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Background The phase III trial KEYNOTE-689 has now established a new standard of care in resectable, locally advanced head and neck squamous cell carcinoma by integrating perioperative PD-1 blockade with surgery and (chemo)radiation. In contrast, four recent phase III trials adding concurrent PD-1 blockade to definitive chemoradiotherapy showed limited oncologic benefit, despite targeting the same disease. These findings suggest that standard therapy directed at tumor-draining lymphatics - when delivered concurrently - may compromise host antitumor immunity and limit response to checkpoint inhibition.We previously demonstrated that surgical ablation or broad-field radiation of tumor-draining lymph nodes (tdLN) eliminates the response to immunotherapy by disrupting dendritic cell trafficking and priming. These results support a model in which successful immunotherapy depends on intact and functional lymphatic-immune architecture. However, the dynamics of immune cell migration from tumors to regional lymphatics in coordinating host responses remain undefined. We hypothesize that rational sequencing of tumor-directed, lymphatic-sparing radiotherapy with immunotherapy reprograms migratory dendritic cells to enhance T cell priming in sentinel lymph nodes (SLN) and promote durable antitumor immunity.Methods and Results To explore this, we developed a novel mouse reporter model allowing inducible, spatiotemporal labeling of immune effectors. Coupled with SLN mapping, this system enables tracking and characterization of migratory immune effectors. Using CITE-sequencing, we defined the ‘cancer immune migratome’ - a diverse repertoire trafficking from tumors to SLNs during an active antitumor immune response. We then develop a tumor-directed, lymphatic-sparing radiotherapy model to modulate the tumor microenvironment. Sequential administration of tumor-directed radiotherapy followed by PD-1 checkpoint inhibition achieves complete and durable responses (figure 1). Analysis of the cancer immune migratome during immunoradiotherapy revealed a crucial role for an activated population of CCR7+ dendritic cells. These migratory dendritic cells are reprogrammed, via rational sequencing of radiation and immunotherapy, from their canonical role in immune tolerance to actively drive antitumor immunity. This reprogramming enhances T cell priming, clonotypic expansion, and, ultimately, the successful tumor response to immunoradiotherapy. Disruption of sentinel lymphatic channels or selective blockade of CCR7+ DC entry into the SLN abrogates the response to immunoradiotherapy (figure 2).Conclusions This work represents the first characterization of the migrating immune repertoire from tumor to SLN during an active host antitumor response, revealing a unique immunologic niche defined by distinct cellular phenotypic and transcriptional profiles. Overall, this work supports rationally sequencing immune-sensitizing, lymphatic-preserving, tumor-directed radiotherapy followed by immune checkpoint inhibition to optimize tumor response to immunoradiotherapy by driving activated dendritic cells to draining sentinel lymph nodes.Abstract 676 Figure 1Tumor-directed immunoradiotherapy enhances dendritic cell antitumor surveillance across the tumor and sentinel lymph node. (A) Schematic of experimental design: WT mice with 4MOSC1 tumors were treated with tdRT→αPD-1, followed by SLN mapping and RNA-seq. Shown are normalized enrichment scores (NES) of immune pathways (X-axis: NES; Y-axis: -log10 FDR q-value). (B) ELISA for CCL19 in the TME post-treatment (n=5/group, p = 0.0008). Mean ± SEM; two-sided unpaired t-test. (C) Flow cytometry of activated DCs (MHCII+ CD11c+ CCR7+) in SLNs post-treatment (n=5/group, p = 0.0432). (D) Schematic of CITE-seq experiment: ROSA26 x Ai9 mice were treated with tdRT→αPD-1 and tamoxifen, followed by SLN mapping. Sorted tdTomato+ SLN cells were subjected to CITE-seq (n=2/group). (E) (left) UMAP of DC clusters; (right) Seurat analysis showing CCR7, CD40, CD86, and MHCII expression across subsets. (F) Multiplex immunofluorescence of SLNs (CD11c, CCR7, CD11b, CD40, DAPI) in control vs. tdRT-treated mice. (G) DC-3 population shows enrichment in migration, phagocytosis, antigen presentation, and interferon pathways. (H) Flow cytometry of CDC1s (MHCII+ XCR1+) in SLNs post-treatment (n=5/group, p = 0.0398). (I) (left) Schematic of Batf3’/’ model lacking cDC1s; (right) tumor growth curves with/without tdRT→αPD-1 (n=5-6/group, p = ns). Mean ± SEM; linear regressionAbstract 676 Figure 2CCR7+ dendritic cell trafficking and MMP9-dependent entry into the sentinel lymph node are critical for immunoradiotherapy efficacy. (A) Schematic: WT mice bearing 4MOSC1 tumors were treated with tdRT→αPD-1 followed by SLN mapping and RNA-seq. Shown are normalized enrichment scores (NES) of immune-related pathways (X-axis: NES; Y-axis: -log10 [FDR q-value]). (B) ELISA of CCL19 in the TME post-treatment (n=5/group, p=0.0008). Mean ± SEM; two-sided unpaired t-test. (C) Frequencies of activated DCs (MHCII* CD11c CCR7’) in SLNs by flow cytometry (n=5/group, p=0.0432). (D) Schematic: ROSA26 x Ai9 mice treated with tdRT→αPD-1 and tamoxifen, followed by SLN mapping and CITE-seq of sorted tdTomato+ cells (n=2/group). (E) (Left) UMAP of dendritic cell clusters; (Right) expression of CCR7, CD40, CD86, and MHCII across subsets. (F) Multiplex IF of SLNs showing CD11c, CCR7, CD11b, CD40, and DAPI in control vs. tdRT-treated mice. (G) Gene expression in DC-3 subset showing activation of migration, phagocytosis, antigen presentation, and interferon signaling. (H) CDC1 frequencies (MHCII* XCR1*) in SLNs by flow cytometry (n=5/group, p=0.0398). (I) (Left) Schematic of Batf3-/- model lacking cDC1s; (Right) tumor growth in control vs. tdRT→αPD-1-treated mice (n=5-6/group, p=ns). Mean ± SEM; linear regression