Figure 4.

PIMs regulate Th1/Th17 signaling axis during early Th cell differentiation

(A) Based on bulk RNA-seq data, STAT1 was predicted as a positive upstream regulator of the differentially expressed genes at 6 h upon PIM TKD using the IPA “upstream regulator” prediction tool (Z score < −2, which indicates predicted upstream regulators; gray arrows represent effects not predicted.

(B) The downregulation of STAT1 RNA levels was confirmed at 24 h and 72 h in PIM TKD Th17 cells by qRT-PCR.

(C and D) The RNA expression of Th1-related factors TBX21 (C) and STAT4 (D) was validated in PIM TKD at 6 h and 24 h of Th17 cell differentiation by qRT-PCR. FC normalized to the Scr control was plotted for four biological replicates. Boxplots represent median and interquartile range, and whiskers extend to maximum and minimum values (B–D). Statistical significance is calculated from four biological replicates using two-tailed Student’s t tests (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001).

(E and F) scRNA-seq was performed at 6 h in PIM TKD and control Th17 cells using the single-cell fixed RNA profiling. Cells double-negative for PIM^–/TBX21 but RORA positive (E) and single cells double-positive for PIM/TBX21 but RORA-negative (F), were colored in blue and projected into a two-dimensional map using UMAP.

(G) Differential expression analysis was performed between Scr (control) and PIM^– in PIM TKD samples at 6 h of differentiation (FDR of <0.05, log2FC of >0.24) for one biological replicate. Scaled scRNA-seq dot plot depicting the differentially expressed genes of interest on the x-axis. The color scale represents the average expression of a given gene in the cluster, and the size of the dot represents the percent of cells that express a given gene.

(H) Violin plots showing the expression of CD82 and STAT1 in Scr and enriched PIM^– cells of PIM TKD samples at 6 h. See also Figures S4 and S5 and Table S2.