FIGURE 2.
Composition 8203 potentiates DLN for Treg response. (A) MPLA preferentially attracts MMΦs to DLN. Shown is flow cytometry of DLN CD11c+CD40+ cells one day after a single injection of 10 μg of MPLA, or a PBS control, into a footpad. (B) Rapamycin (“Rapa”) blocks MPLA-induced influx of Teff cells to DLN. Shown are dose-response curves generated by nonlinear regression. Left: absolute cell counts for MMΦs, DCs, Treg cells, and Teff cells one day after a single injection into a footpad of a graded dose of MPLA. Right: the same set of cells after injection of 3 μg (based on the left panel) of MPLA mixed with a graded dose of Rapa. Each dose point is mean ± SD of four experiments. *p ≤ 0.03 and **p ≤ 0.01, for Teff and MMΦ counts, respectively, at MPLA doses of ≥1 μg versus 0; ***p ≤ 0.03, Teff count at Rapa doses of ≥6 μg versus 0 (one-way ANOVA). (C) Composition 8203 preferentially attracts MMΦs and Treg cells to DLN. DLN cells were counted one day after a single injection into a footpad of PBS, Dex (8 μg), or 8203. Data are mean ± SD of four experiments. *p ≤ 0.001 and **p ≤ 0.02, for absolute and relative MMΦ counts, respectively, of 8203 injected versus any other; ***p ≤ 0.01, relative Treg count of 8203 injected versus PBS injected (one-way ANOVA). (D) Composition 8203 shows similar effects in C57BL/6 Foxp3-eGFP mice. DLN cells were counted one day after a single injection into a footpad of PBS or 8203. Data are mean ± SD of four experiments. *p = 0.005, **p = 0.00009, and ***p = 0.04 (two-sided t test). (E) Role of each component of 8203. DLN cells were counted one day after a single injection into a footpad of 8203 or a composition missing one of the three components. Data are mean ± SD of four experiments. *p ≤ 0.02 and ***p ≤ 0.03, for absolute and relative MMΦ counts, respectively, in group 2 versus any other group; **p ≤ 0.0002 and ****p ≤ 0.0009, absolute Teff count and relative Treg count, respectively, in group 3 versus any other group (one-way ANOVA). (F) Dexamethasone is required for converting MMΦs (IL-10lo) into Dex-MΦs (IL-10hi) (9). MMΦs were isolated from the DLN by flow sorting one day after injection at a footpad with either 8203 or “203” (20 μg rapamycin/3 μg MPLA, as a negative control for dexamethasone), and cultured at 250,000 cells/ml in U-bottom wells in the presence of 0.1 μg/ml of LPS. At 20 h, the medium was analyzed for IL-10 by a CBA assay. Data are mean ± SD of four experiments. *p ≤ 0.00002 (two-sided t test). (G) Effect of 8203 on MMΦ influx lasts for three days after a single injection. Shown is a nonlinearly fitted decay curve for the relative count of MMΦs (% of total CD11c+CD40+ cells) in the DLN. Each point is mean ± SD of four experiments. *p ≤ 0.02, for days 1 and 2 versus any other day (one-way ANOVA). (H) Composition 8203 allows normal uptake and processing of whole protein Ag. Mice were injected at a footpad with 5 μg of DQ OVA in either PBS or 8203; after 2 days, MMΦs and DCs in the DLN were analyzed for mean fluorescence intensity (MFI) resulted from intracellular cleavage of DQ OVA. ΔMFI = MFI from DQ OVA injected − MFI from PBS (control) injected. Data are mean ± SD of four experiments. *p ≤ 0.004 (two-sided t test).