Impaired HA-specific T follicular helper cell and antibody responses to influenza vaccination are linked to inflammation in humans

  1. Danika L Hill
  2. Carly E Whyte
  3. Silvia Innocentin
  4. Jia Le Lee
  5. James Dooley
  6. Jiong Wang
  7. Eddie A James
  8. James C Lee
  9. William W Kwok
  10. Martin S Zand
  11. Adrian Liston
  12. Edward J Carr
  13. Michelle A Linterman

  • Curated by eLife

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    Evaluation Summary:

    This paper will be of significant interest to immunologists interested in understanding the determinants of antibody responses to vaccination. It uses tetramers to specifically identify and track CD4 T cell responses to influenza vaccination in younger and older adults, in an effort to understand why older individuals tend to have lower antibody responses to immunisation. The combination of tetramers, RNA sequencing and TCR clonotype tracking provides a powerful dataset to address fundamental questions of CD4 T cell immunology.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

Antibody production following vaccination can provide protective immunity to subsequent infection by pathogens such as influenza viruses. However, circumstances where antibody formation is impaired after vaccination, such as in older people, require us to better understand the cellular and molecular mechanisms that underpin successful vaccination in order to improve vaccine design for at-risk groups. Here, by studying the breadth of anti-haemagglutinin (HA) IgG, serum cytokines, and B and T cell responses by flow cytometry before and after influenza vaccination, we show that formation of circulating T follicular helper (cTfh) cells was associated with high-titre antibody responses. Using Major Histocompatability Complex (MHC) class II tetramers, we demonstrate that HA-specific cTfh cells can derive from pre-existing memory CD4 + T cells and have a diverse T cell receptor (TCR) repertoire. In older people, the differentiation of HA-specific cells into cTfh cells was impaired. This age-dependent defect in cTfh cell formation was not due to a contraction of the TCR repertoire, but rather was linked with an increased inflammatory gene signature in cTfh cells. Together, this suggests that strategies that temporarily dampen inflammation at the time of vaccination may be a viable strategy to boost optimal antibody generation upon immunisation of older people.

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  1. Version published to 10.7554/elife.70554 on eLife
  2. eLife

    Author Response:

    Reviewer #1:

    In this manuscript Hill et al, analyze immune responses to vaccination of adults with the seasonal influenza vaccine. They perform a detailed analysis of the hemagglutinin-specific binding antibody responses against several different strains of influenza, and antigen-specific CD4+ T cells/T follicular cells, and cytokines in the plasma. Their analysis reveals that: (i) tetramer positive, HA-specific T follicular cells induced 7 days post vaccination correlate with the binding Ab response measured 42 days later; (ii) the HA-specific T fh have a diverse TCR repertoire; (iii) Impaired differentiation of HA-specific T fh in the elderly; and (iv) identification of an "inflammatory" gene signature within T fh in the elderly, which is associated with the impaired development of HA-specific Tfh.

    The paper addresses a topic of considerable interest in the fields of human immunology and vaccinology. In general the experiments appear well performed, and support the conclusions. However, the following points should be addressed to enhance the clarity of the paper, and add support to the key conclusions drawn.

    We thank the reviewer for their supportive evaluation of the manuscript, and have provided the details of how we have addressed each the points raised below.

    1. Abstract: "(cTfh) cells are the best predictor of high titre antibody responses.." Since the authors have not done any blind prediction using machine learning tools with independent cohort, the sentence should be rephrased thus: "cTfh) cells are were associated with high titre antibody responses."

    We agree that this phrasing better reflects the presented data. The sentence in the abstract (page 2) now reads “we show that formation of circulating T follicular helper (cTfh) cells was associated with high titre antibody responses.”

    1. Figure 1A: Please indicate the age range of the subjects.

    Figure 1 has been updated to include the age range of the subjects.

    1. Almost all the data in the paper shows binding Ab titers. Yet, typically HAI titers of MN titers are used to assess Ab responses to influenza. Fig 1C shows HAI titers against the H1N1 Cal 09 strain. Can the authors show HAI titers for Cal 09 and the other A and B strains contained in the 2 vaccine cohorts? Do such HAI titers correlate with the tetramer positive cells, similar to the correlations show in Fig 2e.

