O15.04 Metabolic reprogramming enhances stem-like and functional properties of lung cancer-derived TILs

which catalyzes the transfer of a biotinylated LPETG substrate onto a G5 tag in an acceptor cell, which can be detected by flow cytometry.We took advantage of this system by using OT-I SrtA+ mice as T cell donors, and Cd40G5/G5 mice as tumor bearing hosts.At 72 hours post-therapy, flow cytometry revealed profound remodeling of the myeloid compartment, including expansion of inflammatory Ly6Chigh macrophages.Multiplex cytokine profiling of antigen restimulated tumor lysates showed increased production of GM-CSF, CCL3, CCL4, CCL5, and pro-inflammatory mediators linked to myeloid recruitment and activation.uLIPSTIC labeling demonstrated that OT-I cells formed direct physical contacts with multiple macrophage and dendritic cell subsets.Using EL4 tumors lacking OVA, labeling was largely lost, indicating antigen dependence.To test whether cross-presentation drives these interactions, we generated mixed bone marrow chimeras containing Cd40G5/G5 B2m+/+ and Cd40G5/G5 B2m-/-cells; showing stronger labeling of the H2-Kb + myeloid compartment but an incomplete abrogation of labeling in the B2M-/-deficient compartment, suggesting subsequent antigen independent interactions after an initial priming event.To capture this interplay with deeper resolution, we performed single cell RNA sequencing coupled with CITE-seq on sorted OT-I and myeloid cells.Therapy induced distinct inflammatory macrophage and dendritic cell states marked by strong proinflammatory and interferon signatures.Trajectory analysis suggested an in situ reprogramming of TME infiltrating populations upon therapy.Assignment of the barcoded anti-biotin to specific clusters allowed us to characterize the transcriptional status of myeloid cells who engaged in interactions with therapeutic T cells, including a Nos2+ macrophage subset, mreg-DCs, and ISG DCs.Finally, conditional depletion of myeloid populations with DTR hosts or treatment with myeloid-depleting antibodies impaired tumor clearance, demonstrating their functional support of T cell therapy.Collectively, this work provides a mechanistic map of antigen-driven T cell-myeloid crosstalk and identifies myeloid remodeling as a critical determinant of adoptive T cell therapy efficacy.