Figure 7. Splenic macrophages uptake Ag-SP and express PD-L1 in an IL-10-dependent manner.

Effect of Ag-SP infusion on splenic macrophage ratio. Five groups of SJL/J mice (4–5 mice per group) received IgG2a control antibody, anti-IL-10 alone, OVA_323–339-SP + IgG2a antibody, OVA_323–339-SP + anti-IL-10 antibody, or no treatment. All antibodies were given 30 min. prior to OVA₃₂₃-SP infusion. 3h after infusion, animals were sacrificed and splenocytes stained with a cocktail of antibodies as described in the Materials and Methods. (A) Splenic APC populations were enumerated using the gating strategy shown; black population indicates the ungated isotype control for each dot plot. (B) Percentages of CD4⁺ DCs, CD8α⁺ DCs, and plasmacytoid DCs (pDC) did not change in any of the treatment groups, but percentages of macrophages increased in an IL-10-dependent fashion. F4/80⁺ splenic macrophages uptake Ag-SP and express PDL-1. Spleens from CD45.1⁺ C57BL/6 mice receiving either PBS (C) or 5×10⁷ CD45.2⁺ PKH-26-labeled, OVA₃₂₃-SP (D) were harvested 2.5 hours after i.v. administration. Gate R1 represents recipient cells that have taken up donor Ag-SP, while Gate R2 represents intact Ag-Sp. Numbers adjacent to gate represent the percentage of cells within the gate (D). Relative CD45.2 expression on gates R1 (grey line) and R2 (black line) (E). Cells from gate R1 are 85% CD11b⁺ and 11.6% CD11c^high (F). Cells from gate R3 are 77.5% F4/80^int and 11.3% F4/80^high. The majority of the cells in gate R3 were CD11c^int which is consistent with the phenotype of splenic MZ macrophages (G). >73% of cells from R3 (i.e. those that are of recipient origin, the majority being F4/80⁺ macrophages) that have engulfed Ag-SP express PD-L1 (H). PD-L1 expression increases in the CD8α⁺ DC and F4/80⁺ macrophage populations and expression is reversed by anti-IL-10 in macrophages (I). Data is representative of 2 separate experiments. Asterisks denotes a significant change in APC subset ratio/expression compared to animals treated with IgG2a control antibody (*p<0.05). PD-L1 Expression is Required for Ag-SP Tolerance. PD-L1 blockade prevents Ag-SP tolerance induction. SJL/J mice were treated with anti-PD-L1 or control IgG2a antibody as detailed in the Materials and Methods. Mice were tolerized with OVA₃₂₃-SP or PLP₁₃₉-SP on day −7. Animals were immunized with PLP_139–151/CFA on day 0, and DTH assessed on day 7. Asterisks denote a significant reduction in DTH responses (*p<0.01) as compared to MOG_35–55-SP controls. Results are representative of two separate experiments of at least five mice per group.