Uta E. Höpken

The impact of CCR7 and CXCR5 on lymphoid organ development and systemic immunity

Summary:  The development of secondary lymphoid organs is a complex process dependent on a coordinated interaction of cells of hematopoietic and non‐hematopoietic origin. In this context, chemokines and cytokines belonging to the tumor necrosis factor (TNF)/lymphotoxin (LT) family are critical signaling molecules during the initial steps of lymph node and Peyers patch organogenesis. Homeostatic chemokines, such as CXCL13, CCL21, and CCL19, as well as their corresponding receptors, CXCR5 and CCR7, have now been shown to closely cooperate in the development of lymphoid organs and the maintenance of lymphoid tissue microarchitecture. We summarize recent data on the function of CXCR5 and CCR7 and their intricate connection to the TNF/LT system in order to refine the current model of lymphoid organ development.

Role for CCR7 Ligands in the emigration of newly generated T lymphocytes from the neonatal thymus

Most T lymphocytes are generated within the thymus. It is unclear, however, how newly generated T cells relocate out of the thymus to the circulation. The present study shows that a CC chemokine CCL19 attracts mature T cells out of the fetal thymus organ culture. Another CC chemokine CCL21, which shares CCR7 with CCL19 but has a unique C-terminal extension containing positively charged amino acids, failed to show involvement in thymic emigration. Neonatal appearance of circulating T cells was defective in CCL19-neutralized mice as well as in CCR7-deficient mice but not in CCL21-neutralized mice. In the thymus, CCL19 is predominantly localized in the medulla including endothelial venules. These results indicate a CCL19- and CCR7-dependent pathway of thymic emigration, which represents a major pathway of neonatal T cell export.

Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation

The location of embryonic lymph node development is determined by the initial clustering of lymphoid tissue–inducer (LTi) cells. Here we demonstrate that both the chemokine CXCL13 and the chemokine CCL21 attracted LTi cells at embryonic days 12.5–14.5 and that initial clustering depended exclusively on CXCL13. Retinoic acid (RA) induced early CXCL13 expression in stromal organizer cells independently of lymphotoxin signaling. Notably, neurons adjacent to the lymph node anlagen expressed enzymes essential for RA synthesis. Furthermore, stimulation of parasymphathetic neural output in adults led to RA receptor (RAR)-dependent induction of CXCL13 in the gut. Therefore, our data show that the initiation of lymph node development is controlled by RA-mediated expression of CXCL13 and suggest that RA may be provided by adjacent neurons.

Distinctive role of CCR7 in migration and functional activity of naive-and effector/memory-like Treg subsets

Foxp3+CD25+CD4+ Treg play a fundamental role in the maintenance of self tolerance and the control of inflammatory reactions. Previous data demonstrated a division of labor between naive‐ and effector/memory‐like Treg subsets, which is largely based on their lymph node‐recirculating and inflammation‐seeking migration behavior, respectively. The chemokine receptor CCR7 is expressed on both types of Treg subsets, albeit at different levels. Whether it fulfills similar or distinct roles in these subsets has not been studied so far. We here show that the recirculation of naive‐like Treg through LN and, to some extent, the gut is dependent on CCR7. Lack of CCR7 not only prevents recirculation, but also almost completely abolishes the ability of naive‐like Treg to control the priming phase of an immune response. In contrast, CCR7 deficiency in effector/memory‐like Treg promotes their accumulation in inflamed sites, compatible with a role of CCR7 for exit from the tissue. Local Treg accumulation was accompanied by an enhanced suppression of inflammation. Together, our findings provide conclusive evidence that CCR7 expression on Treg differentially controls in vivo function of the naive‐ and effector/memory‐like subsets.

Systemic immunoregulatory and pathogenic functions of homeostatic chemokine receptors.

