Figure 3. DCs require RAR signaling to induce RA producing enzymes when cultured with Pdpn⁺ CD31⁻ SI LP SCs.

a Aldefluor staining of splenic DCs (gated CD45⁺MHCII^hiCD11c⁺) after 24h culture alone (Untreated) or together with SI SCs (SCs cond.). DEAB (100 µM) was added at the time of the staining together with aldefluor where indicated. Data show one representative experiment of more than 5 experiments with identical results.

b FACs analysis of IL-17A (left panel) or α4β7(right panel) in PMA/Ionomycin re-stimulated OTII cells primed with splenic DCs for 4–5 days. When indicated, DEAB (10 µM) was added to the DC-T cell culture and maintained during the whole length of the experiment. Analysis shows the result from one representative experiment of at least two experiments with similar results.

c mRNA levels of the RA producing enzymes RALDH1 (aldh1a1) RALDH2 (aldh1a2) and RALDH3 (aldh1a3) were measured by qRT-PCR in splenic DCs after 24hr culture in media alone or media supplemented with SC supernatants (SCs sups, obtained as in Fig1a). Data shown correspond to RNA from three independent experiments that were quantified at the same time.

d Aldefluor staining of splenic DCs (gated CD45⁺MHCII^hiCD11c⁺) after 24h culture alone or together with sorted SCs (SCs cond, left panel) or their supernatants (SCs sups, right panel). The global RAR global antagonist BMS204493 (RAR inh) was added to the culture at concentration of 20nM or otherwise indicated. Data shown are from one representative experiment of at least three experiments.