Development Of Tertiary Lymphoid Organs Research Articles

A18 DEVELOPMENT OF CHRONIC ILEITIS IN THE TNFΔARE MOUSE IS ASSOCIATED WITH A LOSS OF MESENTERIC LYVE1+ MACROPHAGES

Abstract Background Crohn’s disease (CD) is a form of inflammatory bowel disease that causes transmural inflammation of any part of the gastrointestinal (GI) tract but preferentially the terminal ileum and/or the colon. This chronic inflammation is not limited to the GI tract but also affects the mesentery of inflamed regions where it induces the formation of tertiary lymphoid organs (TLOs). The homeostatic LYVE1+ macrophages (LYmΦ) have been identified in many different organs including the mesentery. They have been shown to typically align along blood vessels where they participate in the control of collagen deposition and during inflammation, regulate vascular permeability and immune cell recruitment. Here we investigate the role of the mesenteric LYmΦ in a transgenic model of CD, the TNFΔARE mouse which develops terminal ileitis over time and expresses similar mesenteric alterations (TLOs) as seen in CD. Aims To determine whether the mesenteric LYmΦ population is altered during the development of chronic inflammation in the TNFΔARE mouse and whether they participate in the development of TLOs. Methods Confocal immunofluorescent imaging of the terminal ileal mesentery of TNFΔARE mice and littermate controls were preformed to identify changes to the populations of LYmΦ during the development of chronic inflammation. Myeloperoxidase activity was used to assess terminal ileitis. Results During the initial stage of ileitis (8 weeks) LYmΦs are present across the terminal ileal mesentery in both TNFΔARE and age-matched WT littermate controls with a subpopulation aligning along the collecting lymphatic vessels of the mesentery (n=6). At 20 weeks, while the ileitis progresses, aggregates of CD45+ cells have formed within the TNFΔARE mice where LYmΦs also accumulate (n=5). At 28 weeks when the ileitis worsens, the LYmΦs are greatly reduced from the terminal ileal mesentery as well as from the CD45+ cell aggregates (n=5). WT littermate controls never develop large CD45+ cell aggregates (n=5 for each time point). Antibodies to the LYVE1 cytosolic and extracellular domains confirmed the loss of the LYVE1 receptor over receptor cleavage, however, there was no correlation of altered collagen-1 deposition within the mesentery at 28 weeks. Conclusions Here we show a spatial relationship between LYmΦs and collecting lymphatic vessels as well as a decrease in LYmΦs during the development of chronic ileitis in the TNFΔARE mouse. Whether they contribute to TLO formation, or their loss exacerbates terminal ileitis is still unknown and under current investigation. Funding Agencies CCC

Induction and Anti-Tumor Function of Tertiary Lymphoid Organs

To induce the development of tertiary lymphoid organs (TLO) in a mouse model of melanoma and to evaluate TLO's functions in antitumor immunity. Lymphotoxin-beta receptor (LTβR) was overexpressed in NIH3T3 cells through the lentivirus system and the overexpression efficiency of LTβR in LTβR-NIH3T3 cells was examined. Western blot and qPCR were used to examine the non-canonical nuclear factor (NF)-κB signaling pathway in NIH3T3 cells overexpressing LTβR. B16-OVA melanoma mouse model was constructed to explore the induction of TLO and anti-tumor functions of TLO in LTβR-NIH3T3 cells. LTβR was overexpressed in NIH3T3 cells through the lentivirus system, and flow cytometry showed that the proportion of GFP + cells reached 99%. The overexpression of LTβR activated the non-canonical NF-κB signaling pathway in NIH3T3 cells. Findings from the mouse tumor model suggest that the injection of LTβR-NIH3T3 cells successfully induced the development of lymphoid tissue around the tumor and enhanced the tumor infiltration of T cells and MHCⅡ + macrophages, significantly inhibiting tumor growth and prolonging the survival of tumor-bearing mice. LTβR-NIH3T3 cells promoted anti-tumor immunity by inducing TLO development, which may provide new perspectives for tumor immunotherapy.

