Activation of CD4⁺ T cells by splenic DC.

<p>Antigen presenting ability of DC subsets purified from spleens of Act-mOVA mice was compared. L-DC, CD8⁺ and CD8^- cDC, and neutrophils as a control, were sorted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162358#pone.0162358.g001" target="_blank">Fig 1</a> following enrichment of splenocytes by depletion of red blood cells, and T and B lymphocytes using magnetic bead technology. Diluting numbers of APC were plated following treatment with and without LPS (10 μg/ml) for 2 hours. This was followed by addition of 10⁵ CFSE-labelled OT-II (TCR-tg) CD4⁺ T cells, purified from mouse spleen through depletion of B cells, CD8⁺ T cells, DC and myeloid cells using magnetic bead protocols. Cells were cultured at T cell:APC ratios of 33:1, 100:1, 300:1 and 1000:1 for 72 hours. CD4⁺ OT-II T cells were then gated as PI^-Thy1.2⁺Vα2⁺CD8^- cells, and assessed flow cytometrically for CFSE dilution as an indicator of T cell proliferation. OT-II T cells cultured alone served as controls (con). Graphs show % proliferating OT-II cells. Three independent replicate experiments were conducted.