    In this manuscript we have deliberately focussed on the immune response to the H1N1 Cal09 strain, as it is the only influenza strain in the vaccine common to both cohorts. The HAI titre for this strain is now shown as supplementary figure 4. In addition, the class II tetramers were specifically selected to recognise unique epitopes in the Cal 09 strain (J. Yang, {..} W. W. Kwok, CD4+ T cells recognize unique and conserved 2009 H1N1 influenza hemagglutinin epitopes after natural infection and vaccination. Int Immunol 25, 447-457, 2013) because of this we do not think it is appropriate to correlate HAI titres for the non-Cal 09 strains with tetramer positive cells. We agree that showing the correlation of cTfh and other immune parameters with the HAI titres for Cal 09 is important and have included this as supplementary figure 7. The new data and text are presented below:

    Figure 1-figure supplement 4: HAI responses before and after vaccination A) Log2 HAI titres at baseline (d0), d7 and d42 for cohort 1 (n=16) and B) cohort 2 (n = 21). C) Correlation between HAI and A.Cali09 IgG as measured by Luminex assay for cohort 1 and 2 combined. p-values determined using paired Wilcoxon signed rank-test, and Pearson’s correlation.

    Text changes. Page 4. “The increase in anti-HA antibody titre was coupled with an increase in hemagglutination inhibitory antibodies to A.Cali09, the one influenza A strain contained in the TIVs that was shared across the two cohorts and showed a positive correlation with the A.Cali09 IgG titres measured by Luminex assay (Fig. 1C, Figure 1-figure supplement 4).”

    Figure 2-figure supplement 1: Correlations between HAI assay titres and selected immune parameters. Correlation between vaccine-induced A.Cali09 HAI titres at d42 with selected immune parameters in both Cohort 1 and Cohort 2 (n=37). Dot color corresponds to the cohort (black = Cohort 1, grey = Cohort 2). Coefficient (Rho) and p-value determined using Spearman’s correlation, and line represents linear regression fit.

    Results text Changes: Page 5. “Similar trends were seen when these immune parameters were correlated to HAI titres against A/Cali09 (Fig Figure 2-figure supplement 1).”

    1. Fig 2d to i: what % of all bulk activated Tfh at day 7 are tetramer positive? The tetramer positive T cells constitute roughly 0.094% of all CD4 T cells (Fig 2d), of which 1/3rd are CXCR5+, PD1+ (i.e. ~0.03% of CD4 T cells). What fraction of all activated Tfh is this subset of tetramer positive cells? Presumably, there will also be Tfh generated against other viral proteins in the vaccine, and these will constitute a significant fraction of all activated Tfh.

    This is an important point, as the tetramers only recognise one peptide epitope of the Cal.09 HA protein, so there will be many other influenza reactive CD4+ T cells that are responding to other Cal 09 epitopes as well as other proteins in the vaccine. The analysis suggested by the reviewer shows that the frequency of Tet+ cells amongst bulk cTfh cells ranges from 0.14%-1.52% in cohort 1, and from 0.022-2.7% in cohort 2. These data have been included as Figure Figure 1-figure supplement 6C, D in the revised manuscript. In addition, Tet+ cells as a percentage of bulk cTfh cells were reduced in older people compared to younger adults. This data has been included in Figure 5-figure supplement 1C in the revised manuscript.

    Figure 1-figure supplement 6: Percentage of cTfh cells that are Tet+ and CXCR3 and CCR6 expression on HA-specific CD4+ T cells. A) Representative flow cytometry gating strategy for CXCR5+PD-1+ cTfh cells on CD4+CD45RA- T cells, and the proportion of HA-specific Tet+ cells within the CXCR5+PD-1+ cTfh cell gate. B) Percentage Tet+ cells within the CXCR5+PD-1+ cTfh cell population. Within-cohort age group differences were determined using the Mann-Whitney U test.

    Results text, page 4: These antigen-specific T cells had upregulated ICOS after immunisation, indicating that they have been activated by vaccination (Fig. 1F, G). In addition, a median of one third of HA-specific T cells upregulated the Tfh markers CXCR5 and PD1 on d7 after immunisation (Fig. 1H, I). The tetramer binding cells represented between 0.022-2.7% of the total CXCR5+PD-1+ bulk population (Fig Figure 1-figure supplement 6A, B).

    Figure 5-figure supplement 1C: Age-related differences in cytokines and HA-specific CD4+ T cell parameters. C) Percentage Tet+ cells within the CXCR5+PD-1+ cTfh cell population. Within-cohort age group differences were determined using the Mann-Whitney U test.

    Results text, page 8: Across both cohorts, the only CD4+ T cell parameters consistently reduced in older individuals at d7 were the frequency of polyclonal cTfh cells and HA-specific Tet+ cTfh cells, with the strongest effect within the antigen-specific cTfh cell compartment (Fig. 5H-J, Figure 5-figure supplement 1C).