Tertiary lymphoid organs are associated with the progression of kidney damage and regulated by interleukin-17A.

Background: Tertiary lymphoid organs (TLOs) occur after multiple chronic kidney injuries. interleukin-17A (IL-17A) has been reported to associate with the development of TLOs in inflammatory diseases. However, regulation of the renal TLOs and its clinical significance to the pathogenesis of chronic kidney injury are unknown.Methods: To evaluate the clinical significance and regulation of renal TLOs, we analyzed the progression of patients with kidney damage based on the existence and absence of TLOs in a larger multicenter cohort. We also blocked the recruitment of lymphocyte cells into the kidney by FTY720 (fingolimod) in vivo. Besides, we used aged IL-17A genetic knocked out mice and IL-17A-neutralizing antibody to explore the role of IL-17A in renal TLOs formation.Results: We demonstrated that renal TLOs of IgA nephropathy patients were associated with disease severity and were independent risk factors for renal progression after adjustment for age, sex, mean arterial pressure, proteinuria and, baseline eGFR and MEST-C score, especially in the early stage. Plasma levels of TLO-related chemokines CXCL13, CCL19, and CCL21 were higher in patients with renal TLOs. Inhibiting the formation of renal TLOs by FTY720 could reduce the intrarenal inflammation and fibrosis, and early intervention was found to be more effective. IL-17A was increased in renal TLOs models, and genetic depletion of IL-17A or treatment with anti-IL-17A antibody resulted in a marked reduction of the TLOs formation as well as alleviation of renal inflammation and fibrosis.Conclusion: These results indicate that TLOs are associated with the progression of kidney damage and regulated by IL-17A and may be effective targets for the treatment of kidney damage.

Identification of key genes and pathways contributing to artery tertiary lymphoid organ development in advanced mouse atherosclerosis.

Atherosclerosis is a leading cause of mortality worldwide. Artery tertiary lymphoid organ (ATLO) neogenesis is affected by abdominal aorta atherosclerosis, which may lead to an immune response. The present study obtained microarray data to investigate the gene expression differences underlying the potential pathogenesis of atherosclerosis and to elucidate the mechanisms underlying ATLO development. Microarray studies of the aorta, plaques, adventitia, blood, spleen, renal lymph nodes and ATLO were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified in aorta clusters and ATLO clusters. Kyoto Encyclopedia of Genes and Genomes enrichment and Gene Ontology (GO) analyses were conducted to predict the biological functions of DEGs. The results demonstrated that interleukin 7 receptor (Il7r), C-X-C motif chemokine ligand (Cxcl)16, Cxcl13, Cxcl12, C-C motif chemokine receptor 2, C-C motif chemokine ligand (Ccl)8, Ccl5 and Ccl12 may function through pathways associated with ‘cytokine-cytokine receptor interaction’ and ‘chemokine signaling pathway’ in ATLO. Gene expression alterations were validated by reverse transcription-quantitative polymerase chain reaction. Il7r appeared to be the central gene involved in these events, and chemokines and/or chemokine receptors were visualized by GO enrichment. A protein-protein interaction network was constructed, which suggested that Il7r had a core function in all clusters. Taken together, the results indicated that Il7r upregulation may serve an important role in ATLO development via ‘cytokine-cytokine receptor interaction’ and ‘chemokine signaling pathway’. This may provide novel perspectives for understanding ATLO development and the regulation of the immune response in atherosclerosis.

