ABSTRACT Durable immunity to cancer is sustained by memory T cells. In contrast to circulating memory subsets, which traffic in and out of the blood, tissue-resident memory (TRM) cells are transcriptionally programed for prolonged residence and recall function within tissue. Collaborative studies between our laboratories were among the first to identify a requirement for TRM cells in immunity to cancer. Using a melanoma-associated vitiligo (MAV) mouse model that closely mimics the vitiligo that develops in immune checkpoint inhibitor-treated melanoma patients who benefit from prolonged disease-free survival, we showed that skin TRM cells are necessary and sufficient for long term protective immunity against melanoma in the dermis. However, mechanisms for controlling TRM cell persistence and identity as well as the contribution of TRM cells to tumor immunity at sites of frequent metastasis remain unclear. In this application, we examine an unexpected mechanism for TRM cell maintenance in the skin and reveal a new subset of vitally important TRM cells that persist in tumor-draining lymph nodes. In the skin of mice with MAV, as well as melanoma patients with vitiligo, immunofluorescent imaging revealed that TRM cells form lymphoid aggregates containing large populations of CD11c-expressing myeloid cells. While prior work indicates that CD11c+ dendritic cells (DCs) are critical for initiating immune responses but dispensable for reactivating TRM we find that depletion of CD11c-expressing cells results in rapid disaggregation and loss of CD8 TRM cells in the skin. We further show that the CXCR6/CXCL16 axis is required for TRM cell persistence and tumor protection in the skin. These findings identify a critical requirement for CXCL16-expressing myeloid cells in coordinating the organization and retention of CXCR6-expressing TRM in the tissue, which will be examined in Specific Aim 1. The importance of the CXCR6/CXCL16 axis and persisting self antigen in controlling TRM cell function and plasticity will be tested in Specific Aim 2. Finally, parallel mechanisms will be explored in lymph nodes (LNs) where our preliminary studies led us to discover a novel population of tumor-specific T cells that is crucial for protection against melanoma growth in lymph nodes. The presence of LN TRM cells has not previously been shown in the setting of cancer. A role for APCs and chemokines in maintaining such responses is essentially unknown, and will be the focus of Specific Aim 3. This proposal will thus test the overarching hypothesis that tumor-specific TRM cells— both in skin and draining lymph nodes—rely on key interactions with APCs and chemokines for their proper positioning, maintenance, and anti-tumor function.