利妥昔单抗对原发免疫性血小板减少症患者树突细胞功能的影响

The immune effects of rituximab on dendritic cells derived from patients with primary immune thrombocytopenia

目的探讨利妥昔单抗对原发免疫性血小板减少症（ITP）患者树突细胞（DC）功能的影响，探讨其治疗机制。方法取小剂量利妥昔单抗治疗有效的IrrP患者治疗前后的外周血单个核细胞（PBMC）与重组人粒-巨噬细胞集落刺激因子（rhgM—CSF）、重组人白细胞介素4（rhIL-4）于37℃、5％CO，条件下共孵育诱导培养DC；第5天加入肿瘤坏死因子-α（TNF-α）继续培养48h，获得成熟DC。倒置显微镜下观察DC形态；流式细胞术检测DC表型；ELISA法检测DC培养上清人白细胞介素12（IL-12p70）和转化生长因子-β1（TGF-β1）的浓度；噻唑蓝（MTT）法检0n,0DC刺激自体T淋巴细胞的增殖能力。结果①治疗后DC较治疗前DC细胞膜缺乏典型的树枝状突起、体积较小、核多居中规则；②治疗后DCHLA—DR、CD80、CD83和CD86表达水平[（56．37±3．95）％、（36．41±2．82）％、（30．45±4．61）％和（41．98±4．17）％]明显低于治疗前[（73．71±7．61）％、（55．14±7．30）％、（80．91±7．09）％和（59．03±3．43）％]（P值均〈0．05），IL-12p70水平[（50．17±14．52）％]低于治疗前[（66．87±4．29）％]，TGF-β1水平[（9．70±0．31）％]高于治疗前[（2．70±0．36）％]（P值均〈0．05）；③治疗后DC诱导的T细胞增殖指数较治疗前明显减低。结论小剂量利妥昔单抗治疗后ITP患者DC表型及分泌IL-12p70的能力明显降低、分泌TGF—β1能力增高、对自体T细胞增殖的刺激能力减低。小剂量利妥昔单抗下调DC的免疫活性可能是其治疗ITP的机制之一。

Objective To explore the changes of surface antigen and function of rituximab on dendritic cells derived from patients with Primary immune thrombocytopenia（ITP） to further understand the effective mechanism of immunotherapy. Methods The peripheral blood mononuelear cells （PBMCs） were isolated from remission patients with ITP befoe and after low-dose rituximab infusion, and the PMNCs were stimulated for 5 days by rhGM-CSF and rhlL-4 in 5% CO2 air at 37 ℃ incubator. Then all of DCs were cultured with TNF-α for 48 hours. The morphology of DCs was monitored under inverted microscope daily, and the surface antigens of the DCs were analysed by flow cytometry ,meanwhile the levels of IL-12p70 and TGF-β1 in supernatants were detected by ELISA, mix lymphocyte reaction was performed by MTT assay. Results （1）Rituximab-treated-DCs showed no obvious tree-like protruding compared with untreated-DCs. The former cells were small and most of nucleus were centric. （2）The expressions of ttLA-DR, CD80, CD83 and CD86 on rituximab-treated-DCs [ 56.37 ± 3.95 ） %, （ 36.41 ± 2.82 ） %, （30.45 ± 4.61 ） % and （41.98 ± 4.17 ） %, respectively] were significantly lower than those untreated-DCs [ （ 73.71 ± 7. 61 ） %, （ 55. 14 ＋ 7.30） %, （ 80.91 ± 7.09 ） % and （ 59.03 ± 3.43 ） %, respectively ] （ all P 〈 0.05 ）, the concentration of IL-12p70 was signifieantlylower, [（66. 87 ±4.29）% vs （50. 17 ± 14. 52）%], while that of TGF-β1 [（9.70 ± 0.31 ） % ] higher than the untreated-DCs [ （2.70 ± 0.36） % 1 （ P 〈 0.05 ）. （3）The abilities to activate T cells proliferation of rituximab-treated-DCs reduced compared with untreated-DCs. Conclusion The surface antigen of ITP-DCs and the concentration of IL-12p70 redueed after the low-dose rituximab infusion. The abilities to activate T cells proliferation reduced while the concentration of TGF-β1 increased. Rituximab may achieve its therapeutic effect on ITP by downregulating the immunoreactivity of DCs.