28 MESENTERIC TERTIARY LYMPHOID ORGANS INTEGRATED INTO THE INTESTINAL-DRAINING LYMPHATIC NETWORK ARE RESISTENT TO ANTI-TNF THERAPHY IN CROHN’S DISEASE MOUSE MODEL

Characterization of Crohn’s disease (CD) includes susceptible mucosal surfaces via abnormal intestinal permeability caused by either deregulated interactions between the bacterial flora and surface epithelial cells or an overwhelming host immune response. However, historical and recent studies demonstrate lymphatic vessel obstruction and lymphangitis as a critical component of CD. A hallmark of CD is lymphatic vessel swelling, poor drainage, with submucosal edema derived from the dysfunctional lymphatics. Given that lymph flow is essential for lipid absorption and antigen transport, we recently show obstructed mesenteric lymphatic drainage in CD patient biopsies through the development of tertiary lymphoid organs (TLO) on mesenteric collecting vessels. However, this feature of the disease is unknown in inflammatory bowel disease (IBD) mice models. We sought to investigate intestine-draining lymphatics in IBD using whole-mount image and confocal microscopy. We define the existence of TLO associated with mesenteric lymphatic vessels in a well-establish mouse model of chronic ileitis (TNFΔARE model). We generated an inducible lymphatic endothelial cell reporter (Prox1creER-tdTomato) containing TNFΔARE deletion, which develops ileitis. These allow us to confirm mesenteric lymphangiogenesis and association with TLO in the obstruction of intestinal draining lymphatic collectors in mice. Surprisingly, anti-TNF treatment of TNFΔARE mice fails to regress established mesenteric TLO. We investigate lipid and immune cell trafficking using photoactivatable systems to investigate lymphatic network remodeling during IBD progression. These findings suggest that chronic inflammatory imbalance promotes lymphatic remodeling and TLO formation outside the intestine. Our future directions focus on whether tertiary lymphoid tissues prevent dendritic cell migration and their upstream tolerogenic control.

High Endothelial Venules and Other Blood Vessels: Critical Regulators of Lymphoid Organ Development and Function.

The blood vasculature regulates both the development and function of secondary lymphoid organs by providing a portal for entry of hemopoietic cells. During the development of lymphoid organs in the embryo, blood vessels deliver lymphoid tissue inducer cells that initiate and sustain the development of lymphoid tissues. In adults, the blood vessels are structurally distinct from those in other organs due to the requirement for high levels of lymphocyte recruitment under non-inflammatory conditions. In lymph nodes (LNs) and Peyer’s patches, high endothelial venules (HEVs) especially adapted for lymphocyte trafficking form a spatially organized network of blood vessels, which controls both the type of lymphocyte and the site of entry into lymphoid tissues. Uniquely, HEVs express vascular addressins that regulate lymphocyte entry into lymphoid organs and are, therefore, critical to the function of lymphoid organs. Recent studies have demonstrated important roles for CD11c+ dendritic cells in the induction, as well as the maintenance, of vascular addressin expression and, therefore, the function of HEVs. Tertiary lymphoid organs (TLOs) are HEV containing LN-like structures that develop inside organized tissues undergoing chronic immune-mediated inflammation. In autoimmune lesions, the development of TLOs is thought to exacerbate disease. In cancerous tissues, the development of HEVs and TLOs is associated with improved patient outcomes in several cancers. Therefore, it is important to understand what drives the development of HEVs and TLOs and how these structures contribute to pathology. In several human diseases and experimental animal models of chronic inflammation, there are some similarities between the development and function of HEVs within LN and TLOs. This review will summarize current knowledge of how hemopoietic cells with lymphoid tissue-inducing, HEV-inducing, and HEV-maintaining properties are recruited from the bloodstream to induce the development and control the function of lymphoid organs.

Novel CXCL13 transgenic mouse: inflammation drives pathogenic effect of CXCL13 in experimental myasthenia gravis.

