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. 2009 Nov 10;4(11):e7766. doi: 10.1371/journal.pone.0007766

Figure 1. Activated CD4+ T cells secreted IL-2 to help primary CD8+ T-cell activation.

Figure 1

(A) Naïve CD8+ T cells (CD62LhiCD44lo) from B6 mice spleen with purity above 93% were obtained through B cell depletion, CD8+ T cells enrichment by panning and further enriched by MACS sorting with anti-CD62L MACS beads. (B) Expression of activation markers, CD69 and CD25. 3×106/mL of naïve CD8+ T cells (left) were stimulated by anti-CD3/CD28 antibodies for 0 hour and splenocytes (middle) or naïve CD8+ T cells (right) in 24-well culture plate were stimulated by anti-CD3/CD28 antibodies for indicated time, 24 and 48 hours and harvested, followed by staining with FITC anti-CD25, PE anti-CD69 and PE-Cy5 anti-CD8 antibodies, and analysis by flow cytometry. Dotted line: Isotype control. (C) Naïve CD8+ T cells (1×105/well) in a 96-well culture plate were stimulated in the presence or absence of mitomycin C-treated splenocytes (APC) for 96 hours, followed by MTT assay (numbers represent OD at 570 nm). The effect of CD4-depleted splenocytes was also checked (#). (D) Culture supernatants of anti-CD3/CD28 stimulated splenocytes for the indicated time, 0, 2, 4 and 24 hours (0 h-, 2 h-, 4 h- and 24 h-Sup) were collected. Naïve CD8+ T cells (3×105/well) in the 96-well culture plate were then incubated with the culture supernatant with (▪) or without (□) stimulation by anti-CD3/CD28 antibodies. Following 96 hours of stimulation, the cell viability was measured by MTT assay. (E and F) Naïve CD8+ T cells (3×105/well) in the 96-well culture plate were stimulated in the presence of culture supernatants of 0 h-, 2 h-, 4 h- and 24 h-stimulated CD8− splenocytes (E) or 0 h-, 2 h-, 4 h- and 24 h-stimulated CD4+ T cells (F) for 96 hours. Anti-mouse IL-2 antibody (2.5 µg/mL) was used to neutralize murine IL-2 in culture supernatant. The cell viability was measured by MTT assay. The data represent three independent experiments. #, The groups in which the cells received anti-IL-2 or isotype control antibody were not included. *, **, significantly statistical difference by student t-test, p<0.05. APC, antigen-presenting cells. CD8−, CD8+-depleted splenocytes. Spl, splenocytes. Sup, culture supernatant. Ab, antibody. Ctrl, isotype control of rat anti-murine IL-2 antibody, rat IgG2a.