Generation of Th2 microenvironments in draining LNs. (A–I) Naïve CD45.2 4get.OT-II T cells were transferred to CD45.1 mice and injected with the indicated adjuvants plus OVA in the ear pinnae. Mice were treated intraperitoneally with α-CD62L blocking antibody 6 h after immunization and dLNs were harvested and assessed by confocal imaging and histocytometry at 48 h. (A) Representative images depicting Th2 macro-clustering at the T–B border in Papain OVA immunized dLNs. (B) Macro-clustered and non-clustered regions are highlighted in insets with the indicated markers shown. Gata3 signal is masked outside of Ki67+IRF4+ activated cells for visual clarity. (C) Representative images depicting OT-II responses in dLNs after CpG OVA immunization. (D) Histocytometry analyses of OT-II localization at the T–B border and the ratio of macro-clustered versus total OT-II cells. (E–I) Tissue sections were analyzed for the association of Th2 cells with myeloid cells using CytoMAP. (E) Representative confocal image depicting Th2 macro-clusters and Gata3 staining with different myeloid cell markers. (F) Spatial distribution analysis of indicated myeloid cell and activated (IRF4+Ki67+) T cell subsets. (G) 50-μm raster scan neighborhoods of a representative dLN were plotted on a X,Y positional plot and color-coded using neighborhood clustering, as shown in panel H. (H) Heatmap demonstrating the cellular composition for each neighborhood cluster (color depicted at the top). Th2 region (dark red) enriched with CD301b+ DCs is highlighted (arrowhead). (I) Cell–cell spatial correlation of the indicated T cell subsets and myeloid cell populations. (J–L) Naïve CD45.1 OT-II T cells were transferred into CD11c-Cre+ IRF4fl/fl or CD11c-Cre− IRF4fl/fl CD45.2 mice and then immunized with papain OVA in the ear pinnae, treated with α-CD62L blocking antibody at 6 h, and dLNs were harvested and assessed by flow cytometry or confocal microscopy at 48 h. (J) CD44+ OT-II cell number is shown with representative histogram and quantification of Gata3 gMFI, IRF4 gMFI, and BCL6 gMFI of CD44+ OT-II. (K) Representative images depicting OT-II macro-clustering in Cre+ and Cre− dLNs with the indicated markers shown. (L) Histocytometry analyses of Gata3 expression, OT-II localization at the T–B border, and the ratio of macro-clustered versus total OT-II. 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. ****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. Figures A–I are representative of five independent experiments. Figures K and L are representative of three independent experiments.
Figure 1.

Generation of Th2 microenvironments in draining LNs. (A–I) Naïve CD45.2 4get.OT-II T cells were transferred to CD45.1 mice and injected with the indicated adjuvants plus OVA in the ear pinnae. Mice were treated intraperitoneally with α-CD62L blocking antibody 6 h after immunization and dLNs were harvested and assessed by confocal imaging and histocytometry at 48 h. (A) Representative images depicting Th2 macro-clustering at the T–B border in Papain OVA immunized dLNs. (B) Macro-clustered and non-clustered regions are highlighted in insets with the indicated markers shown. Gata3 signal is masked outside of Ki67+IRF4+ activated cells for visual clarity. (C) Representative images depicting OT-II responses in dLNs after CpG OVA immunization. (D) Histocytometry analyses of OT-II localization at the T–B border and the ratio of macro-clustered versus total OT-II cells. (E–I) Tissue sections were analyzed for the association of Th2 cells with myeloid cells using CytoMAP. (E) Representative confocal image depicting Th2 macro-clusters and Gata3 staining with different myeloid cell markers. (F) Spatial distribution analysis of indicated myeloid cell and activated (IRF4+Ki67+) T cell subsets. (G) 50-μm raster scan neighborhoods of a representative dLN were plotted on a X,Y positional plot and color-coded using neighborhood clustering, as shown in panel H. (H) Heatmap demonstrating the cellular composition for each neighborhood cluster (color depicted at the top). Th2 region (dark red) enriched with CD301b+ DCs is highlighted (arrowhead). (I) Cell–cell spatial correlation of the indicated T cell subsets and myeloid cell populations. (J–L) Naïve CD45.1 OT-II T cells were transferred into CD11c-Cre+ IRF4fl/fl or CD11c-Cre− IRF4fl/fl CD45.2 mice and then immunized with papain OVA in the ear pinnae, treated with α-CD62L blocking antibody at 6 h, and dLNs were harvested and assessed by flow cytometry or confocal microscopy at 48 h. (J) CD44+ OT-II cell number is shown with representative histogram and quantification of Gata3 gMFI, IRF4 gMFI, and BCL6 gMFI of CD44+ OT-II. (K) Representative images depicting OT-II macro-clustering in Cre+ and Cre− dLNs with the indicated markers shown. (L) Histocytometry analyses of Gata3 expression, OT-II localization at the T–B border, and the ratio of macro-clustered versus total OT-II. 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. ****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. Figures A–I are representative of five independent experiments. Figures K and L are representative of three independent experiments.

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