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Protective neutralizing influenza antibody response in the absence of T follicular helper cells

Abstract

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.

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Figure 1: Systemic vaccination induced Bcl6-independent humoral response to protect against H5N1.
Figure 2: The TFH cells and GC are not required for the production of protective IgG2 antibodies, but are required for affinity maturation.
Figure 3: TH1 cells regulate protective anti-IAV IgG2 production.
Figure 4: Bcl6 is dispensable for IAV-specific TH1 induction.
Figure 5: TH1 cells are responsible for anti-IAV IgG2 production.
Figure 6: TH1 cells interact with activated B cells in TFH-cell-deficient mice.
Figure 7: IL-21 contributes to TH1-cell-dependent virus-specific IgG2 production.

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Acknowledgements

We thank Y. Ito and A. Komano for helpful advice about the IAV infection experiments. We thank Y. Harada, Y. Motomura, H. Fujimoto, Y. Hachiman and Y. Suzuki for technical support and animal maintenance. We thank P. Burrows for helpful comments on the manuscript. We thank B. Malissen (CIML) for the Cd3e-deficient mice. This work was supported by a Grant-in-Aid for Scientific Research (A) (24249058) to M.K., and by Takeda Science Foundation, Uehara Foundation and The Naito Foundation.

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Authors and Affiliations

Authors

Contributions

K.M. and M.K. designed and conceptualized the research and analyzed the data. A.S.-I., Y.H., Y.U., T.K., Y.T. and K.M. performed H1N1 experiments. K.M., S.F., T.M. and Y.K. performed H5N1 experiments. T.W., A.H. and O.O. performed Ig sequencing analysis. K.I. and M.O.-H. performed statistical data analysis of RNA sequencing. H.H., Y.A. and Y.T. established and provided H1N1 Narita virus. T.T. established the Bcl6fl/fl mice. M.K. established the Il21fl/fl mice, and the Il4, Il21 and Ifng reporter mice. K.M., M.K. and T.T. prepared the manuscript.

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Correspondence to Masato Kubo.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gene expression profile of CD4+ T cells and CTL responses in Bcl6-deficient mice.

(a) Gene expression profile in the resting CD4+ T cells were analyzed by an Affymetrix microarray chip (MouseGenome 430 2.0 array). Differential expressions were found in 10 genes that indicated. (b) Gene expression profile of OT-II derived CD4+ T cells in the response to OVA. The heat map represents expression of signature genes of TH1, TH2, TFH, and TH17 subsets. (c) Flow cytometry analysis of NP tetramer binding CD8+ T cells. Spleen cells were obtained from PR8-infected WT and Bcl6ΔT mice at day10 post infection and were cultured with NP peptide pulsed EL-4 cells in presence of IL-2. After 5-day culture, cells were stained with NP tetramer and anti-CD8a mAb. The graph shows the percentage of NP tetramer binding CD8 cells in WT and Bcl6ΔT mice (n=4). (d) For the killing assay, NP peptide pulsed EL-4 cells were labeled with CFSE and then co-cultured with spleen cells. After 6hr-culture, killing rate was determined with the EL-4 cell number. The percent lysis was calculated based on the background lysis of EL-4 cells without NP peptide. Mean of the specific lysis were indicated at different effector/target ratios (n=4). Error bars represent S.D.

Supplementary Figure 2 Persistent protective response and GC-independent IgG2 memory B formation in Bcl6-deficient mice.

(a) Naive mice were intravenously treated with sera from WT, or Bcl6ΔT mice collected at 2 weeks or 1 year after vaccination and then infected with a lethal dose of Narita (2.5 LD50/mouse). Body weight was measured in the mice receiving sera from the unvaccinated C57BL/6 mice (UV) (closed circle), the WT mice at 2-week (2wk) (open circles) (n=5) and 1-year (1y) (filled blue circle) (n=4) post vaccination, and Bcl6ΔT mice at 1-year post vaccination (filled red circles) (n=4). Statistical analysis was performed using Mann-Whitney U test. **P<0.01. *P<0.05. (b) Circles represent IgG2b+ B cell number of HA binding CD38+ memory cells and GL-7+ GC B cells. (c and d) The memory B cells were transferred into Rag1-deficient mice with Narita primed T cells. Circles represent the frequency of virus-specific IgG2b (c) and IgG2c (d) in the spleen at day 7 after IAV challenge (n = 4).

Supplementary Figure 3 Generation of HA-specific IgG-producing plasmablasts in Bcl6-deficient mice.

