Location of embryonic lymph node development is determined by the initial clustering of lymphoid tissue inducer cells. We demonstrate that both CXCL13 and CCL21 attracted E12.5–E14.5 lymphoid tissue inducer cells and that initial clustering exclusively depended on CXCL13. Retinoic acid induced early CXCL13 expression in stromal organizer cells independent of lymphotoxin signaling. Notably, neurons adjacent to the lymph node anlagen expressed enzymes essential for retinoic acid synthesis. Furthermore, stimulation of parasymphathetic neural output in adults led to a retinoic acid receptor-dependent induction of CXCL13 in the gut. Therefore, our data show that initiation of lymph node development is controlled by retinoic acid-mediated expression of CXCL13 and suggest that retinoic acid may be provided by adjacent neurons.

Objective. In ϳ25% of synovial tissues from rheumatoid arthritis (RA) patients, infiltrates of T cells, B cells, and follicular dendritic cells (FDCs) are spatially organized into structures resembling lymph nodes with germinal centers. The remainder of the tissues lack FDCs and show either a diffuse or an aggregated T cell and B cell infiltrate. To gain more insight into this specific disease process, we sought to identify the genes expressed in RA tissues with ectopic lymphoid structures.Methods. Gene expression profiling of RA synovial tissues was determined by complementary DNA microarray analysis and quantitative real-time polymerase chain reaction. The presence of lymphoid follicles and localization of interleukin-7 (IL-7) in synovial tissue sections was determined by immunofluorescence staining using specific antibodies.Results. Findings of gene expression analysis confirmed previous reports that tissues with lymphoid structures showed elevated expression of CXCL13, CCL21, CCR7, and lymphotoxin ␣ and ␤ messenger RNA. In addition, the tissues also showed enhanced expression of the chemokines CXCL12 and CCL19 and the associated receptors CXCR4 and CXCR5, which are important for the attraction of T cells, B cells, and dendritic cells. Pathway analysis revealed increased expression of genes involved in JAK/STAT signaling, T cell-and B cell-specific pathways, Fc receptor type I signaling in mast cells, and IL-7 signal transduction in the tissues with ectopic lymphoid follicles, accompanied by increased expression of IL-7 receptor ␣ (IL-7R␣)/IL-2R␥ chains and IL-7. Protein expression of IL-7 in RA tissues was localized within fibroblast-like synoviocytes, macrophages, and blood vessels and was colocalized with extracellular matrix structures around the B cell follicles.Conclusion. Activation of the IL-7 pathway may play an important role in lymphoid neogenesis, analogous to its role in the development of normal lymphoid tissue.

The formation of lymph nodes is a complex process crucially controlled through triggering of LT␤R on mesenchymal cells by LT␣ 1 ␤ 2 expressing lymphoid tissue inducer (LTi) cells. This leads to the induction of chemokines to attract more hematopoietic cells and adhesion molecules to retain them. In this study, we show that the extravasation of the first hematopoietic cells at future lymph node locations occurs independently of LT␣ and that these cells, expressing TNF-related activation-induced cytokine (TRANCE), are the earliest LTi cells. By paracrine signaling the first expression of LT␣ 1 ␤ 2 is induced. Subsequent LT␤R triggering on mesenchymal cells leads to their differentiation to stromal organizers, which now also start to express TRANCE, IL-7, as well as VEGF-C, in addition to the induced adhesion molecules and chemokines. Both TRANCE and IL-7 will further induce the expression of LT␣ 1 ␤ 2 on newly arrived immature LTi cells, resulting in more LT␤R triggering, generating a positive feedback loop. Thus, LT␤R triggering by LTi cells during lymph node development creates a local environment to which hematopoietic precursors are attracted and where they locally differentiate into fully mature, LT␣ 1 ␤ 2 expressing, LTi cells. Furthermore, the same signals may regulate lymphangiogenesis to the lymph node through induction of VEGF-C.

