Differences in Influenza-Specific CD4 T-Cell Mediated Immunity Following Acute Infection Versus Inactivated Vaccination in Children

Abstract

Background: Early childhood influenza infections imprint influenza-specific immune memory, with most studies evaluating antibody specificity. In this study, we examined how infection versus inactivated influenza vaccination (IIV) establish pediatric CD4 T-cell mediated immunity to influenza and whether this poises the immune system to respond differently to IIV the following year.

Methods: We tracked influenza-specific CD4 T-cell responses in 16 H3N2 infected and 28 IIV immunized children following both initial exposure and after cohorts were revaccinated with IIV the following fall. PBMCs were stimulated with peptide pools encompassing the translated regions of the H3 HA and NP proteins and were then stained to assess CD4 T-cell specificity and function.

Results: Compared to IIV, infection primed a greater magnitude CD4 T-cell response specific for the infecting HA and NP proteins, with more robust NP-specific immunity persisting through year 2. Post infection, CD4 T cells preferentially produced combinations of cytokines that included interferon-γ. Interestingly, age-specific patterns in CD4 T-cell reactivity demonstrated the impact of multiple influenza exposures over time.

Conclusions: These data indicate that infection and vaccination differentially prime influenza-specific CD4 T-cell responses in early childhood, with these differences contributing to the lasting immunologic imprinting established following early influenza infection.

Clinical trials registration: NCT02559505.

Keywords: CD4 T cells; cellular immune response; influenza immunity; pediatrics.

© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Figures

Figure 1.

Design of the study. Pediatric subjects between 3 months and 7 years of age were enrolled and vaccinated with IIV (n = 28) or enrolled upon presentation with an acute influenza infection (n = 16). Blood was obtained for PBMCs at baseline, day 8 to 14, and day 20 to 28 post exposure. Subjects were then followed for the remainder of the influenza season and were revaccinated with IIV the following fall, with blood again obtained at baseline, day 8 to 14, and day 20 to 28 post vaccination. Abbreviations: IIV, inactivated influenza vaccination; PBMC, peripheral blood mononuclear cell; V, visit.

Figure 2.

Compared to the vaccination cohort, the percentages of IFN-γ-producing H3- and NP-specific CD4 T cells were greater by visit 2 post-H3N2 infection, with NP-specific cells in acute cohort subjects preferentially boosted upon vaccination the following fall. PBMCs from either infected or vaccinated subjects were stimulated with pools of overlapping peptides encompassing the coding regions of the H3 and NP proteins and were stained by intracellular cytokine staining. Results were gated on CD4+ T cells expressing CD69 and IFN-γ. A repeated ANCOVA was applied to examine differences in IFN-γ production between acutely infected and vaccinated subjects, controlling for age as a continuous variable. Filled circles represent CD4 T-cell responses of individual subjects following year 1 acute infection, while open squares represent CD4 T-cell responses post year 1 vaccination. Horizontal lines are means of the individual responses, while bars represent estimated percentages controlled for age as an overlaid graph. Abbreviations: ANCOVA, analysis of covariance; IFN-γ, interferon- γ; PBMC, peripheral blood mononuclear cell.

Figure 3.

There are differences in CD4 T-cell functionality between subjects that were acutely infected with H3N2 influenza or vaccinated with IIV in study year 1. PBMCs were stimulated and stained as previously described with gating as shown in Supplementary Figure 1. Combination gates were created using FlowJo v10 software (TreeStar) to highlight single, double, and triple cytokine producing cells. A repeated ANCOVA analysis was applied to examine differences in polyfunctionality between acutely infected and vaccinated subjects, controlling for age as a continuous variable. Filled circles represent CD4 T-cell responses of individual subjects acutely infected in year 1, while open squares represent CD4 T-cell responses in subjects vaccinated in year 1. Horizontal lines are means of the individual responses, while bars represent estimated percentages controlled for age as an overlaid graph. Data obtained following stimulation of PBMCs with the H3 HA protein are shown in (A) and (B), and with NP-specific responses depicted in (C) and (D). Abbreviations: ANCOVA, analysis of covariance; IIV, inactivated influenza vaccination; PBMC, peripheral blood mononuclear cell.

Figure 4.

The specificity of the influenza-specific CD4 T-cell response varies depending on subject age. Subjects were banded into 2 groups consisting of those A, H3-specific CD4 T-cell response at visit 3 (left) and visit 6 (right) and (B) CD4 T-cell reactivity to NP at the same 2 time points. Data are presented as scatter plots demonstrating individual reactivity, overlaid by bar graphs showing the age-adjusted mean concentration estimate. Statistical analysis was completed using a repeated ANOVA analysis, controlling for age as a group: <3 years vs 3–8 years. C and D, Stacked bar graphs highlighting differences in reactivity by age: (C) CD4 T-cell reactivity at visit 3 (day 20–28 post infection or vaccination in year 1) and (D) reactivity at visit 6 (day 20–28 post vaccination in study year 2). Abbreviations: ANOVA, analysis of variance; IFN-γ, interferon- γ; yo, years old.

Figure 5.

Individual subjects acutely infected with influenza in study year 1 demonstrated an increased fractional response to NP compared to H3. Processed data from intracellular cytokine staining is represented as a stacked bar for each subject, with reactivity to H3 HA in black and NP in gray. Individual subject identities are indicated on the bottom of the graph, arranged by age group. Subjects shown had an adequate quantity of PBMCs to stimulate with both the H3 HA and NP proteins. A, Results from visit 3 (day 20–28 post initial exposure) and (B) visit 6 (day 20–28 following revaccination). Abbreviations: IFN-γ, interferon- γ; PBMC, peripheral blood mononuclear cell.

Figure 6.

Acutely infected subjects showed significantly increased proliferation following infection compared to noninfected vaccinated children. PBMCs were stimulated with complete peptide pools comprising the coding sequences of the H3 HA (A) or NP (B) proteins and then were stained using intracellular cytokine staining. An increase in antigen-specific Ki67+ CD4 T cells was present at approximately day 10 post infection (visit 2) when compared to the same time point post vaccination. This increase was not maintained following vaccination of both cohorts in study year 2. Statistical analysis was performed using a repeated ANCOVA analysis, controlling for age as a continuous variable. Filled circles represent Ki67+CD69+ CD4 T-cell responses of individual subjects following year 1 acute infection; open squares represent data following vaccination. Horizontal lines are means of the individual responses, while overlaid bars are the estimated percentages controlled for age. Abbreviations: ANCOVA, analysis of covariance; PBMC, peripheral blood mononuclear cell.