Figure S5.
Role of LFA-1 in T helper cell responses. (A) 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 flow cytometry at 48 h. Endogenous CD44+Ki67+BCL6− CD4 T cells were assessed for Gata3 and IRF4 coexpression with the indicated treatment. (B and C) Naïve CD45.2 4get.OT-II cells were transferred to CD45.1 mice and injected with CpG OVA in the ear pinnae or front footpad. Mice were treated intraperitoneally with α-CD62L 6 h after immunization, treated intraperitoneally with α-LFA1 blocking antibody or IgG isotype control 24 h after immunization, and dLNs were harvested and assessed by flow cytometry at 48 h. Representative plots (B) and quantification (C) of CD44+ OT-II cell number, frequency of CXCR3+ cells, and Tbet gMFI on CD44+ OT-II are shown. (D and E) Naïve ICAM-1.KO or ICAM-1.WT OT-II cells were transferred to CD45.1 B6 recipients and injected with papain OVA in the ear pinnae or front footpad, treated intraperitoneally with α-CD62L 6 h after immunization, and dLNs were harvested and assessed by flow cytometry at 48 h. (D) Frequency of ICAM-1 expression on OT-II cells from ICAM-1.WT and ICAM-1.KO mice. (E) Quantification of the number of CD44+ OT-II cells, and Gata3 and IRF4 gMFI of CD44+ OT-II cells of the indicated cell type. (F) Proposed model for initiation of Th2 differentiation in skin-draining LNs. Papain immunization of the skin elicits the maturation, costimulatory molecule expression, and migration of antigen-bearing cDC2 to draining LNs where they induce Th2 responses within dedicated microenvironments localized at the T–B border. Th2 differentiation is driven by prolonged T–DC contacts leading to T cell activation via low levels of pMHC and high levels of costimulatory molecules on cDC2s which subsequently drives integrin-mediated macro-clustering, efficient cytokine exchange, and localized Th2 differentiation. Certain skin sites, such as the paw, induce reduced expression of costimulatory molecules on migratory cDC2s which leads to T cell proliferation but reduced macro-clustering and Th2 differentiation within corresponding draining LNs. 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). WT = wild type; KO = knockout. A–C are representative of four independent experiments. D and E are representative of two independent experiments.

Role of LFA-1 in T helper cell responses. (A) 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 flow cytometry at 48 h. Endogenous CD44+Ki67+BCL6− CD4 T cells were assessed for Gata3 and IRF4 coexpression with the indicated treatment. (B and C) Naïve CD45.2 4get.OT-II cells were transferred to CD45.1 mice and injected with CpG OVA in the ear pinnae or front footpad. Mice were treated intraperitoneally with α-CD62L 6 h after immunization, treated intraperitoneally with α-LFA1 blocking antibody or IgG isotype control 24 h after immunization, and dLNs were harvested and assessed by flow cytometry at 48 h. Representative plots (B) and quantification (C) of CD44+ OT-II cell number, frequency of CXCR3+ cells, and Tbet gMFI on CD44+ OT-II are shown. (D and E) Naïve ICAM-1.KO or ICAM-1.WT OT-II cells were transferred to CD45.1 B6 recipients and injected with papain OVA in the ear pinnae or front footpad, treated intraperitoneally with α-CD62L 6 h after immunization, and dLNs were harvested and assessed by flow cytometry at 48 h. (D) Frequency of ICAM-1 expression on OT-II cells from ICAM-1.WT and ICAM-1.KO mice. (E) Quantification of the number of CD44+ OT-II cells, and Gata3 and IRF4 gMFI of CD44+ OT-II cells of the indicated cell type. (F) Proposed model for initiation of Th2 differentiation in skin-draining LNs. Papain immunization of the skin elicits the maturation, costimulatory molecule expression, and migration of antigen-bearing cDC2 to draining LNs where they induce Th2 responses within dedicated microenvironments localized at the T–B border. Th2 differentiation is driven by prolonged T–DC contacts leading to T cell activation via low levels of pMHC and high levels of costimulatory molecules on cDC2s which subsequently drives integrin-mediated macro-clustering, efficient cytokine exchange, and localized Th2 differentiation. Certain skin sites, such as the paw, induce reduced expression of costimulatory molecules on migratory cDC2s which leads to T cell proliferation but reduced macro-clustering and Th2 differentiation within corresponding draining LNs. 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). WT = wild type; KO = knockout. A–C are representative of four independent experiments. D and E are representative of two independent experiments.

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