IL6 suppresses vaccine responses in neonates by enhancing IL2 activity on T follicular helper cells

Abstract

The inability of neonates to develop CD4⁺FoxP3^-CXCR5^hiPD-1^hi T follicular helper (T_FH) cells contributes to their weak vaccine responses. In previous studies, we measured diminished IgG responses when IL-6 was co-injected with a pneumococcal conjugate vaccine (PCV) in neonatal mice. This is in sharp contrast to adults, where IL-6 improves vaccine responses by downregulating the expression of IL-2Rβ on T_FH cells and protecting them from the inhibitory effect of IL-2. In this study, we found that splenic IL-6 levels rapidly increased in both adult and neonatal mice following immunization, but the increase in neonatal mice was significantly more than that of adult mice. Moreover, immunized neonatal T_FH cells expressed significantly more IL-2 as well as its receptors, IL-2Rα and IL-2Rβ, than the adult cells. Remarkably, IL-6 co-injection with PCV vaccine further increased the production of IL-2 and the expression of its receptors by neonatal T_FH cells, whereas excess IL-6 had totally opposite effect in immunized adult mice. Underscoring the role of IL-6 in activating the IL-2 mediated suppression of vaccine responses, immunization of IL-6 knock-out neonates led to improved antibody responses accompanied by expanded T_FH cells as well as lower levels of IL-2 and IL-2 receptors on T_FH cells. Moreover, CpG containing PCV improved T_FH response in neonates by suppressing the expression of IL-2 receptors on T_FH cells and inhibiting IL-2 activity. These findings unveil age-specific differences in IL-6 mediated vaccine responses and highlight the need to consider age-related immunobiological attributes in designing vaccines.

Fig. 1. Germinal center response to PPS14-TT vaccination in neonatal and adult mice.

Adult and neonatal mice were immunized with PPS14-TT and splenocytes were analyzed by FACS. a Representative dot plots from 0, 1, 3, 5, and 7 dpi depict the fluorescence minus one (FMO) controls and the percentages of IL-6⁺ cells gated on total splenocytes. Mean percentages of IL-6⁺ cells among splenocytes are plotted (n = 3). FMOs for each age group are the same for Day 1 and Day 5 because the samples for these days were analyzed on the same day. Also, FMOs for adult and neonates are the same for Day 3 because cells from adult and neonatal mice were pooled due to insufficient number of cells for each age group on this time point. Experiment was performed two times. b–e Adult and neonatal mice were immunized i.p. with PPS14-TT and 24 h post immunization (hpi) splenocytes were analyzed for p-STAT3 levels by FACS. b Representative FACS plots from 24 hpi splenocytes depict the percentages of p-STAT3⁺ cells on total CD4⁺ cells from adult and neonatal mice. Mean percentages of p-STAT3⁺ cells among total CD4⁺ cells are plotted (n = 5). c Pre-gated CD4⁺ cells were further gated for total T_FH (PD-1⁺CXCR5⁺) and pre-T_FH (PD-1^intCXCR5^int) populations in adult and neonatal mice. d The percentages of total T_FH (PD-1⁺CXCR5⁺) cells expressing FoxP3⁻p-STAT3⁺ were analyzed. Representative FACS plots depict the percentages of FoxP3⁻p-STAT3⁺ cells on total T_FH cells. Mean percentages of FoxP3⁻p-STAT3⁺ cells among total T_FH cells are plotted (n = 5). e The percentages of PD-1^intCXCR5^int cells expressing FoxP3⁻p-STAT3⁺ were analyzed. Representative FACS plots depict the percentages of FoxP3^-p-STAT3⁺ cells on PD-1^intCXCR5^int cells. Mean percentages of FoxP3⁻p-STAT3⁺ cells among CD4⁺PD-1^intCXCR5^int cells are plotted (n = 5). Experiment was performed three times. Unpaired student’s t-test and One-Way ANOVA were used for all comparisons; data represented as mean ± SEM are shown. P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ****P < 0.0001 and ns (non-significant).

