Unit: Lymphoid Tissue Development

Director: Gérard EBERL

Adaptive immune responses are initiated in large and highly organized clusters of lymphocytes and antigen presenting cells, the secondary and tertiary lymphoid tissues (LTs). The development of secondary LTs, i.e. lymph nodes and Peyer's patches, is programmed and initiated in the sterile environment of the fetus. A cascade of events is triggered by lymphoid tissue inducer (LTi) cells, which recapitulates the inflammatory reaction, and culminates in the formation of complex structures carrying both specialized hematopoietic and stromal cells. The formation of tertiary LTs follows a similar pathway, but is induced after birth by commensal microbes, infection or injury. Chronic inflammation also leads to the spatial organization of the lesion and to tertiary LT formation, which may establish a positive feedback loop of lymphocyte activation. The aim of our laboratory is to understand the mechanisms that lead to the formation of LTs, during fetal life, in response to commensals, during infection and injury, and during the inflammatory reaction. The long-term goal is to develop new and specific tools for the control of chronic inflammatory disease, such as inflammatory bowel disease or rheumatoid arthritis, or conversely, to induce the formation of lymphoid tissues to enhance local immunity against infection or cancer.

1. Understanding the fetal development of secondary lymphoid tissues

G Eberl, M Lochner, L Peduto, D Bouskra

In the fetus, lymphoid tissue inducer (LTi) cells are recruited to sites of secondary lymphoid tissue (LT) development through the action of chemokines and integrins. LTi cells activate the local lymphoid stromal (LS) cells through membrane lymphotoxin LTα1β2 binding the LTβR, and induce the expression of additional chemokines and adhesion molecules. Although essential for the development of secondary LTs, fetal LTi cells remain poorly characterized, owing to the difficulty of identifying and isolating these cells from fetal tissues. We have previously characterized a nuclear hormone receptor, RORγt, which is specifically expressed by LTi cells. We have generated BAC-transgenic Rorc(γt)-egfpTG transgenic mice, in which LTi cells are the only cells expressing EGFP (BACs are large fragments of genomic DNA that are stably propagated in bacteria, can be modified, purified and used to generate transgenic mice). In these mice, we visualize the cellular events that unfold during LT development using high resolution immunofluorescence histology (see Fig. 1). In particular, we monitor the migration, differentiation and activation of LTi cells, the recruitment of lymphocytes and dendritic cells to nascent LTs, and the cross-talk between hematopoietic cells and stromal cells. Furthermore, LTi cells are isolated and studied both at the cellular and molecular levels.

2. Understanding the post-natal formation and function of tertiary lymphoid tissues

D. Bouskra & M. Lochner

During the first weeks of life, bacterial colonization of the murine colon and distal ileum induces the formation of numerous intestinal tertiary LTs (tLTs), termed isolated lymphoid follicles (ILFs) containing mainly B cells. We have recently provided evidence that clusters of intestinal LTi-like cells, the cryptopatches (CPs), are sensors of the local bacterial flora, as well as of intestinal infection and inflammation. CPs may differentiate into ILFs in order to increase the numbers of intestinal B cells required to maintain intestinal homeostasis. Tertiary LTs also form in chronic inflammatory lesions, as well as during acute lung Influenza and chronic stomach Helicobacter infections. Similar to the development of secondary LTs, the formation of mature tLTs requires LTα1β2 and TNF. We investigate the nature of the host/commensal and host/pathogen interactions that induce the formation of ILFs during colonization of the intestine with different strains of bacteria or pathogens, during inflammation, and in mice deficient for critical factors of innate or adaptive immunity. The impact of the CP/ILF system on intestinal homeostasis is measured in mice specifically deficient in tLTs.

3. Identifying lymphoid stromal cells initiating and supporting lymphoid tissue development

L. Peduto

Although lymphoid stromal (LS) cells remain poorly characterized, they are critical to the process of LT formation through the expression of several structural chemokines, including CXCL13, CCL19 and CCL21, adressins, including ICAM-1 and VCAM-1, and the lymphotoxin receptor LTβR. These factors are necessary for the productive cross-talk between LTi cells and LS cells. Using different BAC-transgenic mice, we are visualizing LS cells and monitoring their localization, activation and differentiation during LT development, as well as their cross-talk with LTi cells. Specific LS cell populations are isolated and characterized both at the cellular and molecular levels, or ablated in vivo using conditional activation of suicide genes.

4. Modulating pathologic inflammation through the control of lymphoid tissues

M. Lochner

Several observations document the role of intestinal lymphoid tissues in the progression of inflammatory bowel disease (IBD), and treatments that block the formation of LTs can prevent disease progression. Furthermore, it has been demonstrated that RORγt-deficient mice lack LTi-like cells and CPs, and cannot form intestinal tLTs. As RORγt is a nuclear hormone receptor and hence may bind small agonist or antagonist compounds, it will be possible to screen for such compounds and use them to modulate the function of LTi-like cells, inhibit the formation of tLTs and modulate the progression of inflammatory disease. Both chemical- and transgene-induced IBD models are used in Rorc(γt)-egfpTG transgenic mice to monitor the formation of intestinal tLTs, assess their impact on disease, and inhibit their formation and further disease progression.

5. Inducing therapeutic lymphoid tissues

L. Peduto

Several members of the tumor necrosis factor (TNF) family, including TNF, LTα and LTβ, have been shown to play an essential role both in inflammation and in the development of lymphoid tissues. Transgenic expression of these factors induces local inflammation and the formation of tLTs. We are assessing whether tLTs are capable of preventing, constraining and eliminating tumors in mouse models of colonic, pancreatic or prostate cancers. Our study may serve as a basis for the development of a new type of immunotherapy based on the Induction of tLTs in the direct vicinity of solid tumors.

Figure 1. Cryptopatches (CPs) and isolated lymphoid follicles (ILFs) in intestines of adult mice. CP and ILF cells similar to fetal lymphoid tissue inducer cells were stained with anti-EGFP antibody (green) in frozen section of intestines from Rorc(γt)-egfpTG transgenic mice. Dendritic cells were stained with anti-CD11c (red) and B cells with anti-CD45/B220 (blue). A small intestine CP is shown on the left (400x) and a colon ILF on the right (100x).

Keywords: Lymphoid Tissues, Inflammation, Stromal Cells, Lymphotoxin, Transgenesis

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