Figure 4.

Subcutaneous co-delivery of antigen and CCL25 induces the development of ${\alpha }_4{\beta }_7^{\text{high}}$ Th1 T cells. (A) Dylight 488-labeled CCL25 (0.06 mg/kg) was subcutaneously injected into mice. Two hours later, the dLNs were harvested. Tissue sections from dLN were stained with hamster anti-mouse CD11c antibody (clone N418, BioLegend), or rat anti-mouse CD31 Antibody (MEC13.3, BioLegend) overnight at 4°C. Following three washes in PBS, the sections were incubated with the secondary antibody Alexa Fluor® 546 goat anti-hamster IgG or Alexa Fluor® 555 goat anti-rat IgG (Life Technologies) for 30 min at room temperature followed by three washes. Sections were mounted on microscopy slides with 4′,6-diamidino-2-phenylindole (DAPI) mounting medium (Vectashield). Images taken by wide field fluorescence microscopy are shown. Scale bar, 20 μM. (B–D) Female Marilyn-rag2^−/− mice were immunized by subcutaneous administration of 5 × 10⁶ male-derived splenocytes in saline solution or the presence of 0.06 mg/kg CCL25. One week later T cells were separately harvested from axillary (draining LNs, dLN), PP, mesenteric LN (non-draining LN, ndLN), and spleen. Marilyn (MY) T cells were identified by gating on the CD4⁺Vβ6⁺ T cell population. The number of ${\alpha }_4{\beta }_7^{\text{high}}$ (B), IFN-γ- (C) or IL-17-expressing (D) T cells was measured by flow cytometry. The mean number of T cells from two independent experiments of identical design is shown below each set of dot-plots (±SD, n = 3). (E–J) OT-II naïve T cells from (10⁷/mouse) were labeled with CFSE (4 μM) and injected intravenously into syngeneic recipients, which were immunized 3 h later by s.c. administration of 0.5 μg OVA-DEC plus 50 μg poly IC adjuvant (InvivoGen) re-suspended in saline solution or in the presence of CCL25 (0.06 mg/kg). T cells were separately harvested from draining LN (axillary, dLN), mesenteric LNs (mLNs), Peyer's Patches (PPs) and spleen 3 (E–G) and 5 days (H–J) later. Expression of α₄β₇ (E,H) and IFN-γ (F,I) or IL-17 (G,J) by CFSE^low T cells was assessed by flow cytometry by gating on CD4⁺Vα2⁺ T cells (OTII TCR). Staining with an isotype-matched control antibody in draining lymph nodes of mice injected with adjuvant alone are shown on top of each set of dot plots. The mean values obtained in at least 3 experiments of identical design are shown below each set of representative dot plots (±SD). ^*p < 0.05.