体外耗竭 CD8(+)CD57(+)T 细胞可促进低危骨髓增生异常综合征患者骨髓集落细胞的恶性生长。

In vitro deprivation of CD8(+)CD57(+)T cells promotes the malignant growth of bone marrow colony cells in patients with lower-risk myelodysplastic syndrome.

机构信息

Department of Hematology, Sixth Hospital affiliated to Shanghai Jiaotong University, PR China.

出版信息

Exp Hematol. 2010 Aug;38(8):677-84. doi: 10.1016/j.exphem.2010.04.002. Epub 2010 Apr 13.

DOI:10.1016/j.exphem.2010.04.002

PMID:20394797

Abstract

OBJECTIVE

CD8(+)T lymphocytes have inhibitory effects on the proliferation of malignant clones in myelodysplastic syndrome (MDS). The exact CD8(+)T subset involved in the regulation of MDS and the target clones of CD8(+)T lymphocytes has not been studied. We investigated the effect of activated CD8(+)T (CD8(+)CD57(+)) lymphocytes on colony formation (in particular, malignant colony formation) during MDS in vitro.

MATERIALS AND METHODS

Bone marrow mononuclear cells (BMNCs) from a total of 59 MDS patients were subjected to magnetic-activated cell sorting to separate CD8(+)CD57(+)T lymphocytes. BMNCs were cultured without CD8(+)CD57(+)T cells or cocultured with a 1:4 ratio of CD8(+)CD57(+)T cells to study the association between stem/progenitor cell colony formation and the existence of activated CD8(+) T cells, as well as the polarization of T cells towards Tc1. In addition, the fluorescence in situ hybridization method was used to detect bone marrow cells carrying abnormal karyotypes, and the proportion of abnormal cells among BMNCs was calculated before and after T-cell deprivation culture in vitro. Crossing cultures between MDS patients and normal volunteer was performed. The impact of effector CD8(+)T cells on the malignant growth of BMNCs was closely examined.

RESULTS

After deprivation of CD8(+)CD57(+)T cells, BMNCs from 33 MDS patients formed colonies in the culture media. The average number cells in the granulocyte and monocyte colony-forming units (CFU-GM) was 40.3/4 x 10(5), and the average number of cells in the erythroid colony-forming unit (CFU) was 10.4/4 x 10(5). These totals were significantly lower than those in the normal control group after deprivation of CD8(+)CD57(+)T cells (CFU-GM 83.4/4 x 10(5) cells, erythroid CFU 32.8/4 x 10(5) cells; p < 0.001). After add-back of CD8(+)CD57(+) T cells (four times), none of the BMNCs cultures from any of the 59 MDS patients formed colonies in vitro. Additionally, in 33 MDS patients whose BMNCs formed colonies after T-cell deprivation, the bone marrow Tc1/Tc2 ratio was positively correlated with CFU-GM count (r = 0.443, p < 0.05). Crossing cultures indicated that CD8(+)CD57(+) T cells from MDS patients cocultured with BMNC from normal donor did not show inhibition to colony-forming. In 15 MDS patients with abnormal karyotypes, deprivation of CD8(+)CD57(+)T cells significantly increased the proportion of abnormal cells from 43.8% to 56.3% in BMNC culture (p < 0.001).

CONCLUSION

Effector CD8(+)T lymphocytes inhibit bone marrow hematopoiesis in MDS patients; target cells were primarily cells with abnormal karyotypes.

摘要

目的

CD8(+)T 淋巴细胞对骨髓增生异常综合征（MDS）恶性克隆的增殖具有抑制作用。但确切涉及 MDS 调节的 CD8(+)T 亚群以及 CD8(+)T 淋巴细胞的靶细胞尚未研究。我们研究了体外 MDS 中激活的 CD8(+)T（CD8(+)CD57(+)）淋巴细胞对集落形成（特别是恶性集落形成）的影响。

材料和方法

共对 59 例 MDS 患者的骨髓单个核细胞（BMNC）进行磁激活细胞分选，分离 CD8(+)CD57(+)T 淋巴细胞。在无 CD8(+)CD57(+)T 细胞的情况下培养 BMNC，或与 CD8(+)CD57(+)T 细胞以 1:4 的比例共培养，以研究干细胞/祖细胞集落形成与激活的 CD8(+)T 细胞的存在之间的关系，以及 T 细胞向 Tc1 的极化。此外，采用荧光原位杂交法检测骨髓细胞携带异常核型，并计算体外 T 细胞剥夺培养前后 BMNC 中异常细胞的比例。进行 MDS 患者与正常志愿者之间的交叉培养，密切观察效应 CD8(+)T 细胞对 BMNC 恶性生长的影响。

结果

在剥夺 CD8(+)CD57(+)T 细胞后，33 例 MDS 患者的 BMNC 在培养基中形成集落。粒细胞-单核细胞集落形成单位（CFU-GM）的平均细胞数为 40.3/4x10(5)，红细胞集落形成单位（CFU）的平均细胞数为 10.4/4x10(5)。这些总数明显低于正常对照组在剥夺 CD8(+)CD57(+)T 细胞后的数值（CFU-GM 83.4/4x10(5)细胞，红细胞 CFU 32.8/4x10(5)细胞；p<0.001）。在添加 4 倍 CD8(+)CD57(+)T 细胞后，59 例 MDS 患者中没有任何 BMNC 培养物在体外形成集落。此外，在 33 例 T 细胞剥夺后 BMNC 形成集落的 MDS 患者中，CFU-GM 计数与 Tc1/Tc2 比值呈正相关（r=0.443，p<0.05）。交叉培养表明，MDS 患者的 CD8(+)CD57(+)T 细胞与正常供体的 BMNC 共培养时，对集落形成没有抑制作用。在 15 例具有异常核型的 MDS 患者中，剥夺 CD8(+)CD57(+)T 细胞可显著增加 BMNC 培养中异常细胞的比例，从 43.8%增加到 56.3%（p<0.001）。