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BackgroundHigh densities of tertiary lymphoid structures (TLSs) are associated with improved clinical outcomes in various malignancies, including human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC). However, the role of TLSs in shaping antitumor immunity in HPV-induced cervical cancer (CESC) remains unclear. Therefore, we analyzed the density, composition, and prognostic impact of TLSs in patients with CESC as well as patients with HNSCC.MethodsMultiplex immunofluorescence, immunohistochemistry, and spatial transcriptomics were used to analyze TLS density and composition in HNSCC and CESC tissue sections with respect to patient prognosis. The spatial approach was supplemented by flow cytometry-based analysis of the polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) phenotype in freshly resected primary tumor tissues.ResultsAlthough both indications were associated with HPV infection, we confirmed a positive correlation between TLS density and improved overall survival only in patients with HNSCC. The TLS composition differed markedly between HNSCC and CESC samples, with a shift toward high regulatory T cell (Treg) and PMN-MDSC abundance in CESC samples. The highest Treg and PMN-MDSC levels were observed in patients with CESC who died of the disease. CESC-infiltrating PMN-MDSCs showed high arginase 1 expression, which correlated with diminished T-cell receptor (TCR)¦Æ chain expression in CESC-infiltrating T cells. Additionally, the high number of PMN-MDSCs in TLSs was associated with the absence of HPV-specific T cells in CESC.ConclusionsUnlike in HNSCC, the composition of TLSs, rather than their quantity, was associated with the overall survival of patients with CESC. High numbers of Tregs and PMN-MDSCs infiltrating immature TLSs prevail in patients with CESC who succumbed to the disease and seem to affect tumor-specific immune responses.

Highlights?CD56bright NK cells correlate with better survival outcomes in bladder cancer.?Deletion of CD56 impairs NK cell cytotoxicity against bladder cancer cells.?CD56 deletion affects NK cell migration and adhesion to tumor cells.?CD56 expression on BCa cells may predict efficacy of NK cell-based immunotherapy. Bladder cancer (BCa) exhibits favorable responses to immunotherapy, but a significant percentage of patients fail to show a response owing to an inadequate tumor-immune landscape. We previously showed that NK cells are one of the predominant tumor-infiltrating lymphocytes in BCa and correlate with improved patient survival. However, that link was observed only with CD56bright NK cells while the CD56dim subset exhibited reduced cytotoxicity and higher accumulation in advanced BCa stages. The role of CD56 in NK cell functionality in BCa, however, remains unclear. Using flow cytometry and cytotoxicity assays, we demonstrated a significant decrease in cytotoxicity and activation of NK92 cells against BCa upon CD56 deletion. Further, migration assays and atomic force microscopy showed CD56 deletion impaired NK92 cell migration and adhesion to bladder tumor cells, reducing NK92 cell-mediated apoptosis of BCa cells. Prolonged exposure to bladder tumors led to CD56 loss in NK92 cells, suggesting tumor-induced NK92 cell dysfunction via CD56 reduction, consistent with our previous findings. Confocal microscopy revealed an overlap of CD56 and phosphorylated Pyk2, a critical kinase at the tumor-immune synapse, potentially mediating the downstream cytotoxicity effects. Blocking Pyk2 phosphorylation decreased CD56-mediated NK92 cell activation and reduced NK92 cell-mediated cytotoxicity against BCa. Finally, we showed that CD56 is also expressed by BCa cells and may be a predictive biomarker for NK cell-based immunotherapy, with its shedding indicating a mechanism for NK cell evasion. Our study identifies a novel innate-immune axis in BCa, leading to a better understanding of intratumoral NK cell biology and advancing NK cell-targeted treatments. Graphical abstractImage, graphical abstract

BackgroundThe characteristics of the tumor immunosuppressive microenvironment represent a major challenge that limits the efficacy of immunotherapy. Our previous results suggested that cryo-thermal therapy, a tumor ablation system developed in our laboratory, promotes macrophage M1-type polarization and the complete maturation of DCs to remodel the immunosuppressive environment. However, the cells that respond promptly to CTT have not yet been identified. CTT can cause extensive cell death and the release of danger-associated molecular patterns and antigens. Neutrophils are the first white blood cells recruited to sites of damage and acute inflammation. Therefore, we hypothesized that neutrophils are the initial cells that respond to CTT and are involved in the subsequent establishment of antitumor immunity.MethodsIn this study, we examined the kinetics of neutrophil recruitment after CTT via flow cytometry and immunofluorescence staining and explored the effect of neutrophils on the establishment of systemic antitumor immunity by in vivo neutrophil depletion and in vitro co-culture assays.ResultsWe found that CTT led to a rapid and strong proinflammatory neutrophil response, which was essential for the long-term survival of mice. CTT-induced neutrophils promoted the activation of monocytes via reactive oxygen species and further upregulated the expression of IFN-¦Ã and cytotoxic molecules in T and NK cells. Adoptive neutrophil transfer further enhanced the antitumor efficacy of CTT in tumor models of spontaneous and experimental metastasis.ConclusionThese results reveal the important role of neutrophil?monocyte interactions in the development of anti-tumor immunity and highlight that CTT could be used as an immunotherapy for targeting neutrophils and monocytes to enhance antitumor immunity.