3D4 cells exhibit transcriptional features inconsistent with alveolar macrophage identity

The 3D4 cell lines are widely used as porcine alveolar macrophage models in immunological research. However, our preliminary experiments revealed that 3D4/21 cells failed to respond to lipopolysaccharide stimulation. We challenged both 3D4/21 and 3D4/2 clones with various Toll-like receptor (TLR)4 (LPS, Ultrapure LPS, Kdo2-Lipid A) and TLR2/TLR1 (Pam3CSK4) ligands. Strikingly, all stimulants failed to activate pro-inflammatory responses (IL1B and TNF expression). To clarify the lack of response of 3D4 cells, we isolated primary porcine alveolar macrophages (PAM) from piglets using lung lavage, and analyzed the transcriptome of unstimulated or Kdo2-Lipid A-stimulated 3D4/21 cells compared with equally treated PAM. We identified extensive transcriptomic differences, with 10,718 differentially expressed genes between untreated 3D4/21 cells and PAM (adjusted p-value < 0.05). Key alveolar macrophage markers and lineage-determining factors (SPI1 and IRF8) were weakly expressed or absent in 3D4/21 cells. Important pathogen recognition genes (TLR1, TLR2, TLR4, TLR6, TLR7, TLR8, TLR9, CGAS, IFI16, and NOD2) showed very low abundance or complete absence compared with PAM. Accordingly, 3D4/21 cells failed to respond to Kdo2-Lipid A stimulation, while PAM showed 2945 regulated genes. Functional annotation revealed that genes preferentially expressed in 3D4/21 cells were enriched for epithelial characteristics. Human Lung Cell Atlas analysis corroborated that 3D4/21 cells originate from respiratory epithelial cells. Similar patterns were observed in 3D4/2 cells, with low macrophage markers (CD163, SPI1, CD14, TLR2, and TLR4) and high epithelial markers (TP63, EGFR, and KRT5). Our data strongly suggest that 3D4 cell lines were misclassified as myeloid cells and actually possess respiratory epithelial characteristics.