Abnormal overexpression of CXCL13 is observed in many inflamed tissues and in particular in autoimmune diseases. Myasthenia gravis (MG) is a neuromuscular disease mainly mediated by anti-acetylcholine receptor autoantibodies. Thymic hyperplasia characterized by ectopic germinal centers (GCs) is a common feature in MG and is correlated with high levels of anti-AChR antibodies. We previously showed that the B-cell chemoattractant, CXCL13 is overexpressed by thymic epithelial cells in MG patients. We hypothesized that abnormal CXCL13 expression by the thymic epithelium triggered B-cell recruitment in MG. We therefore created a novel transgenic (Tg) mouse with a keratin 5 driven CXCL13 expression.The thymus of Tg mice overexpressed CXCL13 but did not trigger B-cell recruitment. However, in inflammatory conditions, induced by Poly(I:C), B cells strongly migrated to the thymus. Tg mice were also more susceptible to experimental autoimmune MG (EAMG) with stronger clinical signs, higher titers of anti-AChR antibodies, increased thymic B cells, and the development of germinal center-like structures. Consequently, this mouse model finally mimics the thymic pathology observed in human MG.Our data also demonstrated that inflammation is mandatory to reveal CXCL13 ability to recruit B cells and to induce tertiary lymphoid organ development.

Control of the T follicular helper-germinal center B-cell axis by CD8⁺ regulatory T cells limits atherosclerosis and tertiary lymphoid organ development.

The atheromodulating activity of B cells during the development of atherosclerosis is well documented, but the mechanisms by which these cells are regulated have not been investigated. Here, we analyzed the contribution of Qa-1-restricted CD8(+) regulatory T cells to the control of the T follicular helper-germinal center B-cell axis during atherogenesis. Genetic disruption of CD8(+) regulatory T cell function in atherosclerosis-prone apolipoprotein E knockout mice resulted in overactivation of this axis in secondary lymphoid organs, led to the increased development of tertiary lymphoid organs in the aorta, and enhanced disease development. In contrast, restoring control of the T follicular helper-germinal center B-cell axis by blocking the ICOS-ICOSL pathway reduced the development of atherosclerosis and the formation of tertiary lymphoid organs. Moreover, analyses of human atherosclerotic aneurysmal arteries by flow cytometry, gene expression analysis, and immunofluorescence confirmed the presence of T follicular helper cells within tertiary lymphoid organs. This study is the first to demonstrate that the T follicular helper-germinal center B-cell axis is proatherogenic and that CD8(+) regulatory T cells control the germinal center reaction in both secondary and tertiary lymphoid organs. Therefore, disrupting this axis represents an innovative therapeutic approach.

M1 macrophages act as LTβR-independent lymphoid tissue inducer cells during atherosclerosis-related lymphoid neogenesis

The goal of this study was to characterize the role of inflammatory macrophages in the induction of the vascular smooth muscle cell (VSMC)-mediated formation of aortic tertiary lymphoid organs (TLOs). Mouse bone marrow-derived M1 macrophages acted as lymphoid tissue inducer cells. Indeed, they expressed high levels of tumour necrosis factor (TNF)-α and membrane-bound lymphotoxin (LT)-α, two inducing cytokines that triggered expression of the chemokines CCL19, CCL20, and CXCL16, as did M1 supernatant. The blockade of LTβR signalling with LTβR-Ig had no effect, whereas that of TNFR1/2 signalling reduced chemokine expression by VSMCs in both wild-type (WT) and LTβR KO mice, demonstrating that LTβR signalling is dispensable for the M1-inducing effect. This effect was corroborated by the development of TLOs observed in LTβR KO->apolipoprotein E knockout (ApoE KO) aortic segments after orthotopic transplantation. Furthermore, treatment of ApoE KO mice with anti-TNF-α antibody decreased the number and incidence of aortic TLOs. Finally, lymphoid nodules composed of T and B cells formed in in vivo-implanted scaffolds seeded with VSMCs previously stimulated ex vivo by M1-conditioned medium. These results are the first to identify M1 macrophages as inducer cells that trigger the expression of chemokines by VSMCs independently of LTβR signalling. We propose that the dialogue between macrophages and VSMCs-established across the vascular wall-contributes to the formation of aortic TLOs.

