Evidence That Blunted CD4 T-Cell Responses Underlie Deficient Protective Antibody Responses to Influenza Vaccines in Repeatedly Vaccinated Human Subjects

Abstract

Despite the benefits of yearly influenza vaccination, accumulating evidence suggests that diminished vaccine efficacy may be related to repeated vaccination. Although studied at the level of B-cell responses, CD4 T-cell responses have not yet been examined. In this study, we analyze CD4 T-cell responses to influenza vaccination in subjects who differ in their vaccine history. We find a striking disparity in their responses, with previously vaccinated subjects exhibiting significantly blunted CD4 T-cell responses and diminished antibody responses. These results suggest that limiting CD4 T-cell help mteaserrlie the diminished or altered antibody responses in repeatedly vaccinated subjects.

Keywords: CD4 T cells; cell-mediated immunity; influenza vaccine; influenza virus.

Figure 1.

CD4 T-cell expansion and T follicular helper cell (Tfh) are more robust in subjects not vaccinated in the prior season. Healthy subjects vaccinated with seasonal influenza vaccine were stratified into 2 groups based on self-reported vaccination status from the previous season. Those subjects that received influenza vaccine in the previous season are indicated here as “V” (closed symbols), and subjects that had not received influenza vaccine in the previous season are indicated here as “UV” (open symbols). In A, CD4-enriched populations were stimulated with pools of peptides from H1 (top left), H3 (top right), HA-B (bottom left), or tetanus and rubeola (T + R, bottom right) as a control antigen and evaluated for interferon (IFN)γ production using enzyme-linked immunospot analyses. Shown is the change in the response between day 0 and day 14 (D14-D0) with the average response indicated by a black line and the mean value reported in red. The P value calculated using the Mann-Whitney U test is indicated in the top left corner for each stimulation condition. In B, circulating Tfh cells, defined as CD4⁺CD45RA⁻CXCR5⁺ICOS⁺PD1⁺ cells, were quantified at day 7-postinfluenza vaccination by flow cytometry. Shown is the frequency of cells at day 7. The average, indicated by a black line and the mean value, reported in red text, are shown. The P value calculated using the Mann-Whitney U test is indicated in the top left corner.

Figure 2.

Subjects not vaccinated in the prior season respond with higher gains in hemagglutinin (HA)-specific antibody responses. Subjects that received influenza vaccine in the previous season are indicated here as “V” (closed symbols), and subjects who reported that they had not received influenza vaccine in the previous season are indicated here as “UV” (open symbols). In A, serum antibody from day 0, before vaccination, and day 14 postvaccination is measured by HA-specific immunoglobulin G enzyme-linked immunosorbent assay (ELISA). The relative titers were determined based on the serum dilution at a fixed OD405 signal on the linear portion of the curve. Shown is the fold change between day 0 and day 14 (D14/D0). A black line indicates the geometric mean and the value is reported in red next to it. The p value calculated using the Mann-Whitney U test is indicated in the top left corner for each HA protein. In panel B, the neutralizing antibody response measured at day 0, before vaccination, and day 28-post vaccination was measured by HAI assay. Shown is the fold change between day 0 and day 28 (D28/D0). The geometric mean, indicated by a black line and the value, reported in red text, are shown. The P value calculated using the Mann-Whitney U test is indicated in the top left corner for each HA protein. In C, the correlation between the CD4 T-cell response and the serum antibody response to H1 (left), H3 (middle), and HA-B (right), represented as the change in interferon (IFN)γ-secreting cells between D0 and D14, and the serum antibody response of the matched protein, represented as the fold change (D14/D0), are shown. The r and P values shown in the top left corner of each panel were calculated by the Spearman-rank correlation test.