Treg induction by Flt3L/antigen/rapamycin cocktail is pDC dependent. (A) Experimental timeline of DO11.10-tg Rag-2−/− BALB/c mice that received 2 weekly injections of PDCA-1 antibody (clone 927) to deplete pDCs. Mice were injected 3 times per week for 3.5 weeks with Flt3L/OVA323-339/rapamycin during the course of PDCA-1 antibody administration. Flt3L/OVA323-339/rapamycin was continued for 2 more weeks after PDCA-1 antibody treatment. (B) Treg induction was substantially lower in pDC-depleted mice after Flt3L/OVA323-339/rapamycin treatment. Data are average ± SD (n = 6 per group). Statistical differences were determined by the Student t test. (C) Experimental timeline of BALB/c mice that received 5 IV injections of PDCA-1 antibody (clone 120G8) over 3 weeks. Mice were infused with 1 × 107 CD4+CD25− effector T cells from DO11.10-tg x Rag2−/− mice 1 day after the first PDCA-1 antibody injection. Mice continued to receive Flt3L/OVA323-339/rapamycin combination during the course of PDCA-1 antibody administration. Control mice only received Flt3L/OVA323-339/rapamycin treatment. (D) Induction of OVA specific (KJ1-26+) Treg from transplanted donor (DO11.10) cells was significantly lower in pDC-depleted mice after Flt3L/OVA323-339/rapamycin treatment. Data are average ± SD (n = 8 per group). Statistical differences were determined by the Student t test. (E) Experimental timeline of BDCA-2-DTR mice that received IV injections of DT (3 times per week for 3 weeks) to deplete pDCs. Mice continued to receive Flt3L/OVA323-339/rapamycin combination during the course of pDC depletion. Control mice only received Flt3L/OVA323-339/rapamycin treatment. (F) Increased OVA specific (MR9-4+) Treg in Flt3L/OVA323-339/rapamycin treated control mice as compared with naïve animals, which is abrogated by pDC depletion. Data are average ± SD (n = 4 per group). Statistical differences were determined by 1-way ANOVA with Bonferonni’s multiple comparison posttest analysis.
