CD24 blockade reprograms macrophages and synergizes with CD47 blockade to overcome tumor immune evasion

Although immunotherapy has revolutionized cancer treatment, only a minority of head and neck squamous cell carcinoma (HNSCC) patients derive clinical benefit, underscoring the urgent need to uncover novel immune evasion mechanisms. CD24 serves as a pivotal innate immune checkpoint that delivers a "don't eat me" signal to macrophages; consequently, CD24 blockade suppresses CD24-high tumors by promoting phagocytosis. However, CD24 is also widely expressed across various immune cell subsets. Whether it drives immune evasion through mechanisms distinct from inhibiting tumor-cell phagocytosis remains largely elusive. This study aimed to investigate the role and underlying mechanisms of CD24 in anti-tumor immunity, focusing on CD24-low HNSCC, and to evaluate its synergistic potential with CD47 blockade. CD24 and CD47 expression profiles in HNSCC were analyzed using The Cancer Genome Atlas (TCGA) datasets and flow cytometry. The anti-tumor therapeutic efficacy of anti-CD24 monoclonal antibodies (mAbs), either alone or combined with anti-CD47 mAbs, was evaluated in subcutaneous (SCC7) and orthotopic (MTCQ1) murine models. To dissect how CD24 blockade reprograms macrophages and enhances antigen presentation, we utilized bulk RNA-seq, single-cell RNA sequencing (scRNA-seq), multicolor flow cytometry, immunofluorescence staining, and in vitro co-culture assays with OT-I CD8 + T cells. Furthermore, clodronate-mediated macrophage depletion and antibody-mediated CD8 + T-cell depletion were performed to delineate the hierarchical dependence of the innate and adaptive immune arms. Despite negligible CD24 expression in HNSCC tumor cells, anti-CD24 treatment significantly suppressed HNSCC growth in both orthotopic and subcutaneous tumor models. Mechanistically, CD24 blockade acted as a "molecular switch" to reprogram tumor-associated macrophages (TAMs) from an M2-like phenotype toward an immunostimulatory CD80 + CD86 + MHC-I hi phenotype. This reprogramming enhanced both phagocytic clearance and MHC-I-mediated antigen presentation, which in turn promoted cytotoxic CD8 + T cells activation. Notably, combined anti-CD24 and anti-CD47 treatment synergistically strengthened anti-tumor immunity, triggered a systemic increase in interleukin-2 (IL-2) and interferon-γ (IFN-γ), and potently inhibited HNSCC growth in vivo. These synergistic effects relied on both macrophages and CD8 + T cells. Our findings establish that CD24 blockade activates anti-tumor immunity through a mechanism independent of direct effects on CD24-expressing tumor cells: reprogramming of TAM antigen presentation and consequent CD8 + T cell activation. Dual CD24 and CD47 blockade synergistically suppresses HNSCC growth. By identifying a macrophage-intrinsic mode of action, these findings suggest that CD24 blockade may extend beyond CD24-high tumors to CD24-low disease.