Modulation of ILT3 expression by different maturation-inducing stimuli in M-DC and P-DC subsets: selective up-regulation by 1,25(OH)₂D₃ in M-DCs. (A) Maturation stimuli modulate ILT3 expression in myeloid and plasmacytoid DC subsets. DC subsets were incubated for 72 hours with 1 μg/mL LPS, 5 μg/mL CpG-A or CpG-B sequence, or CD154-transfected J558L cells at a ratio of 1:4. Surface expression of ILT3 and CD83 was determined by cytofluorimetry. The left panels show expression of ILT3 (dark gray) by ex-vivo purified M-DCs and P-DCs. Light gray histograms represent staining with isotype controls. (B) Blocking ILT3 on P-DCs enhances Th1-cell development. DC subsets were cultured for 5 days in the absence of any exogenous stimulus before analysis of ILT3 and CD83 expression by cytofluorimetry. P-DCs were subsequently cocultured with naive CD4⁺ cells for an additional 7 days in the presence of anti-ILT3 antibody or isotype control. The T-helper–cell phenotype was evaluated by cytofluorimetry following intracellular staining for IFN-γ and IL-4, and the percentages of positive cells are shown. A representative experiment out of 3 performed is shown. (C) Selective up-regulation of ILT3 in M-DCs treated with 1,25(OH)₂D₃. DC subsets were cultured for 48 hours without stimulation or with 1 μg/mL LPS or 5 μg/mL CpG-B sequence, in medium alone or medium containing 10 nM 1,25(OH)₂D₃. Surface expression of ILT3 was determined by cytofluorimetry. A representative experiment out of 3 performed is shown.