    Reviewer #2:

    Hill and colleagues present a comprehensive dataset describing the recall and expansion of HA-specific cTFH cells following influenza immunisation in two cohorts. Using class II tetramers, IgG titres against a large panel of HA antigens, and quantification of plasma cytokines, they find that activated and HA-specific cTFH cells were a strong predictor of the IgG response against the vaccine after 6 weeks. Using RNAseq and TCR clonotype analysis, they find that, in 10/15 individuals, the HA-specific cTFH response at day 7 post-vaccination is recalled from the available CD4 T cell memory pool present prior to vaccination. Post-vaccination HA-specific cTFH cells exhibited a transcriptional profile consistent with lymph node-derived GC TFH, as well as evidence of downregulation of IL-2 signaling pathways relative to pre-vaccine CD4 memory cells.

    The authors then apply these findings to a comparison of vaccine immunogenicity between younger (18-36) and older (>65) adults. As expected, they found lower levels of vaccine-specific IgG responses among the older cohort. Analysis of HA-specific T cell responses indicated that tet+ cTFH fail to properly develop in the older cohort following vaccination. Further analysis suggests that development of HA-specific cTFH in older individuals is not caused by a lack of TCR diversity, but is associated with higher expression of inflammation-associated transcripts in tet+ cTFH.

    Overall this is an impressive study that provides clarity around the recall of HA-specific CD4 T cell memory, and the burst of HA-specific cTFH cells observed 7 days post-vaccination. The association between defective cTFH recall and lower IgG titres post-vaccination in older individuals provides new targets for improving influenza vaccine efficacy in this age group. However, as currently presented, the model of impaired cTFH differentiation in the older cohort and the link to inflammation is somewhat unclear. There are several issues that could be clarified to improve the manuscript in its current form:

    We thank the reviewer for their supportive and comprehensive summary of our work. We agree that the link between impaired inflammation and cTfh differentiation is correlative, we have added new data to address this, including mechanistic data to support chronic IL-2 signalling as antagonistic to cTfh development, as well as providing new analyses to address the other points raised.

    1. It is somewhat unclear the extent to which the reduction in HA-specific cTFH in the older cohort is also related to an overall reduction in T cell expansion - cohort 1 shows a significant reduction in total tet+ CD4 T cells post-vaccination as well as in the cTFH compartment, and while this difference may not reach statistical significance, a similar trend is shown for cohort 2.

    We agree that a possible interpretation is a global failure in T cell expansion in the older individuals. To determine whether there is a relationship between the degree of Tet+ CD4+ T cell expansion and cTfh cell differentiation with age, we performed correlation analyses. There is no correlation between the expansion of Tet+ cells and the frequency of cTfh cells formed seven days after immunisation in either age group. This suggests that the impaired cTfh cell differentiation in older persons is most likely caused by factors other than the capacity of CD4+ T cells to expand after vaccination. These data have been added as Figure 5-figure supplement 1D, and included in the results text on page 8.

    Figure 5-figure supplement 1D: Age-related differences in cytokines and HA-specific CD4+ T cell parameters. D) Correlation between Tet+ cells (d7-d0, % of CD4+) and cTfh (d7-d0, % of TET+) in both cohorts for each age-group (18- 36 y.o n=37, 65+ y.o. n= 39). Dot color corresponds to the cohort (black = Cohort 1, grey = Cohort 2). Coefficient (Rho) and p-value determined using Spearman’s correlation, and line represents linear regression fit.

    Text changes, Page 8: There was no consistent difference in the total d7 Tet+ HA-specific T cell population with age for both cohorts (Fig. 5H) and we observed no age-related correlation between the ability of an individual to differentiate Tet+ cells into a cTfh cell and the overall expansion of Tet+ HA-specific T cell population (Figure 5-figure supplement 1D). Thus, our data suggests that the poor vaccine antibody responses in older individuals is impacted by impaired cTfh cell differentiation (Fig. 5J) rather than size of the vaccine-specific CD4+ T cell pool.

    1. Transcriptomic analysis indicates that HA-specific cTFH in the older cohort show impaired downregulation of inflammation, TNF and IL-2-related signaling pathways. The authors therefore conclude that excess inflammation can limit the response to vaccination. In its current presentation, the data does not necessarily support this conclusion. While it is clear that downregulation of TNF and IL-2 signalling pathways occur during cTFH/TFH differentiation, there is no evidence presented to support the idea that (a) vaccination results in increased pro-inflammatory cytokine production in lymphoid organs in older individuals or that (b) these pro-inflammatory cytokines actively promote CXCR5-, rather than cTFH, differentiation of existing memory T cells.

