MSR1+ macrophages passivate antitumor immunity by inducing ITM2A+ CD4T exhaustion differentiation

Background & Aims: Immune-checkpoint inhibitors target the membrane protein; however, the role of membrane proteins in antitumor immunity remains poorly elucidated. In this study, we aimed to explore the role of membrane proteins and unearth potential membrane proteins that can be targeted. Methods: We initially screened prognosis-related membrane proteins based on The Cancer Genome Atlas Program and International Cancer Genome Consortium databases. Whole-gene, T–cell-specific or CD8T–cell-specific integral membrane protein 2A (ITM2A) knockout mice were constructed and used for orthotopic transplantation or as plasmid-derived spontaneous hepatocellular carcinoma (HCC) models to explore the role of ITM2A in the TME. Through scRNA sequencing, TimiGP analysis, molecular dynamics simulations, and biochemical experiments, the molecular mechanisms underlying the MSR1-ITM2A-TCR signaling regulatory axis were explored. Finally, bioengineering technologies were used to design and construct a new CD4T-targeted antibody-drug conjugate (ADC). Results: High ITM2A expression in HCC indicated a superior prognosis, which was associated with richer immune cell infiltration in the TME. The results of the single-cell RNA-sequencing of the HCC model in genetically knocked-out mice suggest that ITM2A influences the TCR signaling of TILs in the TME. This inference was confirmed in conditional ITM2A knockout mice. MSR1 + macrophages induced CD4T exhaustion by disrupting the ITM2A-ZAP70 axis during TCR activation. ADC (ZEA-αCD4) treatment effectively inhibited tumor growth, independent of the classical αPDL1 immunotherapy. Conclusions: MSR1+macrophages promote tumor-infiltrating ITM2A + CD4T exhaustion differentiation by regulating the ITM2A-ZAP70 axis. ZEA-αCD4 specifically targeted MSR1-ITM2A interaction to activate antitumor immunity and improve the efficacy of αPDL1 immunotherapy.