Purpose. This study is aimed at immunologically characterizing sentinel lymph nodes (SNs) in colorectal cancer (CRC) patients and identifying changes in immunological phenotype and function of SNs isolated from the tumor immunosuppressive microenvironment. Methods. A total of 53 pairs of matched SNs and non-SNs (NSNs) were collected by using a lymph node tracer dye. Flow cytometry was performed to detect the immunophenotype of T cells as well as the expression of activation and inhibitory markers. Differential expression and distribution of characteristic immune cell markers were analyzed by multiplex immunohistochemistry (mIHC). Transcriptomics analysis was conducted to compare the differences in the expression of immune-related genes among lymph nodes. The ex vivo culture of lymph nodes was carried out to examine changes in immunological phenotypes and functions. Results. Compared with NSNs, SNs harbored a significantly higher percentage of regulatory T cells (Tregs) but a lower proportion of MoMDSCs. As indicated in the mIHC assays, Tregs, T follicular helper (Tfh) cells, and M2 macrophages were mainly distributed in cortical areas, germinal centers, and subcapsular sinus areas, respectively, while significantly higher numbers of Tregs and Tfh cells were detected in SNs as compared to NSNs. Moreover, GSEA revealed that T cell activation genes and CD8+ T cell exhaustion-related genes are enriched in SNs and NSNs, respectively. The ex vivo culture led to an increase in the proportion of CD4+ cells, while activating T cells in SNs. In addition, SNs displayed a higher increase in the expression of cytokines IFN-γ, TNF-α, and sFas than NSNs. Conclusion. SNs are shown to be in an immune active state in vivo, while highly expressing inhibitory cytokines and suppressive markers. The ex vivo culture enhanced antitumor immunological function of SN-T cells, providing a starting material for adoptive cell therapy for CRC.

中文翻译：

---

结直肠癌患者前哨淋巴结的免疫特性和可塑性

目的。本研究旨在对结直肠癌 (CRC) 患者的前哨淋巴结 (SNs) 进行免疫学表征，并确定从肿瘤免疫抑制微环境中分离出的 SNs 的免疫表型和功能的变化。方法。通过使用淋巴结示踪染料收集了总共 53 对匹配的 SN 和非 SN (NSN)。进行流式细胞术以检测T细胞的免疫表型以及激活和抑制标志物的表达。通过多重免疫组织化学 (mIHC) 分析特征性免疫细胞标志物的差异表达和分布。进行转录组学分析以比较淋巴结间免疫相关基因表达的差异。离体进行淋巴结培养以检查免疫表型和功能的变化。结果。与 NSNs 相比，SNs 的调节性 T 细胞 (Tregs) 的比例显着较高，但 MoMDSCs 的比例较低。如 mIHC 分析所示，Tregs、T 滤泡辅助 (Tfh) 细胞和 M2 巨噬细胞分别主要分布在皮质区、生发中心和包膜下窦区，而在 SNs 中检测到显着更高数量的 Tregs 和 Tfh 细胞与 NSN 相比。此外，GSEA 揭示了 T 细胞活化基因和 CD8+ T 细胞衰竭相关基因分别在 SN 和 NSN 中富集。离体培养导致 CD4+ 细胞比例增加，同时激活 SN 中的 T 细胞。此外，与 NSN 相比，SN 的细胞因子 IFN- γ、TNF - α和 sFas 的表达增加更高。结论。SNs 显示在体内处于免疫活性状态，同时高度表达抑制性细胞因子和抑制性标志物。体外培养增强了 SN-T 细胞的抗肿瘤免疫功能，为 CRC 的过继细胞治疗提供了起始材料。