    We agree with the reviewer that the data presented in figure 7 are correlative, rather than causative. Unfortunately, we do not have access to secondary lymphoid tissues from younger and older people after vaccination to test point (a) above. In order to test the hypothesis that increased inflammatory cytokine production in lymphoid organs limits Tfh cell differentiation we have used Il2cre/+; Rosa26stop-flox-Il2/+ transgenic mice. In this mouse model, IL-2-dependent cre- recombinase activity facilitates the expression of low levels of IL-2 in cells that have previously expressed IL-2. This creates a scenario in which cells that physiologically express IL-2 cannot turn its expression off therefore increasing expression IL-2 after antigenic stimulation (mice reported in Whyte et al., bioRxiv, 2020, doi: https://doi.org/10.1101/2020.12.18.423431).

    Twelve days after influenza A infection, Il2cre/+; Rosa26stop-flox-Il2/+ transgenic mice have fewer Tfh cells in the draining mediastinal lymph node and in the spleen (Fig. 8A-C), this is accompanied by a reduction in the magnitude of the GC B cell response (Fig. 8D-E). These data provide a proof of concept that sustained IL-2 production limit the formation of Tfh cells, consistent with the negative correlation of an IL-2 signalling gene signature and cTfh cell formation in humans (Figure 7). These new data support the conclusion that excess IL-2 signalling can limit the Tfh cell response. These data are presented in Figure 8, and are discussed on page 12 in the results, and pages 12-13 in the discussion.

    Figure 8: Increased IL-2 production impairs Tfh cell formation and the germinal centre response. Assessment of the Tfh cell and germinal centre response in Il2cre/+; Rosa26stop-flox-Il2/+ transgenic mice that do not switch off IL-2 production, and Il2cre/+; Rosa26+/+ control mice 12 days after influenza A infection. Flow cytometric contour plots (A) and quantification of the percentage of CXCR5highPD-1highFoxp3-CD4+ Tfh cells in the mediastinal lymph node (B) and spleen (C). Flow cytometric contour plots (D) and quantification of the percentage of Bcl6+Ki67+B220+ germinal centre B cells in the mediastinal lymph node (E) and spleen (F). The height of the bars indicates the median, each symbol represents one mouse, data are pooled from two independent experiments. P-values calculated between genotype-groups by Mann Whitney U test.

    Results text, page 12: Sustained IL-2 production inhibits Tfh cell frequency and the germinal centre response. To test the hypothesis that cytokine signalling needs to be curtailed to facilitate Tfh cell differentiation turned to a genetically modified mouse model in which cells that have initiated IL-2 production cannot switch it off, Il2cre/+; Rosa26stop-flox-Il2/+ mice (37). Twelve days after influenza infection Il2cre/+; Rosa26stop-flox-Il2/+ mice have fewer Tfh cells in the draining lymph node and spleen (Fig. 8A-C), which is associated with a reduced frequency of germinal center B cells (Fig. 8D-F). This provides a proof of concept that proinflammatory cytokine production needs to be limited to enable full Tfh cell differentiation in secondary lymphoid organs.

    Discussion text, pages 12, 13: These enhanced inflammatory signatures associated with poor antibody titre in an independent cohort of influenza vaccinees. The dampening of Tfh cell formation by enhanced cytokine production was confirmed by the use of genetically modified mice where IL-2 production is restricted to the appropriate anatomical and cellular compartments, but once initiated cannot be inactivated. Together, this suggests that formation of antigen-specific Tfh cells is essential for high titre antibody responses, and that excessive inflammatory factors can contribute to poor cTfh cell responses.

  3. eLife

    Evaluation Summary:

    This paper will be of significant interest to immunologists interested in understanding the determinants of antibody responses to vaccination. It uses tetramers to specifically identify and track CD4 T cell responses to influenza vaccination in younger and older adults, in an effort to understand why older individuals tend to have lower antibody responses to immunisation. The combination of tetramers, RNA sequencing and TCR clonotype tracking provides a powerful dataset to address fundamental questions of CD4 T cell immunology.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript Hill et al, analyze immune responses to vaccination of adults with the seasonal influenza vaccine. They perform a detailed analysis of the hemagglutinin-specific binding antibody responses against several different strains of influenza, and antigen-specific CD4+ T cells/T follicular cells, and cytokines in the plasma. Their analysis reveals that: (i) tetramer positive, HA-specific T follicular cells induced 7 days post vaccination correlate with the binding Ab response measured 42 days later; (ii) the HA-specific T fh have a diverse TCR repertoire; (iii) Impaired differentiation of HA-specific T fh in the elderly; and (iv) identification of an "inflammatory" gene signature within T fh in the elderly, which is associated with the impaired development of HA-specific Tfh.

