Figure 3.

DRD5 signaling is required for gut homing of CD4⁺T cells in inflammatory conditions. Naïve CD4⁺ T cells (CD3⁺CD4⁺CD45RB^high) were isolated from the spleen of Cd45.1^+/+Drd5^+/+ (black bars) or Cd45.2^+/+Drd5^–/– (white bars) mice and then activated with anti-CD3/anti-CD28 mAbs-coated Dynabeads in the presence of IL-2 and RA for 5d to induce gut tropism. Afterward, CD45.1⁺ and CD45.2⁺ cells were mixed in a 1:1 ratio and intravenously injected (2x10⁶ total cells per mouse) into Rag1^–/– recipient mice. Mice were sacrificed 72 hours later and the relative composition (CD45.1⁺ vs CD45.2⁺) and expression of gut-homing molecules on CD4⁺ T cells isolated from different tissues was analyzed. (A) Scheme illustrating the experimental design. (B) Representative contour plots analyzing the relative abundance of CD45.1⁺ vs CD45.2⁺ CD4⁺ T cells in the input or infiltrating different tissues. (C) Quantification of the relative abundance of CD45.1⁺ vs CD45.2⁺ CD4⁺ T cells normalized by the input. Data are the % of CD45.1⁺ or CD45.2⁺ from CD4⁺ T cells in a given tissue divided by the % of CD45.1⁺ or CD45.2⁺ from CD4⁺ T cells in the input (n = 7 mice/group). (D–F) CCR9 and α4β7 expression was analyzed in CD45.1⁺ and CD45.2⁺ CD4⁺ T cells isolated from different tissues. (D) Values represent the percentage of CCR9⁺α4β7^–, CCR9⁺α4β7⁺, or CCR9^–α4β7⁺ cells in the TCRβ⁺CD3⁺CD4⁺ population from the input (n = 4 mice/group). (E, F) Quantification of the percentage (left panels) and the mean fluorescence intensity (right panels) of (E) CCR9 and (F) α4β7 expressed in CD4⁺ T cells isolated from the spleen, MLNs, and cLP (n = 3–8 mice/group). (C–F) Each symbol represents data obtained from an individual mouse. Mean ± SD are indicated. ∗∗P < .01; ∗∗∗∗P < .0001 by 2-way ANOVA followed by Sidak’s post hoc test.