Figure 3. Induction of antigen-specific pTregs, prolonged suppression of antigen-specific immunity, and infectious tolerance after s.c. injection of calcitriol/OVA_323–339 liposomes.

(A) DO11.10 T cells were transferred to BALB/c hosts. Mice were administered liposomes s.c. once on day 0 or twice on days 0 and 6. Splenocytes were analyzed on day 6 (1 injection) or 10 (2 injections). (B) After adoptive transfer of DO11.10 T cells, mice were administered liposomes or the same concentrations of nonencapsulated OVA_323–339 and calcitriol s.c. on days 0 and 6; splenocytes were analyzed on day 12. For intracellular (i.c.) IFN-γ analysis, splenocytes were restimulated overnight with OVA_323–339. (C) Thee days after s.c OVA/QuilA priming, liposomes were administered s.c. After 24 hours, sorted dLN MHCII⁺CD11c⁺ DCs, MHCII⁺CD19⁺ B cells, and CD45^– stromal cells were incubated with DO11.10 CD4⁺ T cells in the presence of IL-2. In vitro positive control DO11.10 CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 in presence of IL-2, TGF-β, anti-IL-4, and anti-IFN-γ. CD25⁺Foxp3⁺ Tregs were enumerated 5 days later. (D) After DO11.10 T cell transfer, mice were immunized with OVA/QuilA (day 0). Liposomes were administered s.c. on days 0 and 7. Splenocytes were analyzed on day 12 and after overnight OVA_323–339 stimulation for i.c. IFN-γ. (E) 5 × 10⁶ DO11.10 in vitro–generated memory T cells were adoptively transferred to BALB/c hosts, which were administered liposomes s.c. on days 0 and 7. Mice were immunized with OVA/QuilA on day 10 and splenocytes were analyzed on day 17. (F) After DO11.10 T cell transfer, mice were primed with OVA/QuilA (day 0). Liposomes were administered on days 0 and 7. Splenocytes were analyzed on day 12 or day 42 and after overnight OVA_323–339 stimulation for i.c. IFN-γ. Mice analyzed later were boosted with OVA/QuilA on day 35. (G) After adoptive transfer of 5 × 10⁶ DO11.10 T cells, BALB/c hosts were administered calcitriol/OVA_323–339 liposomes twice s.c. into the tail base, 1 week apart. A second cohort of 5 × 10⁶ CFSE-labeled DO11.10 T cells was adoptively transferred, and mice were immunized with OVA/QuilA on day 10. Splenocytes were analyzed on day 15. For i.c. IFN-γ and IL-10 analysis, splenocytes were restimulated overnight with OVA_323–339. n = 6–8 per group, representative of 2 replicates. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ANOVA with Tukey’s multiple comparison test (A–C and E) and t tests (D, F, and G).