Figure 2.

PLD3 and PLD4 act upstream of TLR7

(A) mRNA expression levels of PLD3, PLD4, RNase T2, TLR7, and TLR8 in indicated cell types are plotted.

(B) Single-cell RNA-seq data of human PBMCs from Hao et al.¹⁹ were visualized using the UCSC Cell Browser²⁰ for the transcripts PLD3 and PLD4. Color coding represents the range of gene expression. The insert in the upper left corner specifically highlights the pDC population. Annotations for relevant cell populations were retrieved as annotated.

(C) Quantification of PLD3 and PLD4 protein expression in CAL-1 cells, primary plasmacytoid dendritic cells, and primary monocytes. Data are presented as box and whiskers of n = 4–5 independent experiments, statistics indicate a paired two-tailed Student’s t test.

(D) Unmodified CAL-1 cells (WT) or two independent PLD3^−/− × PLD4^−/− CAL-1 clones were unstimulated or stimulated with pR, RNA40^S, RNA40^O, RNA9.2s^O, CpG^O, or CpG^S. After 16 h, IFN-β release was measured by ELISA. Data are depicted as mean ± SEM of n = 3 independent experiments. Statistical analysis was conducted by two-way ANOVA with Dunnett’s multiple comparison tests.

(E) Indicated knockouts of primary human monocytes were unstimulated or stimulated with ssRNA40^O in the presence of CU-CPT9a, R848, and LPS. After 16 h, IL-6 release was measured by ELISA. Each replicate of 3 independent donors is depicted. For statistical analysis, the data of each donor was normalized to WT^NTC and two-way ANOVA was conducted on log-transformed data with Dunnett’s multiple comparison test.