Figure 1. Dynamic expression of the chemokine receptor CCR9 during T lymphopoiesis.

Flow cytometric analysis of CCR9 on bone marrow derived LMPPs and CLPs or thymus-derived ETP, DN2, DN3, DN4 and DP thymocytes. (A) Gating strategy for the identification of LMPPs (Lin/PI⁻CD117⁺SCA1⁺FLT3⁺) and HSCs (Lin/PI⁻CD117⁺SCA1⁺Flt3⁻), which served as a negative control, among bone marrow cells. Gates used for selection are indicated as are the frequency of cells in each gate. (B) CCR9 expression on LMPPs (black) and HSCs (grey shaded), as indicated in the last plots showing gating in (A). (C) Gating strategy for the identification of CLPs (Lin/PI⁻CD117intCD127⁺SCA1⁺FLT3⁺) and pre-pro-NK cells (Lin/PI⁻CD117intCD127⁺SCA1⁺FLT3⁻), which served as a negative control, among bone marrow cells. (D) CCR9 expression on CLPs (black) and pre-pro-NK cells (grey shaded), as indicated in the final gating in (C). (E) Gating strategy for identification of CD4⁻CD8⁻ (DN) thymocyte subsets. Thymocytes were depleted of Lineage positive cells by MACS and then gated on ETPs (Lin/PI⁻CD117⁺CD25⁻), DN2 (Lin/PI⁻CD117⁺CD25⁺), DN3 (Lin/PI⁻CD117⁻CD25⁺) and DN4 (Lin/PI⁻CD117⁻CD25⁻). (F) CCR9 expression (black) on ETP, DN2, DN3 and DN4 thymocytes as indicated. Grey shading is HSC from BM isolated and stained in the same experiment. (G) CD4 and CD8 expression on total thymocytes with the gate used for identification of DP thymocytes. (H) CCR9 expression on DP thymocytes (black) with HSCs as a negative control (grey). In (B), (D), (F), (H) the frequency of CCR9⁺ cells is indicated in the experimental (black) and control (grey) population. One of at least 3 experiments with similar outcomes is shown for all populations.