1. Critical Role for Skin-Derived Migratory DCs and Langerhans Cells in TFH and GC Responses after Intradermal Immunization 
Clément Levin, Olivia Bonduelle, Charles Nuttens, Charlotte Primard, Bernard Verrier, Alexandre Boissonnas, Béhazine Combadière 
Journal of Investigative Dermatology 
Volume 137, Issue 9, Pages (September 2017)
DOI: /j.jid
Copyright © 2017 The Authors Terms and Conditions

2. Figure 1
Skin migratory cells are necessary for TFH and GC B-cell induction after intradermal immunization. (a) Representative gating strategy for the identification of TFH cells (PD-1+CXCR5+) in DLNs, after pre-gating on live singlet CD3+CD4+ T lymphocytes after intradermal immunization with PBS or p24-PLA NPs in control and EC mice. Unless specified otherwise, ears were removed at 1 hour after injection. (b) Absolute numbers of TFH cells in DLNs 7 days after ID immunization in PBS or NPs or NPs + EC conditions (n = 14–18 mice per group). (c) TFH cell numbers in DLNs 7 days after immunization with PBS or coumarin-6 NPs or p24-NPs. (d) TFH-cell numbers in DLNs and nondraining LNs 7 days after immunization with NPs compared with control PBS-injected mice (n = 3). (e) Absolute numbers of GC B cells (left), p24-specific IgA- (middle), or IgG- (right) secreting cells in DLNs 7 days after immunization with NPs in control and EC mice. Antibody-secreting cells were evaluated by ELISPOT assay. B cells were stimulated for 16 hours with HIV-1 p24 overlapping peptides. (f) Anti-p24 IgG titers in serum of mice at 28 days after immunization with NPs in control and EC-treated mice as determined by ELISA. Data are pooled from three independent experiments of three to six mice/group and are shown as mean ± standard error of the mean. One-way analysis of variance with Bonferroni correction. Statistical significance is indicated by **P < 0.01, ***P < 0.001, and ****P < ASC, antibody-secreting cell; C6, coumarin-6; DLN, draining lymph node; EC, ear cut; GC, germinal center; NP, poly-lactic acid nanoparticle; ns, not significant; PBS, phosphate buffered saline; TFH, T follicular helper.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
Similar amounts of antigen in control and EC animals after injection of fluorescent NPs. (a) Histological section of the ear depicting lymphatic vessel network (anti-Lyve-1, red) 4 hours after intradermal injection of 1.2 × 1011 C6-NPs (green). (b) Ears were removed (EC) 1 hour after injection (lower panel) or not removed (upper panel), and skin DLNs were analyzed by microscopy after 4 hours (left) or 24 hours (right). Scale bar = 100 μm. (c) Quantification of total NP+ cells in DLNs of control and EC mice at 4 hours and 24 hours after injection of C6-NPs, as determined by flow cytometry. C6-NP, coumarin-6–labeled nanoparticle; DAPI, 4’,6-diamidino-2-phenylindole; DLN, draining lymph node; EC, ear cut; h, hour; NP, nanoparticle; PBS, phosphate buffered saline.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
Blood-derived inflammatory cells bypass the skin to reach the DLNs but are not involved in the generation of TFH and GC B cells. (a–c) Analysis of inflammatory cell recruitment to DLNs at 1 hour, 4 hours, 8 hours, and 24 hours after intradermal injection of NPs in control or EC mice. (a) Gating strategy for identification of neutrophils (CD11b+, Ly6C+, Ly6G+), inflammatory monocytes (CD11b+, Ly6C+, Ia(b)–), and inflammatory DCs and macrophages (CD11b+, Ly6C+, Ia(b)+, F4/80+/–) by flow cytometry. (b) Relative proportions of each inflammatory cell subset among total CD11b+ cells at indicated times after PBS or NP intradermal injection. (c) Percentages of each subset of inflammatory cells among total CD11b+ cell fraction in control and EC mice. (d) Absolute numbers of TFH (upper panel) and GC B cells (lower panel) in DLNs 7 days after immunization with PBS or NPs in control and EC mice. Ears were removed at 1 hour, 8 hours, 16 hours, or 24 hours after injection. One-way analysis of variance with Bonferroni correction was applied to determine statistical significance. *P < 0.05, **P < 0.01, ****P < Data are compared with PBS-injected mice followed by EC at (b–c) 8 hours or (d) 1 hour and represented as mean ± standard error of mean. Data are pooled from three independent experiments of three mice/group. DC, dendritic cell; DLN, draining lymph node; EC, ear cut; GC, germinal center; h, hour; NP, nanoparticle; ns, not significant; PBS, phosphate buffered saline; TFH, T follicular helper.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
Skin-migratory DCs produce key signals to the DLN. (a) Gating strategy for identification of resident DCs and migratory DC subsets. (b) mRNA expression of genes in sorted resident (red) and migratory (blue) DCs of the DLN at day 3 after ID injection of PBS (hollow bars) or p24-NPs (plain bars). Cells from three to six mice were pooled. Data are representative of two independent experiments. DC, dendritic cell; DLN, draining lymph node; LC, Langerhans cell; ND, no data; NP, nanoparticle; PBS, phosphate buffered saline.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 5
Langerhans cells promote TFH cell polarization and GC formation. (a) Langerin-DT receptor mice were injected intraperitoneally with 1 μg of diphtheria toxin (DT) on day –13 (condition DT1: no LCs) or day –2 (condition DT2: no LCs and CD103+ dermal DCs) before intradermal immunization with PBS or NPs. (b) Efficiency of cell depletion after DT treatment. (Left panel) Analysis of Langerin-green fluorescent protein expression among MHCII+ skin cells 2 days after PBS or DT injection in langerin-DT receptor mice. (Middle and right panels) Percentage of langerin+ and langerin– cells among MHCII+ cells 2 days after PBS or DT injection in langerin-DTR mice. (c) Absolute numbers of TFH cells in two DLNs 7 days after NP or PBS ID immunization in DT1, DT2, or untreated animals. (d) GC staining on the histological section of skin DLNs by anti-GL7 (green), anti-IgD (blue), and anti-CD4 (red). Mice were injected with PBS or NPs with or without DT treatment 2 days before immunization. GC areas are expressed in μm2. Scale bar = 50 μm. (e) Absolute numbers of GC B cells (left panel), and p24-specific IgG- (middle panel) or IgA- (right panel) antibody-secreting cells in two DLNs 7 days after NP or PBS immunization in DT1, DT2, or untreated animals. Statistical significance is indicated by *P < 0.05, **P < One-way analysis of variance with Bonferroni correction. Results are representative of three to five independent experiments of three mice/group. ASC, antibody-secreting cell; DT, diphtheria toxin; GC, germinal center; LC, Langerhans cell; MHC, major histocompatibility complex; NP, nanoparticle; PBS, phosphate buffered saline; TFH, T follicular helper.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions