Spatial profiling of HPV-stratified head and neck squamous cell carcinoma reveals distinct immune niches and microenvironmental architectures

HPV status is a key determinant of prognosis and treatment response in head and neck squamous cell carcinoma (HNSCC). To investigate how HPV influences the tumor-immune-stromal landscape, we performed high-dimensional spatial profiling, including its impact on spatial organization, tertiary lymphoid structures (TLSs), and spatially organized cellular neighborhoods.

Tumor biopsies from HNSCC patients (n = 16; 7 HPV-positive, 9 HPV-negative) were stained with a multiplex immunofluorescence (mIF) panel focused on immune profiling. A deep learning-based analysis pipeline enabled the identification and phenotypic state profiling of 14 cell types. Tissues were segmented into four distinct tumor regions, and spatial neighborhoods and TLSs were identified and analyzed for differential cellular composition, activation states, and spatial interactions between HPV-positive and HPV-negative tumors.

HPV-positive and HPV-negative tumors differ in their tumor microenvironment (TME) composition, tumor cell state and spatial organization. The TME of HPV-positive tumors exhibited a greater abundance of activated lymphocytes, B- and T-cell-enriched spatial neighborhoods, and PD-1-PD-L1 interactions within the tumor area, whereas HPV-negative tumors were dominated by fibroblast- and macrophage-rich niches. T- cells in HPV-positive tumors showed greater activation across neighborhoods and areas, while in HPV-negative tumors T- cells demonstrated enrichment of exhaustion and terminal differentiation markers such as PD-1 and CD57. HPV-positive tumor cells had increased IDO1, HLA-DR, and Ki67 positivity, whereas HPV-negative tumor cells were more frequently CD44 positive, reflecting a more stem-like phenotype. Importantly, TLSs in HPV-positive tumors were located closer to the tumor area and enriched in activated immune cells, including ICOS⁺ CD4 T- cells, memory T- cells, and CD21⁺ B- cells. In contrast, TLSs in HPV-negative tumors were more distant and enriched for immunosuppressive populations such as PD-1⁺/PD-L1⁺ Tregs and macrophages.

HPV status defines distinct spatial immune architectures in HNSCC. HPV-positive tumors harbor immune-activating TLSs and cellular neighborhoods that support antitumor immunity, whereas HPV-negative tumors exhibit suppressive niches and stromal dominance. These findings highlight TLSs, particularly their proximity and composition, as key features of the HPV-stratified TME and potential biomarkers for immunotherapy response.