The CLEC2B-KLRB1 axis acts as an immune checkpoint, governing the exhaustion of CD8+ T cells and their resistance to immune checkpoint blockade (scRNA-seq Data)

Abstract Background: Many cancer patients benefit little from immune checkpoint blockade (ICB), a major obstacle to immunotherapy for decades. Finding alternative immune checkpoints that control CD8+ T cell exhaustion is urgent if we are to improve the efficacy of immunotherapies, particularly in microsatellite stable (MSS) colorectal cancer (CRC) that is resistant to ICB. Methods: Spatial proximity is essential for suppressive ligand-receptor signaling. Here, we mapped the spatial tumor microenvironment of MSS CRC patients at single-cell resolution and analyzed the cells interacting with exhausted CD8+ T cells to identify immune checkpoint ligand-receptor pairs. To investigate the function of this previously unrecognized immune checkpoint, we performed validation studies spanning cellular experiments, mouse models, and clinical patient samples. Results: We found that a subset of MSS CRC exhibits substantial CD8⁺ T cell infiltration, but their function is suppressed. We identified CLEC2B (ligand)-KLRB1 (receptor) as a novel inhibiting ligand-receptor pair for CD8+ T cells. KLRB1 acts as an immune checkpoint receptor, increasing CD8+ T cell exhaustion and facilitating immune escape in various human cancers. Binding of CLEC2B to KLRB1 initiates immunosuppressive signaling in CD8+ T cells. Clinically, CLEC2B-KLRB1 expression correlates positively with cancer progression and poor response to ICB, demonstrating that KLRB1+ CD8+ T cells are a key marker of the poorly responsive ICB subtype. Furthermore, blocking CLEC2B-KLRB1 signaling with antibodies enhances the anti-tumor function of CD8+ T cells, providing a potential immunotherapy target for ICB non-responders. Conclusions: Our study revealed CLEC2B-KLRB1 as a previously unrecognized immune checkpoint axis that drives T cell exhaustion specifically in ICB poor responsive MSS CRC. Blockade of KLRB1 with a therapeutic antibody reinvigorated CD8+ T cell anti-tumor immunity, positioning this axis as a promising target for enhancing immunotherapy efficiency in malignancies including CRC and other ICB-resistant cancers.