Background: Microglia, the resident immune cells of the brain, undergo dynamic functional maturation that requires precise integration of both intrinsic genetic programming and extrinsic environmental signals. This maturation process enables microglia to execute distinct developmental functions across different stages of brain development. A critical aspect of microglial maturation involves their transition from a predominantly phagocytic state to an antigen-presenting phenotype (1). Evidence from rodent models has shown that CD4+ T cells play an instrumental role in orchestrating this developmental transition (2). However, comparable human model systems to investigate this immunological crosstalk have been lacking. To address this gap, we utilised human microglia differentiated from induced pluripotent stem cells (hiPSCs) to interrogate whether cytokines derived from activated CD4+ T cells drive the phenotypic shift of microglia from phagocytic to antigen-presenting states.
Methods: Microglia were differentiated from N=4 independent hiPSC lines (2 male, 2 female, all neurotypical) using the Haenseler et al. (2017) protocol (3), and updated Washer et al. (2022) monoculture media (4). Cells were exposed for 7 days to 10% conditioned media (CM) collected from peripheral blood-derived CD4+ T cells activated in vitro using CD3/CD28 beads in combination with recombinant interleukin (IL)-2 (30 U/ml). Two treatment applications were administered between days 7-14 of microglial maturation. Control conditions included conditioned media from resting (non-activated) T cells and vehicle control (XVIVO-15 medium supplemented with IL-2). Genome-wide transcriptional profiles were assessed using bulk RNA sequencing, with differential gene expression analysis performed using DESeq2 and pathway enrichment evaluated through Gene Ontology (GO) analysis. To functionally validate transcriptional changes, phagocytic capacity was quantified by measuring fluorescent zymosan bead uptake via flow cytometry and compared to a 24h treatment with the same conditions.
Results: Bulk RNA sequencing analysis revealed 786 differentially expressed genes (adjusted p value < 0.05 and log2 fold change > 1) between activated CM-treated and vehicle control conditions. GO pathway enrichment analysis showed significant upregulation of interferon (IFN)-γ signalling pathways (p.adjust < 0.001), major histocompatibility complex (MHC) assembly processes, and antigen processing and presentation machinery (p.adjust < 0.001). Conversely, pathways associated with phagocytosis were significantly downregulated (p.adjust < 0.001), suggestive of the acquisition of antigen-presenting functional capabilities. Functional validation of these data was performed using zymosan bead phagocytosis. This revealed a significant effect of treatment on normalized mean fluorescence intensity (MFI) values, serving as a proxy measure for phagocytosed bead quantity (2-way ANOVA: F(2,6) = 15.07, p = 0.0046, partial eta² = 0.75). Post-hoc comparisons demonstrated that activated CM treatment significantly reduced normalized MFI relative to resting CD4+ T cell media, at both 7 days and 24h treatment timepoints.
Conclusions: Our findings suggest that soluble factors derived from activated CD4+ T cells drive a transcriptional state shift in human microglia from phagocytic to antigen-presenting phenotypes, which is supported by the functional validation of reduced phagocytosis. These results establish a novel human iPSC-based model system for investigating T cell-microglia interactions and reveal how peripheral immune signals can shape microglial identity during brain development.