剔除自体活化CD4 T淋巴细胞改善骨髓增生异常综合征祖细胞体外培养集落形成

Depleting autologous activated CD4 T lymphocytes improves in vitro generation of colony-forming units in progenitor culture in myelodysplastic syndrome

目的探讨自体活化CD4 T(CD4+/CCR5+T)淋巴细胞对骨髓增生异常综合征(MDS)祖细胞体外培养细胞集落形成情况的影响。方法用免疫磁珠体外剔除16例低危MDS患者和5例正常人骨髓单个核细胞(BMNC)中CD4+/CCR5+T淋巴细胞,剔除CD4+/CCR5+T及加回CD4+/CCR5+T(4倍)的BMNC分别接种于甲基纤维素培养基中培养,比较培养后细胞集落生长情况,以及培养前后髓系细胞抗原和克隆细胞比例的变化。结果①MDS组剔除CD4+/CCR5+T较加回CD4+/CCR5+T(4倍)的BMNC培养后,细胞集落显著增加(P<0.05),加回CD4+/CCR5+T(4倍)的BMNC培养后无细胞集落形成。而正常对照组剔除CD4+/CCR5+T和加回CD4+/CCR5+T(4倍)的BMNC培养后,细胞集落计数无统计学差异(P>0.05)。②MDS组剔除CD4+/CCR5+T的BMNC培养后,红系集落计数与Th1(CD4+/IFN-γ+)细胞数量呈正相关(r=0.55,P<0.05)。③细胞培养前后,MDS组CD34阳性细胞比例无明显下降[(1.8±2.4)%vs(1.4±1.6)%](P>0.05),CD33和CD13阳性细胞比例均有显著下降[(20.3±5.8)%vs(12.1±3.7)%和(21.1±6.4)%vs(17.1±5.4)%](P<0.05);而正常对照组CD34、CD33及CD13阳性细胞比例在培养前后均无明显变化(P>0.05)。④FISH分析5例核型异常MDS患者培养前后克隆细胞比例变化,+8及20q-克隆细胞比例增加,而-7克隆细胞比例无明显变化。结论选择性剔除骨髓CD4+/CCR5+T淋巴细胞,可以增加某些类型MDS骨髓祖细胞体外培养细胞集落形成,并改善MDS骨髓早期髓系细胞堆积现象,促进分化。增加的集落细胞以异常克隆细胞或残存正常细胞为主尚不能定论。

Objective To investigate the effects of autologous activated CD4 T（CD4^＋/CCR5^＋ T）lymphocytes on in vitro generation of colony-forming units （CFU） in progenitor culture in myelodysplastic syndrome （MDS）. Methods Bone marrow mononuclear cells （BMNC） from 16 patients with low-grade MDS and 5 normal donors were depleted of CD4 ^＋/ CCR5^＋ T lymphocytes using magnetic sorting. CD4^＋/CCR5^＋ T lymphocyte-depleted and lymphocyte-plused （4-fold） BMNC were seeded onto methylcellulose medium. The generation of CFU after culture, the medullary cell antigen and rate of clonal ceils before and after culture were analyzed and compared. Results Cultures initiated with CD4^＋/CCR5 ^＋ T lymphocyte-depleted BMNC from patients with MDS exhibited significantly increased generation of CFU compared with the corresponding CD4^＋/CCR5^＋ T （4-fold） lymphocyte-plused cultures （P 〈 0. 05）. However, there was no significant difference in the generation of CFU between CD4^＋/CCR5^＋ T lymphocyte-depleted and CD4^＋/CCR5^＋ T （4-fold） lymphocyte-plused BMNC cultures from normal donors （P 〉 0.05）. The number of Thl （ CD4 ^＋/IFN-γ^＋ ） cells in patients with MDS was positively related to that of colony-forming unit erythroid （CFU-E） after culture with CD4^＋/CCR5^＋ T lymphocyte-depleted BMNC from patients with MDS（r = 0.55, P 〈 0.05）. There was no significant decrease in the rate of CD34 positive cells before culture and after culture in patients with MDS [ （ 1.8 ± 2.4） % vs （ 1.4 ± 1.6） % ] （P 〉 0.05 ）, while there was significant decrease in the rates of CD33 and CD13 positive cells [ （20.3 ± 5.8） % vs （ 12.1 ± 3.7） % and （21.1 ± 6.4） % vs （ 17.1 ± 5.4） % ] （ P 〈 0.05 ）. And there was no significant difference in the rates of CD34, CD33 and CD13 positive cells before culture and after culture in normal donors （P 〉 0.05）. FISH analysis was performed to compare the elonal cell proportions in 5 cases with abnormal chromosome before culture and after culture, and it was found that ＋8 and 20q^- clone cells were increased, while there was no significant change in - 7 clone cells. Conclusion In certain subtypes of MDS, selected depletion of CD4^＋/CCR5^＋ T lymphocytes can increase in vitro generation of CFU in progenitor culture, improve prophase medullary cell accumulation in MDS cases and promote differentiation of MDS medullary system. However, whether the increased CFU are consisted of residual normal hemopoiesis or abnomal hemopoiesis are undetermined.