Live-Attenuated Influenza Vaccine Induces Tonsillar Follicular T Helper Cell Responses That Correlate With Antibody Induction

Sarah Lartey, Fan Zhou, Karl A Brokstad, Kristin G-I Mohn, Steffen A Slettevoll, Rishi D Pathirana, Rebecca J Cox, Sarah Lartey, Fan Zhou, Karl A Brokstad, Kristin G-I Mohn, Steffen A Slettevoll, Rishi D Pathirana, Rebecca J Cox

Abstract

Background: Influenza remains a major threat to public health. Live-attenuated influenza vaccines (LAIV) have been shown to be effective, particularly in children. Follicular T helper (TFH) cells provide B-cell help and are crucial for generating long-term humoral immunity. However the role of TFH cells in LAIV-induced immune responses is unknown.

Methods: We collected tonsils, plasma, and saliva samples from children and adults receiving LAIV prior to tonsillectomy. We measured influenza-specific TFH-cell responses after LAIV by flow cytometry and immunohistochemistry. Systemic and local antibody responses were analysed by hemagglutination inhibition assay and enzyme-linked immunosorbent assay.

Results: We report that LAIV induced early (3-7 days post-vaccination) activation of tonsillar follicles and influenza-specific TFH-cell (CXCR5+CD57+CD4+ T cell) responses in children, and to a lesser extent in adults. Serological analyses showed that LAIV elicited rapid (day 14) and long-term (up to 1 year post-vaccination) antibody responses (hemagglutination inhibition, influenza-specific IgG) in children, but not adults. There was an inverse correlation between pre-existing influenza-specific salivary IgA concentrations and tonsillar TFH-cell responses, and a positive correlation between tonsillar TFH-cell and systemic IgG induction after LAIV.

Conclusions: Our data, taken together, demonstrate an important role of tonsillar TFH cells in LAIV-induced immunity in humans.

Keywords: LAIV; TFH cells; antibody responses; influenza; tonsils.

© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.

