C-type lectin receptor agonists elicit functional IL21-expressing Tfh cells and induce primary B cell responses in neonates

Abstract

Introduction: C-type lectin receptor (CLR) agonists emerged as superior inducers of primary B cell responses in early life compared with Toll-like receptor (TLR) agonists, while both types of adjuvants are potent in adults.

Methods: Here, we explored the mechanisms accounting for the differences in neonatal adjuvanticity between a CLR-based (CAF^®01) and a TLR4-based (GLA-SE) adjuvant administered with influenza hemagglutinin (HA) in neonatal mice, by using transcriptomics and systems biology analyses.

Results: On day 7 after immunization, HA/CAF01 increased IL6 and IL21 levels in the draining lymph nodes, while HA/GLA-SE increased IL10. CAF01 induced mixed Th1/Th17 neonatal responses while T cell responses induced by GLA-SE had a more pronounced Th2-profile. Only CAF01 induced T follicular helper (Tfh) cells expressing high levels of IL21 similar to levels induced in adult mice, which is essential for germinal center (GC) formation. Accordingly, only CAF01- induced neonatal Tfh cells activated adoptively transferred hen egg lysozyme (HEL)-specific B cells to form HEL⁺ GC B cells in neonatal mice upon vaccination with HEL-OVA.

Discussion: Collectively, the data show that CLR-based adjuvants are promising neonatal and infant adjuvants due to their ability to harness Tfh responses in early life.

Keywords: C-type lectin receptor agonists; T follicular helper (Tfh); interleukin-21 (IL-21); interleukin-6 (IL-6); neonatal vaccinology; toll-like receptor agonists; vaccine adjuvants.

Figure 1

Neonatal primary Tfh, germinal center B cell, and antibody responses to HA/CAF01 or HA/GLA-SE. Groups of neonatal CB6F1 mice were immunized s.c. with HA formulated with CAF01 or GLA-SE. Age-matched mice receiving HA/PBS or non-injected (naïve) were used as control. (A, B) Draining LNs were harvested at the indicated time points after immunization, and samples were analyzed by flow cytometry. Graphs report the numbers of (A) T follicular helper (Tfh) cells and (B) Germinal Center (GC) B cells. Dots show values per individual mouse, whereas black lines indicate means. (C) HA-specific IgG antibody titers in sera collected before (day 0) and 3 weeks post-immunization (day 20). Values represent mean logarithmic titers (log 10) of five to eight mice per group ± SEM. (A-C) Data were pooled from at least two independent experiments per time point. Statistical analysis were performed using the Mann-Whitney U test: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Transcriptomic profiling of whole draining LNs from neonatal mice responding to HA/CAF01 or HA/GLA-SE. Groups of neonatal CB6F1 mice were immunized s.c. with HA alone or formulated in combination with CAF01 or GLA-SE. Two draining inguinal LNs were collected 24 h and 7 days post-immunization to perform microarray analysis. (A) The Venn diagram depicts the numbers of unique and shared differentially expressed genes (DEGs) between either adjuvant group compared with the HA alone group, at 24 h and 7 days post immunization as indicated. Blue and red numbers indicate the DEGs that were significantly downregulated (blue) and upregulated (red), respectively. (B–F) Heat maps show the expression profiles of selected genes associated with (B) TLR signaling pathways, (C) genes encoding CD molecules, (D) JAK-STAT signaling pathways, (E) genes encoding interferon regulatory factors, and (F) cytokine- and chemokine-related genes, after CAF01 or GLA-SE at the indicated time points.

Figure 3

Co-expression analysis in dLNs revealed shared and unique features of CAF01 and GLA-SE adjuvants. Groups of neonatal CB6F1 mice were immunized s.c. with HA alone or formulated with CAF01 or GLA-SE. Transcriptomic profiles resulting from the comparison HA/CAF01 versus HA/GLA-SE are shown. (A) Venn diagram depicts the numbers of genes that were significantly up-regulated (red) or down-regulated (blue) in the HA/CAF01 group compared with the HA/GLA-SE group in inguinal draining LNs at 24 h and 7 days post immunization. (B–H) Heat maps show the expression profiles of selected genes associated with (B) cytokine and chemokine activity, (C) encoding for CD molecules, (D) granulocyte-associated genes, (E) interferon regulatory factors, (F) TLR signaling genes, (G) apoptosis-related genes and (H) complement components.

