Abstract 5804: Pathologic responses to neoadjuvant immune checkpoint inhibitor (ICI) combinations in head and neck squamous cell carcinoma (HNSCC) associate with specific CD4+ helper and regulatory T cell activation states and clonal plasticity

Abstract Background: The clinical benefits observed with anti-PD-1 therapy in the recurrent and metastatic setting have sparked greater interest in exploring immunotherapy at an earlier stage of the HNSCC treatment regimen. However, response rates in HNSCC patients are limited, and mechanisms of treatment failure remain poorly understood. In an ongoing neoadjuvant trial, we investigate the transcriptional and spatial dynamics of tumor-infiltrating CD4+ T cells in HNSCC patients treated with neoadjuvant nivolumab (anti-PD-1, Nivo) ± ipilimumab (anti-CTLA-4, Ipi) or relatlimab (anti-LAG-3, Rela). Our goal is to elucidate mechanisms of tumor-infiltrating CD4+ T cells associated with positive clinical response to ICI therapy. Methods: Pre- and post-treatment tumor samples from HNSCC patients were processed for single-cell RNA-sequencing, T cell receptor (TCR) sequencing and spatial imaging, with sequencing successfully completed for 39 patients. Specific T cell transcripts defined cellular subpopulations, revealing distinct transcriptional and clonal dynamics. Multispectral imaging of tissue sections was performed to evaluate changes in T cell composition within the tumor microenvironment. Pathologic response was linked to observed cellular dynamics to treatment outcomes. Results: Pathologic response rates were highest in the Nivo+Rela arm (11/15, 73%) versus Nivo+Ipi (7/11, 64%) or Nivo (5/12, 42%). While both combination ICI therapies increased total CD3+ T cell frequencies within tumor and stromal compartments, only Nivo+Ipi led to a significant increase of conventional CD4+ T helper (Tconv) and regulatory T cells (Treg) in both regions. Notably higher CD8+/Treg and Tconv/Treg ratios positively correlated with tumor rejection in the Nivo+Rela arm. In contrast to Nivo+Ipi, Nivo+Rela therapy expanded clonal diversity exclusively within Tconv populations in major responders, with a majority of emerging clones showing effector characteristics. Moreover, Nivo+Rela altered the phenotype of activated Treg to a more naïve or memory-like phenotype in major responders, suggesting a distinct regulatory landscape with implications for long-term tumor control. Conclusion: Neoadjuvant combination ICI therapies directly influence spatial CD4+ T cell composition and TCR clonal dynamics and modulate the frequency and phenotype of Tconv and Treg cells in the tumor in treatment-specific patterns. These immunological shifts correlate with tumor rejection and improve clinical outcomes. Our findings suggest that a dual ICI approach may enhance response rates beyond those achieved with monotherapies. They also indicate a possible biologic basis in CD4+ T cell activity for precision ICI treatment, supporting more personalized ICI strategies in the neoadjuvant setting. Citation Format: Matthias Brand, Aditi Kulkarni, Housaiyin Li, Pragati Upadhyay, Simon I. Chiosea, Patricia M. Santos, Brian R. Isett, Marion Joy, Gabriel L. Sica, Umamaheswar Duvvuri, Seungwon Kim, Mark Kubik, Fei Tu, Tullia C. Bruno, Dario A. Vignali, Anthony R. Cillo, Riyue Bao, Dan P. Zandberg, Jing Hong Wang, Lazar Vujanovic, Robert L. Ferris. Pathologic responses to neoadjuvant immune checkpoint inhibitor (ICI) combinations in head and neck squamous cell carcinoma (HNSCC) associate with specific CD4+ helper and regulatory T cell activation states and clonal plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5804.