The adoptive immune response relies on a precise temporal and spatial positioning of lymphocytes within lymphoid and nonlymphoid tissues. Chemokines, constitutively expressed or induced during inflammation provide a flexible navigation system directing lymphocytes into specific microcompartments. Precision and specificity in this process are achieved by varying patterns of chemokine receptors expressed on the cell surface of lymphocytes in the course of cell differentiation. The chemokine receptors CXCR5 and CCR7 are principal regulators for targeting T cells, B cells, and dendritic cells into secondary lymphoid organs. The analyses of knockout mice have been instrumental in exploring the crucial role of these receptors for the compartmentalization of secondary lymphoid organs into functionally separated T and B cell zones. Aside from the homeostatic recirculation of lymphocytes and inflammatory processes, chemokine receptors are also involved in malignancies such as lymphoproliferative diseases and cancer metastasis. Recent results from our laboratory present evidence for the involvement of CCR7 in the dissemination of neoplastic cells in classic Hodgkin disease. There is also accumulating evidence for the involvement of CXCR5 in the formation of ectopic follicles as observed in lymphomas or autoimmune diseases. In addition, CCR7 and CXCR5 have been identified as useful markers in the classification of functionally distinct subsets of T‐helper cells, which will lead to a better understanding of T cell memory and T cell effector function in lymphoid system homeostasis and disease.

Mycobacterium tuberculosis Triggers Formation of Lymphoid Structure in Murine Lungs

The hallmark of pulmonary tuberculosis is the granuloma, which consists predominantly of lymphocytes and macrophages and promotes immune-cell interaction with the causative pathogen, Mycobacterium tuberculosis. Granuloma formation is a highly organized process, which depends on leukocyte recruitment facilitated by adhesion molecules and chemokines. Thus, during chronic experimental tuberculosis, granulomata display characteristics of lymphoid structures comprising follicular aggregation of B cells, formation of high endothelial venules, presence of follicular dendritic cells, and expression of the homeostatic chemokines CXCL13 and CCL19. CCR7-/- mice, which are deficient in CCL19 and CCL21 signaling, exhibit increased local inflammatory infiltrates but no follicular B-cell aggregation within those lymphoid structures. However, CCR7-deficient mice are fully capable to control pulmonary tuberculosis; at time points later than 6 weeks postinfection, they carry a lower bacterial load in peripheral organs. Our results show that, during chronic pulmonary tuberculosis in mice, the homeostatic chemokine signaling-network contributes to spatial organization of the granulomatous response, which participates in both containment of M. tuberculosis and the latters dissemination to other organs.

Brain antigens in functionally distinct antigen-presenting cell populations in cervical lymph nodes in MS and EAE

Drainage of central nervous system (CNS) antigens to the brain-draining cervical lymph nodes (CLN) is likely crucial in the initiation and control of autoimmune responses during multiple sclerosis (MS). We demonstrate neuronal antigens within CLN of MS patients. In monkeys and mice with experimental autoimmune encephalomyelitis (EAE) and in mouse models with non-inflammatory CNS damage, the type and extent of CNS damage was associated with the frequencies of CNS antigens within the cervical lymph nodes. In addition, CNS antigens drained to the spinal-cord-draining lumbar lymph nodes. In human MS CLN, neuronal antigens were present in pro-inflammatory antigen-presenting cells (APC), whereas the majority of myelin-containing cells were anti-inflammatory. This may reflect a different origin of the cells or different drainage mechanisms. Indeed, neuronal antigen-containing cells in human CLN did not express the lymph node homing receptor CCR7, whereas myelin antigen-containing cells in situ and in vitro did. Nevertheless, CLN from EAE-affected CCR7-deficient mice contained equal amounts of myelin and neuronal antigens as wild-type mice. We conclude that the type and frequencies of CNS antigens within the CLN are determined by the type and extent of CNS damage. Furthermore, the presence of myelin and neuronal antigens in functionally distinct APC populations within MS CLN suggests that differential immune responses can be evoked.

