Aim of the project: The development of the immune system is a well organized process in which several phases can be distinguished. The early formation of lymph nodes and Peyer's patches involves molecules such as Lymphotoxin-alpha (LT-alpha), -beta (LT-beta) and their receptor LTbeta-R since mice deficient for these molecules show a complete lack of lymph nodes and Peyer's patches. It is also demonstrated that in the earliest phase of lymph node development, the lymph node anlages are specifically colonized by an unusual cell population, characterized by the expression of CD4 in the absence of CD3. These cells express LT-beta, and could potentially play a role in the development of the lymph node tissue architecture. Upon in vivo transfer, these cells migrate specifically into B-cell follicles, suggesting a role in the development of FDCs, once they have entered the lymph nodes. We aim to identify the cells that are instrumental to the development of lymphoid organs by creating a knock-in mouse, in which we will be able to follow the LT-beta expressing cells and their progeny. In this way we will also be able to study the the cells that induce lymph node formation, and determine to which lineage they give rise. By developing an essay to study the formation of FDCs, we also want to identify the precursors to these cells. Summary of the results of the past year: Follicular Dendritic Cells (FDC) form a major constituent of lymphoid follicles and have the unique ability to bind and retain ag in the form of ag-ab complexes for prelonged periods of time. By continuous presentation of these immune complexes to follicular B-cells, FDCs are crucial in the induction of B-cell memory. The nature of the cellular and molecular requirements for the maintenance and induction of FDC in adult lymphoid tissues has been studied in gene-targeted mice which lack FDC-clusters. An important role was postulated for several members of the TNF-receptor/ligand family: Ltalpha1beta2 and TNFalpha expressed by B-cells and the Ltbeta-receptor (Ltbeta-R), present on as yet unidentified stromal cells within the lymph nodes. There has been much debate to whether FDCs originate from stromal or hematopoietic precursor cells, and this still remains enigmatic. In this study we have used the murine neonatal lymph node as a model system to study the development of FDCs. We show that in normal neonates the first FDC-clusters appear at day 7 after birth. Lack of FDC development in the first days of life could be due to the absence of precursor cells and/or the absence of signaling via Ltalpha1beta2 and TNFalpha and their receptors. To address these issues, B-cells present in lymph nodes during the first days after birth were analyzed. These B-cells were IgM+/IgD+/B220+/CD19+ and thus appear to be mature cells. Adult splenic B-cells were subsequently stimulated with various physiological stimuli, in order to determine the signals necessary for induction of Ltalpha1beta2 and TNFalpa. By RNAse-protection assays we show that anti-CD40 induces message for Ltalpha, Ltbeta and TNFalpha, IL-4 induces solely Ltbeta message and IL-4 in combination with anti-Ig induces only message for TNFalpha. After overnight stimulation, these adult B-cells were injected into newborn mice. This resulted in the induction of FDC-clusters on day 4 after birth, which always colocalized with the injected B-cells. It can thus be concluded that starting at day 4 FDC can be formed in murine lymph nodes, but only if mature B-cells are present. The precursors to FDCs are thus present from day 4 after birth.