Department of Hematology, Union Hospital, Fujian Medical University, Fuzhou, China.
Immunobiology. 2013 Apr;218(4):548-53. doi: 10.1016/j.imbio.2012.06.014. Epub 2012 Jul 2.
Donor lymphocyte transfusion (DLT) may induce the graft-versus-leukemia (GVL) effect for patients with AML relapsed after transplant. However, AML is a highly diverse disease and the limited overall efficacy of DLT in clinical practice emphasizes the importance of identifying a specific subgroup of patients who might benefit from this treatment approach.
To monitor the cellular immune response after DLT, we developed an active specific immunization strategy using in vitro generated AML-trained T cells to induce a highly specific antileukemic T-cell response and thus established a novel nonradioactive assay system to assess the antileukemia immunity by flow cytometry, correlated with [3H]-thymidine uptake.
The myeloid blasts derived from five patients with AML relapsed post-allogeneic hematopoietic stem cell transplantation (allo-HSCT) were first labeled with CFDA (5,6-carboxyfluorescein diacetate succinimidyl ester). To analyze the growth inhibitory potential of the donor T cells trained by AML progenitor cells, the myeloid blasts were induced to proliferate by means of a cytokine cocktail (50ng/mL of SCF; 25ng/mL of IL-3; 100ng/mL of GM-CSF; 100ng/mL of G-CSF; 2U/mL of EPO; 0.47g/L of transferrin; and 5×10(-5)mmol/L of 2-ME). The T cell mediated growth inhibitory potential was detected after 5 days by flow cytometry and correlated with [3H]-thymidine uptake. The simultaneous use of TO-PRO-dye and calibrate beads allowed not only the cell viability to be known but also allowed quantification of the effector function.
Here, we applied a CFDA dye to track the proliferation and expansion of AML blasts in response to the cytokine cocktail in vitro. AML-trained T cells, expressed high levels of the activation markers CD25 and CD69, and were generated to recognize the leukemic progenitor cells and inhibit cytokine-induced leukemic cell proliferation, which is an active specific immunization strategy circumventing the identification of leukemia-associated antigens. The capability of proliferation inhibition of AML-trained T cells evaluated with our nonradioactive, CFDA-based assay provided comparable results with the classic [3H]-thymidine assay with an even lower ratio of effector to target cells.
Taken together, the novel, nonradioactive, CFDA-based assay was a robust tool to monitor the antileukemic immune response after DLT in myeloid leukemias.
供者淋巴细胞输注（DLT）可能会为移植后复发的 AML 患者带来移植物抗白血病（GVL）效应。然而，AML 是一种高度异质性疾病，DLT 在临床实践中的总体疗效有限，这强调了确定可能受益于这种治疗方法的特定亚组患者的重要性。
为了监测 DLT 后的细胞免疫反应，我们使用体外生成的 AML 训练 T 细胞开发了一种主动特异性免疫策略，以诱导高度特异性的抗白血病 T 细胞反应，从而建立了一种新的非放射性测定系统，通过流式细胞术评估抗白血病免疫，并与[3H]-胸苷摄取相关联。
从五例异基因造血干细胞移植（allo-HSCT）后复发的 AML 患者的髓样白血病中分离出髓样白血病，并用 CFDA（5,6-羧基荧光素二乙酸琥珀酰亚胺酯）进行标记。为了分析由 AML 祖细胞训练的供者 T 细胞的生长抑制潜力，通过细胞因子鸡尾酒（50ng/mL 的 SCF；25ng/mL 的 IL-3；100ng/mL 的 GM-CSF；100ng/mL 的 G-CSF；2U/mL 的 EPO；0.47g/L 的转铁蛋白和 5×10(-5)mmol/L 的 2-ME）诱导髓样白血病增殖。通过流式细胞术在第 5 天检测 T 细胞介导的生长抑制潜力，并与[3H]-胸苷摄取相关联。同时使用 TO-PRO-染料和校准珠不仅可以知道细胞活力，还可以定量测定效应功能。
在这里，我们应用 CFDA 染料来跟踪 AML 白血病在体外对细胞因子鸡尾酒的增殖和扩增。AML 训练的 T 细胞表达高水平的激活标志物 CD25 和 CD69，并被生成以识别白血病祖细胞并抑制细胞因子诱导的白血病细胞增殖，这是一种主动特异性免疫策略，避免了白血病相关抗原的鉴定。我们的非放射性 CFDA 测定法评估 AML 训练的 T 细胞增殖抑制能力提供了与经典[3H]-胸苷测定法相当的结果，并且具有更低的效应细胞与靶细胞比。
总之，新型非放射性 CFDA 测定法是监测髓性白血病中 DLT 后抗白血病免疫反应的强大工具。
