Murine CCR9, a Chemokine Receptor for Thymus-Expressed Chemokine That Is Up-Regulated Following Pre-TCR Signaling

Abstract

Chemokines are likely to play an important role in regulating the trafficking of developing T cells within the thymus. By using anti-CD3ε treatment of recombinase-activating gene 2 (Rag2−/−) mice to mimic pre-TCR signaling and drive thymocyte development to the double positive stage, we have identified murine GPR-9-6 as a chemokine receptor whose expression is strongly induced following pre-TCR signaling. GPR-9-6 mRNA is present at high levels in the thymus, and by RT-PCR analysis its expression is induced as normal thymocytes undergo the double negative to double positive transition. Furthermore we show that TECK (thymus-expressed chemokine), a chemokine produced by thymic medullary dendritic cells, is a functional ligand for GPR-9-6. TECK specifically induces a calcium flux and chemotaxis of GPR-9-6-transfected cells. In addition, TECK stimulates the migration of normal double positive thymocytes, as well as Rag2−/− thymocytes following anti-CD3ε treatment. Hence, GPR-9-6 has been designated as CC chemokine receptor 9 (CCR9). Our results suggest that TECK delivers signals through CCR9 important for the navigation of developing thymocytes.

CCR7 Directs the Migration of Thymocytes into the Thymic Medulla

“…CCR4 may be an especially attractive candidate, because, like CCR7, its expression is markedly induced as a consequence of positive selection. Moreover, CCL22, one of the ligands for CCR4, is also expressed in the medulla, and this ligand is an efficacious attractant for cells undergoing positive selection (i.e., CD69 ϩ cells) (16,20,43,44). CCR9 is expressed by double-positive cells, but its greatest increase in expression may occur after the pre-TCR signal (15)(16)(17).…”

“…Moreover, CCL22, one of the ligands for CCR4, is also expressed in the medulla, and this ligand is an efficacious attractant for cells undergoing positive selection (i.e., CD69 ϩ cells) (16,20,43,44). CCR9 is expressed by double-positive cells, but its greatest increase in expression may occur after the pre-TCR signal (15)(16)(17). The ligand for CCR9, CCL25, is found in both the cortex and the medulla, and thymocytes show apparently equivalent migration toward it both before and immediately after positive selection (15,17,20).…”

“…Thus, passage through the TCR␤ and TCR␣ developmental checkpoints is associated with the up-regulation of distinct chemokine receptors, the ligands for which are regionally localized in the thymus. For example, pre-TCR signaling leads to increased CCR9 expression, which would allow the cells to migrate toward CCL25 (thymusexpressed chemokine) (15)(16)(17)(18). Similarly, CCR4, CCR7, and CCR8 are all up-regulated during positive selection and would permit migration toward medullary CCL22 (macrophage-derived chemokine) or CCL17 (thymus and activation-regulated chemokine), CCL19 (EBI1-ligand chemokine, macrophage inflammatory protein-3␤, and Exodus-3)/21 (secondary lymphoid tissue chemokine, 6-C-kine, Exodus-2, and thymus-derived chemotactic agent 4), and CCL1 (T cell activation gene-3), respectively (8, 19 -22).…”

Transgenic Expression of RasGRP1 Induces the Maturation of Double-Negative Thymocytes and Enhances the Production of CD8 Single-Positive Thymocytes

“…For some experiments additional RAG-2 Ϫ/Ϫ mice were purchased from Taconic Farms. Total RNA was isolated from thymi of 4-wk-old PBS-or anti-CD3⑀-injected RAG-2 Ϫ/Ϫ mice or tissues of adult C57BL/6 mice, separated on a 1% agarose formaldehyde gel, and analyzed by Northern blot analysis as previously described (33). Blots were probed using a full-length murine RasGRP1 cDNA probe (30) or elongation factor-1␣ as a loading control.…”

“…The remaining cells were stained using anti-CD44-PE, anti-CD25-FITC, and streptavidin-Tri-color and then sorted to obtain individual CD44/CD25 triple-negative subsets. RNA and cDNA were prepared from sorted cells and normalized according to hypoxanthine phosphoribosyltransferase (HPRT) expression as previously described (33), after 30 PCR cycles using the primers HPRT 7/8 and 9 (34). PCR of RasGRP1 was performed for 30 cycles (94°C for 1 min, 60°C for 1 min, 72°C for 1 min) using the primers RasGRP 6 (5Ј-CGGATCATCATCTCCTCAGCC-3Ј) and RasGRP 8 (5Ј-CAGTCATCTCGCCCAGAACC-3Ј).…”