Site-specific Th2 responses are maintained across mouse strains and adjuvants. (A) 0.5 × 10⁶ naïve CD45.2 4get.OT-II cells were transferred to CD45.1 mice and injected with papain OVA in the ear pinnae or front footpad and dLNs, spleen, and lung were harvested and assessed by flow cytometry 6 days later. BCL6 gMFI and frequency of CXCR5⁺PD-1⁺ of CD44⁺ OT-II cells are shown. (B) KN2^+/− mice were immunized with papain in the ear pinnae or front footpad and dLNs were assessed by flow cytometry 2 days later. Representative histogram and frequency of KN2⁺Gata3⁺ and KN2 gMFI of CD44⁺Ki67⁺ CD4 cells are shown. (C) B6 or Balb/c mice were immunized in the ear pinnae and front footpad with papain and dLNs were harvested at day 3 for flow cytometry. CD44⁺Ki67⁺ T cell number, and Gata3 and IRF4 gMFI of CD44⁺Ki67⁺ T cells are shown. (D) B6 mice were immunized in the ear pinnae and front footpad with papain or Alum OVA and dLNs were harvested on day 3 for flow cytometry. CD44⁺Ki67⁺ T cell number, and Gata3 and IRF4 gMFI of CD44⁺Ki67⁺ T cells are shown. (E–G) Naïve CD45.2 4get.OT-II cells were transferred to CD45.1 mice, injected with the indicated adjuvants plus OVA in the ear pinnae or front footpad, treated intraperitoneally with α-CD62L 6 h post immunization and dLNs were harvested and assessed by flow cytometry at 48 h. (E) Representative flow plots, CD44⁺ OT-II cell number, frequency of 4get-GFP⁺ cells, and Gata3 and IRF4 gMFI of CD44⁺ OT-II cells after immunization with the indicated adjuvants are shown. (F and G) Mice were treated as above but injected with papain OVA in the ear pinnae, front footpad, hind footpad, dorsal flank skin, or tail base and the corresponding dLNs were harvested for flow cytometry 2 days later. Representative flow plots, CD44⁺ OT-II cell number, and the frequency of 4get-GFP⁺ cells and Gata3 gMFI of CD44⁺ OT-II cells are shown. (H) DBP was topically applied (painted) onto the ear or front footpad skin of B6 mice and dLNs were harvested for flow cytometry 48 h later. The frequency of CD44⁺Ki67⁺ T cells, and Gata3 and IRF4 gMFI of CD44⁺Ki67⁺ T cells is shown. 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). A–G are representative of two to three independent experiments. H is representative of four independent experiments.