Research ArticleInfluenza

Circulating TFH cells, serological memory, and tissue compartmentalization shape human influenza-specific B cell immunity

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Science Translational Medicine  14 Feb 2018:
Vol. 10, Issue 428, eaan8405
DOI: 10.1126/scitranslmed.aan8405
  • Fig. 1 Vaccination induces activation of cTFH cells and transient ASCs.

    (A) Study design and vaccine composition. Yam, Yamagata; Vic, Victoria. (B) Representative fluorescence-activated cell sorting (FACS) plots and (C) numbers of ICOS+PD-1+cTFH1 cells after vaccination (n = 34 to 42). (D) CD38 expression on activated cTFH1 cells. (E) Frequency of CD38hi cells in the ICOS+PD-1+cTFH1 population (n = 26, 2016 cohort). (F) Representative FACS plots and (G) numbers of CD27hiCD38hiASCs after vaccination (n = 42). (H) Expression of CXCR3 and (I) frequency of CXCR3+ASCs after vaccination. (J and K) MFI of CXCR3 (J) and CXCR5 (K) expression on ASCs and non-ASCs at d7 (n = 26, 2016 cohort). (L) Serological response to vaccination, measured as fold change in HAI titers (2014, n = 7; 2015, n = 16; 2016, n = 26). Bars/lines indicate the median. Statistical significance from baseline or between groups was determined using Wilcoxon matched-pairs signed-rank test (**P < 0.005, ***P < 0.001, ****P < 0.0001).

  • Fig. 2 Influenza A– and influenza B–specific B cell pools differ in size and isotype distribution in peripheral blood.

    PBMCs at steady state were stained with rHA probes from the H1, H3, and B viruses to detect influenza-specific IgD B cells. (A) Representative FACS plots. (B) Absolute numbers of rHA+IgD B cells per milliliter of blood. (C and D) Isotype distributions for rHA+ B cell populations. (C) Representative FACS plots of IgG+ versus IgM+ B cells. (D) Pie charts indicate median frequency (n = 42). (E to G) Numbers of (E) IgG+ B cells, (F) IgA+ B cells, and (G) IgM+ B cells within rHA+ populations. (H and I) CD27/CD21 phenotype of rHA+ populations. (H) Representative FACS plots. (I) CD21/CD27 phenotype distributions of rHA+ B cells. (J) Numbers of CD21+CD27+ B cells per milliliter of blood. Bars/lines indicate the median (n = 42). Significance between HA probes was determined using Wilcoxon matched-pairs signed-rank test (*P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001).

  • Fig. 3 IIV induces CD21hiCD27+ and CD21loCD27+ influenza-specific B cells.

    (A) Numbers of rHA+IgD B cells before and after vaccination. Representative FACS plots of rHA+ B cells after vaccination. (B) Numbers of rHA+ B cells per milliliter of blood for each vaccine component. (C) Representative FACS plots and (D) frequency of CD27+CD20rHA+ B cells before and after vaccination. (E) Numbers of isotype-specific rHA+ B cells. (F) Representative FACS plots for d0 and d14. (G) Numbers of CD21hiCD27+ and CD21loCD27+ IgG+rHA+ B cells. (E and G) Pooled data from 2015 and 2016 cohorts (n = 42). Bars indicate the median. Statistical significance for changes from baseline was determined using Wilcoxon matched-pairs signed-rank test (*P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001).

  • Fig. 4 cTFH1 cells correlate with a boosting of influenza-specific memory B cells.

    (A) Correlation between d7 ASCs and total fold change (Δ) (sum of vaccine response to vaccine components) in serum titers after vaccination. (B) Numbers of ASC on d7 in seroconverters (S) (n = 19) and nonseroconverters (NS) (n = 7). (C) Fold change on d28 over baseline in HAI serum titers from pooled data, from three vaccine components of the 2014 cohort (n = 7), at different time points. (D) Correlation between d7 ICOS+PD-1+cTFH1 cells and fold change in serum titers after vaccination. (E) Numbers of ICOS+PD-1+cTFH1 cells on d7 in seroconverters and nonseroconverters. (F) Correlation between d7 ASCs and ICOS+PD-1+cTFH1 cells. (G) Correlation between fold changes in CD21hiCD27+ B cells or CD21loCD27+ B cells on d14 and numbers of ICOS+PD-1+cTFH1 on d7. (A, D, and F) Data from 2015 and 2016 cohorts (n = 42). (G) Data from 2015 cohort (n = 16). (A, D, and F to I) Correlation was assessed using Spearman’s correlation coefficient (rs). (B and E) Bars indicate the median. Statistical significance was determined using the Mann-Whitney test (*P < 0.05).

