IL-12、IL-15对急性髓系白血病患者骨髓来源CD34+白血病细胞转化为NK细胞作用研究
|
摘要
目的应用细胞因子组合体外诱导CD34+白血病细胞转化为自然杀伤(NK)细胞,探讨白介素(IL)-12、IL-15对NK细胞分化增殖、细胞活性、细胞毒作用以及Granzyme B、TNF-α和IFN-γ基因表达的作用。方法提取26例2016年8月至2017年11月初治高白细胞血症急性髓系白血病患者骨髓单个核细胞(BM-MNC),应用A组(SCF、IL-7、IL-12、IL-15)、B组(SCF、IL-7、IL-2、IL-15)两组细胞因子组合分别体外诱导分化5周,流式细胞术检测NK细胞比例及活化标志,实时定量PCR(QPCR)检测两组NK细胞Granzyme B、TNF-α、IFN-γ等基因的表达水平; CCK-8法分别检测两组NK细胞对患者来源白血病细胞和K562细胞的杀伤率。结果 A组NK细胞比例为(71. 22±2. 25)%,显著高于B组的(56. 32±3. 97)%(P <0. 05)。A组细胞增殖倍数与B组比较,差异无统计学意义(P> 0. 05)。A组NK细胞活化标志CD69+、NKp46+、NKG2D+NK细胞比例分别为(70. 72±3. 62)%、(70. 30±3. 45)%、(72. 75±3. 89)%,均显著高于B组的(59. 98±2. 86)%、(60. 38±2. 84)%和(62. 30±4. 25)%(P <0. 05)。Granzyme B、TNF-α和IFN-γ基因的表达水平较诱导前患者BM-MNC显著升高,A组上述基因表达水平分别为13. 36±1. 54、9. 38±0. 51和6. 26±0. 25,均显著高于B组的9. 82±1. 21、7. 06±0. 52和4. 28±0. 23(P <0. 05)。在效靶比为10∶1时,A组NK细胞对K562细胞和患者来源白血病细胞的杀伤率分别为(62. 20±2. 36)%和(70. 78±2. 54)%,均显著高于B组的(56. 77±3. 81)%和(62. 95±1. 94)%(P <0. 05);且相同效靶比时A、B两组NK细胞对患者来源白血病细胞的杀伤率显著高于K562细胞(P <0. 05)。结论 CD34+白血病细胞可在体外诱导分化为NK细胞,且具有杀伤活性。联合应用IL-12、1L-15细胞因子组合能够诱导分化的NK细胞活性,提高对白血病细胞的杀伤率以及Granzyme B、TNF-α和IFN-γ基因表达水平,且诱导分化的NK细胞对白血病细胞的杀伤具有同体特异性。
Objective To apply cytokine combinations to induce CD34 + leukemia cells to transform natural killer( NK cells in vitro,and investigate the effects of interleukin( IL)-12 and IL-15 on NK cell differentiation,cell activity,cytotoxicity,and granzyme B,TNF-α and IFN-γ gene expression. Methods Bone marrow mononuclear cells from 26 early treatment patients with acute myeloid leukemia( AML) from August 2016 to November 2017 were extracted. Two groups of cytokine combinations( group A:SCF,IL-7,IL-12 and IL-15; group B: SCF,IL-7,IL-2 and IL-15) were used to induce differentiation for 5 weeks in vitro. The percentage of NK cells and activation markers were measured by flow cytometry. Real-time quantitative PCR( QPCR) was used to detect the expression of Granzyme B,TNF-α,IFN-γ of NK cells. The killing rates of NK cells from patients with leukemic cells and K562 cells were measured by CCK-8 assay. Results NK cell ratio in group A was( 71. 22 ± 2. 25) %,significantly higher than( 56. 32 ±3. 97) % in group B( P < 0. 05). There was no significant difference between group A and group B in cell proliferation( P > 0. 05). In group A,CD69 +,NKp46 +,NKG2 D + NK cells were( 70. 72 ± 3. 62) %,( 70. 30 ± 3. 45) % and( 72. 75 ± 3. 89) %,significantly higher than( 59. 98 ± 2. 86) %,( 60. 38 ± 2. 84) % and( 62. 30 ± 4. 25) % of group B( P < 0. 05). The expression levels of Granzyme B,TNF-α and IFN-γ were significantly higher than those of pre-induced bone marrow mononuclear cells. The expression level of these genes in group A was 13. 36 ± 1. 54,9. 38 ± 0. 51 and 6. 26 ± 0. 25,significantly higher than 9. 82 ± 1. 21,7. 06 ± 0. 52 and 4. 28 ±0. 23 in group B( P < 0. 05). In the effective target ratio of 10∶ 1,the cell killing rates of K562 cells and leukemia cells from patients in group A were( 62. 20 ± 2. 36) % and( 70. 78 ± 2. 54) %,significantly higher than( 56. 77 ± 3. 81) % and( 62. 95 ± 1. 94) % in group B( P < 0. 05). At the same target ratio,the killing rate of NK cells in leukemia cells derived from patients with group A and B was significantly higher than that of K562 cells( P < 0. 05). Conclusion CD34 + leukemia cells can induce differentiation into NK cells in vitro and have cytotoxic activity. Under the treatment of cytokine combinations including IL-12 and IL-15,NK cell activity,leukemia cell killing rate,and Granzyme B,TNF-α and IFN-γ gene expression levels are higher,and NK cells that induce differentiation are homologous to leukemia cells.
