Rejuvenating conventional dendritic cells and T follicular helper cell formation after vaccination

  1. Marisa Stebegg
  2. Alexandre Bignon
  3. Danika Lea Hill
  4. Alyssa Silva-Cayetano
  5. Christel Krueger
  6. Ine Vanderleyden
  7. Silvia Innocentin
  8. Louis Boon
  9. Jiong Wang
  10. Martin S Zand
  11. James Dooley
  12. Jonathan Clark
  13. Adrian Liston
  14. Edward Carr
  15. Michelle A Linterman  Is a corresponding author
  1. Laboratory of Lymphocyte Signalling and Development, Babraham Institute, United Kingdom
  2. Epigenetics Programme, Babraham Institute, United Kingdom
  3. Bioceros BV, Netherlands
  4. Division of Nephrology, Department of Medicine and Clinical and Translational Science Institute, University of Rochester Medical Center, United States
  5. Autoimmune Genetics Laboratory, VIB and University of Leuven, Belgium
  6. Biological Chemistry, Babraham Institute, United Kingdom
  7. Department of Medicine, University of Cambridge, United Kingdom
8 figures, 2 tables and 2 additional files

Figures

Poor induction of circulating Tfh (cTfh)-like cells in older persons upon influenza vaccination.

(A) Vaccination and venepuncture schedule for the seasonal influenza vaccination study, 18-36yo n = 16, 65-75yo n = 18. (B) Change in antibody titre of anti-A.Tex12 IgG, an influenza A …

Figure 1—source data 1

Poor induction of circulating Tfh (cTfh)-like cells in older persons upon influenza vaccination.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig1-data1-v1.xlsx
Figure 2 with 1 supplement
Germinal centre (GC) responses are impaired in aged mice.

2–3 month-old adult and 22–24 month-old aged C57BL/6 mice were subcutaneously immunised with NP-1W1K (A-C, E-M) or NP-KLH (D) in Alum. Their draining inguinal lymph nodes (LNs) were analysed by flow …

Figure 2—source data 1

Germinal centre (GC) responses are impaired in aged mice.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Gating strategy for GC B and Tfh cell populations.

2–3 month-old adult and 22–24 month-old aged C57BL/6 mice were immunised subcutaneously with NP-1W1K in Alum. Their draining inguinal lymph node cells were analysed by flow cytometry ten days later. …

Figure 3 with 1 supplement
Activation of cDC2s and T cell priming are impaired in aged mice.

(A–B) 1–5 × 106 CellTrace-labelled hen egg lysozyme (HEL)-specific TCR7 (CTV+TCRVβ3+CD45.1+CD4+) cells or (C-D) 5 × 105 ovalbumin (OVA)-specific (CTV+TCRVα2+CD45.1+CD4+) OTII cells were adoptively …

Figure 3—source data 1

Activation of cDC2s and T cell priming are impaired in aged mice.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Migratory cDC2s are the main antigen-bearing cells in draining lymph nodes (LNs).

(A–C) 5 × 104 OVA-specific (CD45.1+TCRVα2+CD4+) OTII cells were adoptively transferred into 2–3 month-old and 22–24 month-old C57BL/6 recipients, which were subsequently immunised subcutaneously …

Figure 3—figure supplement 1—source data 1

Migratory cDC2s are the main antigen-bearing cells in draining lymph nodes.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig3-figsupp1-data1-v1.xlsx
Reduced type I interferon (IFN-I) signalling in cDC2s from aged mice.

(A) Principal component analysis (PCA) of the 1000 genes with the largest variance in sorted GFP+CD11b+ cDC2s cells from adult 2-month-old (blue) and aged 23-month-old (pink) mice (n = 6 per group). …

Figure 4—source data 1

Reduced type I interferon (IFN-I) signalling in cDC2s from aged mice.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig4-data1-v1.xlsx
Poor vaccine responses in older persons correlate with attenuated IFN-I signalling.

(A) Time course of the expression of IFN-I-stimulated genes in whole PBMC after influenza vaccination. Median expression of IFN-I-stimulated genes were calculated for each sample at each day and …

Figure 5—source data 1

Poor vaccine responses in older persons correlate with attenuated IFN-I signalling.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
Lack of IFN-I signalling in DCs impairs Tfh cell differentiation.

(A–B) Quantitation of median fluorescence intensity (MFI) levels of CD80 and CD86 on GFP+ CD11b+ cDC2s in mice treated with anti-IFNAR1 blocking or isotype control antibodies (n = 7 per …

Figure 6—source data 1

Lack of IFN-I signalling in DCs impairs Tfh cell differentiation.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Lack of IFN-I signalling in DCs does not affect early antigen-specific antibody responses.

(A–C) Quantitation of NP-specific IgG1 antibody responses in the serum of Ifnar1fl/fl:Itgaxcre/+ or Ifnar1fl/fl:Itgax+/+ control mice seven days after immunisation with NP-1W1K in Alum by ELISA (n = …

Figure 6—figure supplement 1—source data 1

Lack of IFN-I signalling in DCs does not affect early antigen-specific antibody responses.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig6-figsupp1-data1-v1.xlsx
Figure 7 with 1 supplement
Imiquimod rejuvenates cDC2s in aged mice by enhancing IFN-I signalling.

(A) Schematic representation of the experimental set-up. (B-G) 22–24 month-old mice were immunised subcutaneously with Eα-GFP in IFA. Half of the mice were topically treated with imiquimod cream …

Figure 7—source data 1

Imiquimod rejuvenates cDC2s in aged mice by enhancing IFN-I signalling.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Imiquimod induces IFN-I signalling and boosts cDC2 responses.

