Fig. 8: Electrical stimulation by charged substrate optimizes TCR signaling and potentiates T cell activation.

a Flow cytometric analysis of the frequencies of CD25⁻CD69⁻ cells, CD25⁻CD69⁺ cells, and CD25⁺CD69⁺ cells in OT-I cells cultured on the nanocomposite membranes with varying surface charges. OVA peptides (2 μg/mL) were used to stimulate T cell activation. Blank, cells cultured on a standard dish without nanocomposite membrane (n = 3 biological replicates, mean ± sem, *P = 0.04 (CD25⁻CD69⁻ cells), *P = 0.0266 (CD25⁺CD69⁺ cells), **P = 0.0038, ***P = 0.0007, ****P < 0.0001, one-way ANOVA). b Flow cytometric analysis of the expression of IFNγ in OT-I cells cultured on the nanocomposite membranes with varying surface charges. OVA peptides (2 μg/mL) were used to stimulate T cell activation (n = 3 biological replicates, mean ± sem, *, compared with NC; &, compared with LC; #: compared with MC, ***P = 0.0003, ****P < 0.0001, ^&P = 0.0160, ^{& & & &}P < 0.0001, ^#P = 0.0132, one-way ANOVA). c OT-I cells were isolated and cultured on the nanocomposite membranes with varying surface charges. Cells were treated with OVA peptides (2 μg/mL) for 6 hours and subjected to RNA-seq. GSEA of genes expressed in the HC group and NC group. ES, enrichment score; NES, normalized enrichment score. d OT-I cells were isolated and cultured on the nanocomposite membranes with varying surface charges and treated with OVA peptides (2 μg/mL) for 6 h. T cells were collected and subjected to Western Blot assay to assess the indicated protein expression. e–g Immunological synapse (IS) formation between PE·Cy7 labeled OT-I cells and GFP expressing LLC-OVA cells was detected by Imaging flow cytometry. OT-I cells and LLC-OVA cells were mixed (1:1) on the nanocomposite membranes with varying surface charges for 30 min. Cells were collected and fixed, permeabilized, and then stained with TRITC-labeled Phalloidin, followed by Imaging flow cytometry analysis. Statistical analysis was performed using the Ideas software (v6.0) (n = 4 biological replicates, mean ± sem, **P = 0.0048, two-tailed unpaired Student’s t test) (f) (n = 15 cells, mean ± sem, **P = 0.0013, two-tailed unpaired Student’s t test) (g). h–j The membrane binding of the cytoplasmic domain of the CD3ε chain was measured by dequenching FRET. The FRET donor was the mTFP1 fused to the C terminus of the CD3ε and the FRET acceptor was R18 dye inserted into the plasma membrane. The short linker constructs, KIR2DL3-3 amino acid (3 AA)-mTFP1 and KIR2DL3-50 amino acid (50 AA)-mTFP1 served as the positive control and negative control, respectively. Jurkat cells with different constructs were cultured on the nanocomposite membranes with varying surface charges and treated with or without anti-CD3/anti-CD28 mAb-coated beads for 30 minutes. BP, before photobleaching; AP, after photobleaching. The scale bars represent 10 μm. k The FRET efficiency was calculated as [DonorAfter - DonorBefore]/[DonorAfter] (n = 15 cells, mean ± sem, ****P < 0.0001, one-way ANOVA). Data are representative of two (a, b, d, f) independent experiments. Source data are provided as a Source Data file.