3.7 Tregs in human peripheral blood are transitioned from naive CD4+ T cells
To further investigate the underlying mechanism of Treg induction from blood samples of patients under UVB treatment by scRNA-seq (Figure 5B,C), slingshot analysis was undertaken to infer cell lineages and pseudotimes and revealed a trajectory originating from naive T cells, transitioning into Tmem or Teff cells, and ultimately differentiating into Tregs (Figure 6A). A similar trajectory was also noted in human skin CD4+ T cells, as annotated in Figure S7A,B, although the skin contained few naive T cells (Figure 6B). This trajectory was corroborated by the chronologically colored pseudotime gradient map from Monocle3, with naive T cells represented by a dark violet hue, Tmem and Teff cells by purple to purplish-orange hues, and Tregs by orange to yellow colors (Figure 6C). Marker gene expression patterns along the trajectory confirmed the annotated cell functions (Figure 6D). Treg transcription factors (FOXP3 , IKZF2 , andAHR ) and TCR activation markers (IL2RA , TIGIT , andICOS ) exhibited increased expression throughout the lineage. Notably, the steep upregulation of FOXP3 and IL2RA , encoding Foxp3 and CD25, respectively, confirmed the Treg transition. Tmem and Teff markers (CD44 and CD69 ) peaked at pseudotime Teff cells. Their subsequent decline indicated the transition to a Treg phenotype. Interestingly, the Treg negative regulator CD226 followed a similar trend as Tmem and Teff markers, suggesting its potential involvement in Treg differentiation. Conversely, SATB1 expression decreased gradually throughout the lineage, with the lowest expression in Tregs, indicating a reduction of its suppressive function. These findings collectively demonstrate that naive T cells differentiate into Tmem and Teff cells, which can further differentiate into Tregs.