Objective To apply cytokine combinations to induce CD34 + leukemia cells to transform natural killer( NK cells in vitro,and investigate the effects of interleukin( IL)-12 and IL-15 on NK cell differentiation,cell activity,cytotoxicity,and granzyme B,TNF-α and IFN-γ gene expression. Methods Bone marrow mononuclear cells from 26 early treatment patients with acute myeloid leukemia( AML) from August 2016 to November 2017 were extracted. Two groups of cytokine combinations( group A:SCF,IL-7,IL-12 and IL-15; group B: SCF,IL-7,IL-2 and IL-15) were used to induce differentiation for 5 weeks in vitro. The percentage of NK cells and activation markers were measured by flow cytometry. Real-time quantitative PCR( QPCR) was used to detect the expression of Granzyme B,TNF-α,IFN-γ of NK cells. The killing rates of NK cells from patients with leukemic cells and K562 cells were measured by CCK-8 assay. Results NK cell ratio in group A was( 71. 22 ± 2. 25) %,significantly higher than( 56. 32 ±3. 97) % in group B( P < 0. 05). There was no significant difference between group A and group B in cell proliferation( P > 0. 05). In group A,CD69 +,NKp46 +,NKG2 D + NK cells were( 70. 72 ± 3. 62) %,( 70. 30 ± 3. 45) % and( 72. 75 ± 3. 89) %,significantly higher than( 59. 98 ± 2. 86) %,( 60. 38 ± 2. 84) % and( 62. 30 ± 4. 25) % of group B( P < 0. 05). The expression levels of Granzyme B,TNF-α and IFN-γ were significantly higher than those of pre-induced bone marrow mononuclear cells. The expression level of these genes in group A was 13. 36 ± 1. 54,9. 38 ± 0. 51 and 6. 26 ± 0. 25,significantly higher than 9. 82 ± 1. 21,7. 06 ± 0. 52 and 4. 28 ±0. 23 in group B( P < 0. 05). In the effective target ratio of 10∶ 1,the cell killing rates of K562 cells and leukemia cells from patients in group A were( 62. 20 ± 2. 36) % and( 70. 78 ± 2. 54) %,significantly higher than( 56. 77 ± 3. 81) % and( 62. 95 ± 1. 94) % in group B( P < 0. 05). At the same target ratio,the killing rate of NK cells in leukemia cells derived from patients with group A and B was significantly higher than that of K562 cells( P < 0. 05). Conclusion CD34 + leukemia cells can induce differentiation into NK cells in vitro and have cytotoxic activity. Under the treatment of cytokine combinations including IL-12 and IL-15,NK cell activity,leukemia cell killing rate,and Granzyme B,TNF-α and IFN-γ gene expression levels are higher,and NK cells that induce differentiation are homologous to leukemia cells.
引文
[1] Caligiuri MA. Human natural killer cells[J]. Blood,2008,112(3):461-469.
[2] Geller MA,Miller JS. Use of allogeneic NK cells for cancer im-munotherapy[J]. Immunotherapy,2011,3(12):1445-1459.
[3] Pegram HJ,Ritchie DS,Smyth MJ,et al. Alloreactive naturalkiller cells in hematopoietic stem cell transplantation[J]. LeukRes,2011,35(1):14-21.
[4] Spanholtzl J,Tordoirl M,Eissens D,et al. High log-scale ex-pansion of functional human natural killer cells from umbilicalcord blood CD34-positive cells for adoptive cancer immunotherapy[J/OL]. PLo S One,2010[2017-12-05]. https://www. ncbi.nlm. nih. gov/pubmed/2821405.
[5] Cany J,van der Waart AB,Spanholtz J,et al. Combined IL-15and IL-12 drives the generation of CD34-derived natural killercells with superior maturation and alloreactivity potential followingadoptive transfer[J/OL]. Oncoimmunology,2015[2017-12-05]. http://lib. nbu. edu. cn/asset/detail. aspx? id=203263844766.
