目的：探讨结直肠癌（colorectal cancer，CRC）肿瘤引流淋巴结（tumor-draining lymph node，TDLN）中T细胞的免疫学 特性及其抗肿瘤作用。方法：收集2018年12月至2021年1月贵州医科大学附属医院收治的33例CRC患者的临床资料和淋巴 结标本。采用染料法示踪，配对采集CRC患者TDLN和非肿瘤引流淋巴结（non-TDLN，NTDLN）。用流式细胞术检测已制备成 单细胞悬液的TDLN和NTDLN中免疫细胞亚群和功能表型差异，酶联免疫斑点法比较 TDLN 和 NTDLN 中肿瘤反应性 T 细 胞比例，多色免疫荧光组织化学技术分析两种淋巴结中免疫细胞空间分布情况。体外扩增 TDLN-T 细胞，检测其 T 细 胞亚群和表型变化以及肿瘤免疫反应能力。结果：与NTDLN相比，TDLN中含有更高比例肿瘤反应性T细胞（ P<0.05），调节 性T（Treg）细胞比例较高（P<0.01），但单核样髓源性抑制细胞比例较低（P<0.05），T细胞活化标志物ICOS、CD28和抑制标志物 PD-1、TIGIT比例均显著升高（P<0.05或P<0.01）。Treg细胞和滤泡性T细胞主要分布在淋巴结皮质区和生发中心。TDLN-T细 胞扩增后，以CD8+ T细胞为主（P<0.01），ICOS、CD28 表达升高（P<0.05或P<0.01），肿瘤反应性 T 细胞比例升高（P<0.01）。结论：CRC的TDLN中T细胞处于免疫激活状态，同时高表达免疫抑制标志物；体外培养可以增加TDLN-T细胞活化水平，并提 高抗肿瘤T细胞比例。

Objective: To investigate the immunological characteristics and the anti-tumor potential of T cells in the tumor-draining lymph node (TDLN) of colorectal cancer (CRC). Methods: The clinical data and lymph node specimens of 33 CRC patients who were treated in the Affiliated Hospital of Guizhou Medical University from December 2018 to January 2021 were retrospectively collected for this study. Paired TDLN and non-tumor-draining lymph nodes (NTDLN) were collected from CRC patients by using Dye tracer. Flow cytometry was used to examine the immune cell subsets and functional phenotypic differences in TDLN and NTDLN that prepared as single cell suspension. ELISPOT (enzyme-linked immunoSPOT) method was employed to compare the proportion of tumor-reactive T cells in TDLN and NTDLN. The spatial distribution of immune cells in the TDLN and NTDLN was evaluated by multiplexed immunohistochemistry (mIHC). The TDLN-T cells were amplified ex vivo to evaluate the T cell subsets and phenotypic changes as well as tumor immune response capabilities. Results: Compared with NTDLN, there were higher proportions of tumor[1]reactive T cells and regulatory T cells (Tregs), but lower proportion of monocytic myeloid-derived suppressor cells (Mo-MDSCs) in TDLN (P<0.01 or P<0.05 ); in addition, both the activation markers (ICOS, CD28) and suppression markers (PD-1, TIGIT) were elevated (P<0.05 or P<0.01 ). mIHC results showed that Tregs were mainly distributed in the cortex, and follicular helper T (Tfh) cells were located in the germinal center. After the TDLN-T cells were expanded ex vivo, CD8+ T cells were the predominant phenotype （P<0.01), and the expression of ICOS and CD28 and the proportion of tumor-reactive T cells were increased (P<0.05 or P<0.01 ).T cells in colorectal cancer TDLN are activated and highly expresses immune suppression markers. Ex vivo expansion can enhance the activation of TDLN-T cells and increase the proportion of tumor-reactive T cells.

贵州省科学技术厅资助项目[No.(2019)2788]；国家自然科学基金贵州医科大学培育项目（No.19NSP045）