Tumour-derived factors prime microglia for immune checkpoint-mediated T-cell regulation

Abstract

Tumour-associated microglia are major regulators of the immune microenvironment in brain malignancies, yet the early mechanisms by which tumour-derived signals shape their immune phenotype remain poorly understood. We used quantitative proteomic analysis on microglia upon tumour exposure and observed extensive remodelling of the microglial proteome following exposure to tumour-conditioned media, indicating that soluble mediators represent the dominant drivers of early microglial activation. Microglia reprogramming was characterized by coordinated upregulation of inflammatory mediators together with proteins involved in antigen presentation and T-cell regulation, including MHC-I, PD-L1 and Galectin-9. Flow cytometric validation using multiple murine and human cell models confirmed the increased expression of PD-L1 and MHC-I on microglia following exposure to tumour-derived conditioned media. Subsequently, tumour-conditioned microglia promoted expression of the inhibitory receptors PD-1 and TIM-3 on T-cells after coculture. Importantly, integration of publicly available single-cell RNA sequencing datasets and cell-cell communication analysis further demonstrated that this immune-regulatory program is recapitulated in microglia from patients with brain tumours. Together, our findings identify an early, soluble factor-driven microglial activation program that precedes direct tumour contact and integrates inflammatory activation with immune checkpoint regulation. These results provide a mechanistic framework for the early establishment of an immunosuppressive brain tumour microenvironment and highlight microglial immune checkpoint pathways as potential targets for therapeutic intervention.