It is known that selected populations of lymphoid cells migrate into and from the adult thymus "Mature no 745" blood vessels at the cortico-medullary junction and in the medulla. Likewise, newly developed mature T cells in the thymic parenchyma of RAG2 mutant mice transferred with wild-type BM cells migrate to the PVS, before leaving the thymus to the "Mature no 745." Accumulation of mature T cells was observed after treatment with sphingosine-1 phosphate receptor agonist FTY not only in the medulla but also in the thymic PVS.
These results suggest that the PVS is a transit pathway for progenitor cells to immigrate into the thymus and for mature T cells to emigrate from the thymus. Cell migration into and out of lymphoid organs regulate homeostasis of the immune system 12. During embryonic life, hematopoietic stem cells immigrate from the fetal liver into the bone marrow BM 3.
Hematopoietic stem cells proliferate and differentiate to B cells and mature blood cells within stromal niches, located in between the venous sinuses 14. After maturation, these cells enter the venous sinuses to leave the BM through the blood circulation 5. Arriving at the lymph node, lymphocytes migrate across high endothelial venules to enter the lymphoid parenchyma, while emigrating lymphocytes leave the lymph node via the medullary sinus through efferent Mature no 745 to the circulation 6.
During organogenesis, hematopoietic progenitor cells enter the thymus anlage from the surrounding connective tissue 78. However, in the post-natal thymus, progenitor cells migrate through blood vessels located in the medulla and at the cortico-medullary junction 9.
Within the thymus, T cells develop in niches formed by stromal cells in the thymic parenchyma 10— After full maturation in the parenchyma, T cells arrive in the medulla and at the cortico-medullary junction and leave the thymus to the periphery.
Only small populations of T lineage cells enter or leave the thymus through blood vessels Therefore, to ensure selective migration of the cells into or from the thymus, the blood vessels located at the sites of cell migration in the thymus must harbor specific recognition structures and transition mechanisms.
Some vessels in the thymus are known to have the perivascular spaces PVS compartmentalized with a vascular basement membrane and a second basement membrane bordering the thymic parenchyma 13— Several authors claimed that the PVS Mature no 745 exclusively seen in the medulla and at the cortico-medullary junction of the thymus 131416while others described that the PVS are also found in the thymic cortex So, the distribution of the PVS in the thymus still remains unclear.
Additionally, the presence of lymphoid cells in the thymic PVS has been reported 15—17 However, the phenotype of the lymphoid cells within the thymic PVS and the role of the PVS in cellular transit are still not clearly understood. To clarify the function of the PVS in the thymus, we analyzed the anatomical distribution of the thymic PVS and the phenotype of the lymphoid cells enclosed in the PVS.
We also examined sites where the lymphoid cells immigrate into the Mature no 745 and emigrate from the thymus. We show that the PVS located around vessels in the medulla and at the cortico-medullary junction of the mouse thymus contain hematopoietic progenitor cells and mature T cells, but not immature thymocytes.
Mice were maintained in our animal facility under Specific pathogen free conditions. Thymi were embedded in OCT Mature no 745 and snap frozen. Primary antibodies used in these stainings were mAbs: Heparinized peripheral blood was harvested from the recipient mice, and after the cell counts, peripheral white blood cells were prepared by the treatment with 0.
cells were stained with the mAb for 30 min on ice. The drug was dissolved in physiological saline. B6 mice received daily intra-peritoneal i. Antibodies to type IV collagen and ER-TR5 were used to detect basement membranes and medullary epithelial cells, respectively.
The PVS are identified as regions separated by two basement membranes: In the cortex of the adult thymus, only small blood vessels
Mature no 745 seen Fig.
On the other hand, in the medulla and at the cortico-medullary junction, large venules were observed besides small vessels. The PVS were only present around the large venules which are distributed in the medulla and Mature no 745 the cortico-medullary junction of the thymus Fig. Hematopoietic progenitor cells and mature T cells are present in the PVS located in the cortico-medullary junction of the adult mouse thymus.
A and B Basement membranes of blood vessels were shown by type IV collagen staining. The PVS, indicated in arrows in B, was found preferentially around large blood vessels, present only in the medulla and the cortico-medullary junction.
Other investigators 9 have previously reported that migration of the cells in and out of the thymus occurs in the medulla and at the cortico-medullary junction where the PVS are present. Therefore, we addressed the question of whether the thymic PVS are involved in migration of lymphoid cells. Phenotypes of the cells localized within the PVS of Mature no 745 adult thymus were examined by immunohistochemistry.
As shown in Fig. These results
Mature no 745 that both T cell progenitors, as well as mature T cells selectively localize within the PVS. The PVS are found around large blood vessels distributed in the medulla and at the cortico-medullary junction, and this is where immigration of hematopoietic progenitor cells and emigration of mature T cells occurs. These results suggest that the PVS may serve as a path for cell trafficking between the thymic parenchyma and the blood stream.
