Immunologic characterization of tumor-draining lymph nodes in head and neck squamous cell carcinoma

Immune checkpoint inhibitors (ICIs) have substantially improved outcome in several malignancies, however, their efficacy in head and neck squamous cell carcinoma (HNSCC) remains limited, with durable responses confined to a minority of patients. Biomarker discovery and mechanistic studies have predominantly focused on the tumor microenvironment (TME) and peripheral blood, while tumor-draining lymph nodes (TDLNs), the primary sites of tumor antigen presentation, immune priming, and early checkpoint regulation, have received comparatively little attention. Given that ICIs primarily act by reinvigorating T cell responses initiated in lymphoid tissues, a more complete understanding of anti-tumor immunity could be achieved by investigating the TDLN compartment. In this context, the overarching aim of this thesis was to characterize the immune landscape of TDLNs in oral squamous cell carcinoma (OSCC) and to determine how dendritic cells (DCs), B cells, and T cells contribute to immunosuppression, clinical outcome, and immune checkpoint modulation. To enable investigation of this compartment pre-operatively in a clinically feasible manner, a method using TDLN biopsy (TDLNB) and fine-needle aspiration (FNA) was developed, enabling flow cytometric immunophenotyping of TDLN-resident immune cells. Comprehensive immune profiling across oral and cutaneous squamous cell carcinoma (CSCC) cohorts revealed that distinct, cell-type-specific immune programs within TDLNs were differentially associated with clinical outcome. B cell features, including elevated CD24, CD73, HLA-DR expression, and memory skewing, were linked to increased recurrence risk and reduced survival. In contrast, enrichment of naïve and plasma B cell profiles, together with elevated expression of CXCR5, CD11c, and PD-L1, were associated with favorable outcomes. HLA-DR expression on B cells also emerged as an independent prognostic marker. Beyond B cell markers, suppressive PD-L1high cDC1s in TDLNs were associated with adverse clinical features. T cell analyses revealed that regulatory and checkpoint-enriched subsets, including FOXP3+, TIGIT+, PD-1+, and CTLA-4+ T cells, correlated with recurrence, while CXCR5+, Tim-3+, and LAG-3+ T cells were linked to favorable prognosis. Together, these findings indicate that immune regulation in TDLNs reflects coordinated dysfunction across antigen-presenting and effector lymphocyte compartments. In conclusion, this thesis establishes TDLNs as a clinically relevant and deficiently explored immunological compartment in HNSCC. This work identifies TDLN-derived markers with prognostic and potentially predictive relevance. It suggests that minimally invasive FNA-based sampling could facilitate longitudinal monitoring of these immune features in future biomarker-driven therapeutic strategies. List of scientific papers I. Lagebro V, Piersiala K, Petro M, Lapins J, Grybäck P, Margolin G, Kumlien Georen S, Cardell LO. A Novel Method Using Fine Needle Aspiration from Tumor-Draining Lymph Nodes Could Enable the Discovery of New Prognostic Markers in Patients with Cutaneous Squamous Cell Carcinoma. Cancers (Basel). 2023 Jun 22,15(13):3297. PMID: 37444407, PMCID: PMC10340690. https://doi.org/10.3390/cancers15133297 II. Liljeström V, Piersiala K, Farrajota Neves da Silva P, Petro M, Cardenas E I., Elliot A, Bark R, Margolin G, Kumlien Georen S, Cardell LO. B cells in Tumor-Draining Lymph Nodes Contribute to the Poor Efficacy of Anti-PD-1 Therapy in OSCC Patients. [Submitted] III. Liljeström V, Farrajota Neves da Silva P, Bark R, Elliot A, Marklund L, Margolin G, Kumlien Georen S, Cardell LO, Piersiala K. B Cell dysfunction in tumor-draining lymph nodes predicts relapse in oral squamous cell carcinoma. Cancer Immunol Immunother. 2025 Nov 12,74(12):373. PMID: 41222706, PMCID: PMC12612397. https://doi.org/10.1007/s00262-025-04231-9 IV. Hjalmarsson E, Piersiala K, Lagebro V, Farrajota Neves Da Silva P, Petro M, Starkhammar M, Elliot A, Bark R, Margolin G, Kumlien Georen S, Cardell LO. High expression of PD-L1 on conventional dendritic cells in tumour-draining lymph nodes is associated with poor prognosis in oral cancer. Cancer Immunol Immunother. 2024 Jul 2,73(9):165. PMID: 38954023, PMCID: PMC11219651. https://doi.org/10.1007/s00262-024-03754-x V. Piersiala K, Hjalmarsson E, Lagebro V, Farrajota Neves da Silva P, Bark R, Elliot A, Marklund L, Margolin G, Georen SK, Cardell LO. Prognostic value of T regulatory cells and immune checkpoints expression in tumor-draining lymph nodes for oral squamous cell carcinoma. Front Immunol. 2024 Oct 15,15:1455426. PMID: 39474426, PMCID: PMC11518749. https://doi.org/10.3389/fimmu.2024.1455426