Cellular composition of Th2 microenvironments after papain immunization or N.b. infection. (A) 4get-GFP mice were immunized in the ear pinnae with papain, and auricular dLNs or naïve LNs was harvested for confocal microscopy 3 days later. Representative images and zoom-ins with the indicated markers are shown. (B and C) 4get-GFP mice were inoculated subcutaneously at the tail base with 500 N.b. L3 larvae and the draining inguinal LNs and mesenteric LN (MLN), or LNs from naïve mice, were harvested 3 or 6 days later, respectively, for confocal microscopy. Representative images and zoom-ins depicting Th2 macro-clustering with the indicated markers are shown. (D) Naïve CD45.2 4get.OT-II T cells were transferred to CD45.1 mice and injected with papain OVA in the ear pinnae, treated with α-CD62L blocking antibody at 6 h, and harvested for flow cytometry 48 h later. Representative gating for T follicular helper (Tfh) and T effector (Teff) OT-II cells is shown with the indicated markers. (E) B6 mice were immunized with papain in the ear pinnae and auricular dLNs were harvested 3 days later for flow cytometry. Representative gating for endogenous T cell activation is shown with Gata3, BCL6, and CD25 identifying Tfh and Teff T cells. (F) KN2+/− mice were immunized with papain in the ear pinnae and auricular dLNs or naïve LNs were harvested 4 days later for flow cytometry. Representative flow cytometry plot and quantification of huCD2 and Gata3 expression is shown. (G and H) Representative gating schemes for histocytometry (G) or flow cytometry (H) analysis of myeloid cells. (I and J) CD11c-Cre+ IRF4fl/fl or CD11c-Cre− IRF4fl/fl were immunized with papain in the ear pinnae and dLNs were harvested 2 days later for flow cytometry or confocal microscopy. (I) Quantification of the indicated myeloid cell subsets is shown. (J) Representative images depicting CD301b+ and CD207+ cell localization in dLNs. Data shown represents one independent experiment with at least n = 4 independently immunized lymph nodes from two mice per group. Graphs show mean ± SD and were analyzed using unpaired Student’s t test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). BF = B cell follicles; TZ = T cell zone. Dashed lines represent T–B border. Inset scale bars represent 20 μm. A and D–H are representative of five independent experiments. B, C, I, and J are representative of two to three independent experiments.
Figure S1.

Cellular composition of Th2 microenvironments after papain immunization or N.b. infection. (A) 4get-GFP mice were immunized in the ear pinnae with papain, and auricular dLNs or naïve LNs was harvested for confocal microscopy 3 days later. Representative images and zoom-ins with the indicated markers are shown. (B and C) 4get-GFP mice were inoculated subcutaneously at the tail base with 500 N.b. L3 larvae and the draining inguinal LNs and mesenteric LN (MLN), or LNs from naïve mice, were harvested 3 or 6 days later, respectively, for confocal microscopy. Representative images and zoom-ins depicting Th2 macro-clustering with the indicated markers are shown. (D) Naïve CD45.2 4get.OT-II T cells were transferred to CD45.1 mice and injected with papain OVA in the ear pinnae, treated with α-CD62L blocking antibody at 6 h, and harvested for flow cytometry 48 h later. Representative gating for T follicular helper (Tfh) and T effector (Teff) OT-II cells is shown with the indicated markers. (E) B6 mice were immunized with papain in the ear pinnae and auricular dLNs were harvested 3 days later for flow cytometry. Representative gating for endogenous T cell activation is shown with Gata3, BCL6, and CD25 identifying Tfh and Teff T cells. (F) KN2+/− mice were immunized with papain in the ear pinnae and auricular dLNs or naïve LNs were harvested 4 days later for flow cytometry. Representative flow cytometry plot and quantification of huCD2 and Gata3 expression is shown. (G and H) Representative gating schemes for histocytometry (G) or flow cytometry (H) analysis of myeloid cells. (I and J) CD11c-Cre+ IRF4fl/fl or CD11c-Cre− IRF4fl/fl were immunized with papain in the ear pinnae and dLNs were harvested 2 days later for flow cytometry or confocal microscopy. (I) Quantification of the indicated myeloid cell subsets is shown. (J) Representative images depicting CD301b+ and CD207+ cell localization in dLNs. Data shown represents one independent experiment with at least n = 4 independently immunized lymph nodes from two mice per group. Graphs show mean ± SD and were analyzed using unpaired Student’s t test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). BF = B cell follicles; TZ = T cell zone. Dashed lines represent T–B border. Inset scale bars represent 20 μm. A and D–H are representative of five independent experiments. B, C, I, and J are representative of two to three independent experiments.

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