2–3 month-old mice were immunised subcutaneously with either Eα-GFP in IFA. Half of the mice were topically treated with imiquimod cream over their immunisation sites. 22 hr after immunisation with …

Figure 7—figure supplement 1—source data 1

Imiquimod boosts Tfh cell differentiation in young mice.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig7-figsupp1-data1-v1.xlsx
Figure 8 with 1 supplement
Imiquimod rejuvenates Tfh cell differentiation.

2–3 month-old and 22–24 month-old C57BL/6 mice (A-F, I-K) or 2 month old Ifnar1fl/fl:Itgaxcre/+ and Ifnar1fl/fl:Itgax+/+ littermate controls (G-H) were all subcutaneously immunised with NP-1W1K in …

Figure 8—source data 1

Imiquimod rejuvenates Tfh cell differentiation.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig8-data1-v1.xlsx
Figure 8—figure supplement 1
Imiquimod treatment does not affect early antigen-specific antibody responses.

(A–C) 22–24 month-old mice were immunised subcutaneously with NP-1W1K in Alum. Half of the mice were topically treated with imiquimod cream over their immunisation sites. Seven days later, serum …

Figure 8—figure supplement 1—source data 1

Imiquimod treatment does not affect early antigen-specific antibody responses.

https://cdn.elifesciences.org/articles/52473/elife-52473-fig8-figsupp1-data1-v1.xlsx

Tables

Table 1
Antibodies used for flow cytometry of human PBMC.
AntibodyCompany and cloneDilution
eFluor780 Viability dyeeBioscience1:5000
APC-eFluor780-coupled anti-human CD14eBioscience (61D3)1:50
APC-eFluor780-coupled anti-human CD16eBioscience (eBioCB16)1:50
APC-eFluor780-coupled anti-human CD19eBioscience (HIB19)1:50
BUV395-coupled anti-human CD3BD (UCHT1)1:100
PerCp-Cy5.5-coupled anti-human CD4BD (RPA-T4)1:50
BUV737-coupled anti-human CD45RABD (HI100)1:25
PE-Cy7-coupled anti-human PD1eBioscience (eBioJ105)1:25
BB515-coupled anti-human CXCR5BD (RF8B2)1:25
Table 2
Antibodies and conjugated probes used for flow cytometry and FACS of mouse tissues.
AntibodySupplier (Clone)Dilution
PE/PE-Cy7-coupled anti-mouse Bcl6BD Biosciences (K112-91)1:100
PE-Cy7-coupled anti-mouse CD95BD Biosciences (Jo2)1:200
BV605-coupled anti-mouse IgG1BD Biosciences (A85-1)1:100
PE-Cy7/BUV395-coupled anti-mouse CD3BD Biosciences (145–2 C11)1:300
PE-Cy7/BUV395-coupled anti-mouse CD19BD Biosciences (1D3)1:200-1:300
PE-Cy7/BUV395-coupled anti-mouse B220BD Biosciences (RA3-6B2)1:300
BUV395-coupled anti-mouse CD8aBD Biosciences (53–6.7)1:200
AF647-coupled anti-mouse CD64BD Biosciences (X54-5/71)1:200
PE-Cf594-coupled anti-mouse CD11bBD Biosciences (M1/70)1:200
BV786-coupled anti-mouse CD103BD Biosciences (M290)1:200
PE/BV510-coupled anti-mouse CD86BD Biosciences (GL1)1:300
APC-AF780-coupled anti-mouse PD1eBioscience (J43)1:200
APC/Foxp3-coupled anti-mouse Foxp3eBioscience (FJK-16S)1:100-1:200
AF488/AF700-coupled anti-mouse Ki67eBioscience (SolA15)1:100
Biotin-coupled anti-mouse Gr1eBioscience (RB6-8C5)1:200
eF450-coupled anti-mouse CD38eBioscience (90)1:400
PerCp-Cy5.5-coupled anti-mouse CD172aeBioscience (P84)1:200
eF450-coupled anti-mouse CD24eBioscience (M1/69)1:500
APC/APC-AF870-coupled anti-mouse CD11ceBioscience (N418)1:200
PE-Cy5/APC-coupled anti-mouse CD80eBioscience (16-10A1)1:300
PerCp-Cy5.5-coupled anti-mouse CD45.2eBioscience (104)1:200
AF700-coupled anti-mouse MHC-IIeBioscience (M5/114.12.2)1:400
BV421-coupled anti-mouse CXCR5Biolegend (L138D7)1:100
V500/PE/BV605-coupled anti-mouse CD4Biolegend (RM4-5)1:400-1:800
BV510/BV785-coupled anti-mouse B220Biolegend (RA3-6B2)1:200-1:400
PerCp-Cy5.5-coupled anti-mouse CD44Biolegend (IM7)1:200
AF488-coupled anti-mouse GL7Biolegend (GL7)1:100
BV605/PE-coupled anti-mouse F4/80Biolegend (BM8)1:200
BV650-coupled anti-mouse XCR1Biolegend (ZET)1:200
PE-Cy7-coupled anti-mouse CD40Biolegend (3/23)1:300
AF700-coupled anti-mouse CD45.1Biolegend (A20)1:200
PerCp-Cy5.5-coupled anti-mouse PDCA-1Biolegend (927)1:100
PE-coupled anti-mouse TCR Vβ3BD Biosciences (KJ25)1:100
APC-coupled anti-mouse TCR Vα2eBioscience (B20.1)1:100
APC/PE-Cy7/BV510-coupled Streptavidin StrepavidinStreptavidinBiolegend, eBioscience1:600
Biotin-coupled anti-mouse Eα 52–68 peptideeBioscience (eBioY-Ae)1:200
PE-coupled 1W1K-IAb tetramerNIH Tetramer core facility1:100
PE-coupled anti-mouse pSTAT1BD Biosciences Phosflow (pY701)1:10

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