To analyze the molecular components of basement membranes and cellular components forming the PVS, immunofluorescent staining of the adult thymi was performed Fig.
The components of basement membranes and the cells compartmentalizing the thymic PVS. Immunofluorescent staining of the sections of the adult thymi was performed to detect type I collagen Col I, A: Double basement membranes contain ECM molecules: These results indicate that the individual basement membranes compartmentalize the PVS as previously reported 13—19and distinct types of cell layers are Mature no 745 the insides of the PVS.
To clarify whether progenitor cells in the circulation directly migrate into the thymic PVS, we transferred progenitor cells and analyzed distributions of donor-derived cells in the recipient thymi at various time after cell transfer. The thymi of recipient mice were excised 0. Sections of the thymi from control RAG2 mutant mice and the recipient mice were analyzed by immunohistochemistry.
In the thymi of RAG2 mutant mice, organized medullary regions are not
Mature no 745 10however, the PVS were detected around large blood vessels localized in deep cortex Fig. Although many CDpositive cells Fig. As early as 0. BM-derived hematopoietic progenitor cells in the circulation migrate into the thymic PVS. F Merged image of D and E. I Merged image of G and H. These results seem to show that progenitor cells in the circulation first enter the thymic PVS, and then these cells move into the thymic parenchyma in the course of their migration into the thymus.
Next, we investigated whether "Mature no 745" T cells, developed in the thymic parenchyma, directly migrate into the thymic PVS. The thymi of the recipient RAG2 mutant mice were analyzed for the expression of type IV collagen and T cell markers by immunohistochemistry Fig. It is likely that mature cells, newly developed in the thymic parenchyma, migrate into the PVS before their distribution in the periphery.
Newly developed mature T cells in the thymus migrate into the PVS before leaving the thymus, and then appear in the circulation. The Mature no 745 of mature T cells in the PVS seemed to increase at 4 weeks and then decrease at 8 weeks after cell transfer.
P and Q Peripheral white blood cells were prepared from RAG2 mutant mice transferred with T cell-depleted BM cells of normal B6 mice weekly, from 1 to 8 weeks after the transfer. A few CD4SP and CD8SP cells were detectable 3 weeks after cell transfer, and the numbers of these cells significantly increased 4 weeks after transfer.
Thereafter the percentages of SP cells increased gradually during 8 weeks after transfer. Next, we examined the possibility that mature T cells, detected in the thymic PVS, are from peripheral T cells which had re-entered the thymus.
To this purpose, RAG2 mutant mice were transferred with spleen cells from B6. Twenty-four hours after cell transfer, the thymi of recipient mice were examined for the presence of donor-derived CD However, we were unable to detect CD These results may indicate that mature T cells in the thymic PVS are not from the periphery but from the thymic parenchyma.
It has been shown previously that a lack of sphingosine-1 phosphate receptor 1 S1P 1 results in an absence of T cells in the periphery, because mature T cells are unable to emigrate from the thymus 23 Under these conditions, emigration of T cells from the thymus is blocked
Mature no 745 The sections of the thymi were examined immunohistochemically. Twenty days after the first injection, accumulation of these cells was also seen in the PVS of the thymus.
These results indicate that S1P 1 signaling is involved in the migration of mature T cells into the medulla as well as into the PVS of the thymus. Immunofluorescent staining of frozen sections of the adult thymi of mice i. Arrows indicate the PVS. Cor, cortex and Med, medulla. Hematopoietic progenitor cells are selectively recruited to the thymus, and exclusively mature thymocytes leave the thymus to the circulating blood Moreover, the migration of these cells in or out of the thymus occurs in the medulla and at the cortico-medullary junction 9.
However, the mechanisms underlying the migration of these cells are not fully understood. These results suggest that donor-derived CDpositive cells in the recipient thymic PVS are progenitor cells for T cells. The numbers of donor-derived CDpositive cells in the parenchyma of the thymus increased during 5—24 h after the transfer. This suggests that progenitor cells in the circulation rapidly migrate to the PVS across the walls of the blood vessels in the medulla and at the cortico-medullary junction, before entering the thymic parenchyma.
At present, our results do not exclude the possibility that some progenitor cells directly migrate into the thymic parenchyma from post-capillary venule distributed in the medulla and at the cortico-medullary junction.
On the other hand, donor-derived mature T cells, newly developed in the thymic parenchyma of RAG2 mutant mice transplanted with wild-type BM cells, enter the thymic PVS before leaving the thymus. Using morphological methods the previous reports showed that lymphatics were present in the rodent thymus 16 None of the lymphatic markers, however, were detectable in the young adult mouse thymus our unpublished data. The discrepancy may attributable to genetic Mature no 745, age or breeding conditions of mice.