利用饲养细胞体外扩增高纯度的效应NK细胞
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摘要
目的比较两类饲养细胞(FC)体外扩增自然杀伤(NK)细胞的效果,鉴定出特定的NK细胞群并进行功能性分析,从而确定最优的FC。方法构建两类FC,分别是第1代FC(FC-000,K562-41BBL)和第2代FC(FC-002,K562-41BBL-mbIL15-mbIL21),分别与人外周血单核细胞(peripheral blood mononuclear cells,PBMCs)共培养14 d,收获细胞,流式细胞术鉴定NK细胞表型,利用实时无标记细胞分析仪检测两类NK细胞对A549肿瘤细胞的杀伤能力。结果培养14 d后,FC-002扩增的NK细胞纯度(93. 5%)高于FC-000扩增的NK细胞(70. 2%)(P<0. 05); FC-002扩增的NK细胞增殖倍数(3 325)高于FC-000扩增的NK细胞增殖倍数(10)(P<0. 05)。两者均促进了具有NKG2D、NKp44、CD69功能性受体的NK细胞群增殖,且FC-002扩增的NK细胞具有更高的肿瘤杀伤能力及干扰素-γ表达能力(P<0. 05)。结论利用FC能够在体外有效地扩增高纯度的NK细胞,并能够选择性扩增具有NKG2D、NKp44、CD69功能性受体的NK细胞群,且第2代FC所扩增的NK细胞对于肿瘤细胞具有更强的杀伤能力,并在NK细胞增殖倍数、纯度上均优于第1代FC。
Objective To compare the effect of two kinds of feeder cells( FC) on proliferation of natural killer( NK) cells in vitro. Methods The first generation of FCs( FC-000,k562-41 BBL)and the second generation of FC( FC-002,k562-41 BBL-mbIL15-mbIL21) were prepared. Peripheral blood mononuclear cells( PBMCs) were co-cultured with FC-000 or FC-002 respectively,and cells were harvested after 14 days of co-culture. Cell phenotype was identified by flow cytometry,and the killing ability of NK cells to A549 tumor cells was detected by real-time cell analyzer( RTCA).Results After 14 d of co-culture the purity of NK cells expanded by FC-002 was higher than that by FC-000( 93. 5% vs 70. 2%,P<0. 05),the proliferation folds of NK cells expanded by FC-002 were higher than those by FC-000( 3 325 vs 10,P<0. 05). Both FC-000 and FC-002 were able to promote the expansion of NK cells with functional receptors NKG2 D,NKp44 and CD69,however,the NK cells expanded by FC-002 had higher tumor killing capacity and higher IFN-γ production than thoseby FC-000( P < 0. 05). Conclusion Feeder cells can effectively expand high purity NK cells in vitro,and also selectively expand NK cells with functional receptors. NK cells expanded by the second generation of FC have a stronger killing ability to tumor cells and a higher proliferation ability of NK cells than those by the first generation of FC.
Objective To compare the effect of two kinds of feeder cells( FC) on proliferation of natural killer( NK) cells in vitro. Methods The first generation of FCs( FC-000,k562-41 BBL)and the second generation of FC( FC-002,k562-41 BBL-mbIL15-mbIL21) were prepared. Peripheral blood mononuclear cells( PBMCs) were co-cultured with FC-000 or FC-002 respectively,and cells were harvested after 14 days of co-culture. Cell phenotype was identified by flow cytometry,and the killing ability of NK cells to A549 tumor cells was detected by real-time cell analyzer( RTCA).Results After 14 d of co-culture the purity of NK cells expanded by FC-002 was higher than that by FC-000( 93. 5% vs 70. 2%,P<0. 05),the proliferation folds of NK cells expanded by FC-002 were higher than those by FC-000( 3 325 vs 10,P<0. 05). Both FC-000 and FC-002 were able to promote the expansion of NK cells with functional receptors NKG2 D,NKp44 and CD69,however,the NK cells expanded by FC-002 had higher tumor killing capacity and higher IFN-γ production than thoseby FC-000( P < 0. 05). Conclusion Feeder cells can effectively expand high purity NK cells in vitro,and also selectively expand NK cells with functional receptors. NK cells expanded by the second generation of FC have a stronger killing ability to tumor cells and a higher proliferation ability of NK cells than those by the first generation of FC.
