Abstract
BACKGROUND: The presence of tertiary lymphoid structures (TLS) in solid tumors, including Merkel cell carcinoma (MCC), is associated with a better prognosis and a better response to immunotherapy with immune checkpoint inhibition (ICI). The detailed mechanisms by which TLS influence antitumor immune responses are only partially understood. METHODS: Clinically annotated tumor tissues of 27 patients with MCC were obtained prior to ICI therapy. Tumor samples were subjected to transcriptomic and multiplex immuno-visual profiling, T-cell receptor (TCR) clonotype mapping, as well as-in selected cases-spatial transcriptomics to comprehensively characterize the tumor immune microenvironment. RESULTS: Weighted gene co-expression network analysis (WGCNA) of transcriptomic data in combination with topological overlap measures indicated a higher abundance of TLS in tumors of patients with MCC responding to ICI therapy. This concept was substantiated through immunomorphological analyses, revealing mature B-cell follicle-like structures characterized by high endothelial venules (HEVs). Further supporting HEVs as critical entry points for naïve T cells, the presence of TLS was correlated with a pronounced infiltration of CD4(+) and CD8(+) T cells, exhibiting both naïve and central memory phenotypes. The TCR repertoire of these infiltrates exhibited enhanced richness and diversity with a pronounced reactivity toward Merkel cell polyomavirus-derived T-cell epitopes. Spatially resolved RNA and V(D)J sequencing revealed the expression of genes associated with T-cell recruitment within TLS, alongside the presence of naïve and central memory T-cell markers. Notably, individual clonally expanded TCR transcripts were detected both within TLS and among tumor-infiltrating lymphocytes. The latter were associated with low expression of memory cell markers and high expression of effector cell markers. Additionally, a spatial gradient in the expression of genes linked to immune stress in MCC cells-such as those involved in the interferon-γ response and antigen processing and presentation machinery-originated in proximity to the TLS. CONCLUSION: Our findings are consistent with a key role of TLS in shaping immune interactions within the MCC microenvironment, driving the recruitment of diverse tumor-reactive T cells. These insights hold promise for advancing immunotherapeutic strategies.