Site-specific T cell responses are mediated through non-equivalent expression of costimulatory molecules by migratory cDCs. (A–D) Mice were transferred with naïve 4get.OT-II cells, treated with papain OVA as in Fig. 1 A, and administered α-CD28 blocking antibody or IgG isotype control intraperitoneally 24 h post immunization and dLNs were harvested and assessed by histocytometry or flow cytometry at 48 h. (A) Representative flow plots and quantification of CD44⁺ OT-II cell number, 4get-GFP⁺ frequency, and Gata3 and IRF4 gMFI of CD44⁺ OT-II cells after α-CD28 or isotype control treatment. (B) Representative images depicting OT-II macro-clustering and Th2 differentiation in auricular dLNs treated with isotype control (top) or α-CD28 blocking antibody (bottom). (C and D) Histocytometry analysis of 4get-GFP, Gata3, and pS6 expression, OT-II localization at the T–B border, and ratio of densely clustered Ki67⁺ OT-II cells with the indicated treatment. (E–G) Naïve OT-II cells were cultured in vitro with the indicated concentrations of α-CD3 and α-CD28. α-IFNγ was added to cultures in some conditions (dashed lines). Cells were harvested 48 h later and assessed for expression of Gata3 and IRF4 by flow cytometry. Data shown represents one independent experiment with at least n = 4 independently immunized lymph nodes from two mice per group or three wells per treatment 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). Dashed lines represent T–B border. A–D are representative of at least two independent experiments. E–G are representative of four independent experiments.