Fig. 3

Activation of CD4⁺ T cells by splenic dendritic and myeloid subsets. Antigen presenting ability of myeloid subsets purified from spleens of Act-mOVA mice was assessed. L-DC, eosinophils (Eos), inflammatory monocytes (Infl mono), neutrophils (Neu), resident monocytes (Resi mono) and CD8⁻ cDC (as a control), were sorted as described in Table 1 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 LPS (10 μg/ml) (solid line) and without LPS (dotted line) for 2 h. 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 900:1 for 72 h. 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. Two independent replicate experiments were conducted