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Netrin-1, a known axon guidance molecule, being a secreted laminin-related molecule, has been suggested to be involved in multiple physiological and pathological conditions, such as organogenesis, angiogenesis, tumorigenesis, and inflammation-mediated tissue injury. However, its function in thymocyte development is still unknown. Here, we demonstrate that Netrin-1 is expressed in mouse thymus tissue and is primarily expressed in thymic stromal cells, and the expression of Netrin-1 in thymocytes can be induced by anti-CD3 antibody or IL-7 treatment. Importantly, Netrin-1 mediates the adhesion of thymocytes, and this effect is comparable to or greater than that of fibronectin. Furthermore, Netrin-1 specifically promotes the chemotaxis of CXCL12. These suggest that Netrin-1 may play an important role in thymocyte development.

Our previous studies have defined a differentiation program followed by the newly generated single-positive (SP) thymocytes before their emigration to the periphery. In the present study, we further characterize the development of CD4SP cells in the thymic medulla using mainly intrathymic adoptive transfer assays. By analyzing the differentiation kinetics of the donor cells, which were shown to home correctly to the medullary region following adoptive transfer, we established the precursor-progeny relationship among the four subsets of CD4SP thymocytes (SP1-SP4) and demonstrated that the progression from SP1 to SP4 was unidirectional and largely synchronized. Notably, while the phenotypic maturation from SP1 to SP4 was achieved in 2-3 days, a small fraction of donor cells could be retained in the thymus for a longer period, during which they further matured in function. BrdU incorporation indicated that cell expansion occurred at multiple stages except SP1. Nevertheless, CFSE labeling revealed that only a limited number of cells actually divided during their stay in the medulla. As to the thymic emigration, there was a clear bias toward cells with increasing maturity, but no distinction was found between dividing and nondividing thymocytes. Collectively, these data not only provide solid evidence for a highly ordered differentiation program for CD4SP thymocytes, but they also illustrate several important features associated with the developmental process.

The newly generated single-positive (SP) thymocytes undergo further maturation in the thymic medulla before their emigration to the periphery. The present study was undertaken to validate a developmental program we proposed for CD4SP medullary thymocytes and to explore the mechanisms regulating this process. During mouse ontogeny, the emergence of different subsets of CD4SP thymocytes followed a strict temporal order from SP1 to SP4. Parallel to the transition in surface phenotype, a steady increase in function was observed. As further evidence, purified SP1 cells were able to sequentially give rise to SP2, SP3, and SP4 cells in intrathymic adoptive transfer and in culture. Notably, the development of CD4SP cells in the medulla seemed to be critically dependent on a functionally intact medullary epithelial cell compartment because Relb and Aire deficiency were found to cause severe blockage at the transition from SP3 to SP4. Taken together, this work establishes an ontogenetically and functionally relevant maturation program for CD4SP thymocytes. Precise dissection of this program should facilitate further inquiry into the molecular mechanisms governing normal thymocyte development and its disturbance in pathological conditions.

Mouse platelet basic protein (CXCL7/mPBP) was cloned from thymic stromal cells and further identification indicated that it was expressed in thymic monocytes/macrophages (Mo/Mphis). Recombinant mPBP was chemoattractive for target cells of polymorphonuclear leucocytes, peritoneal Mo/Mphis and splenic lymphocytes with distinct potencies. CXCR2 was identified to be a cognate receptor for mPBP. Mouse thymocyte subsets of CD4-CD8- double-negative (DN), CD4+CD8+ double-positive (DP), CD4+CD8- single-positive (CD4SP) and CD4-CD8+ single-positive (CD8SP) expressed cell surface CXCR2 with different positive percentages and expression levels. mPBP was chemoattractive for thymocyte subsets with the potency order DN>DP> CD8SP>CD4SP, consistent with the levels of CXCR2 expressed on the respective cells. Thus, mPBP in thymus is functionally redundant with chemokine CXCL12/ SDF-1. Moreover, our finding that thymic Mo/Mphis can produce mPBP implies that they may have other functions apart from acting as scavengers in thymus.

SDF-1/CXCR4 plays an important role in promoting survival, expansion, and differentiation of T cell progenitors. The present study investigates the mechanism by which estrogen inhibits SDF-1alpha expression in mouse thymus. Mouse estrogen enhanced transcript (mEET) is endogenously expressed in a mouse thymus epithelial cell line 1 (MTEC1). In MTEC1 cells that express the transfected sense mEET, the SDF-1alpha transcription and its chemotactic activity were profoundly inhibited. Conversely, in MTEC1 that express the transfected anti-sense mEET, the SDF-1alpha transcription and its chemotactic activity were substantially augmented. Moreover, we disclosed that mEET inhibited the production of SDF-1alpha by its suppression of NF-kappaB translocation into nucleus. Using a combinatorial induction of doxycycline (Dox) and 17beta-estradiol (E2) on the sense and anti-sense mEET transfectants, it was demonstrated that an increase of mEET expression enhanced E2-induced inhibition of SDF-1alpha production, while a blockade of mEET expression alleviated E2-induced inhibition of SDF-1alpha production. In conclusion, the E2-imposed suppression of SDF-1alpha production is partly mediated by mEET involved signaling pathway.

The functional maturation process of medullary-type CD4(-)CD8(+) [CD8 single-positive (SP)] thymocytes remains largely uncharacterized. We describe a phenotypic analysis of CD8 SP medullary-type thymocytes and find a remarkable heterogeneity within this thymic cell population. While mature CD8(+) T cells in the periphery are relatively homogeneous (TCRalphabeta(+)CD3(+)Qa-2(+) HSA(-)3G11(-)6C10(-)CD69(-)), CD8 SP medullary-type thymocytes contain discrete subpopulations that can be identified by differential expression of several cell-surface markers. We have identified at least six discrete subpopulations in the subset of TCRalphabeta(+)CD3(+) CD8 SP cells in the thymus. According to the expressed phenotypes, a linear developmental pathway is predicted among these CD8 SP subpopulations as follows: 6C10(+)CD69(+)HSA(hi)3G11(+)Qa-2(-) --> 6C10(-)CD69(+)HSA(hi/int)3G11(+)Qa-2(-) --> 6C10(-)CD69(-)HSA(int)3G11(+)Qa-2(-) --> 6C10(-)CD69(-)HSA(lo)3G11(+)Qa-2(-) --> 6C10(-)CD69(-)HSA(-/lo)3G11(-)Qa-2(-) --> 6C10(-)CD69(-)HSA(-/lo)3G11(-)Qa-2(+). This study provides a framework for understanding CD8 SP T cell maturation in the thymic medulla.