Fig. 2. Neonatal mice have higher IL-2 levels and IL-2 receptors than the adult mice.

a Adult and neonatal mice were immunized with PPS14-TT and splenocytes were analyzed for IL-2 expression 7 dpi by FACS. Representative contour plots depict the percentages of IL-2-expressing FoxP3⁺ and FoxP3⁻ cells on T_FH cells. Mean percentages of IL-2⁺ cells among T_FH cells are plotted (n = 5). b, c Splenocytes were analyzed for IL-2 receptor expression 7 dpi by FACS. Representative contour plots depict percentages of IL-2Rα- (b) or IL-2Rβ- (c) expressing FoxP3⁺ and FoxP3⁻ cells on T_FH cells. Mean percentages of IL-2Rα⁺ and IL-2Rβ⁺ cells among T_FH cells are also plotted (n = 5). d Splenocytes from 7 dpi were stimulated with or without recombinant IL-2 for 15 min, followed by intracellular staining for p-STAT5. Representative contour plots depict the percentages of p-STAT5⁺ cells among FoxP3⁺ and FoxP3⁻ T_FH (pre-gated on CD4⁺CXCR5^hiPD-1^hi) cells. Mean percentages of p-STAT5⁺ cells among T_FH cells are plotted (n = 5). The data are representative of at least two independent experiments. Each experiment was performed twice. Unpaired student’s t-test and One-Way ANOVA were used for all comparisons; data represented as mean ± SEM are shown. P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns (non-significant).

Fig. 3. Germinal center response to IL-6 co-injected PPS14-TT vaccine in neonatal and adult mice.

Adult and neonatal mice were immunized i.p. with PPS14-TT in PBS (PBS) or PPS14-TT + IL-6 (IL-6) and splenocytes were analyzed by FACS at 7 dpi. a Representative dot plots depict the percentages of T_FH (CXCR5^hiPD-1^hi) cells pre-gated on CD4⁺FoxP3⁻ cells. Mean percentages of T_FH cells are plotted (n = 5). b, c Splenocytes from immunized mice were in vitro stimulated with PMA/Ion for 4 h followed by intracellular staining for IL-2 on T_FH cells. b Representative contour plots depict the percentages of IL-2-expressing FoxP3⁺ and FoxP3⁻ cells on T_FH cells. Mean percentages of IL-2⁺ cells among T_FH cells are plotted (n = 5). c Representative dot plots depict the percentage of T_FH (CXCR5^hiPD-1^hi) cells pre-gated on CD4⁺FoxP3⁻ cells. Mean percentages of T_FH cells are plotted (n = 5). d, e Splenocytes from immunized mice were pre-gated on CD4⁺CXCR5^hiPD-1^hi T_FH cells. Representative contour plots depict percentages of IL-2Rα- (d) or IL-2Rβ- (e) expressing FoxP3⁺ and FoxP3⁻ cells on T_FH cells. Mean percentages of IL-2Rα⁺ and IL-2Rβ⁺ cells among T_FH cells are plotted (n = 4–7). f Splenocytes from immunized mice were stimulated with recombinant IL-2 for 15 min, followed by intracellular staining for pSTAT5. Representative contour plots depict the percentage of pSTAT5⁺ cells on T_FH (CXCR5^hiPD-1^hiFoxP3⁻) cells pre-gated on CD4⁺ cells. Mean percentages of p-STAT5⁺ cells among T_FH cells are plotted (n = 6). Experiments were performed two to four times. One-Way ANOVA was used for all comparisons; data represented as mean ± SEM are shown. P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns (non-significant).

Fig. 4. IL-6 KO neonatal mice antibody and germinal center response to PPS14-TT vaccine.

