Cluster C1 consisted of 95% of the control cells, sharing gene expression patterns consistent with AT2 cells. AT2 cells were well defined by expression of known AT2 cell-selective transcripts, including those encoding surfactant proteins and related proteins (SFTPB, SFTPC, NAPSA, LPCAT1, SLC34A2, and ABCA3) known to play critical and cell-selective roles in surfactant homeostasis in the alveolus. Potential regulators for cells within the C1 category include FOXA2, NKX2-1, CEBPA, and SREBF1. Functional classes selectively enriched in AT2 cells were absent in IPF, including genes mediating cytoprotection/detoxification and response to oxidative stress (e.g., SRXN1, CAT, FBVLN5, PRDX6, SOD2, SOD3, and GPX4).

Cells belonging to the C2 cluster express CD326 and CDH1 signature genes, but were not typical of any known epithelial cell type present in the normal lung. C2 cluster cells generally expressed AT2 cell–associated markers at higher levels than IPF basal or goblet/club cells and shared “lipid transport” and “innate immunity” functions with normal AT2 cells; however, C2 cells also expressed markers normally restricted to the proximal airway epithelium. The uniquely enriched functions predicted to be active in the C2 cluster included “activation of myofibroblasts,” “flux of anion,” and “T cell proliferation”. The predicted driving forces (key regulators) for the C2 cluster include CTGF, GF11, and FL11). Although all of the cells expressed CDH1 and EPCAM, the C2 cells did not express clear signature genes associated with any known lung epithelial subtypes, and we termed these cells “indeterminate” IPF cells.

Cells belonging to the C3 category harbored transcripts for TP63, KRT5, KRT14, BMP7, LAMB3, LAMC2, and ITGB, known markers of human basal cells. Signature genes of the C3 cell cluster were involved in “alpha6-beta4 integrin signaling,” “wound healing,” “cell migration,” and “laminin interaction,” consistent with an airway origin for these cells. Transcripts for SOX2, TP63, and TGFB1 were predicted as upstream regulators of cells belonging to the C3 cluster.

IPF goblet-like cells (C4) selectively expressed SPDEF, a transcription factor regulating goblet cell differentiation, MUC5AC, MUC5B, PIGR, AQP3, and SCGB1A1, transcripts that are characteristically expressed by airway secretory cells. Genes associated with “goblet cell morphology” (e.g., SPDEF, TCF7L2, and ELF3) and “O-linked glycosylation of mucins” (e.g., MUC16, MUC20, MUC4, MUC5AC, MUC5B, GALNT6, and GALNT5) were enriched in C4 IPF goblet cells.