Spatial colocalization of neoantigen-expressing tumor cells and cognate T cells in cancer 2309241

Abstract Introduction Tumor neoantigens have emerged as promising candidates for personalized immunotherapies, however, confirming their presentation and immunogenicity remains challenging due to low neoantigen expression and limited detection sensitivity. Moreover, the spatial organization of neoantigen-specific T cell clones relative to their target tumor cells and other cell types within the tumor microenvironment remains incompletely defined due to technological limitations. To bridge this gap, we developed Slide-GoTags. Methods Slide-GoTags integrates single-nucleus spatial transcriptomics with targeted genotyping and TCR sequencing from a single 20 µm tissue section. Slide-tags spatial barcoding and the 10x Genomics 5’ droplet-based platform are followed by targeted RNase H—dependent PCR and long-read sequencing, enabling genotypes and TCR repertoires to be linked to gene expression profiles and spatial coordinates via shared cell barcodes. Results Slide-GoTags was applied to murine and human tumors, revealing colocalization of clonally expanded, neoantigen-specific T cells with tumor cells expressing their cognate neoantigen, validated by known neoantigen-TCR pairs. T cell functional state and clonotype avidity were linked to neoantigen proximity. Moreover, we identified IFN-γ—rich immunogenic niches consistently enriched for neoantigen-specific progenitor-exhausted T cells, marked by enhanced local T cell activation, clonal expansion, and immune-modulatory interactions with antigen-presenting cells within the TME. Spatially resolved receptor—ligand analysis revealed co-stimulatory and inhibitory interactions that shape immune engagement and regulation within these niches. Conclusion In this study, we define the spatial architecture of neoantigen directed T cell immunity shaped by proximity to neoantigen expressing tumor cells, TCR avidity, and receptor-ligand interactions within tumors, providing a framework for spatially informed TCR deorphanization and the development of personalized T cell therapy. Funding Source This work was supported in part by the NIH/NCI (CA276865), the Mark Foundation for Cancer Research Endeavor Award, the Dana-Farber/Harvard Cancer Center Kidney SPORE (P50CA101942), and the Dana-Farber/Harvard Cancer Center Support Grant (P30CA006516). A.N Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)