CD4⁺ and CD4⁻ cDC are equivalent in their ability to activate CD4⁺ T lymphocytes.

<p>(A) Splenic CD4⁺ and CD4⁻ cDC were sorted on the basis of CD11c, CD11b, CD8 and CD4 expression. The presence of contaminating plasmacytoid DC and CD8α⁺ cDC precursors in the CD4⁻ cDC fraction was evaluated by using anti-Siglec-H and Sirp-α mAbs, respectively. (B, C) Both cDC subsets were sensitized with graded doses of OVA peptide (B) or whole OVA (C) and co-cultured for 3 and 5 days with naive CD4⁺ T cells purified from OT-II mice. The production of IFN-γ and IL-13 was quantified by ELISA. Results represent the mean ± SD of a representative experiment out of two.</p

CD4⁺ and CD4⁻ cDC are equivalent in their ability to activate CD8⁺ T lymphocytes.

<p>(A, B) CD4⁺ and CD4⁻ cDC subsets were sensitized with graded doses of OVA peptide (A) or whole OVA (B) and co-cultured for 3 and 5 days with naive CD8⁺ T cells purified from OT-I mice. The production of IFN-γ was quantified by ELISA. Results represent the mean ± SD of a representative experiment out of two.</p

CD4⁺ and CD4⁻ cDC differ <i>in vitro</i> in their capacity to activate iNKT cells.

<p>(A, C) Sorted CD4⁺ and CD4⁻ cDC were exposed to graded doses of α-GalCer and then co-cultured for 48 h with sorted iNKT cells (A) or with the iNKT cell hybridoma DN32.D3 (C). Cytokine production was quantified by ELISA. Results represent the mean ± SD of 3 (A) or 2 (C) independent experiments. (B) CD1d expression on CD4⁺ and CD4⁻ cDC was assessed by flow cytometry. Of note, the staining with the isotype control was identical on both cDC subsets. For clarity, the isotype control on the CD4⁺, but not CD4⁻, cDC subset is shown. Shown is a representative experiment out of three. (D) Sorted CD4⁺ and CD4⁻ cDC were exposed, or not (medium), to α-GalCer (100 ng/ml) and then co-cultured for 6 h with sorted iNKT cells. RNAs were prepared and IL-12p35 (<i>Il12p35</i>) mRNA copy numbers were measured by quantitative RT-PCR. Data are normalized to expression of <i>Gapdh</i> and are expressed as fold increase over average gene expression in vehicle-treated cDC. Of note, the basal level of IL-12p40 transcript in <i>ex vivo</i> sorted cDC is relatively elevated (Ct: 25–26) (Ct of <i>gapdh</i>: 20, Ct of <i>il12p35</i>: 31–32). Data represent the mean ± SD (triplicates) of an experiment out of two performed. (E) α-GalCer-loaded cDC subsets were co-cultured for 48 h with sorted iNKT cells in the presence of a neutralizing IL-12 Ab or an isotype control Ab. Shown is a representative experiment (mean ± SD) out of three performed. * p<0.05; ** p<0.01; *** p<0.001.</p