Figure 4

CAF01-elicited neonatal adaptive responses develop in an IL6-rich environment. (A-H). One week-old CB6F1 mice were immunized s.c. with HA/PBS, HA/CAF01 or HA/GLA-SE and the two draining LNs were collected at day 7 or 10 post-immunization. RNA from total dLNs was used to measure the expression levels of selected genes by RT-PCR. mRNA expression levels of (A) IL6, (B) IL12B, (C) IL10, (D) IL1B, (E) GZMB, (F) IL4 and (G) IL13, and (H) IL21 are shown. Dots show values per individual mouse (N ≥ 4 per group) whereas black bars indicate means ± SEM. Statistical analysis were performed using the Mann-Whitney U test: *P < 0.05, **P < 0.01, ***P < 0.001. (I) Graph showing the correlation between the mRNA expression levels of IL6 and IL21 in dLN of neonates receiving HA/CAF01; data from day 7 (open circle) and 10 (filled circle) post-immunization were pooled. N = 6 mice/group; Data were pooled from two independent experiments per time point.

Figure 5

At day 10 post immunization CAF01 induced high levels of IL6 in draining LNs and higher levels of IFNγ and IL17 than GLA-SE upon HA restimulation ex vivo. Neonatal CB6F1 mice received HA/CAF01 or HA/GLA-SE and dLNs were collected 10 days after immunization and mashed to prepare single cell suspensions. Cytokine secretion in the supernatants was detected by luminex immunoassay after 3 days of restimulation with HA antigen or medium only. Values are expressed as picograms per milliliter (pg/ml); dots show values per individual mouse (N ≥ 5 per group) whereas black bars indicate means. Statistical analyses were performed using the Mann-Whitney U test: *P < 0.05, **P < 0.01. Data were pooled from at least two independent experiments.

Figure 6

Only CAF01 induces neonatal Tfh cells expressing adult-like levels of IL21. (A, B) One week-old CB6F1 mice were immunized s.c. with HA/CAF01 or HA/GLA-SE and dLNs were harvested at day 10 post immunization to quantify Tfh cells by flow cytometry. Graphs report (A) frequencies and (B) numbers of Tfh cells from a representative experiment. Statistical analyses were performed using the Mann-Whitney U test: ***P < 0.001. (C-E) One week-old and adult CB6F1 mice were immunized s.c. with HA/CAF01 or HA/GLA-SE. At day 10 post immunization, the draining LNs were collected to simultaneously isolate highly pure CD4⁺CXCR5^highPD-1^high Tfh cells, CD4⁺CXCR5^dimPD-1^dim effector T cells (CXCR5^dimPD-1^dim) and CD4⁺CXCR5^negPD-1^neg resting T cells (non- Tfh) by flow cytometry cell sorting according to the gates illustrated in panel (C, D) mRNA expression levels of IL21, Bcl6, ICOS, Foxp3, IL4, IL13, IL17 and IFN-γ are shown in the three distinct populations. (E) mRNA expression levels of IL10, IL2, CD25, and CD122 are shown in the three distinct populations in neonatal mice. (C-E) The cells obtained from the two dLNs of either 8 neonates/group or 6 adults/group per experiment were pooled before sorting to recover sufficient number of cells for experimentation. Results from 3 independent experiments including data from a total of 24 neonates and 18 adults are shown. Results are expressed as mean + SEM. Two -way ANOVA with multiple comparisons: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

The initiation of neonatal GC reactions requires fully functional Tfh cells. (A) Experimental schedule of the adult SWHEL B cells adoptive transfer. Naïve congenic 1-week old C57BL/6 recipient mice received each 2 x 10⁶ adult CD45.1⁺ B cells by intraperitoneal injection. Recipient and control neonatal mice were immunized s.c. the following day with 20 µg HEL-OVA formulated in CAF01, GLA-SE or PBS as control. Serum samples and inguinal draining LNs were collected 12 days post immunization. (B–D) The formation of HEL-specific GCs and plasma cells was measured by flow cytometry in the draining LNs of immunized neonates; graphs show the number of (B) SWHEL HEL⁺ CD45.1⁺ GL7⁺ CD95⁺ GC B cells, (C) endogenous HEL⁺ CD45.1^- GL7⁺ CD95⁺ GC B cells, and (D) total frequencies of B220int/lowCXCR4⁺CD138⁺ PCs. (E-G) HEL-specific serum (E) IgG, (F) IgG1, and (G) IgG2c titers were measured by ELISA. Dots show values per individual mouse (N≥ 6 per group) whereas black bars indicate means ± SEM. Statistical analysis were performed using the Mann-Whitney U test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were pooled from at least two independent experiments.