Fig. 4.

Antigen specificity of tolerance induction by tNP. (A) Antigen specificity of tNP therapy. Animals from Fig. 3B were immunized s.c. with 50 µg of KLH on both lateral flanks at the base of the tail (b.t.) on days 239, 253, and 267. Anti-KLH IgG and anti-OVA IgG titers from each animal on day 280 are shown. (B) Treatment with tNP does not result in broad immunosuppression. C57BL/6 mice were immunized with OVA+CpG in the right (front and hind) limbs and with KLH+CpG in the left limbs, whereas treated animals received OVA+CpG+tNP injections in the right limbs and KLH+CpG in the left limbs every 2 wk for 8 wk. Anti-OVA and anti-KLH titers were determined by ELISA (n = 5). (C) Prophylactic induction of antigen-specific tolerance. C57BL/6 mice were treated with a single injection i.v. of tNPs (rapamycin+OVA), NP[Rapa], or vehicle (no treatment) on day –14. Mice were immunized with pOVA i.v. and KLH s.c. on days 0, 14, and 28. Anti-OVA and anti-KLH titers were assessed on day 41. The results summarize three independent experiments (n = 5). (D) Durability of prophylactic induction of antigen-specific tolerance. Two groups of C57BL/6 animals (no treatment and tNP-treated) were immunized biweekly with eight injections with pOVA i.v. from day 0 to day 98. The treated group received tNP (rapamycin+OVA) for the first three immunizations only (days 0, 14, and 28). The start of immunization for a third group of animals was delayed until day 42 (delayed immunization control). In all figures and panels, the bars and the symbols represent the geometric mean ± 95% confidence interval (n = 5). All P values were calculated using a Bonferroni posttest of a regular one-way or two-way ANOVA test (***P ≤ 0.001). (E) In vivo trafficking of tNP. (Left) (i.v. injection) Balb/C animals were immunized with pOVA on days –27 and –13. On day 0, fluorescently tagged Cy7–tNP containing OVA and rapamycin were injected i.v., and the animals were imaged by 3D FMT at 6 h after injection. (Right) (s.c. injection) Naïve Balb/C mice were injected with fluorescently tagged Cy7–tNP s.c. and then imaged 1 h after injection (n = 3). I, iliac; P = popLNs; R, renal. (F) Cellular localization of tNP. Fluorescently tagged NP–Cy5 (red) NPs were injected i.v., and spleens were harvested 24 h later for cryosectioning and immunohistochemical analysis. Shown are representative low and high (Lower Right panel) magnification (10× and 100×, respectively) photomicrographs of the distribution of the NPs among the red pulp (RP), the white pulp (WP), and the marginal zone (MZ) delineated by the localization of macrophages (MPs, F4/80, green), DCs (CD11c, dark blue), and B cells (B220, cyan).