Sialylated Ab-ICs promote enhanced humoral immune responses.

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Sialylated Ab-ICs promote enhanced humoral immune responses. Sialylated Ab-ICs promote enhanced humoral immune responses. (A) The dot plots show gp120-specific Ab titers (left) or avid Ab titers (right) 10 days after the second immunization in BALB/c mice receiving alum alone (green), alum-adjuvanted gp120 (blue), agalactosylated PGT121 (G0-ICs) with alum (aqua), nonsialylated galactosylated PGT121 (G1/G2 NS-ICs) with alum (lavender), or sialylated galactosylated PGT121 (G1/G2 S-ICs) with alum (pink) (n = 3 to 8 mice, two experiments). (B) The dot plots highlight the number of fluorescently labeled ICs on noncognate B cells (gp120+ B cells), macrophages (gp120+ SSM), or FDCs (gp120+ FDCs) from draining lymph node at 1 and 3 hours after injection with nonsialylated IC (NS-IC) or sialylated IC (S-IC) (n = 4 to 8 mice, two experiments). (C) The confocal microscopy images depict the level of IC deposition in a B cell follicle 1 hour after a mouse is immunized with NS-ICs (left) or S-ICs (right) and stained for g120 (blue), macrophages (green), and GL-7 (red). Representative image of three images from two to three mice per condition. (D) The bar graphs show the number (left) and area occupied (right) of gp120+bead+ FDCs in a draining lymph node. (E) The confocal microscopy images depict IC deposition in a B cell follicle 3 hours after immunization and stained with the same targets as (C). Representative image of three images from two to three mice per condition. (F and G) The line graphs demonstrate the percentage of bound Raji or naïve human B cells after incubation with APC bead–labeled NS-ICs (yellow) or S-ICs (pink), with (colored line) or without complement (gray line), with (blue) or without complement (light blue) alone, or α-FcγRIIB blocking Abs (hatched lines) (F, n = 2; G, n =3). (H) The dot plots show the gp120-specific IgG Ab titers (left) and high-avidity gp120-specific Ab titers (right) after immunizing WT or C1q−/− C57BL/6 mice (n = 2 to 8, two experiments) with alum (white), gp120-alum (blue), NS-ICs with alum (yellow), or S-ICs with alum (pink). (I) The dot plot highlights the ratio of high-avidity gp120-specific Abs to total gp120-specific Abs in WT (dark pink) versus C1q−/− (light pink) mice after immunization with S-ICs. All ELISAs were performed in triplicate, and a one-way ANOVA followed by Tukey’s post test was used for (A), (B), and (D). Mann-Whitney and one-way ANOVA with Holm-Sidak’s multiple comparisons test were used for (H). Two-way ANOVA with Dunnett’s correction comparing samples with the negative control (no Ab, no complement) was used for (G). Mann-Whitney test was used in (I). *P < 0.05, **P < 0.01, and ***P < 0.001. The horizontal bars in all panels indicate mean. Error bars represent SEM in (B), (D), (H), and (I) and SD in (A). Giuseppe Lofano et al. Sci. Immunol. 2018;3:eaat7796 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).