with statistically significant differences(P<0.05).The TSCM subgroup proportion(95.46+0.16, n=3) was also higher thanother groups, with statistically significant differences(P<0.05). Conclusion In PBMC, when the ratio of magnetic beads to cells was 1:2,the proliferation state of T cells and the proportion of TSCM in the cell subtypes were significantly superior to other experimental groups.In UCBMC, when the ratio of magnetic beads to cells was 3:1, the proliferation state of T cells and the proportion of TSCM in the cellsubtypes were significantly superior to other experimental groups. The optimal ratio of magnetic beads activation can be selected based ondifferent T-lymphocyte sources in experimental and clinical studies.
Objective Investigate the effect of anti-CD3/CD28 magnetic beads stimulation on T lymphocyte activation under differentmagnetic beads proportion conditions, which influence on the proliferation of T cells and the Stem Central Memory T cells(TSCM) inthe subtypes of CD8+T cells, as the experimental basis for the correlation of T cells. Methods Density gradient centrifugation isolatedPBMC and UCBMC from healthy adult peripheral blood and cord blood, adopt different proportions(magnetic beads than cells: 1:2, 1:1,2:1,3:1) magnetic beads stimulate PBMC and UCBMC, after 7 d induction, by multicolor flow cytometry to detect T cell proliferation andTSCM variation. Results After T lymphocytes activated by magnetic beads stimulation, compared with the blank control group T cellproliferation was significantly increased in the experimental group, and the difference was statistically significant(P<0.05).In PBMC,when the ratio of magnetic beads and cells was 1:2 and activated for 7 days, the cumulative proliferation of T cells was(9.07 + 0.04, n=3)higher than that of groups 1:1, 2:1 and 3:1, with statistically significant differences(P<0.05).The TSCM subtype proportion(86.74%+0.79,n=3) was higher than other groups, with statistically significant differences(P<0.05).In UCBMC, when the ratio of magnetic beads andcells was 3:1 and activated for 7 days, the cumulative proliferation factor of T cells was(8.75+0.08, n=3) higher than that of groups 2:1,1:1 and 1:2, with statistically significant differences(P<0.05).The TSCM subgroup proportion(95.46+0.16, n=3) was also higher thanother groups, with statistically significant differences(P<0.05). Conclusion In PBMC, when the ratio of magnetic beads to cells was 1:2,the proliferation state of T cells and the proportion of TSCM in the cell subtypes were significantly superior to other experimental groups.In UCBMC, when the ratio of magnetic beads to cells was 3:1, the proliferation state of T cells and the proportion of TSCM in the cellsubtypes were significantly superior to other experimental groups. The optimal ratio of magnetic beads activation can be selected based ondifferent T-lymphocyte sources in experimental and clinical studies.
引文
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[26]Gattinoni,L.,et al.Adoptive immunotherapy for cancer:building on success[J].Nat Rev Immunol,2006,6(5):383-93.
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[2]赵晓玲.T淋巴细胞亚群与带状疱疹关系的研究进展[J].世界最新医学信息文摘,2018(73):32-33+74.
[3]祝桂琦,等.γδT细胞在肝细胞癌中作用的研究进展[J].外科理论与实践,2018(03):289-292.
[4]王霞.外周血T淋巴细胞及NK、B淋巴细胞水平检测在皮肤鳞状细胞癌患者病情程度评估中的应用价值[J].临床医药实践,2018(09):686-688.
[5]位秀丽,朱建勇,余春芳,等.CD19-CA R-T细胞的构建及其对CD19+细胞杀伤作用[J].湖北医药学院学报,2018(04),307-310+314+296.
[6]Ahrends,T.,et al.CD4(+)T Cell Help Confers a Cytotoxic T Cell Effector Program Including Coinhibitory Receptor Downregulation and Increased Tissue Invasiveness[J].Immunity,2017,47(5):848-861.e5.
[7]Wohlfarth,P.,N.Worel and G.Hopfinger,Chimeric antigen receptor Tcell therapy-a hematological success story[J].Memo,2018,11(2):116-121.
[8]穆伟,李娜,王皓毅.基因编辑在T细胞治疗中的应用[J].生命科学,2018,30(9):939-949.