Regulation of T follicular helper cells by CD8+ regulatory T cells reduces pro-atherogenict tertiary lymphoid organ formation in apolipoprotein E KO mice

Tertiary lymphoid organs (TLOs), composed of B cell follicles, develop in the adventitia of atherosclerotic arteries. However, it is not known whether TLOs play an effective role in atherogenesis. In order to address this issue it is necessary to modulate the formation/function of these ectopic B cell follicles within the adventitia. Recent studies have shown that the function of germinal center B cells in secondary lymphoid organs (SLO) is tightly controlled by the Qa-1-restricted CD8+ T cells (CD8+ Treg) which regulate T follicular helper (TfH) cells function. We therefore evaluated whether regulation of TfH by CD8+ Treg can control the development of TLOs and, in turn, of atherogenesis in hypercholesterolemic (ApoE°) mice. We firstly found that the frequency of CD8+ Treg in ApoE° mice significantly decreases with age in the spleen and peripheral lymph nodes of this model. We therefore crossed ApoE° mice with the D227K-Tg mice in which, due to a Qa-1 point mutation, the CD8+ Treg are unable to control the development/function of TfH cells. ApoE° were compared with ApoE°/D227K-Tg littermates (n=5-12/group) at 8, 15 and 35 weeks of age. Qa-1 mutation increased the TfH cell population in SLO of ApoE°/D227K-Tg as compared to ApoE° mice. This effect was accompanied by an increase in the percentage of activated/memory CD4+ and CD8+ T cells and of B cells in lymph nodes of ApoE°/D227K-Tg, at 15 and 35 weeks of age. In parallel, lesion development was accelerated: atherosclerotic plaques in the aortic root of ApoE°/D227K-Tg mice were 2-fold greater than those of ApoE° mice. Immunofluorescent analysis of adventitial whole mounts revealed a greater incidence of TLOs (organized B cell follicles) in ApoE°/D227K-Tg than in ApoE° mice. Our data therefore suggest that CD8+ Treg control the development of adventitial pro-atherogenic TLOs in ApoE° mice through their regulatory action on TfH cells.

B Cells Produce CXCL13 in Lymphoid Neogenesis during Chronic Obstructive Pulmonary Disease. The New Kid on the Block?

Tertiary lymphoid organs (TLO) or ectopic lymphoid follicles are lymphoid aggregates with specialized structures that form under chronic inflammatory processes (1). Their denomination comes from the resemblance these structures bear with secondary lymphoid organs (SLOs), and include the presence of defined B- and T-cell areas, follicular dendritic cells, the promotion of lymphatic development, and the acquisition of high endothelial venule properties by blood vessels (1). TLOs, however, differ from SLOs in that they are not developmentally preprogrammed and appear after exposure to antigen. TLOs have been described in a variety of inflammatory and infectious settings, including influenza (2) and Mycobacterium tuberculosis (3) infections as well as autoimmune conditions such as rheumatoid arthritis (4), multiple sclerosis (5), and systemic lupus erythematosus (6). There is accumulating evidence suggesting that TLOs may be involved in local immune responses that may occur under inflammatory conditions. As such, development of TLOs often correlates with disease aggravation and promotion of autoreactive T- and B-cell activation in autoimmune conditions (1). However, in some infectious diseases, as is the case in tuberculosis (7), their presence may be associated with development of protective immune responses and improved pathogen control.

The development of tertiary lymphoid organs in the central nervous system facilitates determinant spreading of the MP4-specific T cell response (P4164)

Abstract Accumulating evidence indicates that B cells aggregate into tertiary lymphoid organs (TLOs) in the central nervous system (CNS) in the human autoimmune disease multiple sclerosis (MS). In patients with secondary-progressive MS the presence of ectopic B cell aggregates has been linked to more severe clinical disease and cortical pathology. Here we demonstrate the formation of TLOs in myelin basic protein (MBP)-proteolipid protein (PLP) fusion protein MP4-induced experimental autoimmune encephalomyelitis (EAE) of C57BL/6 mice. TLOs were a characteristic feature of the chronic disease stage and most prevalent in the cerebellum, while also being present in the spinal cord and cerebrum, but absent in the brain stem. TLOs were characterized by B and T cell compartmentalization, the presence of high endothelial venules and germinal center formation. An association with TH17, but not TH1 cells was evident. In addition, results obtained from ELISPOT analysis of the antigen-specific T cell response suggest that TLOs facilitate determinant spreading of the MP4-specific T cell response to myelin oligodendrocyte glycoprotein (MOG). Our data add novel insights into the contribution of TLOs to disease progression in the context of CNS autoimmunity. We suggest that the blockade of TLO development is a therapeutic strategy that needs to be carefully considered in future studies.

