Circulating CXCR5+PD-1+ response predicts influenza vaccine antibody responses in young adults but not elderly adults

Abstract

Although influenza vaccination is recommended for all adults annually, the incidence of vaccine failure, defined as weak or absent increase in neutralizing Ab titers, is increased in the elderly compared with young adults. The T follicular helper cell (Tfh) subset of CD4 T cells provides B cell help in germinal centers and is necessary for class-switched Ab responses. Previous studies suggested a role for circulating Tfh cells (cTfh) following influenza vaccination in adults, but cTfh have not been studied in elderly adults in whom weak vaccine responses are often observed. In this study, we studied cTfh expressing CXCR5 and programmed death-1 (PD-1). cTfh from elderly adults were present at reduced frequency, had decreased in vitro B cell help ability, and had greater expression of ICOS compared with young adults. At 7 d after inactivated influenza vaccination, cTfh correlated with influenza vaccine-specific IgM and IgG responses in young adults but not in elderly adults. In sum, we have identified aging-related changes in cTfh that correlated with reduced influenza vaccine responses. Future rational vaccine design efforts should incorporate Tfh measurement as an immune correlate of protection, particularly in the setting of aging.

Conflict of interest statement

Disclosures of conflicts of interest

The authors declare no competing financial interests.

Copyright © 2014 by The American Association of Immunologists, Inc.

Figures

Figure 1. Circulating Tfh cells resemble lymphoid Tfh by phenotype and function

A. Circulating Tfh (cTfh) were identified among non-naive CD4+ cells (orange box, gated on live CD3+CD4+ cells) among PBMCs from young and elderly subjects as CXCR5+PD-1+ (red box). Naïve cells (black box, left panel) and non-naive CXCR5− cells (blue box, second panel) are also indicated. B. ICOS expression of cTfh cells was measured for different subsets of CD4+ cells (n=63). Expression of IL-6Rα(CD126; C), CCR7 (CD197; D), and CXCR4 (CD184; E) in the CD4+ subsets were measured by flow cytometry (n=10–11). F. Analysis of mRNA of additional Tfh genes as measured by RT-qPCR for the genes indicated (n=10–11) for sorted naïve (black), non-naive CXCR5− (blue), and cTfh (red) cells for combined young and elderly subjects are shown. G. Autologous naïve B cells were sorted and co-cultured with sorted naïve CD4+ cells (gated as CD45RA+CD62L+), non-naive memory CXCR5− cells, or cTfh cells from young subjects in the presence of SEB for seven days, followed by analysis of total IgM and IgG levels by ELISA. H. Plasma cell (CD19+CD38+CD138+) differentiation after co-culture with naïve (black), non-naïve memory CXCR5−(blue), or cTfh (red) and autologous naïve B cells in the presence of SEF for seven days was assessed by flow cytometry. Data are shown as frequency of CD38-CD138 co-expression among all live cells. Mean and SEM from 3 independent experiments are shown. Not all statistically significant findings are shown.

Figure 2. cTfh from elderly are reduced in frequency and express more ICOS than cTfh from young adults

A. Frequency of cTfh among the total non-naive CD4+ pool at baseline is shown for young (n=28, filled triangles) and elderly adults (n=35, open triangles). B. Representative plots are shown for cTfh from one young and one elderly adult (red box). Frequency of cTfh in non-naïve CD4+ pool (red number) is shown. C. A ratio of elderly-to-young adult cTfh frequency is shown for Cohort 1 (elderly, n=35) and Cohort 2 (elderly, n=26). D. Expression of ICOS was measured by flow cytometry for cTfh cells from young (filled triangles) and elderly adults (open triangles) in Cohort 1 and Cohort 2.

Figure 3. cTfh express Ki67 and CD38 but demonstrate reduced B cell help ability with aging

A. From PBMCs, co-expression of Ki67 and CD38hi for one representative subject for non-naïve CXCR5−(blue box), cTfh (red box), or ICOS+ cTfh cells (purple box) is shown. B. Summary plots for Ki67+CD38hi co-expression among all PBMCs for combined young and elderly adults (left panel, n=23) are shown. Data are further stratified by young (n=8, filled squares) and elderly adults (n=15, open squares) for cTfh (middle panel) and ICOS+ cTfh (right panel). C. Allogeneic naïve B cells from a young adult subject were sorted and co-cultured with sorted naïve or cTfh cells from young (n=16, filled squares) and elderly (n=17, open squares) adults in the presence of SEB for 7 days. IgM and IgG production was measured in the supernatant by ELISA. D. IL-21 production in cTfh is shown for one representative young and elderly donor following 6-hour stimulation with SEB in vitro. E. IL-21 production in CD4 T cell subsets is shown for young (n=6) and elderly (n=5) subjects. Not all statistically significant findings are shown.

Figure 4. After influenza vaccination, cTfh show increased ICOS expression in young but not elderly adults

A–B. Influenza vaccine was administered to young (n=28) and elderly (n=35) adults followed by serial phlebotomy. cTfh were measured by flow cytometry. Plots indicate CD38 vs ICOS expression of cTfh for one young (A) and one elderly (B) subject at days 0 and 7 after vaccination. Red numbers indicate frequency of ICOS+CD38hi-cTfh among non-naive CD4+ T cells for each time-point. Frequency of cTfh after influenza vaccination in young adults (n=28; C) and elderly adults (n=35; F) are shown. ICOS expression in cTfh cells from young adults (D) and elderly adults (G) are shown. Co-expression of ICOS and CD38 in cTfh cells from young adults (E) and elderly adults (H) are shown. Co-expression of Ki67 and CD38 in cTfh is shown for one representative young (I) and elderly (J) subject at days 0 and 7. Summary plots of co-expression of Ki67 and CD38 in cTfh (K) and in ICOS+ cTfh (L) cells are shown. p values indicate results of paired t test analyses comparing days 0 and 7.

Figure 5. After influenza vaccination, cTfh show increased ICOS expression in young but not elderly adults

A–D. Total binding IgM and IgG against influenza vaccine was measured by ELISA from serum from young (n=28) and elderly (n=35) subjects at days 0, 7, and 14 after vaccination. All statistical comparisons are performed against day 0 (*p<0.05, **p<0.01, ***p<0.001). E–H. Change in total binding IgM or IgG at day 7 was plotted as a fold-change against day 0 to day 7 fold-change in ICOS expression of cTfh cells for young and elderly subjects. Pearson r and p value are shown.