Figure 1. Ackr4 is expressed by LECs in the MLN and mesenchymal cells in the intestine.

(A) Overlaid histogram flow cytometry plots showing GFP expression by CD45⁺ (top panels) and CD45^- cells (bottom panels) among single, live, Ter119^- cells in cell suspensions of the MLN, small intestine (SI) and colon of WT and Ackr4^gfp/+ (Ackr4^tm1Ccbl) mice. (B) Left panel: flow cytometry contour plot of stromal cell populations among single, live, CD45^-Ter119^- cells in the MLN, identifying GP38⁺CD31^- fibroblastic reticular cells (FRC), GP38⁺CD31⁺ lymphatic endothelial cells (LEC), GP38^-CD31⁺ blood endothelial cells (BEC) and GP38^-CD31^- ‘double negative’ (DN) cells. Right panels: representative overlaid histogram plots showing GFP expression by stromal cell populations in the MLN of WT and Ackr4^gfp/+ mice. (C) Flow cytometric analysis of stromal cell populations among single, live, CD45^-Ter119^- cells in the SI and colon, identifying EpCAM⁺ epithelial cells (histograms), and GP38⁺CD31^- intestinal mesenchymal cells (iMC), LECs, BECs and DN cells in the EpCAM^- population (contour plots). (D) Representative overlaid histograms showing GFP expression by stromal cell populations in the SI and colon of WT and Ackr4^gfp/+ mice. In A, B and D, the numbers on the histogram plots indicate the percentage of GFP⁺ cells in Ackr4^gfp/+ samples. Flow cytometry plots in A-D are representative of those from at least three individual experiments involving at least three mice of each genotype. (E, F) Percentage of cells expressing GFP in LEC (E) and GP38⁺CD31^- cell (F) populations in the MLN (n=6), SI (n=23) and colon (n=20) of Ackr4^gfp/+ mice. Mean±1SD is indicated. Data are pooled from two or more individual experiments.