Figure 3.

CCR9 signals induce ${\alpha }_4{\beta }_7^{+}$ Th1-like cells. MY (A,B) or OT-II (C,D) naïve T cells (10⁷/mouse) were labeled with CFSE (4 μM) and injected intravenously into WT and ccl25^−/− syngeneic female recipients. Recipient mice received cognate peptide (100 μg Dby or 0.5 μg OVA-DEC peptide) plus ODN adjuvant (50 μg) orally 24 h later. Five days later, T cells were separately harvested from mesenteric LN (draining LN, dLN), inguinal and axillary (non-draining LNs, ndLN) and the spleen. Production of IFN-γ by divided MY (A) and OT-II (C) T cells was assessed by intracellular staining and flow cytometry. T cells were identified by gating on the CD4⁺ Vβ6⁺ (MY) or Vα2⁺ (OTII) populations. Panels (C) and (D) show the production of IFN-γ by ${\alpha }_4{\beta }_7^{+}$ divided MY an OTII T cells, respectively. The mean number of T cells from three independent experiments of identical design is shown below each set of representative dot plots (±SD, n = 3, N = 2). (E,F) Naive T cells were activated either with plastic bound anti-CD3 in culture medium, or under Th1-skewing culture conditions or in the presence of an activating CCR9 antibody (7 μg/ml). In some experiments, T cells were activated in the presence of the anti-CCR9 antibody in the presence of pertussis toxin (PTX, 0.1 μg/ml). Representative dot-plots of IFN-${\gamma }^{+}{\alpha }_4{\beta }_7^{+}$ and T-${\text{bet}}^{+}{\alpha }_4{\beta }_7^{+}$ T cells (gated on CD3⁺CD4⁺ populations) 5 days after activation and the mean number of T cells from three independent experiments of similar design is shown in panels (E,F), respectively (±SD). Of note, Th1-skewing culture led to T-bet expression mainly in ${\alpha }_4{\beta }_7^{-}$ T cells (F). (G) WT and ccr9^−/− naive T cells were activated either with plastic bound anti-CD3 under Th1-skewing culture conditions or in the presence of an activating CCR9 antibody (7 μg/ml). Five days later, expression of T-bet was assessed by flow cytometry. A representative histogram and the mean T-bet MFI from three independent experiments of similar design are shown (±SD). (H) Naive T cells were activated either with plastic bound anti-CD3 and antiCD28 under Th1-skewing culture conditions or in the presence of an activating CCR9 antibody (7 μg/ml) or isotype-matched antibody control. Three days later, expression of phosphorylated (p)STAT1 (Ser₇₂₇) was assessed by flow cytometry. Representative histogram and the mean pSTAT1 MFI from three independent experiments of similar design are shown (±SD). (I,J) Naïve WT T cells were stimulated with plate-bound anti-CD3 and CD28 antibodies in the presence of either 7 μg/mL anti-CCR9 or isotype control antibody cross-linked with 2.5 μg/mL Donkey–anti-goat IgG overnight. T cells were harvested and expression of BAFT mRNA (I) protein (J) by CD4⁺ T cells was assessed by real-time PCR and intracellular staining, respectively. Staining with an isotype-matched control antibody and proliferation and differentiation in draining lymph nodes of mice injected with adjuvant alone are shown on the right-hand side of each set of dot plots. Bar graph show the mean values obtained in three experiments of identical design each performed in triplicates (±SD). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001.