Objective: This study aimed to explore the application of interaction-dependent fucosyl-biotinylation (FucoID), a chemical biology-based proximity labeling technique, in capturing tumor antigen-specific T cells and its clinical value in chronic myelogenous leukemia (CML) . Methods: Flow cytometry and fluorescence microscopy were employed to evaluate the experimental parameters for FucoID in CML. Peripheral blood samples were obtained from 14 newly diagnosed CML patients in the chronic phase. These samples underwent flow cytometry-based sorting and were subsequently labeled with FucoID to facilitate the isolation of tumor cells and T cells, followed by the immunophenotypic identification of tumor antigen-specific T cells. Finally, the diagnostic and therapeutic potential of FucoID in CML was assessed. Results: Initially, the experimental parameters for FucoID in CML were established. The proportion of CD3(+) T cells in patients was (8.96±6.47) %, exhibiting a marked decrease compared with that in healthy individuals at (38.89±22.62) %. The proportion of tumor-specific antigen-reactive T cells was (3.34±4.49) %, which demonstrated interpatient variability. In addition, the proportion of tumor-specific antigen-active T cells in CD4(+) T cells was (3.95±1.72) %, which was generally lower than the proportion in CD8(+) T cells at (5.68±2.18) %. Compared with those in tumor-specific antigen-nonreactive T cells, CCR7(-)CD45RA(-) effector memory T cells and CCR7(-)CD45RA(+) effector T cells were highly enriched in tumor-specific antigen-reactive T cells. Moreover, the intensity of tumor immune reactivity in patients exhibited a significant correlation with white blood cell count (WBC) and hemoglobin (HGB) levels in peripheral blood, while no such correlation was observed with other clinical baseline characteristics. Conclusion: The combination of FucoID and flow cytometry enables the rapid identification and isolation of tumor antigen-specific T cells in CML. The successful application of this method in CML and the implications of our findings suggest its potential clinical value in the field of hematologic malignancies.
目的： 探讨应用化学生物学邻近标记技术（Interaction-dependent fucosyl-biotinylation, FucoID），捕获血液恶性肿瘤慢性髓性白血病（CML）中肿瘤抗原特异反应性T细胞及其临床价值。 方法： 通过流式细胞术及荧光显微镜成像确定FucoID适用于血液肿瘤CML的实验条件。选取14例CML慢性期患者初诊样本，利用流式细胞术及FucoID对其外周血样本进行肿瘤细胞与T细胞的分离、共孵育与邻近标记，实现肿瘤抗原特异反应性T细胞的表型鉴定，并探讨FucoID在CML中的诊治价值。 结果： 研究首先确定了FucoID适用于血液肿瘤CML的实验条件。患者CD3(+) T细胞比例为（8.96±6.47）%，远低于正常供者的（38.89±22.62）%。患者间肿瘤抗原特异反应性T细胞占比存在差异[（3.34±4.49）%]。CD4(+) T细胞中肿瘤抗原特异反应性T细胞比例为（3.95±1.72）%，普遍低于CD8(+) T细胞中肿瘤抗原特异反应性T细胞比例[（5.68±2.18）%]。相比肿瘤抗原非反应性T细胞，肿瘤抗原特异反应性T细胞中高度富集CCR7(－) CD45RA(－)效应记忆性T细胞以及CCR7(－) CD45RA(+)效应性T细胞。患者肿瘤免疫反应性强度与其外周血中WBC及HGB水平显著相关（P值均<0.05），与其他临床基线特征无相关性。 结论： FucoID结合流式细胞术可快速鉴定及分离CML中肿瘤抗原特异反应性T细胞，该方法在CML中的成功应用提示其在血液肿瘤领域具有潜在的临床应用价值。.
Keywords: Biochemical proximity labeling technology; Hematological malignancy; Immunophenotype; Leukemia myeloid, chronic; Tumor antigen-specific T cell.