Tertiary lymphoid organ development coincides with determinant spreading of the myelin-specific T cell response

While the role of T cells has been studied extensively in multiple sclerosis (MS), the pathogenic contribution of B cells has only recently attracted major attention, when it was shown that B cell aggregates can develop in the meninges of a subset of MS patients and were suggested to be correlates of late-stage and more aggressive disease in this patient population. However, whether these aggregates actually exist has subsequently been questioned and their functional significance has remained unclear. Here, we studied myelin basic protein (MBP)-proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), which is one of the few animal models for MS that is dependent on B cells. We provide evidence that B cell aggregation is reflective of lymphoid neogenesis in the central nervous system (CNS) in MBP-PLP-elicited EAE. B cell aggregation was present already few days after disease onset. With disease progression CNS B cell aggregates increasingly displayed the phenotype of tertiary lymphoid organs (TLOs). Our results further imply that these TLOs were not merely epiphenomena of the disease, but functionally active, supporting intrathecal determinant spreading of the myelin-specific T cell response. Our data suggest that the CNS is not a passive "immune-privileged" target organ, but rather a compartment, in which highly active immune responses can perpetuate and amplify the autoimmune pathology and thereby autonomously contribute to disease progression.

Proinflammatory IL-17 induces iBALT development

Proinflammatory IL-17 induces iBALT development

IL-17, a new kid on the block of tertiary lymphoid organs

Tertiary lymphoid organs (TLOs) are structures that resemble lymph nodes that can be found in chronically inflamed tissues in a number of inflammatory conditions. TLOs can host ongoing immune responses that can potentially contribute to disease pathogenesis. The development of TLOs has been thought in large part to recapitulate the development of secondary lymphoid organs, but the regulation of TLO development has not been well understood. In a recent article in Nature Immunology, Troy Randall and colleagues1 identify IL-17, a cytokine that contributes to the pathogenesis of a number of inflammatory diseases, as a novel critical player in TLO formation in inflamed lungs.

Blocking lymphotoxin signaling abrogates the development of ectopic lymphoid tissue within cardiac allografts and inhibits effector antibody responses

Tertiary lymphoid organs (TLOs) may develop within allografts, but their contribution to graft rejection remains unclear. Here, we study a mouse model of autoantibody-mediated cardiac allograft vasculopathy to clarify the alloimmune responses mediated by intragraft TLOs and whether blocking lymphotoxin-β-receptor (LTβR) signaling, a pathway essential for lymphoid organogenesis, abrogates TLO development. TLOs (defined as discrete lymphoid aggregates associated with high endothelial venules) were detectable in 9 of 13 heart allografts studied and were predominantly B cell in composition, harboring germinal-center activity. These are most likely manifestations of the humoral autoimmunity triggered in this model after transplantation; TLOs did not develop if autoantibody production was prevented. Treatment with inhibitory LTβR-Ig fusion protein virtually abolished allograft TLO formation (mean TLOs/heart: 0.2 vs. 2.2 in control recipients; P=0.02), with marked attenuation of the autoantibody response. Recipients primed for autoantibody before transplantation rejected grafts rapidly, but this accelerated rejection was prevented by postoperative administration of LTβR-Ig (median survival time: 18 vs. >50 d, respectively, P=0.003). Our results provide the first demonstration that TLOs develop within chronically rejecting heart allografts, are predominantly B cell in origin, and can be targeted pharmacologically to inhibit effector humoral responses.

