CCR7 and its ligands: balancing immunity and tolerance - Nature

198 downloads 0 Views 650KB Size Report
Apr 1, 2008 - CCL19 and CCL21 are the sole lig- ands for the CC-chemokine receptor 7 (CCR7), which is expressed by various subsets of immune cells1.
REVIEWS

CCR7 and its ligands: balancing immunity and tolerance Reinhold Förster*, Ana Clara Davalos-Misslitz* and Antal Rot‡

Abstract | A key feature of the immune system is its ability to induce protective immunity against pathogens while maintaining tolerance towards self and innocuous environmental antigens. Recent evidence suggests that by guiding cells to and within lymphoid organs, CC‑chemokine receptor 7 (CCR7) essentially contributes to both immunity and tolerance. This receptor is involved in organizing thymic architecture and function, lymph-node homing of naive and regulatory T cells via high endothelial venules, as well as steady state and inflammation-induced lymph-node-bound migration of dendritic cells via afferent lymphatics. Here, we focus on the cellular and molecular mechanisms that enable CCR7 and its two ligands, CCL19 and CCL21, to balance immunity and tolerance. Regulatory T (TReg) cell

A naturally occurring subtype of regulatory T cell, which develops in the thymus and regulates self-reactive T cells in the periphery. TReg cells are characterized by the expression of CD25 (interleukin-2 receptor α‑chain) and the transcription factor FOXP3 (forkhead box P3).

*Institute of Immunology, Hannover Medical School; 30625 Hannover, Germany. ‡ Novartis Institutes for BioMedical Research, Vienna, Brunnerstrasse 59, A‑1235 Vienna, Austria. Correspondence to R.F. e-mail: foerster.reinhold@ mh-hannover.de doi:10.1038/nri2297 Published online 1 April 2008

The interactions of different subsets of immune and non-immune cells at defined sites are required for the efficient function of the immune system. These dynamic cellular encounters depend on the ability of these cells to actively migrate to and within tissues. Chemokines have been established as key regulators of leukocyte trafficking. Although the expression of most chemokines is induced during infection and inflammation, some chemokines, including CC‑chemokine ligand 19 (CCL19) and CCL21, are constitutively expressed and control cell movement during homeostasis1. CCL19 and CCL21 are the sole ligands for the CC‑chemokine receptor 7 (CCR7), which is expressed by various subsets of immune cells1. CCR7 and its ligands are essentially involved in homing of various subpopulations of T cells and antigen-presenting dendritic cells (DCs) to the lymph nodes. Within lymph nodes, T cells establish close physical contacts with DCs, which allow their antigen-specific activation. Although it is well established that these interactions are necessary for the optimal initiation of protective immunity, recent evidence demonstrates that the CCR7-dependent contacts of T cells and DCs are also essential for the induction of peripheral tolerance and the regulation of the immune response by CD4+CD25+ regulatory T (TReg) cells. Furthermore, a series of recent studies have shown that CCR7 is indispensable for the unperturbed thymic T‑cell development and negative selection of self-reactive T cells. On the basis of these recent discoveries, we discuss in this Review the steps of cellular motility that depend on the interaction of CCR7 with its ligands and how this molecular pathway contributes to the development of immunity, its regulation, as well as peripheral and central tolerance.

362 | may 2008 | volume 8

CCL19 and CCL21 and their receptor, CCR7 CCL19 and CCL21 are the only ligands for CCR7. Unlike CCL19, CCL21 has a uniquely long C‑terminal tail containing 32 amino acids of which 12 are basic amino-acid residues2 that allow avid binding to glyco­ saminoglycans and other molecules. This binding may be required for efficient presentation of CCL21 on the surface of endothelial cells2–4 and other cells5. Podoplanin, a proteo­glycan expressed by lymphatic endothelial cells, reticular stromal cells and other cell types might specifically present CCL21 (Ref. 6), and the expression of podoplanin might regulate the availability of CCL21 at these sites. As a result of gene duplication, two mouse genes encode functional CCL21 variants. CCL21-Leu, which contains a leucine at position 65, is expressed in lymphatic vessels of non-lymphoid organs such as the lung, colon, stomach, heart and skin, whereas CCL21-Ser is expressed in lymphoid organs such as the thymus, lymph nodes and spleen. Interestingly, the human genome apparently only has the gene encoding CCL21Leu, but not the gene encoding CCL21-Ser7. In paucity of lymph-node T cells (plt/plt) mice — a naturally occurring strain of mutant mice that carry an autosomal recessive mutation — a large gene locus that contains the genes encoding CCL19 and CCL21-Ser has been deleted, leaving the gene that encodes CCL21-Leu intact7–9. In human and mouse secondary lymphoid organs, CCL21 is produced by fibroblastic reticular cells of the T‑cellrich area and, in mice also, by high endothelial venules (HEVs)10. In non-inflamed lymph nodes, fibroblastic reticular cells seem to be the only source of CCL19 production in both humans and mice11. As human DCs www.nature.com/reviews/immunol

© 2008 Nature Publishing Group

REVIEWS Paucity of lymph-node T cells (plt/plt) mice A spontaneously occurring mutant strain that lost the expression of the CCchemokine receptor 7 (CCR7)ligands CC-chemokine ligand (CCL19) and the lymphoid tissue form of CCL21 (CCL21Ser). Lack of these chemokines results in defective thymic architecture and function, as well as impaired migration of CCR7-expressing T cells and dendritic cells into lymphoid organs, leading to their hypocellularity.

Central memory T (TCM) cell

A memory T-cell subpopulation that lacks immediate effector function but that expresses the lymph-node homing molecules L‑selectin (CD62L) and CCchemokine receptor 7 (CCR7). Tcm cells rapidly develop the phenotype and function of effector T cells on antigen re-stimulation in lymphoid organs.

can also produce CCL19, it is possible that activated DCs that are recruited into lymph nodes under inflammatory conditions may serve as an additional source of CCR7 ligands12. Similar to all chemokine receptors, CCR7 contains seven‑transmembrane-spanning domains and mediates its signals through heterotrimeric G proteins and their downstream effectors. CCR7 is expressed by semimature and mature DCs13, thymocytes during defined stages of their development14 (see later), naive B and T cells15,16, TReg cells17 and a subpopulation of memory T cells known as central memory T (TCM) cells16. CCR7 is also expressed by different non-immune cells, most notably in various malignancies18. Despite their similar affinities to CCR7, CCL19 and CCL21 induce different signalling effects through this receptor. CCL19, but not CCL21, effectively stimulates CCR7 phosphorylation and internalization19,20, leading to receptor desensitization. This implies that CCR7mediated cell responses to CCL19 may have a shorter time-span than responses to CCL21. Also, CCL19 can desensitize the receptor towards subsequent responses to CCL21 ligation, but not vice versa. It is not yet clear how these differential signalling events may impinge on the complex cellular functions of CCR7-bearing cells in the environments that contain both CCL19 and CCL21. CCL19 and CCL21, together with CCL25, also bind with high affinity to another hepta-helical surface protein, termed CC‑X-chemokine receptor (CCX-CKR)21. CCX-CKR does not couple to characteristic G protein

signalling pathways and was shown to internalize its ligands and scavenge22 or possibly also transport them23, thereby acting as a chemokine ‘interceptor’1.

