FOCUS ON EARLY LYMPHOCYTE DEVELROEPVMIEEWNST Journey through the thymus: stromal guides for T-cell development and selection Yousuke Takahama Abstract | Lympho–stromal interactions in multiple microenvironments within the thymus have a crucial role in the regulation of T-cell development and selection. Recent studies have implicated that chemokines that are produced by thymic stromal cells have a pivotal role in positioning developing T cells within the thymus. In this Review, I discuss the importance of stroma-derived chemokines in guiding the traffic of developing thymocytes, with an emphasis on the processes of cortex-to-medulla migration and T-cell-repertoire selection, including central tolerance. “…and yet men are so foolishly venturous as to set out to provide the appropriate microenvironments for lightly on pilgrimage, and to come without a guide.” promoting and regulating further thymocyte develop- John Bunyan ment13–16. Therefore, the lympho–stromal communica- tion is a bilateral coordination, or crosstalk, between The thymus is an organ that supports the differentia- architectural stromal cells and travelling thymocytes14. tion and selection of T cells1–3. The thymic development The travelling thymocytes have to create their own path of T cells consists of several processes that require the by letting their presence be known to the stromal cells dynamic relocation of developing lymphocytes into, and by changing the stromal environment for further within and out of the multiple environments of the thy- development. Despite this ability, however, most of mus (FIG. 1). These processes include: first, the entry of the thymocytes are unable to complete their journey lymphoid progenitor cells into the thymus; second, the through the thymus, so that they are seldom ‘happy generation of CD4+CD8+ double-positive (DP) thymo- tourists’ travelling through various thymic scenes. cytes at the outer cortex of the thymus; third, the positive Consequently, the crosstalk between thymocytes and and negative selection of DP thymocytes in the cortex; thymic stromal cells offers the view that developing fourth, the interaction of positively selected thymo cytes thymocytes resemble ‘path-making pilgrims’ who make with medullary thymic epithelial cells (mTECs) to their way through the thymus, struggling for their life complete thymocyte development and ensure central beyond the thymus. tolerance; and last, the export of mature T cells from the Among such crosstalk signals, chemokines that are thymus4–9 (FIG. 2). produced by thymic stromal cells in individual micro- For developing thymocytes to experience these key environments seem to have a pivotal role in guiding events at the right places and in the right order, the the direction of migrating thymocytes, and develop- developing thymocytes and thymic stromal cells have ing thymocytes seem to find their way by sequentially to communicate with each other both in close prox- expressing different chemokine receptors. Recent stud- imity and remotely. However, this lympho–stromal ies of mice that are deficient for certain chemokines or Division of Experimental communication does not readily fit a typical textbook chemokine receptors have shown the important role Immunology, Institute view of thymic education, in which the stromal ‘teacher’ of chemotactic guidance in controlling T-cell develop- for Genome Research, unilaterally instructs the lymphocyte ‘apprentices’ in the ment in the thymus (TABLE 1). This Review summarizes University of Tokushima, thymus ‘classroom’. In fact, thymocyte development our current understanding of the trafficking of devel- 3-18-15 Kuramoto, involves a stringent repertoire selection in which only oping thymocytes and the crosstalk in the thymus, Tokushima 770-8503, Japan. e-mail: takahama@genome. 1–3% of thymocytes succeed in survival and export and provides a discussion of the role of chemo tactic 10–12 tokushima-u.ac.jp from the thymus . In addition, thymic stromal cells signals in regulating thymocyte trafficking and doi:10.1038/nri1781 need to be closely coached by developing thymocytes development. NATURE REVIEWS | IMMUNOLOGY VOLUME 6 | FEBRUARY 2006 | 127 © 2006 Nature Publishing Group REVIEWS Thymic primordium Entry of T-progenitor cells into the thymus In the postnatal thymus, lymphoid progenitor cells that The primordium refers to an The seeding of the thymus with lymphoid progeni- have just entered the thymic parenchyma are found mainly organ or tissue in its earliest tor cells (BOX 1) occurs as early as embryonic day 11.5 in the area close to the cortico–medullary junction, where recognizable stage of (E11.5) in mice and the eighth week of gestation in the vasculature is well developed, indicating that the development. The primordium 17,18 