Manifestation of Spontaneous and Early Autoimmune Gastritis in CCR7-Deficient Mice

Autoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4(+) T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis.

Prevention of Diabetes by FTY720-Mediated Stabilization of Peri-Islet Tertiary Lymphoid Organs

OBJECTIVEThe nonobese diabetic (NOD) mouse is a well-established mouse model of spontaneous type 1 diabetes, which is characterized by an autoimmune destruction of the insulin-secreting pancreatic β-cells. In this study, we address the role of tertiary lymphoid organs (TLOs) that form in the pancreas of NOD mice during disease progression.METHODSWe developed a model designed to “lock” lymphocytes in the pancreatic lymph node (PLN) and pancreas by the use of FTY720, which blocks the exit of lymphocytes from lymph nodes. A combination of flow cytometry, immunofluorescence, and analysis of clinical scores was used to study the effects of long-term FTY720 treatment on TLO development and development of diabetes.RESULTSContinuous treatment of NOD mice with FTY720 prevented diabetes development even at a time of significant insulitis. Treatment withdrawal led to accelerated disease independent of the PLN. Interestingly, naive T-cells trafficked to and proliferated in the TLOs. In addition, morphological changes were observed that occurred during the development of the disease. Remarkably, although the infiltrates are not organized into T/B-cell compartments in 8-week-old mice, by 20 weeks of age, and in age-matched mice undergoing FTY720 treatment, the infiltrates showed a high degree of organization. However, in naturally and FTY720-induced diabetic mice, T/B-cell compartmentalization was lost.CONCLUSIONOur data show that TLOs are established during diabetes development and suggest that islet destruction is due to a loss of TLO integrity, which may be prevented by FTY720 treatment.

Néogenèse lymphoïde et lymphangiogenèse : deux nouveaux mécanismes impliqués dans la physiopathologie du rejet chronique

Chronic rejection is one of the main causes of late allograft failure and no therapy is currently available to prevent efficiently its development. Improving the comprehension of the mechanisms involved in the pathophysiology of chronic rejection is a mandatory step to propose innovative therapies that would prolong grafts' survival. Using the rat aortic interposition model of chronic vascular rejection, we have demonstrated that the intragraft inflammatory infiltrate progressively organized itself into a functional ectopic lymphoid tissue (tertiary lymphoid organ) supporting the local synthesis of alloantibody. Thus, during chronic rejection the graft is at the same time the target and the site of elaboration of the humoral allo-immune response. This hypothesis has been confirmed in the clinical setting by the analysis of human grafts (kidneys, hearts and lungs) removed for terminal failure due to chronic rejection. This lymphoid neogenesis process, previously identified in other chronic inflammatory diseases, occurs with a strikingly high frequency in chronically rejected grafts, suggesting that an additional mechanism synergizes to initiate the development of tertiary lymphoid organs during chronic rejection. We propose that the defective lymphatic drainage of chronically rejected organs triggers lymphoid neogenesis and we discuss the complex crosstalk between lymphoid neogenesis and lymphangiogenesis that takes place during chronic rejection.

Keystones in lymph node development

New molecular markers are constantly increasing our knowledge of developmental processes. In this review article we have attempted to summarize the keystones of lymphoid tissue development in embryonic and pathological conditions. During embryonic lymph node development in the mouse, cells from the anterior cardinal vein start to bud and sprout, forming a lymph sac at defined sites. The protrusion of mesenchymal tissue into the lymph sacs forms the environment, where so-called 'lymphoid tissue inducer cells' and 'mesenchymal organizer cells' meet and interact. Defects of molecules involved in the recruitment and signalling cascades of these cells lead to primary immunodeficiency diseases. A comparison of molecules involved in the development of secondary lymphoid organs and tertiary lymphoid organs, e.g. in autoimmune diseases, shows that the same molecules are involved in both processes. This has led to the hypothesis that the development of tertiary lymphoid organs is a recapitulation of embryonic lymphoid tissue development at ectopic sites.