CLEC2B-KLRB1 axis acts as an immune checkpoint, governing the exhaustion of CD8<sup>+</sup> T cells and their resistance to immune checkpoint blockade

<h4>Background</h4>Many patients with cancer 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.<h4>Methods</h4>Spatial proximity is essential for suppressive ligand-receptor signaling. Here, we mapped the spatial tumor microenvironment of patients with MSS CRC 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.<h4>Results</h4>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 antitumor function of CD8⁺ T cells, providing a potential immunotherapy target for ICB non-responders.<h4>Conclusions</h4>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 antitumor immunity, positioning this axis as a promising target for enhancing immunotherapy efficiency in malignancies, including CRC and other ICB-resistant cancers.