# LDRT combined with anti-PD-1 promotes tumor regression in head and neck squamous cell carcinoma through tumor metaprogram evolution and immune cell reprogramming

## TL;DR

Low-dose radiotherapy combined with anti-PD-1 therapy improves tumor regression in head and neck cancer by altering tumor and immune cell behavior.

## Contribution

This study reveals how LDRT enhances αPD-1 efficacy through tumor and immune cell reprogramming in HNSCC.

## Key findings

- LDRT and αPD-1 together reduce tumor growth and reshape the tumor microenvironment.
- Combination therapy increases CD8⁺ T-cell clonal expansion and reduces CD4⁺ Treg-Ctla4 frequency.
- An Isg15high macrophage state is linked to CD4⁺ Treg-Ctla4 and is reduced by LDRT-containing regimens.

## Abstract

Anti–PD-1 (αPD-1) therapy shows limited efficacy in head and neck squamous cell carcinoma (HNSCC), partly due to an immunosuppressive tumor microenvironment (TME) and insufficient infiltration of effector T cells. Low-dose radiotherapy (LDRT) has been suggested to modify the TME, potentially enhancing the effects of αPD-1.

We profiled murine HNSCC treated with LDRT (1 Gy) and/or αPD-1 using single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq), complemented by orthogonal functional assays and in situ validation (spatial transcriptomics and multiplex immunofluorescence).

LDRT and αPD-1 synergistically suppressed tumor growth and remodeled the TME. Single-cell analyses showed malignant programs shifting from proliferative states toward stress- and interferon (IFN)-responsive states. Integrated scTCR-seq analysis revealed enhanced clonal expansion of tumor-reactive CD8⁺ T-cell subsets and reduced the frequency of CD4⁺ Treg-Ctla4 under combination therapy. TCR-based metrics (including STARTRAC) indicated higher migration potential and limited intratumoral clonal overlap for CD4⁺ Treg-Ctla4. An Isg15high macrophage state (Macro-Isg15) was positively associated with CD4⁺ Treg-Ctla4 frequency and was more prominent under αPD-1 monotherapy. Ligand–receptor inference highlighted a Macro-Isg15–associated CXCL14–CXCR4 chemokine axis enriched under αPD-1 monotherapy and attenuated in LDRT-containing regimens, suggesting reduced chemokine-driven support for CD4⁺ Treg-Ctla4 accumulation/recruitment.

These findings support an integrated framework in which LDRT augments αPD-1 efficacy by reprogramming malignant states and reshaping immune cell dynamics, including attenuation of immunosuppressive features within the TME. This preclinical evidence provides a rationale for translational evaluation of LDRT-based combination strategies in HNSCC.

The online version contains supplementary material available at 10.1186/s13046-026-03660-3.