The adjuvant LT-K63 can restore delayed maturation of follicular dendritic cells and poor persistence of both protein- and polysaccharide-specific antibody-secreting cells in neonatal mice

Abstract

Ab responses in early life are low and short-lived; therefore, induction of protective immunity requires repeated vaccinations. One of the major limitations in early-life immunity is delayed maturation of follicular dendritic cells (FDCs), which play a central role in mediating the germinal center (GC) reaction leading to production of Ab-secreting cells (AbSCs). We assessed whether a nontoxic mutant of Escherichia coli heat-labile enterotoxin (LT-K63) and CpG1826 as model adjuvants could accelerate FDC maturation and immune response in neonatal mice, using a pneumococcal polysaccharide of serotype 1 conjugated to tetanus toxoid (Pnc1-TT) as a model vaccine. In neonatal NMRI mice, a single dose of Pnc1-TT coadministered with LT-K63 enhanced Pnc1-TT-induced GC reaction. In contrast, CpG1826 had no effect. Accordingly, LT-K63, but not CpG1826, accelerated the maturation of FDC networks, detected by FDC-M2(+) staining, characteristic for adult-like FDCs. This coincided with migration of MOMA-1(+) macrophages into the GCs that can enhance GC reaction and B cell activation. The FDC-M2(+) FDC networks colocalized with enhanced expression of TNF-α, which is critical for the maintenance of mature FDCs and is poorly expressed in neonates. The accelerated maturation of FDC networks correlated with increased frequency and prolonged persistence of polysaccharide- and protein-specific IgG(+) AbSCs in spleen and bone marrow. Our data show for the first time, to our knowledge, that an adjuvant (LT-K63) can overcome delayed maturation of FDCs in neonates, enhance the GC reaction, and prolong the persistence of vaccine-specific AbSCs in the BM. These properties are attractive for parenteral vaccination in early life.

FIGURE 1.

LT-K63 overcomes delayed vaccine-induced GC reaction because of accelerated maturation of FDC network clusters in neonatal mice. (A) PNA- (left), FDC-M2– (middle), and MOMA-1 (right)–stained spleen sections from neonatal mice immunized with 0.5 μg Pnc1-TT (top panel) or 0.5 μg Pnc1-TT with 5 μg LT-K63 (lower panel). Spleens were removed 14 or 10 d after priming of neonatal and adult mice, respectively, when AbSCs and GCs in spleen have been shown to peak (14). Half of the spleen was snap frozen, serial 7-μm cryosection sections were cut at four levels, starting 700 μm into the tissue, and each level was separated by 210 μm. One representative section from each group is shown. Original magnification ×40. Scale bar, 50 μm. (B–E) Consecutive section from all four levels were stained with (B) anti-IgM, (C) anti-IgG, (D) PNA, and results (mean ± SD) are shown for each group of neonatal (filled bars) and adult (open bars) mice. (E) Mean GC/follicle ratio was calculated for each spleen at all four levels to adjust for age difference in spleen size, and results (mean ± SD) are shown for each group. Statistical difference between test groups and controls (LT-K63 and saline-immunized mice) are shown; *p < 0.05, **p ≤ 0.001. Results in (A)–(E) are from three experiments for neonatal mice (n = 24 per group, except for LT-K63–injected mice n = 16) and two experiments for adult mice (n = 16 per group, except for LT-K63–injected mice n = 8), with eight mice per group in each experiment.

FIGURE 2.

LT-K63 enhanced the colocalization of FDC-M1⁺ FDC networks and migrated MOMA-1⁺ MMMs in activated GCs in mice immunized as neonates. To detect FDCs or MMMs, we stained spleen sections with FDC-M1 (red) or MOMA-1 (green), respectively, or DAPI to visualize the nuclei (blue), 14 or 10 d after priming of neonatal (A, B) and adult (C, D) mice with 0.5 μg Pnc1-TT (A, C) or 0.5 μg Pnc1-TT with 5 μg LT-K63 (B, D). Seven-micrometer sections were cut from four levels in the spleen; the first started 700 μm into the tissue, and levels were separated by 210 μm. One representative section from each group is shown. Original magnification ×40. Scale bar, 50 μm. Results shown in (A)–(D) are from two experiments for neonatal and adult mice (n = 16 per group), with eight mice per group in each experiment.