During murine embryogenesis, the formation of Peyer’s patches (PPs) is initiated by CD45+CD4+CD3− lymphoid tissue inducers that trigger adhesion molecule expression and specific chemokine production from an organizing stromal cell population through ligation of the lymphotoxin-β receptor. However, the steps involved in the development of lymph nodes (LNs) are less clear than those of PPs, and the characteristics of the organizing cells within the LN anlagen have yet to be documented. In this study, we show for the first time that the early anlage is bordered by an endothelial layer that retains a mixed lymphatic and blood vascular phenotype up to embryonic day 16.5. This in turn encompasses CD45+CD4+CD3− cells interspersed with ICAM-1/VCAM-1/mucosal addressin cell adhesion molecule-1, lymphotoxin-β receptor-positive, chemokine-producing cells analogous to the organizing population previously observed in PPs. Moreover, these LN organizers also express the TNF family member, TRANCE. Lastly, we show that the ICAM-1/VCAM-1/mucosal addressin cell adhesion molecule-1 cells present in peripheral and mesenteric LN form two discrete populations expressing either intermediate or high levels of these adhesion molecules but that the former population is specifically reduced in PLN. These findings provide a possible explanation for the well-known differences in developmental requirements for nodes at peripheral or mesenteric locations.

For the formation of lymph nodes and Peyer's patches, lymphoid tissue inducer (LTi) 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. As lymphotoxin (LT)alpha1beta2 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 LTalpha1beta2-independent manner. The postnatal development of the splenic white pulp, involving the influx of T cells, depends on LTalpha1beta2 expressed by B cells.

The lymphatic vasculature drains lymph fluid from the tissue spaces of most organs and returns it to the blood vasculature for recirculation. Before reaching the circulatory system, antigens and pathogens transported by the lymph are trapped by the lymph nodes. As proposed by Florence Sabin more than a century ago and recently validated, the mammalian lymphatic vasculature has a venous origin and is derived from primitive lymph sacs scattered along the embryonic body axis. Also as proposed by Sabin, it has been generally accepted that lymph nodes originate from those embryonic primitive lymph sacs. However,we now demonstrate that the initiation of lymph node development does not require lymph sacs. We show that lymph node formation is initiated normally in E14.5 Prox1-null mouse embryos devoid of lymph sacs and lymphatic vasculature, and in E17.5 Prox1 conditional mutant embryos, which have defective lymph sacs. However, subsequent clustering of hematopoietic cells within these developing lymph nodes is less efficient.

The development of lymphoid organs depends on cross talk between hematopoietic cells and mesenchymal stromal cells and on vascularization of the lymphoid primordia. These processes are orchestrated by cytokines, chemokines, and angiogenic factors that require tight spatiotemporal regulation. Heparan sulfate (HS) proteoglycans are molecules designed to specifically bind and regulate the bioactivity of soluble protein ligands. Their binding capacity and specificity are controlled by modification of the HS side chain by HS-modifying enzymes. Although HS proteoglycans have been implicated in the morphogenesis of several organ systems, their role in controlling lymphoid organ development has thus far remained unexplored. In this study, we report that modification of HS by the HS-modifying enzyme glucuronyl C5-epimerase (Glce), which controls HS chain flexibility, is required for proper lymphoid organ development. Glce−/− mice show a strongly reduced size of the fetal spleen as well as a spectrum of defects in thymus and lymph node development, ranging from dislocation to complete absence of the organ anlage. Once established, however, the Glce−/− primordia recruited lymphocytes and developed normal architectural features. Furthermore, Glce−/− lymph node anlagen transplanted into wild-type recipient mice allowed undisturbed lymphocyte maturation. Our results indicate that modification of HS by Glce is required for controlling the activity of molecules that are instructive for early lymphoid tissue morphogenesis but may be dispensable at later developmental stages and for lymphocyte maturation and differentiation.

The recognition that lymphocytes existed in different varieties and that lymphoid organs were important for their differentiation greatly influenced immunological research. The growing awareness that started in the mid-fifties of the previous century has shifted the emphasis of immunology from a molecular, mostly serological science to the cell-oriented modern immunology of today. Matters such as hematopoietic differentiation, cell-cell interaction, cellular activation, as well as migratory behavior of hematopoietic cells received much attention and deepened our insight in the immune system. The relatively recent generation of mutant mice lacking lymphoid organs prompted the realization that the organogenesis of lymphoid organs could be dissected at the cellular and molecular level. Now we can distinguish several phases of development for lymphoid organs, and can assign molecules and cells to be essentially involved in these phases. Future research will identify additional molecules and cells required for the formation of the various lymphoid organs, because the picture is not complete yet.