Lymph nodes are involved in antigen presentation, T cell priming and activation, which are essential for initiating anti-tumor immunity. However, sentinel LNs (or tumor draining LNs) are one of the most common sites of tumor metastasis. The immunological significance of tumor metastatic LNs in the tumor immune responses remains elusive. Furthermore, it remains an open question whether surgical removal of metastatic LNs is an effective approach. The overall goal of this proposal is to understand, and potentially resolve this immunological and clinical paradox. It has been reported that tumor tertiary lymphoid structures (TLSs) are associated with enhanced responses to cancer immunotherapy, indicating that TLSs are involved in promoting the restoration of adaptive antitumor immune responses. Furthermore, lymph node metastases are prognostically important. However, it is poorly defined if tumor lymph node metastasis, compared to other organ site metastasis, alters therapeutic responses in the immune checkpoint inhibitor (ICI). Our published data suggests that specific organ site metastases, such as liver metastasis, affects T cell responses and antitumor immunity in preclinical models and in patients with cancer. Accordingly, we hypothesize that the contribution of lymph node metastases to immune tone and immunotherapy efficacy is critical. Thus, in this research project we will perform clinical studies to further determine the impact of lymph node metastasis on immune tone and ICI responses in patients with cancer, including breast cancer, melanoma, and colorectal cancer in the first 2-year study. Animal models of lymph node metastases will be developed to address the exact impact of lymph node metastases on CD8+ T cells in responses to immunotherapy. Furthermore, the molecular mechanisms and innate/adaptive immune subsets involved in this process will be elucidated, and the immune responses in TDLNs will be assessed and compared to that in TLSs in tumors. Therefore, this research project will shed new light on the contribution of lymph node metastasis to immune tone and immunotherapy efficacy and will have implications for optimizing therapeutic strategies for patients with lymph node metastasis.