Th2 macro-clustering and differentiation is LFA-1 dependent. (A) B6 mice were immunized in the ear pinnae and front footpad with papain or CpG plus EαGFP and harvested for flow cytometry 48 h later. Expression of ICAM-1 on EαGFP+ and EαGFP− or naïve (untreated, UT) migratory DCs in the indicated LNs is shown. (B–E) Mice were transferred with naïve 4get.OT-II cells, treated with papain OVA as in Fig. 1 A, and administered α-LFA1 blocking antibody or IgG isotype control intraperitoneally 24 h after immunization and dLNs were harvested and assessed by histocytometry or flow cytometry at 48 h. (B) Representative flow plots and (C) quantification of the number of CD44+ OT-II cells, frequency of 4get-GFP+ cells, and Gata3 and IRF4 gMFI of CD44+ OT-II cells with the indicated treatment. (D) Representative images depicting OT-II macro-clustering and Th2 differentiation in auricular dLN treated with isotype control (top) or α-LFA-1 blocking antibody (bottom). (E) Histocytometry analysis of 4get-GFP, Gata3, and pS6 expression, OT-II localization at the T–B border, and the ratio of densely clustered OT-II cells with the indicated treatment is shown. (F and G) B6 mice were inoculated subcutaneously at the tail base with 500 N.b. L3 larvae, treated intraperitoneally with α-LFA1 blocking antibody or IgG isotype control on day 2 and 3 after infection, and the inguinal dLNs, or LNs from naïve mice, were harvested on day 4 for confocal microscopy. (F) Representative images and zoom-ins depicting Th2 macro-clustering with α-LFA1 blocking antibody or isotype control. Gata3 signal is masked outside of Ki67+ activated cells for visual clarity. (G) Histocytometry analysis of Gata3 expression in Ki67+ T cells with the indicated treatment. Data shown represent one independent experiment with at least n = 8 independently immunized lymph nodes from four mice per group. Data from multiple pooled experiments are denoted by different symbols within the same 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 A). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Dashed lines represent T–B border. A–C are representative of four independent experiments. D–G are representative of two independent experiments.
Figure 6.

Th2 macro-clustering and differentiation is LFA-1 dependent. (A) B6 mice were immunized in the ear pinnae and front footpad with papain or CpG plus EαGFP and harvested for flow cytometry 48 h later. Expression of ICAM-1 on EαGFP+ and EαGFP− or naïve (untreated, UT) migratory DCs in the indicated LNs is shown. (B–E) Mice were transferred with naïve 4get.OT-II cells, treated with papain OVA as in Fig. 1 A, and administered α-LFA1 blocking antibody or IgG isotype control intraperitoneally 24 h after immunization and dLNs were harvested and assessed by histocytometry or flow cytometry at 48 h. (B) Representative flow plots and (C) quantification of the number of CD44+ OT-II cells, frequency of 4get-GFP+ cells, and Gata3 and IRF4 gMFI of CD44+ OT-II cells with the indicated treatment. (D) Representative images depicting OT-II macro-clustering and Th2 differentiation in auricular dLN treated with isotype control (top) or α-LFA-1 blocking antibody (bottom). (E) Histocytometry analysis of 4get-GFP, Gata3, and pS6 expression, OT-II localization at the T–B border, and the ratio of densely clustered OT-II cells with the indicated treatment is shown. (F and G) B6 mice were inoculated subcutaneously at the tail base with 500 N.b. L3 larvae, treated intraperitoneally with α-LFA1 blocking antibody or IgG isotype control on day 2 and 3 after infection, and the inguinal dLNs, or LNs from naïve mice, were harvested on day 4 for confocal microscopy. (F) Representative images and zoom-ins depicting Th2 macro-clustering with α-LFA1 blocking antibody or isotype control. Gata3 signal is masked outside of Ki67+ activated cells for visual clarity. (G) Histocytometry analysis of Gata3 expression in Ki67+ T cells with the indicated treatment. Data shown represent one independent experiment with at least n = 8 independently immunized lymph nodes from four mice per group. Data from multiple pooled experiments are denoted by different symbols within the same 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 A). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Dashed lines represent T–B border. A–C are representative of four independent experiments. D–G are representative of two independent experiments.

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