Separation of splenic red and white pulp occurs before birth in a LTαβ independent manner

For the formation of lymph nodes and Peyer’s patches, lymphoid tissue inducer cells are crucial in triggering stromal cells to recruit and retain hematopoietic cells. Although LTi cells have been observed in fetal spleen, not much is known about fetal spleen development and the role of LTi cells in this process. Here we show that LTi cells collect in a periarteriolar manner in fetal spleen at the periphery of the white pulp anlagen. Expression of the homeostatic chemokines can be detected in stromal and endothelial cells, suggesting that LTi cells are attracted by these chemokines. Since LTα1β2 can be detected on B cells, but not LTi cells, in neonatal spleen starting at 4 days after birth, the earliest formation of the white pulp in fetal spleen occurs in a LTα1β2 independent fashion. The postnatal development of the splenic white pulp, involving the influx of T cells, depends on LTα1β2 expressed by B cells. 121 Separation of splenic red and white pulp occurs before birth in a LTαβ independent manner Introduction The spleen is composed of a branching splenic artery that eventually ends in venous sinuses. The arterial branches, central arterioles, are surrounded by a layer of lymphoid tissue, the white pulp. It consists of T cell areas and B cell follicles, more or less resembling the organization found in lymph nodes. The arterial blood ends in a sinusoid system in the area surrounding the T and B cell zones, thereby forming an anatomical border between the white and red pulp, the marginal zone. Here sinusoid spaces formed by lining cells continuous with the endothelium of the arterioles and reticular fibroblasts make up a network through which the blood freely percolates on its way to the red pulp and can be scanned for pathogens and debris by macrophages and dendritic cells. The blood runs from the marginal zone through the red pulp cords into the venous sinuses, enabling the spleen to exert its function as a filter of the blood by removal of dysfunctional red blood cells 1 5 For development and organization of lymphoid organs the members of the TNF superfamily are crucial. The organogenesis of Peyer’s patches (PP) and lymph nodes (LN) is dependent on the expression of LTα1β2 and other TNF-family members 6, 7. During fetal and neonatal life, LTα1β2 is expressed by CD45 +CD4+Il7r+CD3lymphoid tissue inducer (LTi) cells in PP and LN anlagen 8, 9. LTi cells are attracted by non-hematopoietic stromal cells that express the homeostatic chemokines CCL19, CCL21, and CXCL13 as well as the adhesion molecules VCAM-1, ICAM-1, and MAdCAM-1 10 12. These molecules are thought to be induced by the interaction of LTi and stromal cells, which leads to lymphotoxin beta receptor (LTβR) and TNFR-I triggering 13. Expression of these molecules favors the subsequent recruitment and retention of more LTi cells as well as other hematopoietic cells 10. For the proper development of the murine neonatal lymphoid part of the spleen, indications that LTα1β2 is involved so far only stem from data obtained after birth, that show LTα1β2 expressing B cells are required for the induction of sufficient CCL21, produced by stromal cells in the T cell zone areas 14. Considering the important role of LTi cells in the organogenesis of other lymphoid organs relatively little is known about their role in spleen development, although their presence has been demonstrated in the fetal spleen as early as E13.5 8, 15. Transfer of in vitro Il7 activated splenic LTi cells was shown to restore the B/T segregation in spleens of LTα-/mice 16, suggesting a role for LTi cells in white pulp development. Since these experiments may not reflect the actual interactions and cellular requirements during fetal and neonatal spleen development we studied the role of LTi cells in the developing splenic white pulp in more detail. Our results show that neonatal splenic LTi cells lack LTα1β2 expression, and that LTα1β2, required for correct formation of the splenic white pulp, is expressed by B cells starting around 4 days after birth. Analysis of the earliest events during spleen development demonstrates that compartmentalization of red and white pulp areas starts to be regulated during embryogenesis. By grafting fetal spleens, distinct white pulp areas, containing donor derived LTi and B cells and red pulp areas, harbouring erythrocytes, can be observed. Therefore the white pulp stroma is already primed before E15.5 to segregate away from the red pulp areas. Moreover, we suggest that this early phase of stroma instruction is independent of the LTβR pathway during murine spleen development.