CCR7 regulates homing of immune cells CCR7-mediated signals control the migration of immune cells to secondary lymphoid organs and subsequently their positioning within defined functional compartments. In this section we outline the contribution of CCR7 to cell homing to the lymph nodes, spleen and Peyer’s patches and summarize the mechanisms characteristic for the migration of different cell subsets. T cells. Most T cells, including naive T cells, Tcm cells and Treg cells, enter the lymph nodes via the HEVs, probably following a programme of sequential steps of interaction and adhesion to endothelial cells (BOX 1). The importance of CCR7 in lymphocyte homing to the lymph nodes has been revealed by gene targeting (BOX 2) . In CCR7-deficient mice, lymph nodes and Peyer’s patches are largely devoid of T cells24. Following adoptive transfer into wild-type recipients, CCR7deficient T cells fail to home to the lymph nodes but are present in the spleen, where they accumulate in the red pulp and are entirely excluded from the lymphoid white pulp. By contrast, CCR7-deficient B cells can migrate both to the lymph nodes and the splenic white pulp24. Although CCR7 has been identified as a lymph-node homing receptor, evidence is accumulating that this receptor is also involved in lymphocyte recirculation. Following their emigration into peripheral

Box 1 | The multi-step process of lymphocyte homing to lymph nodes Naive T cells emigrate from the Tethering CCR7-mediated and rolling activation Arrest Transmigration blood to peripheral lymph nodes in a multistep process, consisting of Naive rolling tethering and rolling, T cell CC‑chemokine receptor 7 (CCR7)mediated activation, firm arrest CCR7 αLβ2-Integrin Transcellular Paracellular CCR7 ligand L-selectin and transendothelial migration. GAG ICAM1 or ICAM2 First, lymphocyte L‑selectin binds PNAd to peripheral-node addressins (PNAd), a group of sialomucins with sialyl-LewisX-related motifs that are present on high endothelial venules Endothelial cell Basement membrane (HEVs). This interaction allows for a fast but transient attachment of T cells to HEVs, which, owing to the lateral shear forces of blood flow, results in cell rolling. Next, rolling lymphocytes bind the CCR7 ligands CCL21 and/or CCL19, which are immobilized by glycosaminoglycans (GAGs) on the luminal surface of HEVs4,87,88. CCR7-mediated signals together with the shear force of Nature Reviews | Immunology blood flow induce conformational changes of αLβ2-integrins on lymphocytes that allow the subsequent firm binding to intercellular adhesion molecule 1 (ICAM1) and ICAM2. CCR7 signalling also activates α4β7-integrins, which bind to mucosal addressin cell-adhesion molecule 1 (MAdCAM) that is expressed on HEVs in mesenteric lymph nodes and Peyer’s patches. At these two sites, tethering and rolling can also be mediated by transient binding of non-activated α4-integrin to MAdCAM1. Next, lymphocytes are thought to perform lateral locomotion on the endothelial surface followed by transendothelial migration, which is possibly induced by CCR7 ligands that are presented on either the luminal or abluminal surfaces of the HEVs. Potentially, lymphocytes choose between two routes of transendothelial migration, the paracellular (migrating around endothelial cells) and the transcellular (piercing through the endothelial cytoplasm). This homing behaviour applies not only to naive T cells but also to regulatory T cells and a subpopulation of memory cells termed central memory T (Tcm) cells, which are generated during an adaptive immune response. In contrast to effector cells, which express homing molecules that allow migration into peripheral sites, Tcm cells express CCR7 and L‑selectin and continuously re-circulate through lymph nodes, where they can respond to secondary antigen encounter and rapidly give rise to effector and memory T cells (for a recent review, see ref. 89).

nature reviews | immunology

volume 8 | may 2008 | 363 © 2008 Nature Publishing Group

REVIEWS Box 2 | Phenotype of CCR7-deficient mice The main feature of CC-chemokine receptor 7 (CCR7)-deficient mice is the grossly altered micro-architecture of their lymphoid organs. In CCR7-deficient animals the medullary areas of the thymus are smaller but more numerous than in wild-type mice and are occasionally misplaced to the outer rim of the organ14,84. The lymph nodes are lymphopaenic and deficient in CD11c+MHC class IIhi dendritic cells (DCs), whereas the structural organization of the paracortical areas of lymph nodes, the white pulp of the spleen and the Peyer’s patches is scrambled24. Additionally, CCR7-deficient mice may infrequently lack defined lymph nodes90 but, paradoxically, consistently develop ectopic lymphoid structures in mucosal sites such as the lungs, stomach and intestine. These lymphoid aggregates are highly organized with well segregated B‑ and T‑cell zones and high endothelial venules59,66,69. The cellular sequelae in Ccr7–/– mice leading to the development of these tertiary lymphoid structures are not yet apparent. Conversely, the morphological changes in secondary lymphoid organs are clearly due to the non-redundant role of CCR7 in homing and positioning of their lymphoid and non-lymphoid cellular constituents13,24,50,59. CCR7-deficient mice show impaired lymph-node homing and positioning of naive, central memory and regulatory T cells, as well as a defect in migration of dendritic cells from the skin, intestinal lamina propria and lungs31,33. CCR7 expression and function is also required for thymocyte differentiation and maturation14,67,68,84. As a consequence of the multiple defects in lymphoid organ homing and positioning, the interactions of T‑cell subsets with different haematopoietic and non-haematopoietic cells are perturbed in Ccr7–/– mice, thereby preventing normal T‑cell development and function. Consequently, CCR7-deficient mice, on one hand, display delayed induction of adaptive immune responses and, on the other hand, show impairment in central and peripheral tolerance and defective function of TReg cells31,33,46,50,68. Ineffective tolerance to self antigens leads to the development of multi-organ autoimmunity in CCR7-deficient mice66,67.

