Increased costimulation promotes enhanced cytokine signaling and Th2 differentiation. (A–D) Naïve CD45.1 OT-II T cells were transferred to B6 mice and injected with papain OVA in the ear pinnae or front footpad. Mice were treated intraperitoneally with α-CD62L blocking antibody 6 h after immunization and dLNs were harvested and assessed by flow cytometry at 48 h. (A) Representative flow plots of Gata3 and CD25 expression on CD44+ or naïve OT-II cells in auricular dLNs. (B) Representative histograms and quantification of pSTAT5 and pSTAT6 staining for the indicated cell subsets within the same sample. (C) Representative histograms and quantification of pSTAT5 and pSTAT6 expression on CD44+ OT-II cells in auricular and brachial LNs. (D) pSTAT5 and pSTAT6 correlation with Gata3 gMFI. (E–H) Mice were treated as in A but treated intraperitoneally with α-CD28 blocking antibody, α-IL-2 blocking antibody, or IgG isotype control 24 h after immunization, and dLNs were harvested 48 h later. (E and F) Representative flow cytometry plots (E) and quantification (F) of the frequency of pSTAT5+ and pSTAT6+ cells, gMFI of pSTAT5 and pSTAT6, and Gata3 expression in CD44+ OT-II cells. (G) Representative images depicting OT-II macro-clustering and Th2 differentiation in auricular dLN treated with isotype control (top) or α-IL-2 blocking antibody (bottom). Gata3 signal is masked outside of Ki67+IRF4+ activated cells for visual clarity. (H) Histocytometry analysis of percent activated OT-II cells coexpressing 4get-GFP and Gata3, OT-II localization at the T–B border, and the ratio of densely clustered OT-II cells with the indicated treatment. Data shown represent one independent experiment with at least n = 6 independently immunized lymph nodes from three mice per group. Graphs show mean ± SD and were analyzed using unpaired Student’s t test. Paired t tests were performed when comparing responses within the same experimental tissue (panel B). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Dashed lines represent T–B border. Figures are representative of two to three independent experiments.
Figure 7.

Increased costimulation promotes enhanced cytokine signaling and Th2 differentiation. (A–D) Naïve CD45.1 OT-II T cells were transferred to B6 mice and injected with papain OVA in the ear pinnae or front footpad. Mice were treated intraperitoneally with α-CD62L blocking antibody 6 h after immunization and dLNs were harvested and assessed by flow cytometry at 48 h. (A) Representative flow plots of Gata3 and CD25 expression on CD44+ or naïve OT-II cells in auricular dLNs. (B) Representative histograms and quantification of pSTAT5 and pSTAT6 staining for the indicated cell subsets within the same sample. (C) Representative histograms and quantification of pSTAT5 and pSTAT6 expression on CD44+ OT-II cells in auricular and brachial LNs. (D) pSTAT5 and pSTAT6 correlation with Gata3 gMFI. (E–H) Mice were treated as in A but treated intraperitoneally with α-CD28 blocking antibody, α-IL-2 blocking antibody, or IgG isotype control 24 h after immunization, and dLNs were harvested 48 h later. (E and F) Representative flow cytometry plots (E) and quantification (F) of the frequency of pSTAT5+ and pSTAT6+ cells, gMFI of pSTAT5 and pSTAT6, and Gata3 expression in CD44+ OT-II cells. (G) Representative images depicting OT-II macro-clustering and Th2 differentiation in auricular dLN treated with isotype control (top) or α-IL-2 blocking antibody (bottom). Gata3 signal is masked outside of Ki67+IRF4+ activated cells for visual clarity. (H) Histocytometry analysis of percent activated OT-II cells coexpressing 4get-GFP and Gata3, OT-II localization at the T–B border, and the ratio of densely clustered OT-II cells with the indicated treatment. Data shown represent one independent experiment with at least n = 6 independently immunized lymph nodes from three mice per group. Graphs show mean ± SD and were analyzed using unpaired Student’s t test. Paired t tests were performed when comparing responses within the same experimental tissue (panel B). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Dashed lines represent T–B border. Figures are representative of two to three independent experiments.

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