Crucial roles of interleukin-7 in the development of T follicular helper cells and in the induction of humoral immunity

Abstract

T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses.

Importance: Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

Augmented TIV-specific antibody responses by codelivery of IL-7 or IL-7-mFc. (A) BALB/c mice were immunized with 0.6 μg TIV alone or with either 1.8 μg IL-7 or 1.8 μg IL-7-mFc (n = 8/group). (B) BALB/c mice were immunized with 0.6 μg TIV alone or in combination with the indicated dose of IL-7-mFc (n = 8/group). (C) Mice were intramuscularly injected with 0.6 μg TIV into the right limb, and 1.8 μg IL-7-mFc was either added to the same injection as a mixture or administered into the opposite limb (n = 8/group). At 1 or 3 wpi, serum levels of IgG, IgG1, or IgG2a specific to TIV were determined individually by endpoint titration ELISA. Data, shown as means ± standard errors of the means (SEM), are representative of three independent experiments. TIV, trivalent inactivated influenza virus vaccine consisting of H1N1 A/New Caledonia/20/99, H3N2 A/Fujian/411/2002, and B/Shanghai/361/2002. *, P < 0.05 by Student's t test.

FIG 2

Dose-sparing effect by codelivery of Fc-fused IL-7. BALB/c mice (n = 8/group) (A) or cynomolgus monkeys (n = 4/group) (B) were immunized with the indicated doses of TIV in combination with IL-7-mFc or IL-7-hFc, respectively. Serum IgG responses specific to TIV protein were determined by endpoint titration ELISA at 1 wpi in mice or at 1 to 4 wpi in cynomolgus monkeys. Data, shown as means ± SEM, are representative of three independent mice experiments (A) or a single monkey experiment (B). *, P < 0.05 compared to the group treated only with TIV by Student's t test.

FIG 3

Induction of cross-reactive IgG responses and enhanced protective efficacy against heterologous influenza virus challenge by cotreatment of IL-7-mFc. (A and B) BALB/c mice (n = 8/group) (A) or cynomolgus monkeys (n = 4/group) (B) were immunized with the indicated doses of TIV in combination with IL-7-mFc or IL-7-hFc, respectively. Serum IgG responses specific to inactivated PR8/H1N1 virus particles were determined by endpoint titration ELISA at 1 wpi in mice or at 1 to 4 wpi in cynomolgus monkeys. (C and D) BALB/c mice were immunized with the indicated doses of TIV in the presence or absence of IL-7-mFc (n = 8/group). At 8 days after immunization, mice were challenged intranasally with a lethal dose of PR8/H1N1 virus (2 × 103 PFU). Mice were observed daily to monitor body weight (C) and survival rate (D). Data, shown as means ± SEM, are representative of three independent mice experiments (A) or a single monkey experiment (B). *, P < 0.05 compared to the TIV-only group by Student's t test. PR8/H1N1, H1N1 A/Puerto Rico/8/34. Data are representative of two independent experiments with similar results. †, P < 0.05 compared to the TIV-only group by a log-rank test.

FIG 4

Expansion of Tfh cells and GC B cells, as well as GC formation by TIV cotreatment with IL-7-mFc. BALB/c mice were immunized with 0.6 μg TIV alone or in combination with IL-7-mFc. CD4-depleting antibody (α-CD4) was treated 1 day prior to immunization with the vaccine (n = 6/group). (A) At 1 wpi, HA-specific IgG responses were determined by endpoint titration ELISA. (B to E) BALB/c mice were immunized with 0.6 μg TIV with or without 1.8 μg IL-7-mFc (n = 10/group). (B) At 1 wpi, draining lymph node cells were stimulated with 10 μg/ml TIV HA for 24 h, and the percentage of Th1 (IFN-γ+), Th2 (IL-4+), or Th17 (IL-17+) cells was measured by intracellular cytokine staining. Tfh cells (CXCR5+ Bcl-6+ and CXCR5+ PD-1+) among CD4+ T cells (C) or germinal center B cells (FAS+ GL-7+) among B220+ B cells (D) were analyzed by flow cytometry. (E) Representative images of PNA staining (red) for detecting PNA+ GCs (depicted by arrows) in spleen by immunohistochemical analysis are shown. Data, shown as means ± SEM, are representative of two independent experiments. P < 0.05 (*) and P < 0.01 (**) by Student's t test.

FIG 5

Effect of IL-7-mFc cotreatment on the generation of Th subsets in OT-II transfer model. C57BL/6 mice were intravenously injected with OT-II cells and then immunized with 10 μg OVA protein in the presence or absence of 30 μg IL-7-mFc (n = 5/group). At 1 wpi, splenocytes were isolated and restimulated for 6 h with 1 μM OVA232-339 peptide. (A) Gated CD4+ T cells were analyzed by intracellular cytokine staining for IFN-γ, IL-4, or IL-17 production. The percentage of Tfh cells in OT-II cells (B) and GC B cells in B220+ cells (C) was determined as previously described. (D) Serum levels of OVA-specific IgG were determined by endpoint titration ELISA. Data, shown as means ± SEM, are representative of two independent experiments. **, P < 0.01 by Student's t test.

FIG 6

Comparison of mRNA expression levels between antigen-only and IL-7-mFc cotreatment. C57BL/6 mice were transferred with naive OT-II cells (5 × 105) and then immunized with 10 μg OVA (with alum) in the presence or absence of 30 μg IL-7-mFc (n = 4/group). The transferred OT-II cells were sorted from spleen of the immunized mice on day 2 or day 4 after immunization. mRNA levels of the indicated molecules were determined by quantitative real-time PCR. Data are representative of three independent experiments. NA, data not available owing to the failure of detecting the mRNA level of either group.

FIG 7

Effect of the IL-6/IL-21 blockade on the generation of Tfh cells and GC B cells by IL-7-mFc cotreatment. BALB/c mice were immunized with 10 μg TIV with or without 30 μg IL-7-mFc. In addition, the combination of anti-IL-6 and anti-IL-21 neutralizing antibodies was injected into the immunized mice as described in Materials and Methods (n = 5/group). At 1 wpi, draining lymph node cells were analyzed to evaluate the percentage of Tfh cells (A) and GC B cells (B). Data, shown as means ± SEM, are representative of two independent experiments. P < 0.05 (*) and P < 0.01 (**) by Student's t test.

FIG 8

Effects of the IL-7 blockade on Tfh cell and GC B cell development during TIV immunization. BALB/c mice first were immunized with 10 μg TIV and then treated with anti-IL-6, anti-IL-7, or anti-IL-21 neutralizing antibody, either alone or in combination, as described in Materials and Methods (n = 5/group). At 1 wpi, draining lymph node cells were analyzed to determine the frequency of Tfh cells (A) and the frequency and the number of GC B cells (B). (C) At 1 wpi, serum levels of IgG to TIV were determined by endpoint titration ELISA. Data, shown as means ± SEM, are representative of two independent experiments. **, P < 0.01 by Student's t test.