  • Fig. 5 Preexisting antibodies negatively correlate with the serological response and the magnitude of the CD21lo B cell response.

    (A) HAI titers at baseline in seroconverters (S) and nonseroconverters (NS). Seroconversion was defined as a change in HAI titer ≥4. Dotted line indicates a HAI of 40. Bars indicate the geometric mean. Statistical significance was determined using the Mann-Whitney test (**P < 0.005). (B) Correlation between HAI titers at baseline and fold change in serum titers after vaccination. Correlations between total HAI titers at baseline and (C) total fold change in serum titers (d28/d0), (D) numbers of ASCs, or (E) ICOS+PD-1+cTFH1 cells on d7. Correlation between total HAI titers at baseline and the fold change in (F) CD21hiCD27+ or (G) CD21loCD27+ IgG+ B cells on d14. Correlation was assessed using Spearman’s correlation coefficient (rs).

  • Fig. 6 Vaccination fails to induce CD8+ and innate T cell responses.

    (A and B) IFN-γ/TNF production after overnight stimulation with live IAV-H1N1 or IBV-B/Phuket. (A) Representative FACS plots of H1N1 stimulation. (B) Numbers of IFN-γ+ cells per milliliter of blood after IAV or IBV stimulation (n = 7, 2015 cohort). “No virus” control counts were subtracted. Significance for changes between time points was determined using the Friedman test (*P < 0.05). (C) IAV-specific CD8+ T cells were detected by peptide/MHC-I tetramers (A2-M158–66, A1-NP44–52, and B8-NP225–233). FACS plots are gated on CD3+ cells, and percentages are based on CD8+ T cells. (D) Longitudinal tracking of % tetramer+CD8+ T cells (n = 3). Arrows indicate vaccination. (E) FACS plots of CD27/CD45RA expression on tetramer+CD8+ T cells. (F) Longitudinal tracking of activated CD27+CD45RAtetramer+CD8+ T cells (n = 3). (G) T cell subsets were assessed for activation and differentiation markers. Bars indicate the median. Data are from randomly selected donors (n = 14, 2014 to 2015 cohorts).

  • Fig. 7 Vaccine-induced influenza-specific B cells are not maintained in peripheral blood.

    Responses in (A) antibody serum titers, (B) numbers of IgDrHA+ B cells, (C) pie graphs of isotype distribution within each rHA+ population, (D) changes in isotype-specific class-switched rHA+ B cells, and (E) numbers of CD21hi and CD21lo influenza-specific B cells at baseline, d28, and >d350 (n = 9, 2015 cohort). Significance for changes from baseline was determined using Wilcoxon matched-pairs signed-rank test (*P < 0.05, **P < 0.005). (F to H) Numbers of (F) total IgDrHA+ B cells and (G) CD21hi and (H) CD21loIgG+rHA+ B cells in individuals vaccinated in 2015 and 2016 (n = 8).

  • Fig. 8 Influenza-specific B cells show distinct patterns of tissue compartmentalization.

    (A) Frequency of CD19+CD10 B cells across human tissues. (B) CD21/CD27 phenotype and (C) isotype of B cells across tissues. (D) Frequency of rHA+ (H1/H3) IgD B cells across tissues. (E) Phenotype distribution and (F) isotype distribution of rHA+ B cells across tissues. For isotype distributions, the frequency of each isotype was normalized to the sum of all three isotypes. (G) Representative FACS plots are shown. (A and D) Bars indicate median. (B, C, E, and F) Lines indicate mean, and error bars show SEM. Adult blood, n = 5; cord blood, n = 5; bone marrow, n = 5; tonsil, n = 5; lymph node, n = 3; lung, n = 3; spleen, n = 7. (H) PBMCs from paired blood and spleen samples were analyzed. Frequency of each CD21/CD27 population within total B cells for each blood and spleen pair (n = 5) is shown. Significance was assessed using a paired t test (*P < 0.05; **P < 0.005). ND, not determined.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/428/eaan8405/DC1

    Fig. S1. Gating strategy for circulating ASCs and activated cTFH1 cells.

