A multistep adhesion cascade for lymphoid progenitor cell homing to the thymus

Abstract

Homing of bone marrow (BM)-derived progenitors to the thymus is essential for T cell development. We have previously reported that two subpopulations of common lymphoid progenitors, CLP-1 and CLP-2, coexist in the BM and give rise to lymphocytes. We demonstrate that CLP-2 migrate to the thymus more efficiently than any other BM-derived progenitors. Short-term adoptive transfer experiments revealed that CLP-2 homing involves P-selectin/P-selectin glycoprotein ligand 1 interactions, pertussis toxin-sensitive chemoattractant signaling by CC chemokine ligand 25 through CC chemokine receptor 9, and binding of the integrins alpha4beta1 and alphaLbeta2 to their respective ligands, vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Preferential thymus-tropism of CLP-2 correlated with higher chemokine receptor 9 expression than on other BM progenitors. Thus, CLP access to the thymus is controlled by a tissue-specific and subset-selective multistep adhesion cascade.

Fig. 1.

CLP-2 are enriched in the thymus after adoptive transfer of lin⁻ BM cells. lin⁻ BM cells from pTα-hCD25 mice were adoptively transferred into Rag2^−/−γc^−/− mice. After 20 h, thymi (A) and BM (B) were harvested, and single-cell suspensions were stained for B220 and c-kit. Data are shown as the percentage of cells in the input population versus the percentage of those populations in the target organ.

Fig. 2.

P-selectin and PSGL-1 mediate lymphoid progenitor homing to the thymus. lin⁻ BM cells from pTα-hCD25 mice were pretreated with anti-L-selectin or control mAbs and adoptively transferred into Rag2^−/−γc^−/− mice. Anti-P-selectin, anti-E-selectin, or control mAbs were injected into recipient mice 15 min before transferring lin⁻ BM cells. After 20 h, thymi (A) and BM (B) were harvested, and single-cell suspensions were stained for Ly5.1 and hCD25 (n = 6 mice). (C) CFSE-labeled lin⁻ BM cells from PSGL-1^−/− mice together with TRITC-labeled lin⁻ BM cells from WT controls were mixed in a 1:1 ratio and injected i.v. into Rag2^−/−γc^−/− cells. After 20 h, spleen, thymi, and BM were harvested, and single-cell suspensions were analyzed for the presence of TRITC⁺ and CFSE⁺ lin⁻ BM cells (n = 5 mice).

Fig. 3.

Chemokine receptor expression and chemotaxis of lymphoid progenitors. (A) From left to right: BM pro-B cells, CLP-2, CLP-1, and early thymic immigrants (double-negative 1 c-kit⁺hCD25⁺) were sorted and analyzed by semiquantitative RT-PCR for the expression of multiple CCRs. cDNA quantities were normalized according to GADPH or actin expression over three subsequent 5× dilutions. (B and C) lin⁻ BM cells from pTα-hCD25 mice were added to inserts that were placed in wells containing medium alone or the following chemokines: CCL5, CCL2, CCL19, CCL21, CXCL12, or CCL25. Responding cells were harvested 2 h later, stained for hCD25, B220, and c-kit, and analyzed by flow cytometry. (B) Migration as percentage of the input. (C) Chemotactic index. (n = 3 experiments.)

Fig. 4.

CCR9/CCL25 is involved in lymphoid progenitor homing to the thymus. lin⁻ BM cells from pTα-hCD25 mice were i.v injected into Rag2^−/−γc^−/− mice pretreated with anti-CXCL12, anti-CCL21, anti-CCL25, or control mAbs. After 20 h, thymi (A) and BM (B) were harvested, and single-cell suspensions were stained for Ly5.1 and hCD25 (n = 8 mice). (C) CFSE-labeled lin⁻ BM cells from CCR9^−/− mice together with TRITC-labeled lin⁻ BM cells from WT controls were mixed in a 1:1 ratio and injected i.v. into Rag2^−/−γc^−/− cells. After 20 h, spleen, thymi, and BM were harvested, and single-cell suspensions were analyzed by flow cytometry for the presence of TRITC⁺ and CFSE⁺ lin⁻ BM cells (n = 6 mice).

Fig. 5.

A nonredundant role of α4β1 and αLβ2 in lymphoid progenitor homing to the thymus. lin⁻ BM cells from pTα-hCD25 mice were pretreated with anti-α4, anti-β2, both, or control mAbs and adoptively transferred into Rag2^−/−γc^−/− mice treated or not with anti-VCAM-1 or anti-ICAM-1 mAbs. Twenty hours later, thymi (A) and BM (B) were harvested, and cell suspensions were stained for Ly5.1 and hCD25 (n = 4 mice).

Fig. 6.

Homing to the thymus is mediated by a unique cascade of adhesion and signaling events. Cells expressing PSGL-1 or other P-selectin ligands and probably also α4β1 can initiate the adhesion cascade to home to the thymus. Subsequently, a Gα_i-coupled activating stimulus is required to activate the integrins α4β1 and αLβ2, which allow the firm adhesion of the progenitors through interactions with VCAM-1 and ICAM-1, respectively. This activation depends on signaling through the CCL25/CCR9 pair. A still-unknown Gα_i-independent mechanism is partially involved in the homing of lymphoid progenitors to the thymus. The unique combination of these events efficiently results in the selective recruitment of lymphoid progenitors.