引文
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[15]NI J,MILLER M,STOJANOVIC A,et al.Sustained effector function of IL-12/15/18-preactivated NK cells against established tumors[J].J Exp Med,2012,209(13):2351-2365.
[16]LEONG J W,CHASE J M,ROMEE R,et al.Preactivation with IL-12,IL-15,and IL-18 induces CD25 and a functional high-affinity IL-2 receptor on human cytokineinduced memory-like natural killer cells[J].Biol Blood Marrow Transplant,2014,20(4):463-473.
[17]WAGNER J,PFANNENSTIEL V,WALDMANN A,et al.A two-phase expansion protocol combining interleukin(IL)-15 and IL-21 improves natural killer cell proliferation and cytotoxicity against rhabdomyosarcoma[J].Front Immunol,2017,8:676.
[18]LANIER L L.NKG2D receptor and its ligands in host defense[J].Cancer Immunol Res,2015,3(6):575-582.
[19]TORELLI GF,ROZERA C,SANTODONATO L,et al.A good manufacturing practice method to ex vivo expand natural killer cells for clinical use[J].Blood Transfus,2015,13:464-471.
[20]PAHL J,CERWENKA A.Tricking the balance:NKcells in anti-cancer immunity[J].Immunobiology,2017,222(1):11-20.
[21]DENMAN C J,SENYUKOV V V,SOMANCHI S S,et al.Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells[J].PLoSOne,2012,7(1):e30264.
[22]RANSON T,VOSSHENRICH C A,CORCUFF E,et al.IL-15 is an essential mediator of peripheral NK-cell homeostasis[J].Blood,2003,101(12):4887-4893.
[23]CHESTER C,FRITSCH K,KOHRT H E.Natural killer cell immunomodulation:targeting activating,inhibitory,and co-stimulatory receptor signaling for cancer immunotherapy[J].Front Immunol,2015,6:601.
[24]GRANZIN M,WAGNER J,KOHL U,et al.Shaping of natural killer cell antitumor activity by ex vivo cultivation[J].Front Immunol,2017,8:458.
[25]MOLFETTA R,QUATRINI L,SANTONI A,et al.Regulation of NKG2D-dependent NK cell functions:the Yin and the Yang of receptor endocytosis[J].Int JMol Sci,2017,18(8):E1677.
[26]NIELSEN N,PASCAL V,FASTH A E,et al.Balance between activating NKG2D,DNAM-1,NKp44and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts[J].Immunology,2014,142(4):581-593.
[27]刘贤俊,刘海亮,景波,等.人不同组织间充质干细胞免疫调控能力的比较[J].同济大学学报(医学版),2017,38(6):12-17.
[2]COOPER M A.Teach your NK cells well[J].Immunity,2016,45(2):229-231.
[3]O'SULIIWAN T E,SUN J C,LANIER L L.Natural killer cell memory[J].Immunity,2015,43(4):634-645.
[4]FEHNIGER T A,COOPER M A.Harnessing NK cell memory for cancer immunotherapy[J].Trends Immunol,2016,37(12):877-888.
[5]VENKATASUBRAMANIAN S,CHEEKATLA S,PAIDIPALLY P,et al.IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis[J].Mucosal Immunol,2017,10(4):1031-1042.
[6]DOMOGALA A,MADRIGAL J A,SAUDEMONTA.Natural killer cell immunotherapy:from bench to bedside[J].Front Immunol,2015,6:264.
[7]BERG M,LUNDQVIST A,MCCOY P JR,et al.Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells[J].Cytotherapy,2009,11(3):341-355.
[8]KOEHL U,BREHM C,HUENECKE S,et al.Clinical grade purification and expansion of NK cell products for an optimized manufacturing protocol[J].Front Oncol,2013,3:118.