tissues, some T cells can reach lymph nodes by entering the draining lymphatics, a step that also involves CCR7 (Refs 25,26). Dendritic cells. DCs reside as sentinels in the skin and mucous membranes, including the respiratory, alimentary and urogenital tracts27. Following activation by infectious or inflammatory insults, DCs undergo maturation that leads to profound changes in their antigen uptake, processing and presentation capabilities. DC maturation is characterized by the upregulated expression of MHC class II molecules and co-stimulatory molecules such as CD80, CD83 and CD86, as well as CCR7 (Refs 12,28–30). Little is known about the mechanisms that regulate the trafficking of mature DCs to the lymph nodes via the afferent lymphatics. Although gene targeting has shown that CCR7 is essential for DC mobilization, it is still not entirely clear where and how CCR7 and its ligands are involved in this process. Wild-type and CCR7-deficient mice have similar numbers of DCs in their peripheral organs, indicating that CCR7 has no role in the recruitment of DC progenitors to the skin and mucosal surfaces. Conversely, Ccr7–/– DCs fail to leave dermal tissue and migrate into the draining lymph nodes in response to in vivo mobilization stimuli, such as contact sensitization induced by fluorescein isothiocyanate (FITC) skin painting24. Furthermore, DCs differentiated from the bone marrow of CCR7-deficient mice do not migrate to the draining lymph nodes following their subcutaneous injection or intra-tracheal instillation13,31,32. Important insights regarding the contribution of CCR7 to homeostatic DC trafficking have been obtained by analysing mice kept under specific pathogen-free or germ-free conditions. Whereas 2–4% of all cells isolated from lymph nodes in wild-type mice are CD11c +MHC class II hi DCs, this population is missing in CCR7-deficient mice13. Further detailed studies confirmed that the continuous inflammation-independent turnover of DCs from skin, intestine and lung depends on CCR7 (Refs 31,33–35). 364 | may 2008 | volume 8

In vivo studies of DC migration revealed an important difference between plt/plt and Ccr7–/– mice. As outlined earlier, plt/plt mice still express CCL21-Leu in non-lymphoid organs, allowing for the mobilization of DCs in the periphery. Consequently, FITC skin painting or the subcutaneous injection of wild-type DCs into plt/plt mice results in the mobilization of DCs to the draining lymph nodes36. However, in both cases, fewer DCs migrate to the draining lymph nodes in plt/plt mice compared with wild-type mice. Therefore, it seems likely that lymph-node-derived CCL19 and/or CCL21-Ser may also be involved in driving DC migration into the lymph node. It is also possible that these two chemokines are required for guiding DCs within the lymph node, from the subcapsular sinus to their final destination: the T‑cell area. Recent evidence suggests that CCL19 and CCL21 might not only drive the migration of DCs but also more directly affect their ability to prime T cells. The exogenous addition of CCL19 to cell cultures of bone-marrow DCs induced their maturation, and, in DC–T-cell co-culture systems, increased T‑cell proliferation37. Also, it has been shown that CCR7 ligands increase the antigen uptake of mature DCs38.

CCR7 in lymph-node homing and positioning Once naive T cells have entered the lymph node, they migrate on reticular stromal cells within the paracortical T‑cell rich area in an apparently random ‘walk’ pattern of motion39. Two-photon microscopy has shown that CCL19 and CCL21, expressed by reticular stromal cells9,11 can influence the migration speed of naive T cells but not migration directionality 40–42. Following adoptive transfer into wild-type mouse recipients, CCR7-deficient T cells that entered the popliteal lymph node displayed a 30% reduction in velocity, as well as a 50% reduction of the motility coefficient, representing the ability to move away from the starting point of movement. A comparable reduction was observed for naive wild-type T cells that www.nature.com/reviews/immunol

© 2008 Nature Publishing Group

REVIEWS had been adoptively transferred into plt/plt recipients. Remarkably, intravenous application of CCL21 could completely restore the intranodal migration velocity of T cells40. In vitro studies showed that in a shear-free environment, surface-immobilized CCL21 molecules induce integrin clustering but not activation, and that the resulting CCL21-induced T‑cell migration does not depend on integrin-mediated adhesion43. There is a notable dichotomy of chemokine-receptor usage within lymph nodes. CCR7 delivers signals that direct cells to the T‑cell areas, whereas CXCR5 guides cells to B‑cell follicles. Following activation, follicular B cells downregulate the CXCR5 and upregulate CCR7 (Ref. 15). This shift in chemokine-receptor expression allows for the transient mobilization of follicular B cells towards the T‑cell zone, where activated B cells can receive help from CD4+ T helper (TH) cells. Likewise, at a later stage of the immune response, a small subpopulation of CD4+CXCR5+ follicular TH cells is generated. These cells express low levels of CCR7, allowing them to migrate into the B‑cell follicles to provide help for antibody production and immunoglobulin class switching44,45. Taken together, these results demonstrate that CCR7 represents an important lymph-node homing receptor for DCs, as well as for a defined subpopulation of T cells, and that CCR7 together with CXCR5 determines the positioning of immune cells in the functional microenvironments of the secondary lymphoid organs.

Immunoglobulin class switching A process in B cells by which the class of a secreted immunoglobulin is changed (for example, from IgM to IgG) without altering its antigen specificity.

Contact hypersensitivity A form of delayed-type hypersensitivity (type IV), in which T cells respond to antigens that are introduced through skin contact. This step requires dendritic-cell mobilization from the skin to the draining lymph nodes to prime the antigen-specific T cells.

Severe combined immunodeficiency (SCID) mice Spontaneous mutant mice in which T and B cells are absent. SCID mice lack functional lymphocytes because they have a deficiency that impairs rearrangement of different immunoglobulin and T-cell receptor genes. Lack of T and B cells leads to defects in cellmediated and humoral immune responses.

CCR7 in immunity and peripheral tolerance Immunity to pathogens and alloantigens. Considering the multifaceted effects of CCR7 and its ligands on immune cells and their concomitant importance for the organization of the paracortical areas of the lymph node, it was not surprising to see weak and delayed adaptive immunity in CCR7-deficient mice following a single administration of a model antigen24. More refined studies have shown that although CCR7-deficient mice develop normal antibody responses against replicating vesicular stomatitis virus (VSV) or against high amounts of recombinant VSV glycoproteins, they mount largely impaired humoral immune responses when antigen levels are low. As DCs are not involved in generating VSV immunity, these data indicate that CCR7-mediated interactions between B cells and Th cells are of particular relevance when antigen is sparse46. The need for CCR7-mediated cell interactions can also be bypassed in other cases of adaptive immunity to pathogens. Accordingly, plt/plt mice as well as Ccr7–/– mice mount neutralizing immune responses towards lymphocytic choriomeningitis virus, obviously due to antigen presentation at ectopic sites, including the splenic marginal zone and the superficial cortex of lymph nodes47,48. Similar effects may be responsible for the relative resistance of CCR7-deficient mice towards infection with Listeria monocytogenes. In this model, the priming of naive but not memory MHC-class-Iarestricted CD8+ T cells requires CCR7, whereas naive MHC-class-Ib-restricted CD8+ T cells or MHC-class-IIrestricted CD4+ T cells are less dependent on the presence of this chemokine receptor49. It was also noted that

