Fig 1: MiR-15a and miR-15b promote CD8+T cell activation,proliferation and induce CD8+T cell-mediated NB cytotoxicity(A–D) Representative flow cytometric plots of human peripheral CD8+ T cells showing the expression of intracellular Granzyme B (A), intracellular Perforin (B), surface CD3 and CD8 (C), and intracellular Ki-67 (D) after coculture (E:T=1:1) with miR-15a and miR-15b expressing SK-N-AS (A–D, top panels), and NB-19 (A–D, bottom panels) cells for 24 h. (E) Representative flow cytometric plots of miR-15a and miR-15b expressing SK-N-AS (E, top panel), and NB-19 (E, bottom panel) showing the expression of intracellular active caspase-3 upon coculture (E:T=1:1) with activated human CD8+ T cells for 24 h. (F) A quantification graph showing normalized luciferase activity in SK-N-BE(2) cells expressing miR-15a and miR-15b upon coculture (E:T=5:1) with activated CD8+ T cells for 24 h. Untouched CD8+ T cells were isolated from PBMCs of healthy human blood donors by negative selection using the MojoSort human CD8+ T cell Isolation Kit (BioLegend). For 7 days, CD8+ T cells were activated and expanded using human T-activator CD3/CD28 Dynabeads and used for coculture experiments with NB cells. NB cells were transfected with miRs for 24 h and used for coculture experiments with activated CD8+T cells for an additional 24 h. Non-targeting control (ctrl) miR cells served as a control group. Cells were permeabilized (intracellular), fixed, and stained for respective antibodies described in the materials and methods section and analyzed by flow cytometry. The percentage of Granzyme B+ (A), Perforin+ (B), CD3/CD8+ (C), ki67+ (D), and cleaved caspase-3+ cells (E) are shown in each of their respective plots. Data represent the mean ± standard error of three to five independent biological experiments. Statistical analyses were performed using a two-sided unpaired t-test.