Fig. 3: IL-16 promotes the differentiation of Th1 cells.

a, b Naive CD4⁺ T cells were magnetically sorted from mouse spleen and cultured under Th1 condition in the absence or presence of 50 ng/ml IL-16. Cells were harvested on day 5 and then subjected to RNA sequencing. The number of differentially expressed genes (DEGs, defined as p < 0.05, fold change > 1.5) was shown by volcano plot (a), GO enrichment analysis was performed using the IL-16-upregulated Th1 marker genes (b). c–e Naive CD4⁺ cells were cultured under Th1 condition in the absence or presence of 50 ng/ml IL-16 for 5 days, the mRNA levels of Ifng (IFN-γ-encoding gene) and Tbx21 (T-bet-encoding gene) in Th1 cells were evaluated by QPCR (c, n = 3/group), the percentages of IFN-γ⁺CD4⁺ cells (Th1 cells) were analyzed by flow cytometry (d, n = 4/group), the levels of IFN-γ in T-cell culture supernatant were analyzed by ELISA (e, n = 5/group). f Naive CD4⁺ T cells were sorted from human PBMCs and subsequently cultured under Th1 conditions in the absence or presence of 50 ng/ml IL-16. The levels of IFN-γ in T-cell culture supernatant were analyzed by ELISA (n = 5/group). g Th1 cells were differentiated in the presence of 10 μg/ml αIL-16 or control IgG, and the levels of IFN-γ in T-cell culture supernatant were analyzed by ELISA (n = 5/group). h Th1 cells were treated with PBS or 100 ng/ml IL-16 for 5 days, cell proliferation was evaluated by CFSE dilution assay (n = 4/group). i Naive CD8⁺ T cells were magnetically sorted from the mouse spleen and stimulated with 50 ng/ml IL-16 for 4 days, the expression of perforin and GZMB was evaluated by QPCR (n = 3/group). Unpaired, two-tailed Student’s t-test (c–g) was used. Data are presented as mean ± SD.