    Fig. S2. Specific activation of cTFH1 cells after IIV.

    Fig. S3. Gating strategy for influenza-specific B cells in PBMC.

    Fig. S4. Validation of rHA probe staining.

    Fig. S5. BCR analysis of single rHA+ B cells.

    Fig. S6. Frequency of IgA+ cells in IgGIgDIgMrHA+ B cells in healthy adults.

    Fig. S7. Numbers of isotype-specific rHA+ (H3N2/Swi) B cells.

    Fig. S8. rHA+ B cell kinetics by ELISPOT.

    Fig. S9. CD20 expression by CD21loCD27+ B cells and ASCs.

    Fig. S10. Gating strategy for ex vivo live virus ICS.

    Fig. S11. Vaccination does not induce CD8+ and innate T cell responses.

    Fig. S12. Gating strategy for T cell phenotyping.

    Fig. S13. Fold change in influenza-specific B cells during repeated vaccination.

    Fig. S14. Validating of rHA probes in human tissues.

    Fig. S15. B cell isotype distributions in paired tissue samples.

    Table S1. Details of vaccination cohorts.

    Table S2. Cohorts of human tissues.

    Table S3. FACS panel for ASCs and cTFH cells.

    Table S4. FACS panel for influenza-specific T cells after influenza virus infection.

    Table S5. FACS panel for phenotyping T cells after vaccination.

    Table S6. FACS panel for influenza-specific B cells in 2015 cohort.

    Table S7. FACS panel for influenza-specific B cells in 2016 cohort.

    Table S8. FACS panel for influenza-specific B cells in human tissues.

    Table S9. Primary data.

  • Supplementary Material for:

    Circulating TFH cells, serological memory, and tissue compartmentalization shape human influenza-specific B cell immunity

    Marios Koutsakos, Adam K. Wheatley, Liyen Loh, E. Bridie Clemens, Sneha Sant, Simone Nüssing, Annette Fox, Amy W. Chung, Karen L. Laurie, Aeron C. Hurt, Steve Rockman, Martha Lappas, Thomas Loudovaris, Stuart I. Mannering, Glen P. Westall, Michael Elliot, Stuart G. Tangye, Linda M. Wakim, Stephen J. Kent, Thi H. O. Nguyen,* Katherine Kedzierska*

    *Corresponding author. Email: kkedz{at}unimelb.edu.au (K.K.); tho.nguyen{at}unimelb.edu.au (T.H.O.N.)

    Published 14 February 2018, Sci. Transl. Med. 10, eaan8405 (2018)
    DOI: 10.1126/scitranslmed.aan8405

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Gating strategy for circulating ASCs and activated cTFH1 cells.
    • Fig. S2. Specific activation of cTFH1 cells after IIV.
    • Fig. S3. Gating strategy for influenza-specific B cells in PBMC.
    • Fig. S4. Validation of rHA probe staining.
    • Fig. S5. BCR analysis of single rHA+ B cells.
    • Fig. S6. Frequency of IgA+ cells in IgGIgDIgMrHA+ B cells in healthy adults.
    • Fig. S7. Numbers of isotype-specific rHA+ (H3N2/Swi) B cells.
    • Fig. S8. rHA+ B cell kinetics by ELISPOT.
    • Fig. S9. CD20 expression by CD21loCD27+ B cells and ASCs.
    • Fig. S10. Gating strategy for ex vivo live virus ICS.
    • Fig. S11. Vaccination does not induce CD8+ and innate T cell responses.
    • Fig. S12. Gating strategy for T cell phenotyping.
    • Fig. S13. Fold change in influenza-specific B cells during repeated vaccination.
    • Fig. S14. Validating of rHA probes in human tissues.
    • Fig. S15. B cell isotype distributions in paired tissue samples.
    • Table S1. Details of vaccination cohorts.
    • Table S2. Cohorts of human tissues.
    • Table S3. FACS panel for ASCs and cTFH cells.
    • Table S4. FACS panel for influenza-specific T cells after influenza virus infection.
    • Table S5. FACS panel for phenotyping T cells after vaccination.
    • Table S6. FACS panel for influenza-specific B cells in 2015 cohort.
    • Table S7. FACS panel for influenza-specific B cells in 2016 cohort.
    • Table S8. FACS panel for influenza-specific B cells in human tissues.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S9 (Microsoft Excel format). Primary data.

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