(a) Flow cytometry analysis of HA-binding CD138+ B cells. Spleen cells from Narita immunized WT, Bcl6ΔT and Bcl6ΔB mice were stained with B220, and CD138 antibodies and APC labeled HA at the indicated days after immunization. HA+CD138+ B cells were sorted for the IgG sequencing analysis. (b) The bar graphs show the absolute number of HA+ (top) and CD138+ cells (bottom) (n = 3).

Supplementary Figure 4 Gene signature of CXCR3+CXCR5+ TFH cells and the expression of CXCR3 and CXCR5 in IFN-γ+ cells

(a) Construction map of the Venus-targeted Ifng locus (Top). The IRES-Venus cassette was inserted immediately after the stop codon of Ifng gene. Splenocytes from Ifng Venus reporter mice were stimulated with anti-TCRβ antibody in the presence of IL-12 and anti-IL-4 antibody (for TH1), or IL-4 and anti-IFN-γ antibody (for TH2). Five days after the stimulation, ex-vivo induced TH1 and TH2 cells were re-stimulated with anti-TCRβ antibody, and intracellular staining of IFN-γ and IL-4 (bottom) were carried out. (b) Heatmap of TH1, TH2, TH17, and TFH signature genes, and heatmap of common signature genes of TH1 and TFH in CXCR5+ and CXCR3+CXCR5+ cells. CXCR5 single positive and CXCR3 CXCR5 double positive CD4+ T cells were sorted from vaccinated C57BL/6 mice at day14 post vaccination. The RNA sequencing analysis of sorted populations was carried out by Hiseq. (c) Comparison analysis of CXCR3+CXCR5+ and CXCR5+ TFH cells. Fold-change of gene expression in CXCR3+CXCR5+ versus CXCR5+ cells are plotted as histogram. Major TFH signature genes are shown in red. (d) Flow cytometry analysis of Venus expression was examined in splenic CD4+T cells from vaccinated ifng Venus reporter mice at 14 days after immunization. The CD4+ T cells were separated into 3 fractions based on the magnitude of Venus expression (Negative (Neg), intermediated (Med) and High). Each population was analyzed for PD-1, CXCR5, and CXCR3 expression. The bar graph shows cell number of TFH and CD4+T cells in Neg, Med, and High fractions (n=3).

Supplementary Figure 5 Gene signature of CXCR3+CXCR5+ T cell population

(a) Flow cytometry analysis of PD-1 and CXCR5 expression (top) and CXCR3 and CXCR5 expression (bottom) by CD4+T cells at the indicated time points. Splenic CD4+T cells from vaccinated WT or Bcl6ΔT mice at the indicated days after vaccination were stained for PD-1, CXCR3, and CXCR5. (b) Heatmap of TH1, TH2, TH17, and, TFH signature genes in CXCR5+, CXCR3+CXCR5-, and CXCR3+CXCR5dull cells. The indicated populations were sorted from the vaccinated WT or Bcl6ΔT mice at day14 post vaccination. The RNA sequencing analysis of sorted populations were carried out by Illmina Hiseq.

Supplementary Figure 6 Establishment of and characterization of the Il21ΔT mice

(a) Construction of the LoxP-flanked Il21 allele (Il21f/f) mice. Exons 1 and 2 of the Il21 gene locus were flanked by LoxP sites (open triangles) by homologous recombination. The flanked region was removed from the mouse germline by crossing with Cd4-cre mice. Arrows show binding sites of PCR primers. (b) Electrophoretic analysis of PCR fragments of DNA in the Il21 locus of the sorted CD4+ T cells from WT and Il21ΔT mice. Arrows indicate PCR products derived from the Il21 locus. (c) Percentage of CD8+ and CD4+ T cells in thymus of Il21ΔT mice. (d) The expression of Il21 mRNA in splenic CD4+ T cells and Peyer’s patches. Il21 mRNA expression was measured in CD4+ T cells from Il21ΔT mice by qPCR. (e) Expression of GL-7 and Fas were analyzed in B220+ B cells from WT or Il21ΔT mice immunized with Narita. The bar graph shows the percentage of GC B cells in the B220+ population (n=5).

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Miyauchi, K., Sugimoto-Ishige, A., Harada, Y. et al. Protective neutralizing influenza antibody response in the absence of T follicular helper cells. Nat Immunol 17, 1447–1458 (2016). https://doi.org/10.1038/ni.3563

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