Fig 2: Intermediate level of Piezo1 is optimal for CAR T cell mechano-metabolic and effector functions.(a) UMAP projection of scRNA-seq data showing clustering of CAR T cells based on Piezo1 expression levels, categorized into Low, Intermediate, and High groups (Piezo1low, Piezo1int, Piezo1high) by k-means clustering (k = 3). (b) Bar plot showing the proportion of Piezo1low, Piezo1int, Piezo1high expression groups within activated and non-activated CAR T cell populations. (c) Correlation of Piezo1 with downstream calcium–NFAT signaling genes. Pearson correlation matrix of Piezo1 expression with NFAT family transcription factors (NFATC1–4) and CaMKII isoforms (CAMK2D, CAMK2G). (d) Heatmap showing z-score normalized expression of selected genes related to mechanosignaling (left) and metabolic pathways (right) across Piezo1 expression groups. Piezo1int group is associated with increased expression of effector-associated genes, cytoskeletal regulators, mTOR signaling components, glycolytic genes (e.g., HK2, ALDOA), and OXPHOS genes (e.g., COX4I1, NDUFS1). (e) Violin plots showing single-cell expression of selected effector and signaling genes across Piezo1 expression groups. Piezo1high cells show elevated expression of IL2RA, CD69, IFNG, TNF, and GZMB. (f) Radar plot depicting the distribution of T cell functional states (resting, early activation, effector, exhausted) across Piezo1 expression groups. (g) Representative staining images of Piezo1, CD69 and Granzyme B in single CAR T cells, and (h) Scatter plot of Granzyme B expression versus Piezo1 levels (top), and CD69 expression versus Piezo1 levels (bottom) measured by a microfluidic digital ELISA single-cell phenotyping platform. Cells are divided into 10 domains according to their Piezo1 level using K-mean clustering. Domains 1–2 are denoted as Piezo1low, domains 3–5 are denoted as Piezo1int, and domain 5–10 are denoted as Piezo1high (n = 707, 2326, 1663 cells for Low, Intermediate, and High Piezo1 clusters respectively). (i) Percentage of positive cells expressing Granzyme B and CD69 in the clustered cells. A threshold was determined according to the data from negative control. Cells with signals larger than the threshold are considered positive (n = 3). Data are shown as the mean ± s.e.m. Statistical tests used one-way ANOVA with Tukey’s post hoc test.