LAMP3+ migratory dendritic cells establish immune-accessible tumor microenvironment in non-Hodgkin lymphoma

Effective anti-tumor immunity requires functional tumor infiltrating lymphocytes (TILs) to migrate into the tumor nest, recognize tumor antigens, and exert effector function despite suppressive tumor microenvironment (TME). Compared to immune-accessible “hot” tumors, immune-inaccessible “cold” tumors lack immune cell infiltration and resist immunotherapy. Concomitant viral infections in the tumor further shape TME heterogeneity, promoting an immunoregulatory state that limits anti-tumor immunity. We aim to dissect spatial heterogeneity in the TME to identify immune determinants of T cell infiltration and effector function that underlie hot versus cold tumors in non-Hodgkin lymphoma (NHL). Using pre-treatment snap-frozen tumors with and without viral infections (EBV and HIV) from 12 NHL patients (6 HIV+ and 6 HIV−, including 3 ABC-DLBCL, 7 GCB-DLBCL, and 2 EBV+ Burkitt lymphoma) and lymph nodes from 17 donor without NHL (9 HIV+ and 8 HIV−), we performed single-cell multi-omics (paired ATAC-seq and RNA-seq in the same nucleus) and profiled 415,548 cells, including 127,455 cancer cells and 40,730 TILs. From matched tissues, we performed spatial transcriptomics (10x Xenium immuno-oncology panel with 100 custom probes, including HIV- and EBV-specific probes) in 69 tissue sections. Across HIV+ and HIV− NHL tumor sections, we found spatially discrete tumor regions with high type I Interferon stimulated gene (ISG) expression that did not correlate with local EBV or HIV expression, excluding viral-induced type I IFN responses as the primary drivers of ISG gradients. Joint single-cell multi-omics and spatial transcriptomics profiling revealed that LAMP3+ migratory dendritic cells (LAMP3+ mDCs) established discrete ISG-high niches characterized by CXCL9 and CXCL10 expression from fibroblastic reticular cells, increased infiltration of CXCR3+ cytotoxic CD8 T cells, and ISG-high cancer cells that upregulated MHC class I and II with reduced proliferation. In contrast, in tumor nests without LAMP3+ mDCs, TIL infiltration was minimal, and cancer cells did not express ISG, had decreased MHC I and MHC II expression, and maintained proliferation capacity. Ligand-receptor analysis with NicheNet (single-cell) and CellNEST (spatial transcriptomics) revealed that LAMP3+ mDCs supported both TIL cytotoxicity via IL-15 trans-presentation and TIL exhaustion through PD-L1-PD-1 engagement. In addition to TIL exhaustion, gene-regulatory networks revealed that HIV-induced suppressive TME was shaped by MAF, which drove CD4+ T cells towards a type I regulatory (Tr1) phenotype ( MAF , IKZF2 , TIGIT , CTLA4 , IL2RB , and IL12RB2 ). Overall, we identified LAMP3+ mDC-defined niches that drives type I IFN gradients and coordinates immune effector recruitment and function. Our study nominates LAMP3+ mDCs as immunotherapy targets to promote TIL migration into cold tumors and enhance anti-tumor immunity.