Figure 2. Intravenously infused Ag-SP are rapidly removed from the spleen and trigger IL-10 production.

(A–C) SJL/J mice were tolerized with 5×10⁷ PKH76-labeled OVA₃₂₃-SP. Groups of 3–5 mice were sacrificed at 0, 3 and 18 h post-infusion. At least 20, 8µM sections were examined from each animal. PKH76-labeled sub-cellular debris present at 3 h (B) post-infusion was completely absent by 18 h (C). (D–E) A separate cohort of at least 4 animals was treated with 5×10⁷ CFSE-labeled OVA₃₂₃-SP and mice were sacrificed 30 min and 3 h post-infusion. Numerous CFSE-labeled Ag-SP were observed at 30 min (D) but were completely absent by 3 h post infusion (E). IL-10 is secreted in response to Ag-SP infusion. Groups of at least 4 mice were infused with 5×10⁷ OVA₃₂₃-SP, recipient spleens harvested at 0, 10, 60 and 180 min post-infusion and IL-10 levels in supernatants of individual homogenized spleens (run in triplicate) measured using ELISA. *IL-10 levels significantly higher than baseline (p<0.01). (F). IL-10-deficient mice can not be tolerized with OVA_323–339 (G). Wild type (B6) and IL-10-deficient (IL-10gko) C57BL/6 mice were tolerized i.v. with 5×10⁷ syngeneic splenocytes from IL-10gko mice coupled with MOG_35–55 (irrelevant peptide control) or OVA_323–339 on day −5. On day 0, the mice were immunized with 200 µg OVA_323–339/CFA and DTH responses to OVA_323–331 ear challenge determined on day 7 (G). IL-10 neutralization prevents Ag-SP tolerance induction (H). Anti-IL-10 or control IgG2a antibody was given 30 min prior and 18 h after MOG₃₅-SP or OVA₃₂₃-SP infusion on day −5. Animals were immunized with OVA_323–339/CFA on day 0, and DTH assessed on day 7. Asterisks denote a significant reduction in DTH responses (*p<0.0005) as compared to MOG_35–55-SP controls. Data in all panels are representative of at least 3 experiments of at least 4 mice per group.