Background The complex interplay between cancer cells and the immune microenvironment critically influences treatment response in head and neck squamous cell carcinoma (HNSCC). This study aimed to delineate single-cell RNA signatures associated with myeloid and B cells in HNSCC and to elucidate their roles in modulating response to immune checkpoint inhibition (ICI).

Methods We analyzed a publicly available single-cell RNA sequencing dataset (GSE232240, n=17) from pre- and post-treatment HNSCC tumors of patients treated with anti-PD-1 and anti-CTLA-4 therapy. Comparative analysis between non-responders (n=7) and responders (n=10) enabled extraction of signatures predominantly expressed in ICI non-responders. Cell-cell communication analysis was performed to evaluate differences in immune cell interactions in responders versus non-responders. Putative ICI resistance signatures were evaluated in bulk RNA sequencing data from an independent HNSCC cohort (TCGA, n=521). LASSO regression was employed to create separate models for pre- and post-treatment samples. Individual genes were evaluated for correlation with overall survival using TCGA data.

Results Integrative single-cell profiling revealed crosstalk between B cells and myeloid cells in ICI responders that was not present in non-responders. Functional inference highlighted dysregulation of immunomodulatory pathways, including CD40 and FLT3, in non-responders, with diminished cytotoxic T cells and NK cells interaction with myeloid cells. We identified a transcriptional signature that strongly correlated with ICI response both at pre-treatment (AUC = 0.861) and post-treatment (AUC = 0.88). Increased expression of B cell-associated genes (e.g., MS4A1, CD79A) associated with ICI response in that transcriptional signature also correlated with improved overall survival in TCGA data. Conversely, elevated expression of non-responder signature genes (e.g., GSN, SERPINH1, SERPINF1) was linked to poor overall survival in the TCGA cohort, suggesting a role for aberrant fibroblast activation and extracellular matrix remodeling in HNSCC progression.

Conclusions This study provides novel insights into potential immune-mediated determinants of treatment response and disease progression in HNSCC. Identification of myeloid and B cell signatures associated with resistance to immune checkpoint blockade may inform the development of rational combination therapies to enhance treatment efficacy in HNSCC.

Ethics Approval The original published study (GSE232240) was conducted with ethical approval from the Medical Research Ethics Committee of The Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital (MREC AVL; reference number 4).

Consent This study utilized publicly available data from a previously published study (GSE232240). In the original publication, written informed consent was obtained from all participating patients in accordance with the ethical standards of the institutional and/or national research committee.