Background <p>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.</p> Methods <p>We profiled murine HNSCC treated with LDRT (1&#xa0;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).</p> Results <p>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 <i>Isg15</i>^<i>high</i> 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.</p> Conclusion <p>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.</p> Graphical abstract <p></p>