The characterization of tumor immune microenvironment after neoadjuvant immunotherapy in head and neck squamous cell cancer using multiplex immunohistochemistry

• Higher baseline tumor infiltration with CD4 + T cells, CD8 + T cells, Tregs, PD1 + T-cells and PDL1 + cells was associated with PR after neoadjuvant immunotherapy. • Higher expression of TIM3 and LAG3 in T cells after neoadjuvant treatment were found in the NPR patients relative to the PR patients. • Tumor infiltration lymphocyte analysis indicated that CD8 + T cell infiltration in tumor marginal area was significantly higher in the PR group. Optimizing clinical decision-making in head and neck squamous cell carcinoma (HNSCC) is challenging due to the ambiguous understanding of the immune cell dynamics and immune checkpoints regulation in the disease after the administration of neoadjuvant immunotherapy (NIT). HNSCC biopsy samples collected before and after the neoadjuvant treatment are classified into the pathologic response (PR) and the non-pathologic response (NPR) groups according to treatment responses and the expression of immune cells and checkpoints was labeled using multiplex immunohistochemistry (m-IHC). The populations of CD4 + T cells, CD8 + T cells, regulatory T cells (Treg), PD-1, and PD-L1 were particularly higher in the PR group than the NPR group in pre-treatment tissues, with the p-values of log-transformed positive cell density <0.05. Almost all markers showed a lower expression in the PR patients after treatment, resulting lower post/pre-treatment ratios of positive cell densities in the PR patients relative to the NPR patients. Following treatment, TIM3 + T cells and LAG3 + T cells exhibited significantly diminished levels in the PR cohort relative to the NPR cohort, with post/pre-treatment expression ratios showing significant differences (P < 0.05). Tumor infiltration lymphocyte analysis revealed that the PR group exhibited a considerably higher average density of CD8 + T cells infiltrating in the tumor marginal zone. The presence of T cells demonstrated significant predictive capability for responses to neoadjuvant immunotherapy in HNSCC patients. Furthermore, TIM3 + T cells and LAG3 + T cells were found to be remarkably lower in the partial response (PR) cohort than in the non-partial response (NPR) cohort post-treatment. This research contributes critical understanding of the physiological changes occurring in immune cell responses.