Early Rise of Blood T Follicular Helper Cell Subsets and Baseline Immunity as Predictors of Persisting Late Functional Antibody Responses to Vaccination in Humans

Abstract

CD4+ T follicular helper cells (T(FH)) have been identified as the T-cell subset specialized in providing help to B cells for optimal activation and production of high affinity antibody. We recently demonstrated that the expansion of peripheral blood influenza-specific CD4(+)IL-21(+)ICOS1(+) T helper (T(H)) cells, three weeks after vaccination, associated with and predicted the rise of protective neutralizing antibodies to avian H5N1. In this study, healthy adults were vaccinated with plain seasonal trivalent inactivated influenza vaccine (TIIV), MF59(®)-adjuvanted TIIV (ATIIV), or saline placebo. Frequencies of circulating CD4(+) T(FH)1 ICOS(+) T(FH) cells and H1N1-specific CD4(+-)IL-21(+)ICOS(+) CXCR5(+) T(FH) and CXCR5(-) T(H) cell subsets were determined at various time points after vaccination and were then correlated with hemagglutination inhibition (HI) titers. All three CD4(+) T cell subsets expanded in response to TIIV and ATIIV, and peaked 7 days after vaccination. To demonstrate that these T(FH) cell subsets correlated with functional antibody titers, we defined an alternative endpoint metric, decorrelated HI (DHI), which removed any correlation between day 28/day 168 and day 0 HI titers, to control for the effect of preexisting immunity to influenza vaccine strains. The numbers of total circulating CD4(+)T(FH)1 ICOS(+) cells and of H1N1-specific CD4(+)IL-21(+)ICOS(+) CXCR5(+), measured at day 7, were significantly associated with day 28, and day 28 and 168 DHI titers, respectively. Altogether, our results show that CD4(+) T(FH) subsets may represent valuable biomarkers of vaccine-induced long-term functional immunity.

Trial registration: ClinicalTrials.gov NCT01771367.

Conflict of interest statement

Competing Interests: FSp, ES, EB, LZ, RC, NC, FSc, DR, FC, DM, SB, and GDG were employee of Novartis Vaccines & Diagnostics S.r.l. at the time of the study. FSp, ES, EB, LZ, NC, FSc, DR, DM, SB, and GDG are now employee of GSK Vaccines S.r.l. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. CONSORT Flow diagram.

Fig 2. B-cell and functional antibody responses after seasonal influenza vaccination.

(A) Absolute number of plasmablasts (CD19+CD20- CD38+) in 106 live PBMCs acquired. (B) HI Geometric mean titers (GMT) for A H1N1 and H3N2, and B influenza strains at baseline (D0), 7 days (D7), 28 days (D28), and 168 days (D168) after a single dose of influenza vaccine. Data show three cohorts: saline placebo (n=7), TIIV (n=18), and ATIIV (n=17). (C) Geometric mean ratio (GMR) for all vaccine strains. Non-parametric Wilcoxon’s signed rank test was used for statistical analyses: *p < 0.05, **p < 0.01, and ***p < 0.001 compared to day 0; §p < 0.05, §§p < 0.01 and §§§p < 0.001 compared to saline placebo.

Fig 3. Expansion of ICOS+ and PD-1+ TFH1 cells after TIIV and ATIIV vaccination.

(A) Number of CD4+ T cells expressing CXCR5 and ICOS in human PBMCs after seasonal influenza vaccination. (B and C) Number of TFH1 cells expressing ICOS and PD-1. Data show three cohorts: saline placebo (n=7), TIIV (n=18) and ATIIV (n=17) at baseline (D0), day 7 (D7) and day 28 (D28) after a single dose of influenza vaccine. Data are shown for each participant and expressed as number of cells in 106 live PBMCs acquired. Non-parametric Wilcoxon’s signed rank test was used for statistical analyses: *p < 0.05, **p < 0.01, and ***p < 0.001 compared to day 0; §p < 0.05, §§p < 0.01 and §§§p < 0.001 compared to saline placebo.

Fig 4. H1N1-specific CD4+IL-21+ICOS+ TH cells expand 7 days after seasonal influenza vaccination.

Numbers of CD4+IL-21+ICOS+ TH cells in PBMCs stimulated overnight with A/California/7/2009 (H1N1) antigen. Data show three cohorts: saline placebo (n=7), TIIV (n=18) and ATIIV (n=17) at baseline (D0), day 7 (D7) and day 28 (D28) after a single dose of influenza vaccine. Data are shown for each subject and expressed as number of cells in 106 live CD4+ T cells acquired; mean ± SEM is shown. Non-parametric Wilcoxon’s signed rank test was used for statistical analyses: *p < 0.05, **p < 0.01, and ***p < 0.001 compared to day 0; §p < 0.05, §§p < 0.01 and §§§p < 0.001 compared to saline placebo.

Fig 5. H1N1-specific CD4+IL-21+ICOS+ TH cells subsets expressing or not CXCR5 expand after influenza vaccination.

Number of CD4+IL-21+ICOS+ TH cells, showing a CXCR5+ (black) or CXCR5- (gray) phenotype, in vaccinated participants after overnight stimulation with A/California/7/2009 (H1N1) antigen or SEB. Data show saline placebo (n=7), and merged TIIV (n=18) and ATIIV (n=17) cohorts at baseline (D0), day 7 (D7) and day 28 (D28) after a single dose of influenza vaccine. Data are expressed as number of cells in 106 live CD4+ T cells; mean ± SEM is shown. Non-parametric Wilcoxon’s signed rank test was used for statistical analyses: *p < 0.05, **p < 0.01, and ***p < 0.001 compared to day 0.

Fig 6. TFH1 ICOS+ cells predict functional antibody responses.

Correlations between the number of total circulating CD4+ TFH1 ICOS+ cells and the maximum DHI responses observed across the three influenza strains represented in the vaccine, measured at (A) day 28 and (B) day 168 after immunizzation. Dashed lines represent the least squares regressions fit to the data. R: Pearson product-moment correlation coefficient. p: correlation-associated p value.

Fig 7. H1N1-specific CD4+IL-21+ICOS+CXCR5+ TFH cells predict functional antibody responses.

Correlations between the number of H1N1-specific CD4+IL-21+ICOS+CXCR5+ TFH cells and H1N1-specific DHI responses measured at (A) day 28 and (B) day 168 after immunizzation. Dashed lines represent the least squares regressions fit to the data. R: Pearson product-moment correlation coefficient. p: correlation-associated p value.