nature reviews | immunology

following repeated systemic antigen administration of, for example, tetanus toxoid, full-blown humoral and cell­ ular immunity also develops in Ccr7–/– mice50. Moreover, in chronic experimental models of contact hypersensitivity, autoimmune encephalitis and allergic asthma, as well as in an adoptive transfer model of inflammatory bowel disease in severe combined immunodeficiency (SCID) mice, the elicited immune responses and ensuing pathological changes develop in the absence of CCR7 or its ligands, albeit with delayed kinetics50–53. Although, as illustrated by these examples, the requirement for CCR7 expression in mounting an immune response against different pathogens and antigens may vary, evidence is emerging that some pathogens can exploit CCR7 and its ligands for their infection strategies. For example, CCL19 and CCL21 have been shown to increase the permissiveness of human resting memory CD4+ T cells to HIV‑1 infection54. Also, pathogens such as L. monocytogenes use the CCR7‑mediated migration of DCs to transfer to the draining lymph nodes, from where they can spread to other organs55. Cumulatively, these results imply that the requirement for CCR7 can be bypassed in immune reactions that are induced by copious amounts of antigens. Tolerance to environmental antigens. As outlined above, the continuous migration of DCs from the periphery represents an essential step for tolerance induction towards environmental or food antigens. DCs constantly sample self and foreign antigens while residing in the periphery. Even in the absence of apparent inflammatory stimuli, these cells migrate towards afferent lymphatic vessels, albeit at low frequencies. The oak-leaf-shaped endothelial cells of the initial segments of lymphatic vessels are not tightly connected to each other, but rather contain ‘buttons’ of adherens and tight junctions, which create overlapping flaps between endothelial cells56. It has been suggested that fluids enter the lymphatic circulation through the openings between these buttons56 and it seems possible that DCs take the same route, as cells of the initial lymphatic vessels express CCL21. The origin and the underlying mechanisms of this spontaneous mobilization of DCs remain obscure. However, the migration of these so-called ‘semi-mature’ or ‘tolerogenic’ DCs via the afferent lymphatic vessels into the draining lymph nodes relies entirely on their expression of CCR7 (Ref. 13). Therefore, CCR7-deficient mice were ideal to test the hypothesis that DC‑mediated transport of harmless antigens is indeed necessary for the induction of peripheral tolerance. Feeding wild-type mice with the model antigen ovalbumin (ova) results in the systemic non-responsiveness of these animals towards OVA when this antigen is reapplied subcutaneously or intravenously. Microsurgical approaches identified the mesenteric lymph node as the place where the orally administered antigen is presented to cognate T cells33. Oral tolerance could not be induced in CCR7-deficient mice, possibly owing to the impaired migration of ova-laden DCs from the lamina propria to the mesenteric lymph nodes33. volume 8 | may 2008 | 365

© 2008 Nature Publishing Group

REVIEWS Further insights into the mechanisms of antigen transport that are required for tolerance induction came from studies in which antigen was delivered into the respiratory tract by inhalation or intratracheal instillation. Under these experimental conditions, the transport of the fluorochrome-labelled antigen can be monitored in vivo and ex vivo (FIG. 1). Again, wild-type but not CCR7‑deficient mice entered a state of systemic non-responsiveness following exposure to ova via the respiratory route31. Reporter T cells, which recognize defined epitopes of ova, were adoptively transferred into wild-type and CCR7-deficient mice. In wild-type recipients that received ova aerosols, these cells showed abortive proliferation. By contrast, in Ccr7–/– mice, the ova aerosol had no effect on these T cells, which were neither deleted nor anergized and also failed to upregulate any activation marker. This was rather surprising as the lymph-node-resident DCs, which are also present in Ccr7–/– mice, avidly took up soluble antigen draining from the lung, but obviously failed to present it to CD4+ or CD8+ T cells31. This defect in tolerance induction by aerogenic antigen in Ccr7–/– mice could be overcome by the intratracheal adoptive transfer of immature wild-type bone-marrow-derived DCs before exposure to the ova aerosol31. Taken together, these data indicate that DCs that reside at mucosal sites induce tolerance under homeostatic conditions by continuously sampling innocuous antigens and transporting them in a CCR7-dependent manner to draining lymph nodes for efficient tolerogenic presentation to T cells. CCR7 in the development and function of TReg cells. Another efficient mechanism of peripheral tolerance to self and foreign antigens involves the suppression of immunity by forkhead box P3 (FOXP3)+CD4+CD25+ regulatory T (TReg) cells57. Naturally occurring TReg cells develop in the thymus in the absence of CCR7. The total number of FoxP3+ T cells in the thymi of Ccr7–/– mice is identical to that in wild-type mice50. Also, the in vitro suppressive activity of CCR7-deficient TReg cells is intact, as they are equipotent with their wild-type counterparts in inhibiting T‑cell proliferation in vitro50,58,59. Conversely, the suppressive function of Ccr7–/– TReg cells in vivo is profoundly defective. This is primarily due to their inability to home to lymph nodes and position themselves within the T‑cell zone50,58,59. Thus, the disrupted architecture of the T‑cell zone observed in Ccr7–/– mice extends also to defective positioning of TReg cells. Because this region of the lymph node is the major site of TReg-cell-mediated in vivo suppression, CCR7 and its ligands hold the key to TReg-cell-mediated tolerance. The exact means of TReg-cell suppressive activity within the lymph nodes remains contentious. It is very likely that different mechanistic principles cumulatively contribute to their function (FIG. 2). First, following CCR7-mediated homing to the T‑cell zone of the lymph node, TReg cells proliferate and expand on contact with their cognate antigen50. Second, the CCR7-dependent presence of TReg cells in the lymph node during antigen recognition by TH cells results in reduced numbers of activated TH cells50. As demonstrated by the adoptive co-transfer of T‑cellreceptor-transgenic TReg cells and TH cells, this effect, in 366 | may 2008 | volume 8