[9]PHAN M T,LEE S H,KIM S K,et al.Expansion of NK cells using genetically engineered K562 feeder cells[J].Methods Mol Biol,2016,1441:167-174.
[10]HUI Y,RUI H T,et al.A new ex vivo method for effective expansion and activation of human natural killer cells for anti-tumor immunotherapy[J].Cell Biochem Biophys,2015,73(3):723-729.
[11]SINHA C,CUNNINGHAM L C.An overview of the potential strategies for NK cell-based immunotherapy for acute myeloid leukemia[J].Pediatr Blood Cancer,2016,63(12):2078-2085.
[12]PIETRA G,VITALE C,PENDE D,et al.Human natural killer cells:news in the therapy of solid tumors and high-risk leukemias[J].Cancer Immunol Immunother,2016,65(4):465-476.
[13]LOPEZ-LASTRA S,DI SANTO J P.Modeling natural killer cell targeted immunotherapies[J].Front Immunol,2017,8:370.
[14]BJORKLUND A T,CARLSTEN M,SOHLBERG E,et al.Complete remission with reduction of high-risk clones following haploidentical NK-cell therapy against MDS and AML[J].Clin Cancer Res,2018,24(8):1834-1844.
[15]NI J,MILLER M,STOJANOVIC A,et al.Sustained effector function of IL-12/15/18-preactivated NK cells against established tumors[J].J Exp Med,2012,209(13):2351-2365.
[16]LEONG J W,CHASE J M,ROMEE R,et al.Preactivation with IL-12,IL-15,and IL-18 induces CD25 and a functional high-affinity IL-2 receptor on human cytokineinduced memory-like natural killer cells[J].Biol Blood Marrow Transplant,2014,20(4):463-473.
[17]WAGNER J,PFANNENSTIEL V,WALDMANN A,et al.A two-phase expansion protocol combining interleukin(IL)-15 and IL-21 improves natural killer cell proliferation and cytotoxicity against rhabdomyosarcoma[J].Front Immunol,2017,8:676.
[18]LANIER L L.NKG2D receptor and its ligands in host defense[J].Cancer Immunol Res,2015,3(6):575-582.
[19]TORELLI GF,ROZERA C,SANTODONATO L,et al.A good manufacturing practice method to ex vivo expand natural killer cells for clinical use[J].Blood Transfus,2015,13:464-471.
[20]PAHL J,CERWENKA A.Tricking the balance:NKcells in anti-cancer immunity[J].Immunobiology,2017,222(1):11-20.
[21]DENMAN C J,SENYUKOV V V,SOMANCHI S S,et al.Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells[J].PLoSOne,2012,7(1):e30264.
[22]RANSON T,VOSSHENRICH C A,CORCUFF E,et al.IL-15 is an essential mediator of peripheral NK-cell homeostasis[J].Blood,2003,101(12):4887-4893.
[23]CHESTER C,FRITSCH K,KOHRT H E.Natural killer cell immunomodulation:targeting activating,inhibitory,and co-stimulatory receptor signaling for cancer immunotherapy[J].Front Immunol,2015,6:601.
[24]GRANZIN M,WAGNER J,KOHL U,et al.Shaping of natural killer cell antitumor activity by ex vivo cultivation[J].Front Immunol,2017,8:458.
[25]MOLFETTA R,QUATRINI L,SANTONI A,et al.Regulation of NKG2D-dependent NK cell functions:the Yin and the Yang of receptor endocytosis[J].Int JMol Sci,2017,18(8):E1677.
[26]NIELSEN N,PASCAL V,FASTH A E,et al.Balance between activating NKG2D,DNAM-1,NKp44and NKp46 and inhibitory CD94/NKG2A receptors determine natural killer degranulation towards rheumatoid arthritis synovial fibroblasts[J].Immunology,2014,142(4):581-593.
[27]刘贤俊,刘海亮,景波,等.人不同组织间充质干细胞免疫调控能力的比较[J].同济大学学报(医学版),2017,38(6):12-17.