ICOS(+)PD-1(+)CXCR3(+) T follicular helper cells contribute to the generation of high-avidity antibodies following influenza vaccination

Abstract

The immune mechanism leading to the generation of protective antibody responses following influenza trivalent inactivated vaccine (TIV) vaccinations remains largely uncharacterized. We recently reported that TIV vaccination induced a transient increase of circulating ICOS(+)PD-1(+)CXCR3(+) T follicular helper (cTfh) cells in blood, which positively correlated with the induction of protective antibody responses measured at day 28. However, whether and how these T cells directly contribute to antibody response remains unclear. In this study, we analyzed the changes after TIV vaccination in the amount and the avidity of the polyclonal antibodies specific for the HA1 subunit of the pandemic H1N1 virus, and analyzed the correlation with the increase of ICOS(+)PD-1(+)CXCR3(+) cTfh cells. We found that both the amount and the avidity of specific antibodies rapidly increased during the first 7 days after TIV. Importantly, the increase of ICOS(+)PD-1(+)CXCR3(+) cTfh cells strongly correlated with the increase in the avidity of antibodies, particularly in subjects who did not have high affinity antibodies at baseline. We propose that ICOS(+)PD-1(+)CXCR3(+) Tfh cells directly contribute to the generation of high-avidity antibodies after TIV vaccinations by selectively interacting with high affinity B cells at extrafollicular sites.

Figures

Figure 1. pH1N1 Ab maturation occurs within 7 days after TIV.

(a) The amount (Max RU) and the avidity (Kd) of serum polyclonal Abs specific for pH1N1 HA1 at day 0, 7, and 28 post-TIV were determined by surface plasmon resonance. Paired t-test. ***p-value < 0.001, n = 26. (b) The correlation between Max RU and Kd of serum polyclonal Abs against pH1N1 HA1, and HI and VN titers. Results of day 0 and day 28 post-TIV. Spearman R and p-value are indicated. n = 52. (c) The correlation between the fold increase of Max RU/the fold decrease of Kd at day 28 and the increase of HI and VN titers at day 28. Spearman R and p-value are indicated. n = 26.

Figure 2. The increase of ICOS+PD-1+CXCR3+ cTfh cells correlates with the increase in Ab avidity.

(a) The correlation between the increase of ICOS+PD-1+CXCR3+ cTfh cells at day 7 post-TIV and the fold increase of Max RU/the fold decrease of Kd at day 7. Spearman R and p-value are indicated. n = 26. (b) The three groups were defined in the subjects by using the results of Kd and HI titers at day 0. (c) The correlation between the increase of ICOS+PD-1+CXCR3+ cTfh cells at day 7 post-TIV and the fold increase of Max RU/the fold decrease of Kd at day 7 in each group.

Figure 3. cTfh cells at day 7 post-TIV react to multiple influenza antigens.

The CD154 assay was performed with PBMCs at day 7 and day 60 post-TIV and overlapping peptide libraries derived from hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 and 2 (M1, M2), nonstructural protein 1 and 2 (NS1, NS2), polymerase proteins (PA, PB1, PB2) of the 2009 pandemic H1N1 strain. PBMC samples were obtained from at least 4 subjects and two assays were performed per sample on different days (indicated by closed and open symbols). The frequency of CD154+cytokine+ cells within CXCR5+CD4+ T cells and CXCR5−CD4+ T cells is indicated. (a) The CD154 assay was performed with PBMCs at day 7 and day 60 post-TIV and overlapping peptide libraries. The frequency of CD154+cytokine+ cells within CXCR5+CD4+ T cells is indicated. Mann-Whitney test. p-value ** < 0.01, *** < 0.001. (b) The results of the CD154 assay performed with PBMCs at day 7 and day 60 post-TIV and influenza vaccine (Fluzone®) or control (cont. PBS). The frequency of CD154+cytokine+ cells within CXCR5+CD4+ T cells and CXCR5−CD4+ T cells is indicated. (c) The results of the CD154 assay performed with PBMCs at day 7 and 60 post-TIV and overlapping peptide libraries. The frequency of CD154+cytokine+ cells within CXCR5−CD4+ T cells is indicated.

Figure 4. A model for the generation of high-avidity Abs in TIV vaccination.

(a) Healthy individuals have a broad range of pre-existing antigen-experienced B cells that recognize a specific HA1 epitope at different affinities. When vaccination successfully induces a preferential expansion of high affinity clones and their differentiation into Ab-producing cells, vaccination induces an increase in the avidity of Abs. (b) High affinity B cells have an advantage over low affinity cells to capture and present antigens to ICOS+PD-1+CXCR3+ Tfh cells for cognate interactions. These interactions occur at extrafollicular sites, and induce high affinity clones to undergo extensive proliferation and differentiation into Ab-producing cells. ICOS+PD-1+CXCR3+ Tfh cells are composed of heterogeneous populations that recognize wide range of influenza proteins, which contributes to increase the chance to engage cognate interactions with any high affinity clones. Such selective activation and expansion of high affinity B cells contribute to the rapid increase of Ab avidity.