turn, has multiple facets. On one hand, TReg cells suppress the concurrent antigen-induced proliferation of TH cells. On the other hand, TReg cells actively reduce the number of TH cells in the lymph node by either interfering with their homing or inducing their apoptosis50. To this end, it is possible that TReg cells and TH cells simply compete for T‑cell homing and/or survival signals in lymph nodes. Notably, CCR7 ligands can provide both homing and survival cues11. After being downregulated in the lymph node during an immune response60, CCR7 ligands may become too scarce to support homing and survival of all T cells that express CCR7. Furthermore, wild-type but not CCR7-deficient TReg cells inhibit the antigen-induced activation and/or differentiation of Th cells50. Third, peripheral naive T cells may directly undergo differentiation into TReg cells 61,62. This conversion establishes the lymph node as an additional site, other than the thymus, where regulatory T‑cell development may take place. All TReg-cell silencing mechanisms mentioned here, regardless of the targeting of DCs or T cells, take place in the T‑cell zone of the lymph node, and are therefore inevitably dependent on the CCR7-mediated lymph-node homing of the three major cellular components involved in suppression: TReg cells, TH cells and DCs. However, it should be noted that, especially during inflammation, these cell populations might home to the lymph nodes using receptors other than CCR7, and other antigenpresenting cells, such as B cells, might also contribute to the expansion and function of TReg cells63. Natural TReg cells have been subdivided into two different subpopulations based on their surface expression of CD103: ‘naive-like’ TReg cells are CD103– and ‘effectorlike’ TReg cells are CD103+ (Ref. 64). Naive-like TReg cells act as suppressors in the lymph node, whereas effectorlike TReg cells have been shown to migrate to peripheral tissues, where they can contain T‑cell-mediated inflammation. It was suggested that these two distinct TReg-cell phenotypes represent two developmental stages of the same lineage65. Accordingly, TReg-cells would emerge from the thymus as naive-like CCR7+CD103– cells. These cells would then need to home to the lymph nodes, by means of CCR7, to undergo the switch to the effector-like phenotype, which is also accompanied by the upregulation of inflammatory chemokine receptors such as CCR2, CCR4 and CCR6. However, this hypothesis has been challenged by the finding that CD103+ effector-like TReg cells predominate in CCR7-deficient mice, suggesting that this T-cell phenotype can develop in the complete absence of CCR7-dependent lymphnode homing50. Yet, it is noteworthy that most of the CD103+ effector-like TReg cells express CCR7 (Ref. 58) and thus may use it to migrate to the lymph node where they receive putative signals required for their activation and subsequent suppressive function. In the absence of CCR7-mediated lymph-node homing, CD103+ TReg cells migrate to peripheral inflammatory lesions but are unable to suppress the pathological changes in, for example, the skin lesions of contact hypersensitivity50. The regulatory competence of CD103+ TReg cells from Ccr7–/– mice can be restored by their activation in vitro, which results in effective suppression of contact hypersensitivity after www.nature.com/reviews/immunol

© 2008 Nature Publishing Group

REVIEWS

Epithelial cell

Basement membrane

Mucus

Goblet cell

Alveolus

Inhaled antigen

Bronchial DC Interstitial DC CCR7

Precursor DC

CCL21 Afferent lymph vessel Activated T cell

Blood vessel

Bronchial lymph node T-zone reticular cell

Subcapsular sinus

CCL19 CCL21 HEV

Naive T cell

TCR

Peptide–MHC complex Regulatory T cells

B-cell follicle

Anergic T cells Apoptotic cells

Medulla

Figure 1 | CCR7 in the induction of tolerance to inhaled antigens. Dendritic cell (DC) precursors enter the lung via blood vessels and give rise to sessile interstitial and bronchial DCs. Inhaled antigens are taken Nature up by bronchial that are Reviews | DCs Immunology located below the basement membrane. Some DCs spontaneously upregulate CC‑chemokine receptor 7 (CCR7) and migrate towards the initial segments of lymphatic vessels that express CC‑chemokine ligand 21 (CCL21). Following CCR7mediated entry into the lymphatics, DCs are passively carried with the afferent lymph into the draining lymph node. Under experimental conditions, intratracheally applied DCs migrate from the bronchus and/or alveolus into afferent lymphatics. It is as yet unknown whether the migration of DCs from the subcapsular sinus into the T‑cell area of the bronchial lymph nodes depends on CCR7. Within the lymph-node paracortex, DCs present antigens to naive T cells, which enter the lymph node via the high endothelial venules (HEVs). T cells randomly migrate on reticular cells, which express CCL19 and CCL21. These chemokines enhance the velocity of T‑cell locomotion within the lymph node and thus increase the likelihood of their encounter with DCs presenting cognate antigen. T cells that recognize inhaled innocuous antigens either undergo apoptosis, become anergic or gain regulatory capacities. Some T cells that are present in the lung parenchyma also rely on CCR7 for their entry into the afferent lymphatics. TCR, T-cell receptor. nature reviews | immunology

volume 8 | may 2008 | 367 © 2008 Nature Publishing Group

REVIEWS Antigen Afferent lymph vessel

DC Lymph node

T-zone reticular cell

CCL21 CCR7

TReg cell HEV

Peptide–MHC complex TCR

e

CCL21

d a

B-cell follicle

b

Naive T cell

c

Effector T cells

Medulla

Figure 2 | CCR7 in function of regulatory T cells. Virtually all naturally occurring forkhead box P3 (FOXP3)+CD4+CD25+ regulatory T (TReg) cells in blood express CC‑chemokine receptor 7 (CCR7) and use it to enter the lymph node via the high endothelial venules (HEVs). On homing to the lymph node and migrating within paracortical lymph-node areas, TReg cells Nature Reviews | Immunology establish contacts with antigen-laden dendritic cells (DCs) that have homed via the afferent lymphatics. Consequently TReg cells proliferate and expand when presented with cognate antigen (a); interfere with the concurrent antigen-induced proliferation of naive T helper (TH) cells (b); and actively reduce the number of effector cells and suppress their differentiation (c). To exert these suppressive activities, TReg cells may alternatively target DCs (d) or T cells (b,c) or both, whereas the means and mechanisms of suppression (that is, the requirements for cell contacts or soluble mediators) are not entirely clear. Upon homing to the lymph node and coming into contact with DCs, naive T cells may also undergo direct conversion into TReg cells (e) in a process that requires low amounts of cognate antigen, low-level co-stimulation and interleukin‑10 or transforming growth factor‑β, but not interleukin‑2.

adoptive transfer58. In conclusion, Ccr7–/– mice have defective in vivo TReg-cell function, which may also explain how experimental chronic immune reactions result in a more severe outcome in these animals than in their wild-type counterparts50.

Sjögren’s syndrome An autoimmune disorder in which immune cells attack and destroy exocrine glands. The hallmarks of Sjögren’s syndrome are dry eyes and dry mouth. The disease is often associated with arthritis.

CCR7, autoimmunity and lymphoid neogensis Recent studies have demonstrated that a lack of CCR7 is associated with the manifestation of spontaneous autoimmunity 66,67. The autoimmune phenotype of CCR7‑deficient mice is characterized by lymphocyte infiltrates in several peripheral organs, as well as increased titres of circulating auto-antibodies against a multitude of tissue-specific antigens that lead to IgG deposition in renal glomeruli66,67. Mechanistically, the autoimmunity that emerges in Ccr7–/– mice might result from inefficient negative selection of autoreactive T cells in the thymus67,68 (see later), incomplete maintenance of peripheral tolerance31,33 or defective function of TReg cells50,59.

