Pediatric AML blasts drive immunosuppressive reprogramming and metabolic adaptation in macrophages, impairing T cell activation and anti-leukemic immunity Free

Despite transformative impact of immunotherapy in cancer care, patients with acute myeloid leukemia (AML) continue to experience limited clinical benefits. This inefficacy is partly due to an incomplete understanding of the AML microenvironment (ME), including the role of innate immune cells. Among these, macrophages, which are highly plastic cells and comprise a substantial proportion of tumor-infiltrating immune cells, are key players in this context. In the AML ME, macrophages frequently adopt an anti-inflammatory, pro-tumorigenic (M2-like) phenotype rather than a pro-inflammatory, anti-tumorigenic (M1-like) state. Although their critical role in AML progression and therapy resistance is increasingly recognised, the nature of their interaction with AML blasts remains poorly defined.

Our previous work demonstrated that the bone marrow of pediatric AML patients is dominated by M2-like macrophages. Building on this, we investigated how macrophages and leukemic blasts from pediatric AML bone marrow influence each other's phenotype and function.

Using co-culture systems, we found that AML blasts reprogram both undifferentiated and pro-inflammatory macrophages into an anti-inflammatory, immunosuppressive phenotype. By day 6 of co-culture, these reprogrammed macrophages lost their ability to inhibit the growth of t(8;21) and KMT2A-rearranged patient-derived xenograft (PDX) and primary AML cells. Flow cytometry and immunofluorescence analyses revealed a phenotypic shift characterized by downregulation of immune-stimulatory markers (CD80, HLA-DR) and upregulation of immune-suppressive markers (CD206, CD163) along with reduced secretion of pro-inflammatory cytokines (TNF, IFNγ, and IL-1β). Transcriptomic profiling confirmed this immunosuppressive reprogramming, showing significant downregulation of key genes in the NF-κB signaling pathway (NFKB1, IRF1, TRAF1, and IL1B), all associated with M1 polarization.

Notably, transwell assays showed direct cell–cell contact is required for macrophage reprogramming, as physical separation prevented the M2-like transition. Confocal microscopy revealed the formation of tunnelling nanotubes (TNTs) between AML PDX cells and macrophages, suggesting a route for direct intercellular communication. Mitochondrial labelling demonstrated predominant transfer of mitochondria from AML cells to macrophages. Inhibition of TNT formation reduced expression of M2-associated markers, implicating mitochondrial transfer as a driver of macrophage reprogramming. Transcriptomic and Seahorse metabolic analyses further demonstrated a shift in macrophage metabolism toward increased oxidative phosphorylation (OXPHOS) following co-culture with AML cells.

To assess functional consequences, we evaluated macrophage-mediated T cell responses. A bioluminescence-based T cell activation assay showed that pro-inflammatory macrophages lost their ability to stimulate T cells after co-culture with AML cells. In cytotoxicity assays, they failed to support T cell–mediated killing of AML cells and suppressed the cytotoxic activity of the bispecific T cell engager blinatumomab against t(8;21) AML cells. Consistent with an immunosuppressive phenotype, reprogrammed macrophages also upregulated expression of the immune checkpoint molecules TIM-3 and LAG-3.

In conclusion, our study highlights the plasticity of macrophages in the pediatric AML bone marrow microenvironment and their critical role in shaping immune responses. We propose that mitochondrial transfer via TNTs is a key mechanism through which AML blasts reprogram macrophages, fostering immune suppression. Disrupting this AML-macrophage interaction may restore immune surveillance and improve the efficacy of immunotherapies.