In Vivo Differentiated Cytokine-Producing CD4+ T Cells Express Functional CCR7

Chemokines and their receptors fulfill specialized roles in inflammation and under homeostatic conditions. CCR7 and its ligands, CCL19 and CCL21, are involved in lymphocyte recirculation through secondary lymphoid organs and additionally navigate lymphocytes into distinct tissue compartments. The role of CCR7 in the migration of polarized T effector/memory cell subsets in vivo is still poorly understood. We therefore analyzed murine and human CD4+ cytokine-producing cells developed in vivo for their chemotactic reactivity to CCR7 ligands. The responses of cells producing cytokines, such as IFN-γ, IL-4, and IL-10, as well as of subsets defined by memory or activation markers were comparable to that of naive CD4+ cells, with slightly lower reactivity in cells expressing IL-10 or CD69. This indicates that CCR7 ligands are able to attract naive as well as the vast majority of activated and effector/memory T cell stages. Chemotactic reactivity of these cells toward CCL21 was absent in CCR7-deficient cells, proving that effector cells do not use alternative receptors for this chemokine. Th1 cells generated from CCR7−/− mice failed to enter lymph nodes and Peyer’s patches, but did enter a site of inflammation. These findings indicate that CD4+ cells producing effector cytokines upon stimulation retain the capacity to recirculate through lymphoid tissues via CCR7.

The chemokine receptor CCR7 controls lymph node‐dependent cytotoxic T cell priming in alloimmune responses

The chemokine receptor CCR7 and its ligands regulate migration and colocalization of T cells and mature dendritic cells to and within secondary lymphoid organs. The requirement of CCR7 in efficient priming of allospecific cytotoxic CD8+ T cells is poorly characterized. Here, we demonstrate a role for CCR7 in the initiation of an alloimmune response and in the development of transplant rejection. Remarkably, in a model of acute allogeneic tumor rejection, CCR7–/– mice completely failed to reject subcutaneously injected MHC class I mismatched tumor cells and cytotoxic activity of allospecific T cells was severely compromised. When solid tumors derived from wild‐type mice were transplanted, recipient CCR7–/– mice were capable of rejecting the allografts. In contrast, tumor allografts transplanted from CCR7–/– donors onto CCR7–/– recipients showed allograft survival up to 28 days, suggesting a critical function of CCR7 on donor‐type passenger leukocytes in the initiation of cytotoxic CD8+ T cell responses. In a heterotopic heart transplantation model CCR7 deficiency resulted in significantly prolonged but not indefinite allograft survival. Additional prolongation of graft survival was observed when hearts from CCR7–/– mice were used as donor organs. Our results define a key role for CCR7 in allogeneic T cell priming within the context of draining lymph nodes.

The chemokine receptor CXCR5 is pivotal for ectopic mucosa-associated lymphoid tissue neogenesis in chronic Helicobacter pylori-induced inflammation.

Ectopic lymphoid follicles are a key feature of chronic inflammatory autoimmune and infectious diseases, such as rheumatoid arthritis, Sjögrens syndrome, and Helicobacter pylori-induced gastritis. Homeostatic chemokines are considered to be involved in the formation of such tertiary lymphoid tissue. High expression of CXCL13 and its receptor, CXCR5, has been associated with the formation of ectopic lymphoid follicles in chronic infectious diseases. Here, we defined the role of CXCR5 in the development of mucosal tertiary lymphoid tissue and gastric inflammation in a mouse model of chronic H. pylori infection. CXCR5-deficient mice failed to develop organized gastric lymphoid follicles despite similar bacterial colonization density as infected wild-type mice. CXCR5 deficiency altered Th17 responses but not Th1-type cellular immune responses to H. pylori infection. Furthermore, CXCR5-deficient mice exhibited lower H. pylori-specific serum IgG and IgA levels and an overall decrease in chronic gastric immune responses. In conclusion, the development of mucosal tertiary ectopic follicles during chronic H. pylori infection is strongly dependent on the CXCL13/CXCR5 signaling axis, and lack of de novo lymphoid tissue formation attenuates chronic immune responses.