Analysis of tolerance induction by the sialylated antigens NP–PA–NeuGc and NP–PA–bNeuGc, and responses in siglec-deficient mice. (A–D) Functional assay of in vivo tolerance induction. Results shown are representative of at least two independent experiments. (A) Mice (n = 4/group) were challenged with NP₆₅–PA–NeuGc on day 0 and rechallenged with NP₂₀₀–PA on day 17. IgM and IgG3 antibody levels were assessed at the indicated time points. Control mice received either NP₆₅–PA on day 0 and were rechallenged with NP₂₀₀–PA as in the experimental group, or received just NP₂₀₀–PA without pretreatment (green lines). #, a significant reduction in NP₆₅–PA–NeuGc–pretreated mice at 7 d after challenge with NP₂₀₀–PA (P = 8.84 × 10⁻⁵) compared to nonpretreated animals at 7 d after NP₂₀₀–PA challenge (green line). (B) Mice (n = 4/group) were challenged with NP–PA–bNeuGc on day 0 and rechallenged with NP–PA on days 14 and 31. Control mice received NP-Ficoll on day 0 and were rechallenged with NP–PA as in the experimental group. (C and D) Tolerance induction in siglec-deficient mice. Serum IgM and IgG3 anti-NP responses of mice that were primed on day 0 with either NP–PA–bNeuGc or NP–PA and boosted on days 17 or 18 with NP–PA. (C) Responses in CD22^−/− mice (n = 8 NP–PA; n = 9 NP–PA–bNeuGc). Secondary challenge was done on day 17. Because of the known weak TI-2 responses in CD22^−/− mice, all conjugate injections were performed with a double dose of antigen (40 µg/mouse). (D) Responses in Siglecg^−/− mice (n = 8 NP–PA; n = 9 NP–PA–bNeuGc). Secondary challenge was done on day 18. Shown are means + SD. *, P < 0.05; **, P < 0.01; and ***, P < 0.005 using the two-tailed Student’s t test.