K562冻融抗原负载的树突状细胞诱导抗慢性粒细胞白血病的免疫效应

An in vitro dendritic cells-induced T lymphocytes cytotoxicity study activated by thaw frozen K562 lysates antigen in chronic myeloid leukemia

目的研究K562冻融抗原负载的健康供者来源的树突状细胞(DC)诱导细胞毒性T淋巴细胞(CTL)体外杀伤慢性粒细胞白血病(CML)细胞的毒性效应。方法利用健康供者外周血单个核细胞诱导分化为DC,采用反复冻融法从K562中提取的可溶性相关抗原负载DC;流式细胞学检测负载抗原前后DC表面分子表达的变化;ELISA法检测DC上清中IL-12和IFNγ的含量;混合淋巴细胞反应(MLR)测定DC体外刺激T细胞增殖的能力;乳酸脱氢酶法检测K562冻融抗原负载DC诱导的抗原特异性CTL对CML细胞的杀伤作用。结果与未经抗原负载的DC相比,经K562抗原负载的DC表面分子表达明显上调,CD1a(27·40±5·00)%、(15·40±2·34)%,CD80(61·35±5·35)%、(42·00±2·77)%,CD83(93·30±3·48)%、(25·15±4·02)%,CD86(85·25±4·39)%、(37·25±3·20)%,CD40(89·80±7·18)%、(35·95±4·06)%,HLA-DR(49·50±5·45)%、(17·15±3·61)%,DC分泌IL-12和诱导T细胞分泌IFNγ的能力增加(P<0·05),具有很强的刺激T细胞增殖能力,且刺激强度在24h最强,48h降低,冻融抗原负载DC后激活的CTL在体外对K562的杀伤率为77·35%,显著高于未经抗原负载的DC(P=0·001)。结论经K562细胞冻融抗原负载DC激活的CTL在体外具有更强的增殖能力和杀伤CML细胞的作用。

Objective To investigate the anti-chronic myeloid leukemia（CML） effect of cytotoxicity T lymphocytes（CTL） activated by dendritic cells （DC） pulsed with freeze thaw K562 lysates antigan. Methods DC were achieved by healthy donors peripheral blood monocytes which were induced by granulocyte-macrophage colony stimulating factor （GM-CSF） and IL-4. On the sixth day, these cells were collected. One part of them was usecl to prove that they were DC from morphology and phenotypes. The other part was pulsed with K562 lysates and the lysate-loaded DC were compared with immature DC from phenotypes . The expression of IL-12 and IFNγ in supernatant, the mixed lymphocytes reaction and the specific cytotoxicity against leukemia cells were tested. Results Freeze thaw K562 lysate up-regulated the expression of various differentiation antigens of DC, such as CD1a （27.40± 5.00） % vs （ 15.40 ± 2.34） %, CD80（61.35 ±5.35）% vs （42.00 ±2.77）%,CD83 （93.30 ±3.48）% vs （25.15 ±4.02）%,CD86 （85.25±4.39）% vs （37.25 ±3.20）%,CD40（89.80 ±7. 18）% vs （35.95 ±4.06）% and HLA-DR （49.50 ± 5.45） % vs （ 17.15 ± 3.61 ） %. Simultaneously, the expression of IL-12 rose after DC were loaded with tumor lysates after 24 hours （ P 〈 0. 05 ）. The T cells from healthy donors and CML patients, proliferated by antigen loaded DC, could induce IFNγ increase（P 〈0. 01 ）. The proliferation of T cells had distinct relation with the time of antigen loaded with DC. These T cells had specific cytotoxicity against K562 and HLA partial matching CML cells. Conclusions DC pulsed with K562 lysates can stimulate T cells, and can keep high immunocompetence with specific cytotoxicity against K562.