368 | may 2008 | volume 8

Interestingly, CCR7-deficient mice spontaneously develop organized tertiary lymphoid structures at mucosal sites such as the lung, stomach and colon59,66,69. Such lymphoid structures are frequently observed in lesions of Sjögren’s syndrome, rheumatoid arthritis and autoimmune thyroditis, and are thought to maintain local chronic autoimmune responses that favour disease progression70. To what extent the ectopic lymphoid structures that are present in CCR7-deficient mice contribute to the establishment and maintenance of autoimmunity is still unknown. Paradoxically, CCR7 has an ambivalent role in the development of tertiary lymphoid structures. On the one hand, lack of CCR7 signalling leads to spontaneous lymphoid neogenesis primarily in mucosal organs, which demonstrates that CCR7 is not required for this process. On the other hand, transgenic expression of CCR7L in the thyroid and pancreas, but not skin, leads to the formation of tertiary lymphoid structures 71–74. Also, the ectopic www.nature.com/reviews/immunol

© 2008 Nature Publishing Group

REVIEWS Capsule Subcapsular epithelium cTEC

Subcapsular zone

DN3 DN4

DN2

DP

DN1–DN2

CD69– CCR7–

CCR7+ or CCR7– DN1

BM-derived progenitor cell

Blood vessel

CD69+ CCR7+ or CCR7–

DP

SP

Cortex

CD69+ CD24hi CCR7– or CCR7low

Cortico– medullary junction

mTEC

DC

SP CD69– CD24low CD62hi CCR7+

SP CD69+ CD24int CCR7+

Medulla

Figure 3 | CCR7 in thymocyte migration. Bone-marrow (BM)-derived thymocyte progenitors enter the adult thymus through the venules at the cortico–medullary Nature Reviews | Immunology junction. They lack CD4 and CD8 expression and are therefore termed double negative (DN) cells. DN1 thymocytes (CD25–CD44hi) differentiate in proximity to the site of thymic entry. Differentiation to the DN2 (CD25+CD44hi) stage occurs while cells migrate into the mid cortex. CC‑chemokine receptor 7 (CCR7) may be involved in this migratory step, as its expression has been detected in some of the CD25intCD44hi (DN1–DN2) cells. DN3 thymocytes (CD25hiCD44low) differentiate during their migration from the mid to the outer cortex and accumulate in the subcapsular zone where they subsequently develop into DN4 cells (CD25–CD44–). Transition from the DN to the double positive (DP) stage is accompanied by a reverse in the direction of migration, with DP thymocytes (CD4+CD8+CD69–) travelling across the cortex towards the medulla. Positively selected DP cells (CD4+CD8+CD69+) enter the medulla, where they complete maturation and give rise to CD4+ or CD8+ single positive (SP) cells. CCR7 is expressed by a small subpopulation of DP cells and may be involved in their migration from the cortex to the medulla. The most immature SP cells (CD24hiCD69+) represent the next developmental stage and express no or only very low levels of CCR7. By contrast, CCR7 is highly expressed by SP CD24intCD69+ cells and mature SP CD24lowCD69–CD62 ligand (CD62L)hi cells that reside in the medulla. On these cells, CCR7 might provide a retention signal that allows these cells to complete maturation before being released into the periphery. During this journey, thymocytes interact with different subpopulations of resident cells such as dendritic cells (DCs) and cortical and medullary thymic epithelial cells (cTECs and mTECs), enabling positive selection as well as the deletion of auto-reactive thymocytes.

expression of CCL21 observed in autoimmunity and infection has been associated with the development of tertiary lymphoid structures in different organs 75–77. CCR7 mediates this effect, as the formation of tertiary lymphoid structures is not observed in the thyroid of CCR7-deficient mice that ectopically express CCL21 (Ref. 78). Similarly, lymphoid neogenesis is absent in the joints of CCR7-deficient animals following induction of antigen-induced arthritis79. The mechanisms by which the presence or absence of CCR7 can promote the formation of tertiary lymphoid structures remain to be clarified. nature reviews | immunology

CCR7 in thymus architecture and function The thymus is the key organ responsible for the maintenance of the peripheral T‑cell pool. In recent years, several studies have demonstrated that chemokine receptors, including CCR7, and their ligands are important in coordinating migratory events into, within and out of the thymus14,80,81. During embryogenesis, CCL21 has been detected in the thymic primordium of embryonic day 11.5 (E11.5) to E12.5 embryos, suggesting that this chemokine might be involved in the recruitment of fetal haematopoietic progenitors into the developing organ82,83. Supporting this idea, CCR7-deficient mice and plt/plt mice have reduced numbers of thymocytes at E13.5 (Ref. 83). More recent data also demonstrate that mice that overexpress CCX-CKR (the interceptor for CCL19, CCL21 and CCL25) on thymic epithelial cells have reduced numbers of haematopoietic precursors in the thymic anlage23. In the adult thymus, the presence of CCL19 and CCL21 is not restricted to a given compartment, as they are detectable in the cortex and in the medulla14. Consequently, CCR7 ligands seem to be able to guide the migration of developing thymocytes through both thymic compartments (FIG. 3). Early progenitors lack CD4 and CD8 expression and are referred to as double negative (DN) cells. CCR7 is prominently expressed by a CD44hiCD25int DN subpopulation, which probably reflects a transitory state between DN1 (CD44+CD25–) and DN2 (CD44+CD25+) thymocytes (termed DN1– DN2 cells)14. Approximately half of these cells express CCR7, indicating that this receptor could be involved in the migration of cells outwards from the cortico– medullary junction. Accordingly, in the absence of CCR7 signalling, part of the DN1–DN2 population undergoes developmental arrest at this transitional stage. However, it should be mentioned that at this early developmental stage, intrathymic precursors are still not committed to any particular T‑cell lineage, and, therefore, it remains unclear to what extent DN1–DN2 cells actually contribute to the pool of developing T cells. A crucial role for CCR7 in the translocation of positively selected CD4+CD8+ double positive (DP) thymocytes from the cortex into the medulla has recently been suggested84. However, as only approximately 25% of positively selected DP cells (CD4+CD8+CD69+) express CCR7 (Ref. 68), it is still unclear to what degree positively selected thymocytes require CCR7 to transit from the cortex to the medulla. The most abundant expression of CCR7 is observed in single positive (SP) populations14,68, which are present at a high frequency in the medulla. Remarkably, the most immature CD4+ SP cell subpopulation (CD4+CD8– CD24hiCD69+), which probably corresponds to cells undergoing negative selection, express no or only low levels of CCR7. By contrast, cells that escape negative selection (CD4+CD8–CD24intCD69+) as well as mature cells (CD4+CD8–CD24–CD62L+) express high amounts of CCR7 (Ref. 68). The expression of CCR7 by thymocytes at a late maturational stages could have a role in the retention of SP cells in the medulla while they complete functional maturation. Alternatively, CCR7 expression could be involved in the positioning of mature volume 8 | may 2008 | 369

© 2008 Nature Publishing Group

REVIEWS thymocytes near the blood vessels before they exit the thymus80. A direct role for CCR7 in thymic egress of mature thymocytes has been demonstrated for newborn mice only85. Presumably owing to impaired migration of thymocytes, Ccr7–/– and plt/plt mice show altered thymus morphologies accompanied by reduced numbers of thymocytes and impaired T‑cell development14,84. Interestingly, the thymus of mice deficient for CCL19 has a normal architecture and cellular composition11. This observation indicates that in contrast to peripheral lymphoid organs, in which CCL19 and CCL21 exert different functions, the lack of CCL19 function in the thymus seems to be compensated by that of CCL21. Disruption of thymus morphology is frequently associated with the breakdown of central tolerance and development of autoimmunity86. As discussed above, the lack of CCR7 signalling during thymic development of T cells may also contribute to the manifestation of autoimmunity in plt/plt and CCR7-deficient mice66,67. In line with this observation, defective negative selection has recently been demonstrated in CCR7-deficient mice68. Conversely, it has been reported that negative selection of thymocytes is normal in plt/plt mice84. This apparent discrepancy is probably due to the use of different models to study negative selection. An important aspect to be considered is that the defective negative selection of CCR7-deficient thymocytes correlates with impaired T-cell receptor stimulation68, suggesting that functions of the CCR7–CCR7 ligands pathway other than guiding the migration of thymocytes may contribute to the maintenance of central tolerance.