of the thymus is generated at humans , and is mediated by at least two different lymphoid progenitor cells enter the postnatal thymus by the ventral aspect of the third pathways: the vasculature-independent pathway, which transmigrating from the blood to the thymic parenchyma, pharyngeal pouch as early as probably occurs during the early stage of embryonic mainly through the area around the cortico–medullary embryonic day 10.5 in mice. development before vascularization of the thymus, and junction23 (FIG. 2a). Whereas the role of chemokines in the vasculature-dependent pathway, which probably postnatal thymus seeding is unclear, it has been reported Thymic parenchyma The parenchyma refers to the occurs in the late stage of embryogenesis and post natally that thymus seeding of the adult thymus is regulated by functional part of an organ. after vascularization. It is thought that vasculature- the adhesive interaction between platelet (P)-selectin The parenchyma of the thymus independent colonization of the fetal thymus is regulated glycoprotein ligand 1 (PSGL1), which is expressed by cir- is surrounded by the capsule, by the chemotactic attraction of lymphoid progenitor culating lymphoid progenitor cells, and P-selectin, which the trabeculae and the thymic primordium (FIG. 3a) 24 perivascular spaces. cells to the . The partial but is expressed by the thymic endothelium . Interestingly, significant roles of two chemokines, CC-chemokine lig- the entry of lymphoid progenitor cells into the thymus and 21 (CCL21) and CCL25, in this early stage of fetal is not a continuous event but an intermittent and gated thymus colonization have been reported19–21. CCL21 event that occurs in waves during embryogenesis and in and CCL25 are expressed by the fetal thymus primor- adulthood25–27. During embryogenesis, distinct waves of dium, along with several other chemokines19,20. In a thymus-colonized cells give rise to distinct progenies naturally occurring mutant mouse strain plt/plt, which of γδ T cells with different usages of T-cell receptor (TCR)- is deficient for CCL21, or in mice that are deficient for Vγ and Vδ chains, indicating that T-lymphoid progenitor CC-chemokine receptor 7 (CCR7), which is the recep- cells that colonize the fetal thymus differ in developmental tor for CCL21, the number of thymocytes was partially potential from T-lymphoid progenitor cells that enter the but significantly lower than that in normal mice until postnatal thymus27–31. In the adult thymus, the receptivity E14.5 (REF. 20). Mice deficient for CCR9, the receptor to chimerism of total thymocytes (largely of the αβ T-cell for CCL25, showed a three-fold decrease in total thymo- lineage) varies cyclically with a periodicity of 3–5 weeks cyte cellularity until E17.5 (REF. 21). These results indicate in non-irradiated mice, as shown by quantitative intra- that CCL21 and CCL25 are partially involved in fetal venous bone-marrow adoptive-transfer experiments and thymus colonization. Whether the combination of the timed separation of parabiotic adult mice25. CCL21 and CCL25 is sufficient for fetal thymus coloni- zation or whether other chemokines also have a role in Cortex formation and outward traffic this process is unclear. It has also been shown that CXC- Following entry into the thymus, lymphoid progeni- chemokine ligand 12 (CXCL12) and its receptor tor cells begin their development into T cells through CXC-chemokine receptor 4 (CXCR4) are not involved the developmental pathway that is commonly identi- in early fetal thymus colonization22. fied by the expression profiles of CD25 and CD44, a Human b Mouse Cortico– Subcapsular medullary Capsule Trabeculae Hassall’s corpuscles zone Cortex junction Trabeculae Subcapsular Cortex Cortico–medullary Medulla Capsule Medulla zone junction Figure 1 | The thymus architecture. a | The human thymus section from a newborn child was stained with haematoxylin and eosin. b | The thymus section from an adult C57BL/6 mouse was stained for CD4 (green), CD8 (blue), and medullary thymic epithelial cells defined using Ulex europaeus agglutinin 1 (red). Cells in cyan indicate the co-expression of CD4 and CD8, therefore being CD4+CD8+ double-positive cells. 128 | FEBRUARY 2006 | VOLUME 6 www.nature.com/reviews/immunol © 2006 Nature Publishing Group FOCUS ON EARLY LYMPHOCYTE DEVELROEPVMIEEWNST Along this developmental pathway, immature DN thymocytes promote the differentiation of thymic stromal cells and trigger the formation of the cortical- Trabeculae epithelial environment in the thymus15,16,38,39. In mice cTEC Fibroblast that are deficient for thymocyte development beyond the DN1 stage, such as a mouse strain carrying a trans- Developing thymocyte gene encoding human CD3ε, TECs are arrested at the b PSGL1 DC immature stage,