CD301b+ dendritic cells suppress T follicular helper cells and antibody responses to protein antigens
- Yale University School of Medicine, United States
- Howard Hughes Medical Institute, Yale University School of Medicine, United States
- University of Pittsburgh, United States
Figures
Figure 1
Depletion of CD301b+ DCs leads to enhanced antibody responses.
(a,b) WT and Mgl2-DTR mice were injected i.p. with 0.5 µg DT on days −1, +1 and +3. Mice were immunized with 50 µg papain in 20 µl phosphate-buffered saline (PBS) with or without (No OVA at 1°) 5 µg …
Figure 2 with 1 supplement
Depletion of CD301b+ DCs enhances antibody responses in the non-skin dLNs.
(a,b) Mice were immunized with 10 µg OVA in 100 µl alum i.p. and received i.p. injection of 10 µg OVA without adjuvant on day 14. Sera were harvested on day 21 for OVA-specific antibody ELISA. Bars …
Figure 2—figure supplement 1
Phenotype of CD301b+ DCs in LNs and peritoneal cavity.
DT-treated WT or Mgl2-DTR mice were immunized with OVA and papain in the right footpad. Right (dLN) or left (ndLN) popliteal LNs and peritoneal washes were harvested one day after immunization and …
Figure 3
Depletion of CD301b+ DCs leads to enhanced antibody responses to the antigen immunized with weak or no adjuvant.
(a) Immunization and sample collection timeline. (b) Mgl2-DTR mice were treated with 0.5 µg DT or its inactive mutant CRM197 (CRM) on days -1 and +2 and immunized on day 0 with 50 µg papain plus …
Figure 4
Depletion of CD301b+ DCs enhances antibody responses to type 1 immunization when adjuvant is limited.
WT and Mgl2-DTR mice were treated with 0.5 µg DT on days −1 and +2 and immunized in the footpad with 5 µg OVA plus indicated amount of CpG2216 or LPS as in a. All mice received i.p. injection of 10 …
Figure 5
Depletion of CD301b+ DCs enhances the development of antigen-specific B cells with GC B cell phenotype without negatively affecting the affinity of antibodies.
As in a, mice were treated with DT and immunized with 5 µg NP16OVA plus 50 µg papain in the footpad. Two weeks later, all mice received i.p. injection of NP16OVA in PBS. (b) Serum antibodies against …
Figure 6
Depletion of CD301b+ DCs enhances autoreactive antibodies.
WT or Mgl2-DTR mice were treated i.p. with PBS or 0.5 µg DT every third day for 9 days as in a. Sera were harvested at day 60 and examined for anti-nuclear IgG antibodies by ELISA (b). Bars indicate …
Figure 7 with 1 supplement
Depletion of CD301b+ DCs leads to GC B cell expansion in response to immunization with OVA and papain.
(a,b) WT and Mgl2-DTR (DTR) mice were treated with DT on days −1 and +2 and immunized with 50 µg papain and 5 µg OVA in 20 µl PBS in the right footpad. The left non-draining (L) or right draining …
Figure 7—figure supplement 1
Cellular kinetics in CD301b+ DC-depleted mice immunized with papain and OVA.
Cell numbers of each indicated subset were calculated in mice shown in Figure 7b and Figure 8a and b.
Figure 8
GC B cell expansion in CD301b+ DC-depleted mice is accompanied with Tfh expansion.
(a,b) Mice were immunized as in Figure 7a. Tfh (PD-1+CXCR5+) differentiation (a) and the ratio between follicular (PSGL1loPD-1+) and extrafollicular (PSGL1hiPD-1-) cells within CD44hi CD4+ T cells (b…
Figure 9 with 1 supplement
Migratory and inflammatory DC subsets are not required for the enhanced antibody, Tfh and GC B cell development induced by the CD301b+ DC depletion.
(a–d) Mice were treated with 0.5 µg DT and immunized with 50 µg papain and 5 µg OVA in the footpad. At the time of immunization, different DC subsets were either intact (+) or depleted (∆) depending …
Figure 9—figure supplement 1
Depletion of various skin-dLN DC subsets in different DTR mice.
Mice that carry Mgl2-DTR, huLangerin-DTR (huLang-DTR), msLangerin-DTR (msLang-DTR) and/or CD11c-DTR in their genetic construct were treated i.p. with 0.5 µg DT or PBS and DC subsets in the skin-dLNs …
Figure 10 with 2 supplements
Blockade of PD-L1 at the time of immunization enhances Tfh and GC B cell responses in CD301b+ DC-dependent manner.
(a) Pooled skin-dLN cells from naïve WT mice were stained and gated as indicated. Representative flow cytometry plots from three independent experiments are shown. (b–f) WT and Mgl2-DTR mice were …
Figure 10—figure supplement 1
Depletion of CD301b+ DCs does not reduce PD-1 ligand expression on the residual migratory DCs.
(a) WT or Mgl2-DTR mice were treated with DT, then skin-dLNs were harvested 24 hr later for flow cytometry. (b) Mice were treated with DT and anti-PD-L1 or PD-L2 blocking mAb, and immunized with OVA …
Figure 10—figure supplement 2
Differential effects of PD-1 ligand blockade on CD4T and B cell compartments in CD301b+ DC-intact and depleted animals.
(a–c) Tfh and GC B cell development was assessed in the dLN (a,b) or in the contralateral ndLN (c) in mice shown in Figure 10b–d. (d) WT mice were treated with indicated antibodies and immunized as …
Figure 11
Model for CD301b+ DC-mediated suppression of humoral immunity.
(a) Summary of the results shown in this study. (b) Model for Tfh and GCB cell suppression by CD301b+ DCs. CD301b+ DCs express high levels of PD-L1 to inhibit the expansion of Tfh. The impaired Tfh …
Author response image 1
IL-4 production from non-T cells in CD301b+ DC-depleted mice.
Dashed line indicates staining background.
Author response image 2
IgG3 production in mice immunized with OVA plus papain in the footpad.
All mice received intraperitoneal injection of OVA without papain on day 14.
