Th2 macro-clustering and differentiation are site-specific. (A–E) Mice were transferred with naïve 4get.OT-II cells as in Fig. 1 A, injected with the indicated adjuvant plus OVA at different sites in the ear pinnae or front footpad, and corresponding dLNs were harvested and assessed by histocytometry and flow cytometry at 48 h. Representative images depicting OT-II macro-clustering and Th2 differentiation at the T–B border in (A) auricular versus (B) brachial dLNs. (C) Histocytometry analysis of the percent activated OT-II coexpressing 4get-GFP and Gata3, OT-II localization, and the ratio of macro-clustered versus total OT-II in the indicated LNs. (D and E) Representative flow plots and quantification of CD44+ OT-II cell number, frequency of 4get-GFP+ and CXCR3+ cells, and Gata3 and Tbet gMFI of CD44+ OT-II cells. (F and G) 0.5 × 106 naïve 4get.OT-II cells were transferred to recipients and then injected with papain OVA in the ear pinnae or front footpad. 6 days later, cells were labeled intravenously, and dLNs, spleen, and lung were harvested and assessed by flow cytometry. Representative plots and quantification of frequency of 4get-GFP+ and Gata3 gMFI of IV− CD44+ OT-II cells and total CD44+ OT-II cell number are shown. (H and I) Mice were treated as in A but were immunized in the ear pinnae and front footpad with Alum plus OVA. (H) Representative images depicting OT-II macro-clustering and Th2 differentiation at the T–B border in auricular versus brachial dLNs. (I) Histocytometry analysis of the percent activated OT-II coexpressing 4get-GFP and Gata3, OT-II localization, and the ratio of macro-clustered versus total OT-II in the indicated LNs. Data shown represent 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). Au = auricular; Br = brachial; IV = intravenous. Dashed lines represent T–B border. A–E are representative of five independent experiments; F–I are representative of two independent experiments.
Figure 2.

Th2 macro-clustering and differentiation are site-specific. (A–E) Mice were transferred with naïve 4get.OT-II cells as in Fig. 1 A, injected with the indicated adjuvant plus OVA at different sites in the ear pinnae or front footpad, and corresponding dLNs were harvested and assessed by histocytometry and flow cytometry at 48 h. Representative images depicting OT-II macro-clustering and Th2 differentiation at the T–B border in (A) auricular versus (B) brachial dLNs. (C) Histocytometry analysis of the percent activated OT-II coexpressing 4get-GFP and Gata3, OT-II localization, and the ratio of macro-clustered versus total OT-II in the indicated LNs. (D and E) Representative flow plots and quantification of CD44+ OT-II cell number, frequency of 4get-GFP+ and CXCR3+ cells, and Gata3 and Tbet gMFI of CD44+ OT-II cells. (F and G) 0.5 × 106 naïve 4get.OT-II cells were transferred to recipients and then injected with papain OVA in the ear pinnae or front footpad. 6 days later, cells were labeled intravenously, and dLNs, spleen, and lung were harvested and assessed by flow cytometry. Representative plots and quantification of frequency of 4get-GFP+ and Gata3 gMFI of IV− CD44+ OT-II cells and total CD44+ OT-II cell number are shown. (H and I) Mice were treated as in A but were immunized in the ear pinnae and front footpad with Alum plus OVA. (H) Representative images depicting OT-II macro-clustering and Th2 differentiation at the T–B border in auricular versus brachial dLNs. (I) Histocytometry analysis of the percent activated OT-II coexpressing 4get-GFP and Gata3, OT-II localization, and the ratio of macro-clustered versus total OT-II in the indicated LNs. Data shown represent 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). Au = auricular; Br = brachial; IV = intravenous. Dashed lines represent T–B border. A–E are representative of five independent experiments; F–I are representative of two independent experiments.

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