NF-kB/RelB Function During Lymph Node Development

One of the most important steps during the immune response to an infectious agent is the activation of lymphocytes, through their interaction with other cells that present the infectious agent to them. These interactions occur in very specific microenvironments that are organised in lymph nodes. A failure in lymph node development or in the organisation of the specific areas for lymphocyte-antigen presenting cell interactions will result in impaired immune responses to infections and decrease life expectancy of the host. Previous work by different research groups had demonstrated the similarities between chronic inflammation and lymph node formation. In other words, the same molecules and signalling pathways that participate in the formation of secondary lymphoid organs during foetal and early neonatal life are involved in the development of tertiary lymphoid tissues during chronic inflammatory diseases in humans. We have demonstrated that the product of a gene named NF-kB2, although not necessary for lymph node formation, is required for their normal organisation and the recruitment of lymphoid cells to these organs. In contrast, our recent results showed that another protein, RelB, which acts together as a dimer with NF-kB2, plays a critical role in lymph node formation, for lymph nodes developed very poorly in the absence of RelB. However, despite this initial observation, we know virtually nothing about the processes controlled by RelB. This project proposes to investigate RelB function in lymph node development, using a combination of genetics and novel reconstituted cellular systems that have been developed in our laboratories. Our hypothesis is that RelB mediates a proliferation-survival signal in cells that form the structure of the lymph node (stromal cells) and in its absence these cells either fail to proliferate or undergo cell death resulting in the absence of lymph nodes. To test our hypothesis we propose to investigate the following points: i- To determine whether RelB is required in lymph node stromal cells for their proliferation and/or survival, and for the expression of molecules involved lymph node development. ii- To determine what are the RelB-target genes required for cell proliferation and/or survival. iii- To determine whether RelB is required in lymph node inducer cells for their proliferation and/or survival. This project, by identifying the signals and mechanisms that regulate lymph node formation, will strengthen our understanding of the development of these important organs of the immune system. In addition, investigating the interactions between inducer and organiser cells is also relevant to studies investigating the cross-talk between B cells and precursors of follicular dendritic cells (pFDCs) that results in FDC maturation, as well as B cell-FDCs communication during antibody affinity maturation in germinal centres. Indeed, several of the ligands, receptors and transcription factors involved in lymph node formation are common with those required for FDC-B cell interactions. Dissecting the processes of lymph node development will help understanding the mechanisms behind the development of ectopic lymphoid tissues in chronic inflammatory diseases. In particular, this project will establish whether interfering with RelB in a cell specific manner is a suitable alternative to current treatments.

Specific activation of lymphocytes during immune responses occurs in secondary lymphoid tissues such as lymph nodes (LNs). LNs contain microenvironments that facilitate contact between antigen presenting cells and T cells that are essential for the development of adaptive immune responses. Absence of LNs or a disruption of the specific microenvironments leads to impaired immune responses to infections and decrease survival of the host. LN organogenesis depends on the successful interaction between bone marrow derived 'inducer' cells and mesenchymal 'organizer' stromal cells through ligands and receptors of the Tumour Necrosis Factor family that converge in activation of the NF-kB family of transcription factors resulting in dramatic changes in gene expression. This project aims to uncover the specific functions of the transcription factor NF-kB/RelB whose absence leads to a failure in LN development. Our hypothesis is that RelB mediates a proliferation-survival signal in stromal cells downstream of the Lymphotoxin b receptor (LtbR) and in its absence cells either fail to proliferate or undergo apoptosis resulting in the absence of LNs in relb-/- mice. We want to establish whether RelB controls cell proliferation and/or survival of stromal cells, to identify the RelB-target genes in these processes and to assess whether Rel-B is also required in inducer cells for their proliferation-survival. Among the methods that will be used in this project it is important to highlight the isolation and expansion of inducer and organiser cells; the use of LN organ cultures to analyse the interaction between these two cell types and the grafting of neonatal LNs or re-aggregate LNs under the kidney capsule of congenic mice to assess the colonisation of these organs by host lymphocytes. These methods include for the first time in the field of lymphoid tissue organogenesis a reconstituted cellular system that recapitulate the in vivo microenvironment of LN development.