[6] Biota B,Spits H. Development of human lymphoid cells[J]. A-non Rev Immuno1,2006,24(1):287-320.
[7] Curti A,Ruggeri L,D'Addio A,et al. Successful transfer of al-loreactive haploidentical KIR ligand-mismatched natural killercells after infusion in elderly high risk acute myeloid leukemia pa-tients[J]. Blood,2011,118(12):3273-3279.
[8] Min B,Shin H,Kim TM,Heo DS,et al. GMP-compliant,large-scale expanded allogeneic natural killer cells have potentcytolytic activity against cancer cells in vitro and in vivo[J/OL].PLo S One,2013[2018-05-20]. https://www. ncbi. nlm. nih.gov/pubmed/23326467.
[9] Soiffer RJ,Murray C,Shapiro C,et al. Expansion and manipula-tion of natural killer cells in patients with metastatic cancer bylow-dose continuous infusion and intermittent bolus administrationof interleukin 2[J]. Clin Cancer Res,2015,2(3):493-499.
[10] Romee R,Schneider SE,Leong JW,et al. Cytokine activationinduces human memory-like NK cells[J]. Blood,2012,120(24):4751-4760.
[11] Sun JC,Madera S,Bezman NA,et al. Proinflammatory cytokinesignaling required for the generation of natural killer cell memory[J]. J Exp Med,2012,209(5):947-954.
[12] Ikemizu S,Chirifu M,Davis SJ. IL-2 and IL-15 signaling com-plexes:different but the same[J]. Nat Immunol,2012,13(12):1141-1142.
[13] Gill S,Vasey AE,De Souza A,et al. Rapid development of ex-haustion and downregulation of eomesodermin limit the antitumoractivity of adoptively transferred murine natural killer cells[J].Blood,2013,119(24):5758-5768.
[2] Geller MA,Miller JS. Use of allogeneic NK cells for cancer im-munotherapy[J]. Immunotherapy,2011,3(12):1445-1459.
[3] Pegram HJ,Ritchie DS,Smyth MJ,et al. Alloreactive naturalkiller cells in hematopoietic stem cell transplantation[J]. LeukRes,2011,35(1):14-21.
[4] Spanholtzl J,Tordoirl M,Eissens D,et al. High log-scale ex-pansion of functional human natural killer cells from umbilicalcord blood CD34-positive cells for adoptive cancer immunotherapy[J/OL]. PLo S One,2010[2017-12-05]. https://www. ncbi.nlm. nih. gov/pubmed/2821405.
[5] Cany J,van der Waart AB,Spanholtz J,et al. Combined IL-15and IL-12 drives the generation of CD34-derived natural killercells with superior maturation and alloreactivity potential followingadoptive transfer[J/OL]. Oncoimmunology,2015[2017-12-05]. http://lib. nbu. edu. cn/asset/detail. aspx? id=203263844766.
[6] Biota B,Spits H. Development of human lymphoid cells[J]. A-non Rev Immuno1,2006,24(1):287-320.
[7] Curti A,Ruggeri L,D'Addio A,et al. Successful transfer of al-loreactive haploidentical KIR ligand-mismatched natural killercells after infusion in elderly high risk acute myeloid leukemia pa-tients[J]. Blood,2011,118(12):3273-3279.
[8] Min B,Shin H,Kim TM,Heo DS,et al. GMP-compliant,large-scale expanded allogeneic natural killer cells have potentcytolytic activity against cancer cells in vitro and in vivo[J/OL].PLo S One,2013[2018-05-20]. https://www. ncbi. nlm. nih.gov/pubmed/23326467.
[9] Soiffer RJ,Murray C,Shapiro C,et al. Expansion and manipula-tion of natural killer cells in patients with metastatic cancer bylow-dose continuous infusion and intermittent bolus administrationof interleukin 2[J]. Clin Cancer Res,2015,2(3):493-499.
[10] Romee R,Schneider SE,Leong JW,et al. Cytokine activationinduces human memory-like NK cells[J]. Blood,2012,120(24):4751-4760.
[11] Sun JC,Madera S,Bezman NA,et al. Proinflammatory cytokinesignaling required for the generation of natural killer cell memory[J]. J Exp Med,2012,209(5):947-954.
[12] Ikemizu S,Chirifu M,Davis SJ. IL-2 and IL-15 signaling com-plexes:different but the same[J]. Nat Immunol,2012,13(12):1141-1142.
[13] Gill S,Vasey AE,De Souza A,et al. Rapid development of ex-haustion and downregulation of eomesodermin limit the antitumoractivity of adoptively transferred murine natural killer cells[J].Blood,2013,119(24):5758-5768.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |