CRISPR Screen and Single-Cell Multi-Omics Identify a Novel Immune Checkpoint on Tumor-Infiltrating Regulatory T Cells

Abstract

Tumor infiltration regulatory T cells (Tregs) mediate immune escape by highly expressing multiple inhibitory receptors, which is one of the key factors in resistance to existing immune checkpoint inhibitors. However, systematic screening of Treg-specific immune checkpoints within tumors remains lacking. This study aims to integrate CRISPR functional screening with single-cell multi-omics to identify novel immune checkpoints with selective high expression and key inhibitory functions on intratumoral Tregs. A mouse tumor model was constructed to screen candidate genes affecting tumor growth in Treg using in vivo CRISPR-Cas9 libraries; Transcription and chromatin accessibility differences between tumor-infiltrating Treg and peripheral Treg were analyzed by combining single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq); Candidate molecular functions were validated through conditional gene knockout mice, antibody blockade, and tumor-infiltrating lymphocyte functional experiments, and their expression and clinical relevance in human tumor samples were evaluated. Leukocyte immunoglobulin-like receptor B4 (LILRB4) was identified as a novel immune checkpoint molecule selectively highly expressed on tumor-infiltrating Tregs, with expression induced by hypoxia in the tumor microenvironment and IL-10 signaling. The intracellular ITIM motif of LILRB4 enhances Treg’s inhibitory function and stability. Conditional knockout of Lilrb4 in Tregs or use of anti-LILRB4 monoclonal antibodies can significantly reshape the tumor immune microenvironment, enhance CD8 T cell effector function, inhibit tumor growth in various mice (tumor volume reduction by about 60%), and do not induce systemic autoimmunity. Human tumor sample analysis showed that LILRB4 expression on tumor-infiltrating Treg is significantly associated with anti-PD-1 resistance and poor patient prognosis. This study systematically reveals for the first time the function and mechanism of LILRB4 as a Treg-specific immune checkpoint for tumor infiltration, providing a theoretical basis and potential drug targets for developing a new generation of tumor immunotherapies that selectively target intratumoral Tregs while preserving peripheral immune tolerance.