Dendritic cells control tertiary lymphoid structure development and maintenance in cancer

INTRODUCTION: Lymphoid aggregates accumulate in chronic inflammatory lesions, including tumors. They consist of organized assemblies of innate and adaptive immune cells arising in nonlymphoid tissues and are thought to operate as local hubs for lymphocyte recruitment, (re)activation, and maintenance. The most mature aggregates, which develop T and B cell zones with germinal centers, are referred to as tertiary lymphoid structures (TLSs). The presence of TLSs is associated with superior prognosis and response to immune checkpoint blockade (ICB) in cancer, which makes these structures attractive therapeutic targets. RATIONALE: Previous studies have identified DC-LAMP, expressed by mature dendritic cells (DCs), as a specific marker of human TLSs. Studies have also identified that CD11c⁺ cells, which include DCs and other myeloid populations, are required for TLS persistence in mouse lung infection models. However, the precise contribution of major histocompatibility complex class I (MHC-I) and MHC-II antigen presentation, including cross-presentation by specific dendritic cell subsets, to TLS initiation, maintenance, and function in cancer remains unknown. Given the strong association of TLS presence with ICB response, defining the cellular and molecular drivers of TLSs in cancer is now a critical priority. RESULTS: To address the role of DCs in TLS formation and maintenance, we developed a mouse model of non–small cell lung cancer (NSCLC) that forms mature TLSs (KP-HELLO-2) containing B cell follicles with germinal centers and T cell zones with T follicular helper (T_FH) cells and TCF1⁺PD-1⁺ progenitor exhausted CD8⁺ T (T_PEX) cells. We show that, during early stages of tumor development, TLS formation relies on interferon-γ (IFN-γ)–driven maturation of type 1 conventional dendritic cells (cDC1s), migration of cDC1s to tumor-draining lymph nodes (tdLNs), WDFY4-dependent cross-presentation, and ultimately the recruitment of activated T cells to the tumor site. As tumors progress, TLS maintenance becomes independent of T cell egress from tdLNs, coinciding with a significant reduction of cDC1 migration to tdLNs. Instead, mature cDC1s accumulate within intratumoral CCR7 ligand–enriched stromal hubs. Notably, timed depletion of cDC1s or preventing their migration to these stromal hubs after TLSs are formed disrupts TLS functional architecture and maintenance. We found that cDC1-mediated concomitant MHC-II and MHC-I antigen presentation to CD4⁺ and CD8⁺ T cells and intact CD40 signaling are critical for the maintenance of TLSs, the preservation of the T_FH cell pool, germinal center formation, tumor-specific immunoglobulin G (IgG) production, and the differentiation of T_PEX cells. Furthermore, expanding or activating DCs with FLT3L-Fc or CD40 agonist expands TLSs. Using spatial transcriptomics and multiplex imaging across various human tumors, we confirmed that mature DCs expressing high levels of CCR7 accumulate in TLSs close to CCL19⁺ stromal hubs and interact with T_FH cells, T_PEX cells, and B cells. Moreover, Mature cDC1s orchestrate TLS formation and maintenance through concomitant antigen presentation to CD4⁺ and CD8⁺ T cells. (1) TLS formation requires the maturation of cDC1s and their migration to tdLNs followed by (2) the recruitment of primed T cells into the tumor microenvironment. As tumors progress, (3) intratumoral migration of cDC1s into CCR7 ligand–enriched stromal hubs becomes essential for TLS maintenance. This process (4) depends on concomitant antigen presentation to both CD4⁺ and CD8⁺ T cells to sustain the T_FH cell pool, drive T_PEX cell differentiation, and (5) support germinal center (GC) formation and tumor-specific IgG production. the presence of mature DCs or TLSs correlates with improved overall survival in NSCLC and disease-free survival in hepatocellular carcinoma. CONCLUSION: These findings underscore the pivotal role of mature cDC1s in both establishing and maintaining functional TLSs, primarily through concomitant MHC-II and MHC-I antigen presentation to CD4⁺ and CD8⁺ T cells within tumor lesions. They further highlight the therapeutic potential of cDC1-targeted strategies to enhance TLS function and improve antitumor immunity in cancer patients.