ABSTRACT: Plasmacytoid dendritic cells (pDCs) are critical antiviral sentinels known for rapid type I interferon (IFN-I) production. However, their proteomic profile and nutrient dependencies are not well understood. Here, we used absolute quantitative proteomics of ex vivo murine splenic pDCs to characterize their protein landscape. Cross-species comparison with human blood pDCs revealed strong conservation but some differences in metabolic machinery. The transferrin receptor, responsible for transferrin-iron uptake, is the most abundant nutrient transporter in both species with 40,000 copies per murine pDC, and is significantly enriched compared to conventional dendritic cells. Despite pDCs showing constitutive transferrin-iron uptake they have low expression of iron storage proteins and a modest repertoire of iron-dependent enzymes. Also, pDC do not have an increase in iron atoms per cell compared to cDC, which suggests that pDC export excess iron through ferroportin and argue that Tfrc may have iron-independent roles. Indeed, iron chelation did not affect pDC production of type I interferon. These data suggest that Tfrc supports noncanonical functions in pDC biology, possibly related to antigen uptake. This work provides new insights into pDC metabolism and iron biology, advancing understanding of their specialized immune roles. Plasmacytoid dendritic cells (pDCs) are specialized antiviral sentinels defined by rapid type I interferon (IFN‑I) production, yet their metabolic organization and metal‑ion handling remain poorly understood. Here, we establish a high‑confidence, absolute quantitative proteome of murine splenic pDCs directly ex vivo and compare it with conventional dendritic cell subsets and human pDCs. Cross‑species proteomic analysis reveals strong conservation of pDC identity alongside divergence in metabolic pathway usage. pDCs display exceptionally high expression of the transferrin receptor (TFRC) and robust transferrin uptake compared with other immune cells; however, quantitative ironome analysis demonstrates that total cellular iron content is equivalent across dendritic cell subsets. Instead, iron is differentially allocated, with pDCs enriched for iron-sulfur cluster assembly proteins, cDC1s for heme‑binding proteins, and cDC2s for non‑heme iron enzymes. Despite elevated transferrin uptake, pDCs do not accumulate intracellular iron, coincident with expression of the iron exporter ferroportin, suggesting active iron efflux. Functionally, acute chelation or supplementation of extracellular iron does not affect CpG‑A plus IFNα-induced IFNα or TNFα production by pDCs. Together, these data demonstrate that pDCs uncouple surface transferrin receptor abundance from intracellular iron accumulation and effector cytokine production, revealing a distinct organization of iron handling that may support specialized trafficking or sensing functions rather than metabolic iron demand.
Linda Sinclair 