Rot, A. & von Andrian, U. H. Chemokines in innate and adaptive host defense: basic chemokinese grammar for immune cells. Annu. Rev. Immunol. 22, 891–928 (2004). 2. Yoshida, R. et al. Secondary lymphoid-tissue chemokine is a functional ligand for the CC chemokine receptor CCR7. J. Biol. Chem. 273, 7118–7122 (1998). 3. Gunn, M. D. et al. A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. Natl Acad. Sci. USA 95, 258–263 (1998). 4. Stein, J. V. et al. The CC chemokine thymus-derived chemotactic agent 4 (TCA‑4, secondary lymphoid tissue chemokine, 6Ckine, exodus‑2) triggers lymphocyte function-associated antigen 1‑mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules. J. Exp. Med. 191, 61–76 (2000). 5. Friedman, R. S., Jacobelli, J. & Krummel, M. F. Surface-bound chemokines capture and prime T cells for synapse formation. Nature Immunol. 7, 1101–1108 (2006). 6. Kerjaschki, D. Lymphatic neoangiogenesis in human neoplasia and transplantation as experiments of nature. BANTAO J. 4, 60–61 (2006). 7. Vassileva, G. et al. The reduced expression of 6Ckine in the plt mouse results from the deletion of one of two 6Ckine genes. J. Exp. Med. 190, 1183–1188 (1999). 8. Nakano, H. & Gunn, M. D. Gene duplications at the chemokine locus on mouse chromosome 4: multiple strain-specific haplotypes and the deletion of secondary lymphoid-organ chemokine and EBI‑1 ligand chemokine genes in the plt mutation. J. Immunol. 166, 361–369 (2001). 9. Luther, S. A., Tang, H. L., Hyman, P. L., Farr, A. G. & Cyster, J. G. Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse. Proc. Natl Acad. Sci. USA 97, 12694–12699 (2000). 10. Carlsen, H. S., Haraldsen, G., Brandtzaeg, P. & Baekkevold, E. S. Disparate lymphoid chemokine expression in mice and men: no evidence of CCL21 1.

11.

12. 13. 14. 15. 16.

17.

18.

19.

20.

Future perspectives Here, we have drawn a picture of CCR7 function in which this receptor is crucially involved in efficient induction of immune reactions as well as their silencing and regulation. Most of the knowledge on the involvement of CCR7 in the development of immunity and tolerance is derived from mouse models, whereas the data on CCR7 function in humans is rather sparse. Fortunately, expression patterns of CCR7 in mice and humans are similar, corroborating the use of mouse models for the in vivo investigations of this chemokine receptor pathway. The apparently ambiguous role of CCR7 and its ligands in shaping immunity makes it difficult to target these molecules for therapeutic intervention in human immunemediated diseases. However, the recently discovered contribution of CCR7 to the induction and maintenance of tolerance suggests new strategies to target CCR7 for breaking undesired immune tolerance, for example, to malignant tumours. Several types of malignancies express CCR7 and use it for their dissemination and survival. Thus, potential CCR7-targeted cancer therapy might allow us to not only overcome immunological tumour tolerance but may also have direct tumoricidal effects as well as interfere with tumour metastasis through afferent lymphatic vessels. Future studies on CCR7 in human molecular medicine as well as more refined mouse models of site-specific and inducible CCR7 and CCR7-ligand knockout, knockdown and knock-in mice will benefit our understanding of this fundamental molecular pathway and the cellular responses driven by it.

synthesis by human high endothelial venules. Blood 106, 444–446 (2005). Link, A. et al. Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells. Nature Immunol. 8, 1255–1265 (2007). This study shows that fibroblast reticular cells support the intranodal migration of lymphocytes in peripheral lymph nodes. In agreement with this data, reference 41 demonstrates that a subtype of reticular cells express CCL19, CCL21 and IL-7, which support the survival of lymphocytes migrating in the lymph node T-cell zone. Sallusto, F. et al. Distinct patterns and kinetics of chemokine production regulate dendritic cell function. Eur. J. Immunol. 29, 1617–1625 (1999). Ohl, L. et al. CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions. Immunity 21, 279–288 (2004). Misslitz, A. et al. Thymic T cell development and progenitor localization depend on CCR7. J. Exp. Med. 200, 481–491 (2004). Reif, K. et al. Balanced responsiveness to chemoattractants from adjacent zones determines B‑cell position. Nature 416, 94–99 (2002). Sallusto, F., Lenig, D., Förster, R., Lipp, M. & Lanzavecchia, A. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401, 708–712 (1999). Szanya, V., Ermann, J., Taylor, C., Holness, C. & Fathman, C. G. The subpopulation of CD4+CD25+ splenocytes that delays adoptive transfer of diabetes expresses L‑selectin and high levels of CCR7. J. Immunol. 169, 2461–2465 (2002). Shields, J. D. et al. Autologous chemotaxis as a mechanism of tumor cell homing to lymphatics via interstitial flow and autocrine CCR7 signaling. Cancer Cell 11, 526–538 (2007). Bardi, G., Lipp, M., Baggiolini, M. & Loetscher, P. The T cell chemokine receptor CCR7 is internalized on stimulation with ELC, but not with SLC. Eur. J. Immunol. 31, 3291–3297 (2001). Kohout, T. A. et al. Differential desensitization, receptor phosphorylation, β-arrestin recruitment, and

370 | may 2008 | volume 8

21.

22.

23.

24. 25. 26.

27. 28.

29.

30.

31.

