Emerging data indicate a role for innate immune checkpoints in immune evasion, whereby tumours can escape macrophage-mediated phagocytosis through expression of anti-phagocytic signals. A new study now reports that one such ‘don’t eat me’ signal, CD24, orchestrates a novel innate immune checkpoint through interaction with the inhibitory receptor sialic acid-binding Ig-like lectin 10 (Siglec-10) on tumour-associated macrophages (TAMs).

Credit: Simon Bradbrook/Springer Nature Limited

Using RNA sequencing (RNA-seq) data from the TARGET and TCGA databases, Barkal et al. first observed CD24 upregulation across almost all tumour types analysed relative to normal tissues, which was particularly strong when compared with expression of known innate immune checkpoints such as CD47. Importantly, the largest increases in CD24 expression were in triple-negative breast cancer (TNBC) and ovarian cancer, and stratification by CD24 expression revealed improvements in relapse-free survival and overall survival in patients with CD24-low ovarian cancer and breast cancer, respectively. Subsequently, single-cell RNA-seq data and fluorescence-activated cell sorting (FACS) assays in primary ovarian and breast cancer samples revealed that CD24 was specifically overexpressed in the tumour compartment, whereas SIGLEC10 was expressed in a substantial fraction of TAMs, suggesting the possibility of a CD24–Siglec-10 interaction.

Next, the role of CD24–Siglec-10 interactions in regulating macrophage-mediated antitumour immune responses was examined. In a co-culture model coupled to a flow-cytometry-based phagocytosis assay, stable genetic ablation of CD24 (ΔCD24) potentiated the phagocytosis of MCF-7 human breast cancer cells by Siglec-10+ M2-like macrophages. Moreover, live-cell microscopy-based phagocytosis assays illustrated that ΔCD24 MCF-7 cells were more readily engulfed into the phagolysosomes of co-cultured macrophages than wild-type MCF-7 cells. In addition, SIGLEC10 knockout in donor-derived macrophages markedly exacerbated their phagocytic ability towards wild-type MCF-7 cells, further illustrating that the CD24–Siglec-10 interaction exerts an anti-phagocytic effect.

The therapeutic relevance of the findings was then investigated. Using live-cell microscopy-based phagocytosis assays, anti-CD24 antibody treatment was shown to markedly increase engulfment of wild-type MCF-7 cells into the phagolysosome of co-cultured macrophages. Subsequent FACS-based assays confirmed this enhanced phagocytic response upon CD24 blockade in primary TNBCs and ovarian cancers and cell lines, which, interestingly, was greater than the pro-phagocytic effect seen upon anti-CD47 antibody treatment. Crucially, genetic deletion of SIGLEC10 in macrophages greatly reduced the pro-phagocytosis response to CD24 blockade, suggesting the anti-CD24 antibody functions primarily by specifically disrupting the CD24–Siglec-10 interaction.

In addition to MCF-7 cells, CD24 expression was positively correlated with the pro-phagocytic response to CD24 blockade across a panel of CD24+ pancreatic adenocarcinoma, pancreatic neuroendocrine tumour and small-cell lung cancer cell lines, and no effect on the phagocytosis was noted in other CD24− cell lines, indicating that CD24 is a potent ‘don’t eat me’ signal that modulates the antitumour innate immune response.

Finally, to assess the anti-phagocytic role of CD24 in vivo, GFP-luciferase+ MCF-7 cells (wild-type or ΔCD24) were engrafted into NOD SCID gamma (NSG) mice. At 21–28 days, bioluminescence assays revealed a markedly lower tumour burden in the ΔCD24 group than the wild-type group. Importantly, TAM depletion significantly abrogated the aforementioned reduction in tumour burden in mice with ΔCD24, but not wild-type, tumours, suggesting that the antitumour effects were attributable to TAM-mediated phagocytosis. Indeed, mice engrafted with ΔCD24 tumours had a substantial survival advantage over those with wild-type tumours. In support of the therapeutic promise of CD24 blockade, anti-CD24 antibody treatment led to major decreases in tumour growth in mice with established MCF-7 wild-type tumours.

CD24 is a potent ‘don’t eat me’ signal that modulates the antitumour innate immune response

Overall, the findings identify the CD24–Siglec-10 interaction as a novel innate immune checkpoint that modulates antitumour immunity, highlighting the promise of CD24 blockade as an immunotherapy strategy.