Inhibiting CXCR4 Attenuates TOX-Programmed Exhausted Phenotype of CD8+ T cells via Regulating JAK2/STAT3 Pathway [ChIP-Seq]

T-cell exhaustion (Tex) phenotype has limited the efficacy of single-agent immune checkpoint blockade (ICB) in cancer immunotherapy, underscoring the need to target upstream pathways. Clinical trials have indicated that CXCR4 antagonists enhance anti-cancer activity. However, the specific mechanisms through which CXCR4 contributes to immune cell phenotypes are not fully understood. By targeting CXCR4 using antagonists or transgenic mice (Cxcr4flox/flox LckCre), we effectively attenuated the Tex phenotype and enhanced the functional T-cell phenotype in vivo. Additionally, single-cell RNA/TCR-seq analyses demonstrated that CXCR4 inhibition promotes anti-PD-1 immunotherapy by reducing CD8+ Tex phenotype and enriching the abundance of TCR clonotypes in vivo. Mechanically, we found that CXCR4-blocking T cells mitigate Tex phenotype by regulating the JAK2-STAT3 pathway, reducing the interaction between p-STAT3 and TOX. Single-cell ATAC/RNA-seq validated that CXCR4 deficiency epigenetically orchestrates the transition from functional to exhausted phenotypes in CD8+ T cells. Altogether, we demonstrate a mechanism of targeting CXCR4 to orchestrate CD8+ Tex cells in cancers and provide a rationale for the combined CXCR4 antagonists and ICBs in clinical trials. Overall design: In this study, we explored single-cell transcriptome data from normal and tumors to identify CXCR4 expression patterns in T cells and found that CXCR4 is highly expressed in CD8+ PD-1high Tex cells. CXCR4 antagonists and transgenic mice (Cxcr4flox/flox LckCre) in multi-tumor models were used to identify the molecular mechanisms of CXCR4 in maintaining CD8+ Tex phenotypes in tumors. Furthermore, single-cell RNA/TCR-seq and RNA/ATAC-seq were employed to validate the TOX-mediated Tex phenotype triggered by CXCR4 in vitro and in vivo. Our findings provide a novel insight into molecular mechanisms underlying CXCR4 in maintaining the Tex phenotype in tumors, thereby providing a rationale for combination strategies between CXCR4 inhibitors and ICB in cancers.