Figures

Figure 1.
Figure 1.
Illustration of the study design. Forty children (3 to 17 years old) and 37 adults (18 to 51 years old) were enrolled in this study. Thirty-four children and 31 adults were vaccinated with trivalent live-attenuated influenza vaccine (LAIV) (Fluenz; AstraZeneca) during the influenza season 2013–2014. The children and adults were randomized into 3 groups and vaccinated on specific days before their scheduled tonsillectomy: Group 1 (2–5 days, 7 children and 15 adults), Group 2 (6–9 days, 15 children and 8 adults), and Group 3 (10–22 days, 12 children and 8 adults). Age- and gender-matched unvaccinated subjects (6 children and 6 adults) were enrolled as controls. Children (n = 27) under 9 years old received the second dose LAIV at day 28. Tonsils, plasma, and saliva samples were collected during tonsillectomy. In addition, plasma and saliva samples prevaccination (day 0) and 28 days, 56 days, 6 months (day 180), and 12 months (day 360) postvaccination were collected for all vaccinated subjects (Groups 1–3).
Figure 2.
Figure 2.
Activation of follicular T helper cells after live-attenuated influenza vaccine. (A) Representative immunohistochemistry images (original magnification ×4) of sections stained with CD4, Bcl6, or CD57 from a child (7 years old, top) and an adult (24 years old, bottom). Stained sections were counterstained with hematoxylin. Scale bar indicates 400 μm. (B) A summary of inducible T-cell costimulator (ICOS)+ area inside follicles from controls and vaccinees in Groups 1–3 in children (n = 22) and adults (n = 18). Follicles were measured based on CD20 and hematoxylin staining. (C) Representative CD4+ T-cell subsets gating based on CXCR5 and CD57 in a child (4 years old, left) and an adult (40 years old, right). Tonsillar mononuclear cells were pregated as CD3+CD19−CD56−CD4+CD45RA− (pregating in Supplemental Figure S1). Numbers adjacent to outlined areas indicate frequencies of CD4+ T cells in each subset. (D) A summary of the distribution of the CD4+ T-cell subsets in 12 unvaccinated children and adults. (E) The expression of ICOS in median fluorescence intensity (MFI) in CD4+ T-cell subsets: CXCR5+CD57+ (orange), CXCR5+CD57− (blue), and CXCR5−CD57− (red) from unvaccinated children and adults. (F) A summary of ICOS expression in MFI from controls and vaccinees in Groups 1–3 in children (n = 25) and adults (n = 23). The mean values are shown as bars, and each symbol represents 1 subject (B and D–F). Sidak’s multiple comparisons between CD4+ T-cell subsets were performed in two-way analysis of variance ([ANOVA] D and E). Multiple t tests with desired false discovery rate of 1% between vaccinated and unvaccinated subjects were performed in two-way ANOVA (B and F). Data were from 6 independent experiments. ***, P < .001.
Figure 3.
Figure 3.
Live-attenuated influenza vaccine elicits influenza-specific follicular T helper (TFH)-cell responses. (A and B) Influenza-specific inducible T-cell costimulator (ICOS) expression increases (Delta ICOS in median fluorescence intensity [MFI]) in CD4+ T-cell subsets from controls and vaccinees in Groups 1–3 in children (n = 25; A) and adults (n = 23; B). (C) Vaccine-induced total influenza-specific TFH-cell responses were calculated as the elevation in TFH-cell activity (Delta ICOS × CXCR5+CD57+ %). Tonsillar mononuclear cells were rested and stimulated with influenza split antigens from A/California/07/2009-like (H1N1) virus (left) or A/Victoria/361/2011-like (H3N2) virus (center) or B/Massachusetts/2/2012 virus (right). Results from vaccinees (Groups 1–3) are presented as arbitrary values relative to unvaccinated subjects (Control). The mean values are shown as bars, and each symbol represents 1 subject. Multiple t tests with desired false discovery rate of 1% between vaccinated and unvaccinated subjects were performed in two-way analysis of variance. Data were from 6 independent experiments. *, P < .05; **, P < .01; ***, P < .001.
Figure 4.
Figure 4.
Live-attenuated influenza vaccine elicits influenza-specific antibodies within 14 days in children. Hemagglutinin-specific antibodies (hemagglutination inhibition [HI] titer; A) and total influenza-specific antibodies (immunoglobulin [Ig]G; B) were measured using plasma samples before (D0) and after vaccination (DT) from vaccinees in Groups 1–3. (C) Virus-specific local antibodies (IgA) were measured using saliva samples before (D0) and after vaccination (DT) from vaccinees in Groups 1–3. Antibodies were tested against A/California/07/2009-like (H1N1) virus (left) or A/Victoria/361/2011-like (H3N2) virus (center) or B/Massachusetts/2/2012 virus (right). The geometric mean values are shown as bars, and each symbol represents 1 subject. Antibody titers and concentrations were Ln transformed in statistical analyses. Multiple t tests with desired false discovery rate of 1% between pre- and postvaccination titers were performed in two-way analysis of variance. The horizontal dotted lines indicate HI titer of 40 (A). Duplicates were performed in all experiments. *, P < .05; **, P < .01.
Figure 5.
Figure 5.
Live-attenuated influenza vaccine elicits long-term influenza-specific systemic and local antibodies in children. Systemic hemagglutinin-specific antibodies (hemagglutination inhibition [HI] titer; A) and total influenza-specific antibodies (immunoglobulin [Ig]G; B) were measured using plasma samples prevaccination (D0) and 28 days (D28), 56 days (D56), 6 months (D180), and 12 months (D360) postvaccination. Local influenza-specific antibodies (IgA; C) were measured using saliva samples before vaccination (D0) and 28 days (D28) and 56 days (D56) after vaccination. Antibodies were tested against A/California/07/2009-like (H1N1) virus (left) or A/Victoria/361/2011-like (H3N2) virus (center) or B/Massachusetts/2/2012 virus (right). The geometric mean titers are shown as bars, and each symbol represents 1 subject. Antibody titers and concentrations were Ln transformed in statistical analyses. Sidak’s multiple comparisons between prevaccination (D0) and each time point postvaccination (D28, D56, D180, and D360) were performed in two-way ANOVA. The geometric mean HI titers (GMT) at each time point are noted above the graphs, and the horizontal dotted lines indicate HI titer of 40 (A). Duplicates were performed in all experiments. *, P < .05; **, P < .01; ***, P < .001.
Figure 6.
Figure 6.
Pre-existing local antibodies correlate with age and follicular T helper (TFH)-cell responses after live-attenuated influenza vaccine (LAIV). (A) Pre-existing influenza-specific local antibodies (immunoglobulin [Ig]A) in saliva samples from children and adults correlated strongly with their ages. (B) The LAIV-induced TFH-cell responses (Delta inducible T-cell costimulator [ICOS] × CXCR5+CD57+ %) inversely correlate with pre-existing influenza-specific local antibodies (IgA). The TFH-cells activity and antibodies were tested against split antigens from A/California/07/2009-like (H1N1) virus (left) or A/Victoria/361/2011-like (H3N2) virus (center) or B/Massachusetts/2/2012 virus (right). Pre-existing IgA concentrations in saliva samples were Ln transformed in statistical analyses. Linear fitting curve was plotted as dotted line when nonparametric Spearman P < .10. Spearman r and P values are noted for each correlation. MFI, median fluorescence intensity.
Figure 7.
Figure 7.
Influenza-specific follicular T helper (TFH)-cell responses after live-attenuated influenza vaccine (LAIV) correlate with antibody responses. (A) The correlations between LAIV-induced TFH-cell responses (Delta inducible T-cell costimulator [ICOS] × CXCR5+CD57+ %) and systemic antibody fold induction (plasma immunoglobulin [Ig]G D28/D0) from all vaccinated children and adults. (B) Influenza A viruses specific TFH-cell and antibody responses correlate in naive individuals (D0 hemagglutination inhibition [HI] <40), whereas influenza B virus-specific TFH-cell and antibody responses correlate in pre-exposed individuals (D0 HI ≥40). The TFH-cell responses and antibody fold induction were tested against split antigens from A/California/07/2009-like (H1N1) virus (left) or A/Victoria/361/2011-like (H3N2) virus (center) or B/Massachusetts/2/2012 virus (right). Systemic antibody fold inductions (plasma IgG D28/D0) were Ln transformed in statistical analyses. Linear fitting curve was plotted as dotted line when nonparametric Spearman P < .10. Spearman r and P values are noted in each correlation. MFI, median fluorescence intensity.

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