[9]O’Flynn,K.,et al.Different pathways of human T-cell activation revealed by PHA-P and PHA-M[J].Immunology,1986,57(1):55-60.
[10]Jiang,K.,Y.Chen and J.N.Jarvis.Soluble factors from LPS-and PHA-activated PBMC induce MAPK,Stat1 and Stat3 phosphorylation in primary cultures of human term placental trophoblasts:implications for infection and prematurity[J].Placenta,2007.28(5-6):538-42.
[11]Wang,F.,et al.GP73 was upregulated in PBMC stimulated with ConA but failed to promote lymphocyte proliferation[J].Cell Biol Int,2015,39(3):334-40.
[12]Trickett,A.and Y.L.Kwan.T cell stimulation and expansion using antiCD3/CD28 beads[J].Journal of Immunological Methods,2003,275(1-2):251-255.
[13]Kalamasz,D.,et al.Optimization of human T-cell expansion ex vivo using magnetic beads conjugated with anti-CD3 and Anti-CD28 antibodies[J].Journal of immunotherapy(Hagerstown,Md.:1997),2004,27(5):405-418.
[14]李海洋等.胃癌患者外周血T淋巴细胞亚群、PD-1水平的变化与预后的关系分析[J].现代医学,2018(10):1119-1122.
[15]叶丽林.慢性病毒感染CD8 T细胞免疫应答及相关治疗性疫苗研究[J].中国基础科学,2017(03):7-11+19.
[16]姚超与钱程.CAR-T细胞在肿瘤治疗中的机遇与挑战[J].中国肿瘤生物治疗杂志,2017(01):6-11.
[17]Park,J.H.,M.B.Geyer and R.J.Brentjens.CD19-targeted CAR T-cell therapeutics for hematologic malignancies:interpreting clinical outcomes to date[J].Blood,2016,127(26):3312-3320.
[18]Kalos,M.and C.H.June,Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology[J].Immunity,2013,39(1):49-60.
[19]Gattinoni,L.,et al.A human memory T cell subset with stem cell-like properties[J].Nat Med,2011,17(10):1290-7.
[20]高舒平等.干细胞与转化医学研究进展浅析[J].中国实验动物学报,2016(04):439-442.
[21]Sabatino,M.,et al.Generation of clinical-grade CD19-specific CAR-modified CD8+memory stem cells for the treatment of human B-cell malignancies[J].Blood,2016,128(4):519-28.
[22]Gattinoni,L.,C.A.Klebanoff and N.P.Restifo,Paths to stemness:building the ultimate antitumour T cell[J].Nat Rev Cancer,2012,12(10):671-84.
[23]Mahnke,Y.D.,et al.The who’s who of T-cell differentiation:human memory T-cell subsets[J].Eur J Immunol,2013,43(11):2797-809.
[24]Gauthier,J.and I.Yakoub-Agha,Chimeric antigen-receptor T-cell therapy for hematological malignancies and solid tumors:Clinical data to date,current limitations and perspectives[J].Current Research in Translational Medicine,2017,65(3):93-102.
[25]Wherry,E.J.,et al.Lineage relationship and protective immunity of memory CD8 T cell subsets[J].Nat Immunol,2003,4(3):225-34.
[26]Gattinoni,L.,et al.Adoptive immunotherapy for cancer:building on success[J].Nat Rev Immunol,2006,6(5):383-93.
[27]June,C.H.Adoptive T cell therapy for cancer in the clinic[J].J Clin Invest,2007,117(6):1466-76.
[28]Gerlach,C.,et al.Heterogeneous differentiation patterns of individual CD8+T cells[J].Science,2013,340(6132):635-9.
[29]Buchholz,V.R.,et al.Disparate individual fates compose robust CD8+Tcell immunity[J].Science,2013,340(6132):630-5.
[30]Schiller,A.,et al.A Positive Control for Detection of Functional CD4 TCells in PBMC:The CPI Pool[J].Cells,2017,6(4).
[31]Berglund,S.,et al.Advances in umbilical cord blood cell therapy:the present and the future[J].Expert Opin Biol Ther,2017,17(6):691-699.
[32]Garbuzova-Davis,S.,J.Ehrhart and P.R.Sanberg,Cord blood as a potential therapeutic for amyotrophic lateral sclerosis[J].Expert Opin Biol Ther,2017,17(7):837-851.