FIGURE 3.

LT-K63 enhanced TNF-α expression that colocalized with FDC-M2 staining in mice immunized with Pnc1-TT as neonates. Spleen sections were stained with FDC-M2 (red), Ab to TNF-α (green), or DAPI to visualize the nuclei (blue) 14 or 10 d after priming of neonatal (A, B) and adult (C, D) mice with 0.5 μg Pnc1-TT (A, C) or 0.5 μg Pnc1-TT+5 μg LT-K63 (B, D), when induction of GCs in spleen peaks in neonatal versus adult mice (14). Seven-micrometer sections were prepared from four different levels, the first started 700 μm into the spleen and the levels were separated by 210 μm. One representative section from each group is shown. Original magnification ×40. Scale bar, 50 μm. Results shown in (A)–(D) are from two experiments for neonatal and adult mice (n = 16 per group), with eight mice per group in each experiment.

FIGURE 4.

LT-K63 enhanced the TNF-α levels in cell culture supernatants after in vitro TT stimulation of spleen cells from mice immunized with Pnc1-TT as neonates. Spleen cells isolated 3 wk after priming of neonatal mice with Pnc1-TT (open column) or Pnc1-TT+LT-K63 (filled column) were stimulated with or without TT (10 μg/ml) for 48 h (20). TNF-α in the supernatants was measured by ELISA. The results are shown as pg TNF-α/ml (mean ± SD) in TT-stimulated cultures after subtracting TNF-α levels in unstimulated cultures. Student t test was applied, and p < 0.05 was considered statistically significant. Results shown are from two experiments for neonatal mice (n = 14 per group), with seven mice per group in each experiment.

FIGURE 5.

LT-K63 increased the frequency of Pnc1-TT–specific AbSCs. PPS-1– and TT-specific IgG⁺ AbSCs measured by ELISPOT, shown as number of spots (mean ± SD) per 10⁶ cells (A, B), and PPS-1– and TT-specific IgG levels (mean EU/ml ± SD) in serum measured by ELISA (C, D). Results are shown for neonatal (filled bars) and adult (open bars) mice immunized with 0.5 μg Pnc1-TT with or without 5 μg LT-K63, LT-K63, or saline. Spleens were removed 14 or 10 d after priming, when GCs and AbSCs peak in neonatal and adult mice, respectively (14), and single-cell suspension was prepared from half of the spleen. Statistical difference between test groups and controls (LT-K63– and saline-immunized mice) are shown; *p < 0.05, **p ≤ 0.001. Results in (A)–(D) are from three experiments for neonatal mice (n = 24 per group, except for LT-K63 injected mice n = 16) and two experiments for adult mice (n = 16 per group, except for LT-K63–injected mice n = 8), with eight mice per group in each experiment.

FIGURE 6.

CpG1826 does not overcome delayed maturation of FDCs and lack of TNF-α expression in GCs in early life. Spleen sections were stained with FDC-M2 (red), Ab to TNF-α (green), or DAPI to visualize the nuclei (blue) 12 d after priming of neonatal mice with (A) 0.5 μg Pnc1-TT+5 μg LT-K63 or (B) 0.5 μg Pnc1-TT+20 μg CpG1826. Seven-micrometer sections were cut at four different levels; the first started 700 μm into the spleen, and levels were separated by 210 μm. One representative section from each group is shown. Original magnification ×40. Scale bar, 50 μm. Results shown in (A) and (B) are from two experiments in neonatal mice with nine mice per group.

FIGURE 7.

LT-K63 enhanced the induction and long-term persistence of vaccine-specific AbSCs. PPS-1–specific (left) and TT-specific (right) IgG⁺ AbSCs in spleen (A, B) and BM (C, D) measured by ELISPOT, and serum IgG Ab levels (E, F) and AIs (G, H) measured by ELISA at days 14, 23, 39, and 55 after priming of neonatal mice with Pnc1-TT (□), Pnc1-TT+LT-K63 (▪), or saline (○). The results are from two experiments, expressed as number of spots/10⁶ cells (mean ± SD), IgG levels (mean EU/ml ± SD), and AI (mean AI ± SD) in 12 mice/group for each time point.