Background <p>Immune checkpoint inhibitors have traditionally been understood to exert their effects primarily within the tumor microenvironment, particularly targeting CD8 + T cells. However, recent studies have highlighted a pivotal role of tumor-draining lymph nodes in mediating responses to immune checkpoint inhibitor therapy. This study aimed to elucidate the specific mechanisms by which tumor-draining lymph nodes respond to immune checkpoint inhibitor therapy and regulate the tumor microenvironment in human colorectal cancer.</p> Methods <p>We performed single-cell RNA sequencing and T cell receptor sequencing on tumor-draining lymph nodes and tumor tissues from patients with colorectal cancer. Through in-depth analysis of the single-cell data, we established the connection between TDLNs and tumor, and explored the impact of immune checkpoint inhibitor therapy on the immune microenvironment of tumor-draining lymph nodes. In addition, we conducted animal experiments to validate these findings.</p> Results <p>Our findings revealed that immune checkpoint inhibitor treatment induced the expansion of tumor-specific CD8 + effector memory T cells within tumor-draining lymph nodes, which may serve as a source for the progenitor-exhausted CD8 + T cells in the tumor microenvironment. Moreover, conventional dendritic cells type 1 and macrophages within tumor-draining lymph nodes facilitated this process. We also observed that immune checkpoint inhibitor therapy promoted the expansion of tumor-specific CD4 + follicular helper T cells in tumor-draining lymph nodes, which may explain the increase of CD4 + follicular helper T cells in the tumor microenvironment after immune checkpoint inhibitor therapy. These hypotheses were corroborated through experiments in mice.</p> Conclusions <p>Our findings delineate the critical regulatory function of tumor-draining lymph nodes in modulating the tumor microenvironment during Immune checkpoint inhibitor therapy.</p>