(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 …
Poor induction of circulating Tfh (cTfh)-like cells in older persons upon influenza vaccination.
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 …
Germinal centre (GC) responses are impaired in aged mice.
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. …
(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 …
Activation of cDC2s and T cell priming are impaired in aged mice.
(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 …
Migratory cDC2s are the main antigen-bearing cells in draining lymph nodes.
(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). …
Reduced type I interferon (IFN-I) signalling in cDC2s from aged mice.
(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 …
Poor vaccine responses in older persons correlate with attenuated IFN-I signalling.
(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 …
Lack of IFN-I signalling in DCs impairs Tfh cell differentiation.
(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 = …
Lack of IFN-I signalling in DCs does not affect early antigen-specific antibody responses.
(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 …
Imiquimod rejuvenates cDC2s in aged mice by enhancing IFN-I signalling.
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 …
Imiquimod boosts Tfh cell differentiation in young mice.
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 …
Imiquimod rejuvenates Tfh cell differentiation.
(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 …
Imiquimod treatment does not affect early antigen-specific antibody responses.
Antibody | Company and clone | Dilution |
---|---|---|
eFluor780 Viability dye | eBioscience | 1:5000 |
APC-eFluor780-coupled anti-human CD14 | eBioscience (61D3) | 1:50 |
APC-eFluor780-coupled anti-human CD16 | eBioscience (eBioCB16) | 1:50 |
APC-eFluor780-coupled anti-human CD19 | eBioscience (HIB19) | 1:50 |
BUV395-coupled anti-human CD3 | BD (UCHT1) | 1:100 |
PerCp-Cy5.5-coupled anti-human CD4 | BD (RPA-T4) | 1:50 |
BUV737-coupled anti-human CD45RA | BD (HI100) | 1:25 |
PE-Cy7-coupled anti-human PD1 | eBioscience (eBioJ105) | 1:25 |
BB515-coupled anti-human CXCR5 | BD (RF8B2) | 1:25 |
Antibody | Supplier (Clone) | Dilution |
---|---|---|
PE/PE-Cy7-coupled anti-mouse Bcl6 | BD Biosciences (K112-91) | 1:100 |
PE-Cy7-coupled anti-mouse CD95 | BD Biosciences (Jo2) | 1:200 |
BV605-coupled anti-mouse IgG1 | BD Biosciences (A85-1) | 1:100 |
PE-Cy7/BUV395-coupled anti-mouse CD3 | BD Biosciences (145–2 C11) | 1:300 |
PE-Cy7/BUV395-coupled anti-mouse CD19 | BD Biosciences (1D3) | 1:200-1:300 |
PE-Cy7/BUV395-coupled anti-mouse B220 | BD Biosciences (RA3-6B2) | 1:300 |
BUV395-coupled anti-mouse CD8a | BD Biosciences (53–6.7) | 1:200 |
AF647-coupled anti-mouse CD64 | BD Biosciences (X54-5/71) | 1:200 |
PE-Cf594-coupled anti-mouse CD11b | BD Biosciences (M1/70) | 1:200 |
BV786-coupled anti-mouse CD103 | BD Biosciences (M290) | 1:200 |
PE/BV510-coupled anti-mouse CD86 | BD Biosciences (GL1) | 1:300 |
APC-AF780-coupled anti-mouse PD1 | eBioscience (J43) | 1:200 |
APC/Foxp3-coupled anti-mouse Foxp3 | eBioscience (FJK-16S) | 1:100-1:200 |
AF488/AF700-coupled anti-mouse Ki67 | eBioscience (SolA15) | 1:100 |
Biotin-coupled anti-mouse Gr1 | eBioscience (RB6-8C5) | 1:200 |
eF450-coupled anti-mouse CD38 | eBioscience (90) | 1:400 |
PerCp-Cy5.5-coupled anti-mouse CD172a | eBioscience (P84) | 1:200 |
eF450-coupled anti-mouse CD24 | eBioscience (M1/69) | 1:500 |
APC/APC-AF870-coupled anti-mouse CD11c | eBioscience (N418) | 1:200 |
PE-Cy5/APC-coupled anti-mouse CD80 | eBioscience (16-10A1) | 1:300 |
PerCp-Cy5.5-coupled anti-mouse CD45.2 | eBioscience (104) | 1:200 |
AF700-coupled anti-mouse MHC-II | eBioscience (M5/114.12.2) | 1:400 |
BV421-coupled anti-mouse CXCR5 | Biolegend (L138D7) | 1:100 |
V500/PE/BV605-coupled anti-mouse CD4 | Biolegend (RM4-5) | 1:400-1:800 |
BV510/BV785-coupled anti-mouse B220 | Biolegend (RA3-6B2) | 1:200-1:400 |
PerCp-Cy5.5-coupled anti-mouse CD44 | Biolegend (IM7) | 1:200 |
AF488-coupled anti-mouse GL7 | Biolegend (GL7) | 1:100 |
BV605/PE-coupled anti-mouse F4/80 | Biolegend (BM8) | 1:200 |
BV650-coupled anti-mouse XCR1 | Biolegend (ZET) | 1:200 |
PE-Cy7-coupled anti-mouse CD40 | Biolegend (3/23) | 1:300 |
AF700-coupled anti-mouse CD45.1 | Biolegend (A20) | 1:200 |
PerCp-Cy5.5-coupled anti-mouse PDCA-1 | Biolegend (927) | 1:100 |
PE-coupled anti-mouse TCR Vβ3 | BD Biosciences (KJ25) | 1:100 |
APC-coupled anti-mouse TCR Vα2 | eBioscience (B20.1) | 1:100 |
APC/PE-Cy7/BV510-coupled Streptavidin StrepavidinStreptavidin | Biolegend, eBioscience | 1:600 |
Biotin-coupled anti-mouse Eα 52–68 peptide | eBioscience (eBioY-Ae) | 1:200 |
PE-coupled 1W1K-IAb tetramer | NIH Tetramer core facility | 1:100 |
PE-coupled anti-mouse pSTAT1 | BD Biosciences Phosflow (pY701) | 1:10 |
Key Resources Table.