    The paper addresses a topic of considerable interest in the fields of human immunology and vaccinology. In general the experiments appear well performed, and support the conclusions. However, the following points should be addressed to enhance the clarity of the paper, and add support to the key conclusions drawn.

    1. Abstract: "(cTfh) cells are the best predictor of high titre antibody responses.."
      Since the authors have not done any blind prediction using machine learning tools with independent cohort, the sentence should be rephrased thus: "cTfh) cells are were associated with high titre antibody responses."

    2. Figure 1A: Please indicate the age range of the subjects.

    3. Almost all the data in the paper shows binding Ab titers. Yet, typically HAI titers of MN titers are used to assess Ab responses to influenza. Fig 1C shows HAI titers against the H1N1 Cal 09 strain. Can the authors show HAI titers for Cal 09 and the other A and B strains contained in the 2 vaccine cohorts? Do such HAI titers correlate with the tetramer positive cells, similar to the correlations show in Fig 2e.

    4. Fig 2d to i: what % of all bulk activated Tfh at day 7 are tetramer positive? The tetramer positive T cells constitute roughly 0.094% of all CD4 T cells (Fig 2d), of which 1/3rd are CXCR5+, PD1+ (i.e. ~0.03% of CD4 T cells). What fraction of all activated Tfh is this subset of tetramer positive cells? Presumably, there will also be Tfh generated against other viral proteins in the vaccine, and these will constitute a significant fraction of all activated Tfh.

  5. eLife

    Reviewer #2 (Public Review):

    Hill and colleagues present a comprehensive dataset describing the recall and expansion of HA-specific cTFH cells following influenza immunisation in two cohorts. Using class II tetramers, IgG titres against a large panel of HA antigens, and quantification of plasma cytokines, they find that activated and HA-specific cTFH cells were a strong predictor of the IgG response against the vaccine after 6 weeks. Using RNAseq and TCR clonotype analysis, they find that, in 10/15 individuals, the HA-specific cTFH response at day 7 post-vaccination is recalled from the available CD4 T cell memory pool present prior to vaccination. Post-vaccination HA-specific cTFH cells exhibited a transcriptional profile consistent with lymph node-derived GC TFH, as well as evidence of downregulation of IL-2 signaling pathways relative to pre-vaccine CD4 memory cells.

    The authors then apply these findings to a comparison of vaccine immunogenicity between younger (18-36) and older (>65) adults. As expected, they found lower levels of vaccine-specific IgG responses among the older cohort. Analysis of HA-specific T cell responses indicated that tet+ cTFH fail to properly develop in the older cohort following vaccination. Further analysis suggests that development of HA-specific cTFH in older individuals is not caused by a lack of TCR diversity, but is associated with higher expression of inflammation-associated transcripts in tet+ cTFH.

    Overall this is an impressive study that provides clarity around the recall of HA-specific CD4 T cell memory, and the burst of HA-specific cTFH cells observed 7 days post-vaccination. The association between defective cTFH recall and lower IgG titres post-vaccination in older individuals provides new targets for improving influenza vaccine efficacy in this age group. However, as currently presented, the model of impaired cTFH differentiation in the older cohort and the link to inflammation is somewhat unclear. There are several issues that could be clarified to improve the manuscript in its current form:

    1. It is somewhat unclear the extent to which the reduction in HA-specific cTFH in the older cohort is also related to an overall reduction in T cell expansion - cohort 1 shows a significant reduction in total tet+ CD4 T cells post-vaccination as well as in the cTFH compartment, and while this difference may not reach statistical significance, a similar trend is shown for cohort 2.

    2. Transcriptomic analysis indicates that HA-specific cTFH in the older cohort show impaired downregulation of inflammation, TNF and IL-2-related signaling pathways. The authors therefore conclude that excess inflammation can limit the response to vaccination. In its current presentation, the data does not necessarily support this conclusion. While it is clear that downregulation of TNF and IL-2 signalling pathways occur during cTFH/TFH differentiation, there is no evidence presented to support the idea that (a) vaccination results in increased pro-inflammatory cytokine production in lymphoid organs in older individuals or that (b) these pro-inflammatory cytokines actively promote CXCR5-, rather than cTFH, differentiation of existing memory T cells.

  6. Version published to 10.1101/2021.04.07.21255038v1 on medRxiv