Thymic T cell development and progenitor localization depend on CCR7

Abstract

T cell differentiation in the adult thymus depends on sequential interactions between lymphoid progenitors and stromal cells found in distinct regions of the cortex and medulla. Therefore, migration of T cell progenitors through distinct stromal environments seems to be a crucial process regulating differentiation and homeostasis inside the thymus. Here we show that CCR7-deficient mice are distinguished by a disturbed thymic architecture, impaired T cell development, and decreased numbers of the thymocytes. Analysis of developing double negative (CD4-CD8-) pool of wild-type thymus reveals that CCR7 expression is restricted to a CD25intCD44+ subpopulation. Correspondingly, CCR7 deficiency results in an accumulation of this population in mutant thymus. Furthermore, immunohistology shows that in CCR7-deficient mice CD25+CD44+ cells accumulate at the cortico-medullary junction, suggesting that CCR7 signaling regulates the migration of early progenitors toward the outer thymic cortex, thereby continuing differentiation. Results obtained from mixed bone marrow chimeras support this view, since the development of CCR7-deficient thymocytes is also disturbed in a morphologically intact thymus. Thus, our findings establish an essential role for CCR7 in intrathymic migration and proper T cell development.

Figure 1.

Differential expression of chemokine receptors during T cell development. (a) Thymocytes of adult C57BL/6 mice (6–8 wk old) were stained with a cocktail of biotinylated antibodies against the linage markers CD4, CD8, CD3, CD11c, CD11b, CD19, Ly5G, and Ter119 followed by Streptavidin-PerCP to allow gating of linage-negative cells (see histogram on the left) and with anti–CD44-FITC and anti–CD25-PE to identify DN populations as indicated. (b) Expression of CCR7 (CCL19-hIgG), CXCR4 (2B11), and CCR9 (7E7) on the DN populations shown in panel a, in DP and SP cells was revealed by goat anti–human-Cy5 (CCR7) or mouse anti–rat-Cy5 (CXCR4, CCR9). The CCL19–hIgG fusion protein did not bind to thymocytes of CCR7-deficient animals (not depicted). Shaded areas indicate binding of isotype controls.

Figure 2.

Localization of chemokine expression in the thymus of adult C57BL/6 mice. Cryosections from adult thymus of 6–8-wk-old C57BL/6 (a) or plt/plt (b) mice were stained with antibodies to cortical (pan-cytokeratin; clone C11; green) and medullary (MTS10; blue) thymic epithelial cells and to the chemokines CCL19, CCL21, CXCL12, and CCL25 as indicated (red, left). Chemokine staining was amplified using the Tyramide system. To facilitate visualization of chemokine expression, chemokine staining alone is also shown in the right panel. CCL19, CCL21, and CXCL12 expression was found in the medulla and on scattered cells in the cortex, whereas CCL25 was abundantly expressed in the whole thymus. plt/plt mice fail to express CCL19 and CCL21.

Figure 3.

Altered thymus architecture in CCR7-deficient and plt/plt mice. Hematoxylin and eosin staining from representative sections of adult thymi of wild-type (left), CCR7-deficient (middle), and plt/plt (right) animals (6–8 wk old) are shown. Compared to wild-type animals, thymi of CCR7-deficient mice shown numerous small medullary areas distributed through the whole cortex. Note that medulla is even found in the subcapsular zone (arrows). The thymic morphology of plt/plt mice is very similar to that of CCR7-deficient animals.

Figure 4.

Reduced cellularity and impaired thymocyte development in CCR7-deficient mice. (a) Absolute numbers of thymocytes in young (6–8 wk old) and old (6–8 mo old) CCR7-deficient mice are reduced in comparison to age-matched controls. (b) Relative distributions of DP (CD4⁺CD8⁺) and SP (CD4⁺ and CD8⁺) cells in the thymus of young and old wild-type and mutant mice. Proportions of DP cells decreased with age in the majority of CCR7-deficient animals analyzed. (c) Proportions of cells belonging to distinct DN stages among the total DN population in the thymus of wild-type and mutant mice. Increased proportions of DN1 and DN1-2 cells and decreased proportions of DN3 cells were observed in young CCR7-deficient animals in comparison to age-matched controls. In older animals, the proportions of DN1 and DN1-2 cells increased further, whereas the proportions of DN2, DN3, and DN4 cells were markedly reduced in comparison to young animals. Note that ∼50% of the old animals analyzed have virtually no DN3 cells (c). Each dot relates to data obtained from a single animal; bars indicate means. Data for old CCR7-deficient mice shown in panels b and c were derived from each five animals of C57BL/6 and a mixed BALB/c × 129Sv/Ev genetic background. No differences were observed in the variation of the data obtained from mice of both genetic backgrounds (not depicted).

Figure 5.

Accumulation of DN1-2 cells in CCR7-deficient animals. Thymocytes of young (6–8 wk; a–d) and old (6–8 mo; e–h) wild-type (a, c, e, and g) and mutant mice (b, d, f, and h) were stained as described for Fig. 1 a. Young mutants (b) have increased frequencies of DN1-2 cells compared with wild types (a), whereas no obvious difference regarding the distribution of SP and DP thymocytes could be observed (compare c and d) between both strains. Old mutants perform an aggravated phenotype (e and f) also showing decreased numbers of DP thymocytes (g and h). Numbers indicate the percentage of lin⁻ cells within the corresponding gates for a, b, e, and f.

Figure 6.

Reduced cellularity and impaired thymocyte development in plt/plt mice. (a) Similar to CCR7-deficient animals, absolute numbers of thymocytes in young (6–8 wk old) plt/plt mice are significantly reduced in comparison to age-matched controls (P < 0.05). (b) Proportions of cells belonging to distinct DN stages among the total DN population in the thymus of young plt/plt mice. Increased proportions of DN1 (P < 0.05) and DN1-2 (P < 0.0001) cells and decreased proportions of DN3 cells (P < 0.0001) were observed in young plt/plt animals in comparison to age-matched controls.

Figure 7.

CD25⁺CD44⁺ cells accumulate at the CMJ of adult CCR7-deficient animals. Cryosections of thymus from 6–8-wk-old C57BL/6 wild-type and mutant mice were stained with antibodies to CD25 (red) and CD44 (green). DP cells accumulated at the CMJ from CCR7-deficient mice and appear in yellow (arrows). Inserts show higher magnification of CMJs. (Bars, 100 μm).

Figure 8.

Requirement for CCR7 for proper T cell differentiation. Stable bone marrow chimeras were prepared, as described in Materials and Methods, resulting in donor chimerism >98% (a) or <5% (b). The phenotype of chimeric thymi was analyzed 2 mo later by flow cytometry and immunohistology (red, CD25; green, CD44) using mAbs as indicated. (a) Transfer of wild-type bone marrow in irradiated congenic wild-type recipients resulted in a phenotype similar to that observed in wild types (left). CCR7 mutant mice that received bone marrow of congenic wild-type mice also developed a thymus phenotype similar to that of wild-type mice (middle), whereas the thymic phenotype of CCR7-deficient was induced in wild-type recipients after transfer of congenic mutant bone marrow (right). Data shown are representative for three to four mice of each group. The numbers shown in the top panels indicate the percentage of lin⁻ cells within the corresponding gates, whereas the numbers in the middle panels refer to the percentage of all thymocytes. (b) Bone marrow of wild-type (CD45.1) and CCR7-deficient (CD45.2) donors was mixed and transferred to irradiated wild-type recipients. Low levels of mutant donor chimerism in the thymus of wild-type recipients confirms impaired development of CCR7-deficient DN cells compared with that of wild-type DN cells isolated from the same organs.