Neonatal wild-type (C57BL/6J) and IL-6 KO mice were immunized i.p. with PPS14-TT and splenocytes were analyzed by FACS at 7 dpi. a Serum anti-PPS14 IgG titers were determined by ELISA 4 weeks after immunization (n = 8 wild-type and n = 7 IL-6 KO). b Representative dot plots depict the GC B (GL7⁺FAS⁺) cells pre-gated on B220⁺ cells. Mean percentages of GC B cells are plotted (n = 5). c Representative dot plots depict the GC T_FH (CXCR5^hiPD-1^hi) cells pre-gated on CD4⁺FoxP3⁻ cells. Mean percentages of FoxP3⁻ T_FH cells are plotted (n = 5). d Representative dot plots depict the GC T_FR (CXCR5^hiPD-1^hi) cells pre-gated on CD4⁺FoxP3⁺ cells. Mean percentages of T_FR cells are plotted (n = 9). e Splenocytes from immunized mice were in vitro stimulated with PMA/Ion for 4 h and T_FH cells were analyzed. Representative contour plots depict the percentages of IL-2-expressing FoxP3⁺ and FoxP3⁻ cells pre-gated on T_FH (CD4⁺CXCR5^hiPD-1^hi) population. Mean percentages of IL-2⁺ cells among T_FH cells are plotted (n = 5). f, g Splenocytes from immunized mice were pre-gated on CD4⁺CXCR5^hiPD-1^hi T_FH cells. Representative contour plots depict percentages of IL-2Rα- (f) or IL-2Rβ- (g) expressing FoxP3⁺ and FoxP3⁻ cells on T_FH cells. Mean percentages of IL-2Rα⁺ and IL-2Rβ⁺ cells among T_FH cells are also plotted (n = 8). h Splenocytes from immunized mice were stimulated with or without IL-2 for 15 min, followed by intracellular staining for p-STAT5. Representative contour plots depict the percentages of p-STAT5⁺ cells among FoxP3⁺ and FoxP3⁻ T_FH (pre-gated on CD4⁺CXCR5^hiPD-1^hi) cells. Mean percentages of p-STAT5⁺ cells among T_FH cells are plotted (n = 6). Experiments were performed two to seven times. Unpaired student’s t-test and One-Way ANOVA were used for all comparisons; data represented as mean ± SEM are shown. P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ****P < 0.0001 and ns (non-significant).

Fig. 5. Neonatal mice germinal center response to CpG containing PPS14-TT vaccine.

C57BL/6J mice were immunized i.p. with PPS14-TT (PBS) or PPS14-TT + CpG (CpG) and splenocytes were analyzed by FACS at 7 dpi. a Representative dot plots from 24 h post immunization depict the FMO control and the percentages of IL-6⁺ cells on total splenocytes. Mean percentages of IL-6⁺ cells among splenocytes are plotted (n = 5). b Representative dot plots from 24 h post immunization depict the FMO control and the percentages of IL-6⁺ cells on CD11c⁺ cells. Mean percentages of CD11c⁺IL-6⁺ subsets are plotted (n = 5). c Splenocytes from 7 dpi were in vitro stimulated with PMA/Ion for 4 h and T_FH cells were analyzed. Representative contour plots depict the percentages of IL-2⁺ cells among T_FH (CXCR5^hiPD-1^hi) population pre-gated on CD4⁺FoxP3⁻ cells. Mean percentages of IL-2⁺ cells among T_FH cells are plotted (n = 5). d, e Splenocytes from 7 dpi were pre-gated on CD4⁺CXCR5^hiPD-1^hi T_FH cells. Representative contour plots depict percentages of IL-2Rα (d) or IL-2Rβ (e) expressing FoxP3⁺ and FoxP3⁻ cells on T_FH cells. Mean percentages of IL-2Rα⁺ and IL-2Rβ⁺ cells among T_FH cells are also plotted (n = 6). f Splenocytes from 7 dpi were stimulated with or without recombinant IL-2 for 15 min, followed by intracellular staining for p-STAT5. Representative contour plots depict the percentages of p-STAT5⁺ cells among FoxP3⁺ and FoxP3⁻ T_FH (pre-gated on CD4⁺CXCR5^hiPD-1^hi) cells. Mean percentages of p-STAT5⁺ cells among T_FH cells are plotted (n = 5). Experiments were performed twice. Unpaired student’s t-test and One-Way ANOVA were used for all comparisons; data represented as mean ± SEM are shown. P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6. IL-6 KO neonatal mice T_FH response to CpG containing PPS14-TT vaccine immunization.

Neonatal wild-type (C57BL/6J) and IL-6 KO mice were immunized i.p. with PPS14-TT (PBS) or PPS14-TT + CpG (CpG) and splenocytes were analyzed by FACS at 7 dpi. a Representative dot plots depict the percentages of T_FH (CXCR5^hiPD-1^hi) cells pre-gated on CD4⁺FoxP3⁻ cells. Mean percentages of T_FH cells are plotted (n = 5). b Representative contour plots depict the percentages of IL-2⁺ cells among T_FH (CXCR5^hiPD-1^hi) population pre-gated on CD4⁺FoxP3⁻ cells. Mean percentages of IL-2⁺ cells among T_FH cells are plotted (n = 5). Unpaired student’s t-test and One-Way ANOVA were used for all comparisons; data represented as mean ± SEM are shown. P values < 0.05 were considered statistically significant. *P < 0.05 and ns (non-significant).