ERK1/2 activation by the two endogenous ligands for the CC chemokine receptor 7. J. Biol. Chem. 279, 23214–23222 (2004). Gosling, J. et al. Cutting edge: identification of a novel chemokine receptor that binds dendritic cell- and T cell-active chemokines including ELC, SLC, and TECK. J. Immunol. 164, 2851–2856 (2000). Comerford, I., Milasta, S., Morrow, V., Milligan, G. & Nibbs, R. The chemokine receptor CCX-CKR mediates effective scavenging of CCL19 in vitro. Eur. J. Immunol. 36, 1904–1916 (2006). Heinzel, K., Benz, C. & Bleul, C. C. A silent chemokine receptor regulates steady-state leukocyte homing in vivo. Proc. Natl Acad. Sci. USA 104, 8421–8426 (2007). Förster, R. et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99, 23–33 (1999). Debes, G. F. et al. Chemokine receptor CCR7 required for T lymphocyte exit from peripheral tissues. Nature Immunol. 6, 889–894 (2005). Bromley, S. K., Thomas, S. Y. & Luster, A. D. Chemokine receptor CCR7 guides T cell exit from peripheral tissues and entry into afferent lymphatics. Nature Immunol. 6, 895–901 (2005). Banchereau, J. & Steinman, R. M. Dendritic cells and the control of immunity. Nature 392, 245–252 (1998). Dieu, M. C. et al. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J. Exp. Med. 188, 373–386 (1998). Sozzani, S. et al. Differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties. J. Immunol. 161, 1083–1086 (1998). Yanagihara, S., Komura, E., Nagafune, J., Watarai, H. & Yamaguchi, Y. EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation. J. Immunol. 161, 3096–3102 (1998). Hintzen, G. et al. Induction of tolerance to innocuous inhaled antigen relies on a CCR7-dependent dendritic cell-mediated antigen transport to the bronchial lymph node. J. Immunol. 177, 7346–7354 (2006).

www.nature.com/reviews/immunol © 2008 Nature Publishing Group

REVIEWS 32. Martín-Fontecha, A. et al. Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming. J. Exp. Med. 198, 615–621 (2003). This reference demonstrates that DCs have to express CCR7 to migrate from the skin to peripheral lymph nodes under inflammatory conditions, and reference 13 shows that CCR7 is also indispensable for DC migration in noninflammatory, steady-state situations. 33. Worbs, T. et al. Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells. J. Exp. Med. 203, 519–527 (2006). This reference and reference 31 show that CCR7dependent migration of antigen-carrying DCs into the draining lymph nodes is required for the induction of tolerance to ingested and inhaled antigens. 34. Johansson-Lindbom, B. et al. Functional specialization of gut CD103+ dendritic cells in the regulation of tissue-selective T cell homing. J. Exp. Med. 202, 1063–1073 (2005). 35. Jang, M. H. et al. CCR7 is critically important for migration of dendritic cells in intestinal lamina propria to mesenteric lymph nodes. J. Immunol. 176, 803–810 (2006). 36. Gunn, M. D. et al. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J. Exp. Med. 189, 451–460 (1999). 37. Marsland, B. J. et al. CCL19 and CCL21 induce a potent proinflammatory differentiation program in licensed dendritic cells. Immunity 22, 493–505 (2005). 38. Yanagawa, Y. & Onoe, K. CCR7 ligands induce rapid endocytosis in mature dendritic cells with concomitant up-regulation of Cdc42 and Rac activities. Blood 101, 4923–4929 (2003). 39. Bajenoff, M. et al. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25, 989–1001 (2006). 40. Worbs, T., Mempel, T. R., Boelter, J., von Andrian, U. H. & Förster, R. CCR7-ligands stimulate the intranodal motility of T lymphocytes in vivo. J. Exp. Med. 204, 489–495 (2007). References 40–42 demonstrate that CCR7 influences the motility of lymphocytes in lymph nodes. In addition, reference 43 shows that, in the absence of shear forces, lymph-node chemokines promote T-cell motility without triggering firm integrin adhesiveness. 41. Okada, T. & Cyster, J. G. CC chemokine receptor 7 contributes to Gi-dependent T cell motility in the lymph node. J. Immunol. 178, 2973–2978 (2007). 42. Huang, J. H. et al. Requirements for T lymphocyte migration in explanted lymph nodes. J. Immunol. 178, 7747–7755 (2007). 43. Woolf, E. et al. Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces. Nature Immunol. 8, 1076–1085 (2007). 44. Hardtke, S., Ohl, L. & Förster, R. Balanced expression of CXCR5 and CCR7 on follicular T helper cells determines their transient positioning to lymph node follicles and is essential for efficient B‑cell help. Blood 106, 1924–1931 (2005). 45. Arnold, C. N., Campbell, D. J., Lipp, M. & Butcher, E. C. The germinal center response is impaired in the absence of T cell-expressed CXCR5. Eur. J. Immunol. 37, 100–109 (2007). 46. Scandella, E. et al. Dendritic cell-independent B cell activation during acute virus infection: a role for early CCR7-driven B–T helper cell collaboration. J. Immunol. 178, 1468–1476 (2007). 47. Junt, T. et al. Antiviral immune responses in the absence of organized lymphoid T cell zones in plt/plt mice. J. Immunol. 168, 6032–6040 (2002). 48. Junt, T. et al. Impact of CCR7 on priming and distribution of antiviral effector and memory CTL. J. Immunol. 173, 6684–6693 (2004). 49. Kursar, M. et al. Differential requirements for the chemokine receptor CCR7 in T cell activation during Listeria monocytogenes infection. J. Exp. Med. 201, 1447–1457 (2005). 50. Schneider, M. A., Meingassner, J. G., Lipp, M., Moore, H. D. & Rot, A. CCR7 is required for the in vivo function of CD4+ CD25+ regulatory T cells. J. Exp. Med. 204, 735–745 (2007). This study, along with reference 59, demonstrates that CCR7 is required for the migration of TReg cells

51.

52.

53.

54.

55.

56. 57.

58.

59.

60. 61. 62.

63.

64.

65.

66.

67.

68.

69. 70. 71.

72.

into lymph nodes and that this homing is essential for the unimpaired function of TReg cells. Pahuja, A. et al. Experimental autoimmune encephalomyelitis develops in CC chemokine receptor 7‑deficient mice with altered T‑cell responses. Scand. J. Immunol. 64, 361–369 (2006). Mori, S. et al. Mice lacking expression of the chemokines CCL21-ser and CCL19 (plt mice) demonstrate delayed but enhanced T cell immune responses. J. Exp. Med. 193, 207–218 (2001). Grinnan, D. et al. Enhanced allergen-induced airway inflammation in paucity of lymph node T cell (plt) mutant mice. J. Allergy Clin. Immunol. 118, 1234–1241 (2006). Saleh, S. et al. CCR7 ligands CCL19 and CCL21 increase permissiveness of resting memory CD4+ T‑cells to HIV‑1 infection: a novel model of HIV‑1 latency. Blood 110, 4161–4164 (2007). Pron, B. et al. Dendritic cells are early cellular targets of Listeria monocytogenes after intestinal delivery and are involved in bacterial spread in the host. Cell. Microbiol. 3, 331–340 (2001). Baluk, P. et al. Functionally specialized junctions between endothelial cells of lymphatic vessels. J. Exp. Med. 204, 2349–2362 (2007). Sakaguchi, S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nature Immunol. 6, 345–352 (2005). Menning, A. et al. Distinctive role of CCR7 in migration and functional activity of naive- and effector/memorylike Treg subsets. Eur. J. Immunol. 37, 1575–1583 (2007). Kocks, J. R., Davalos-Misslitz, A. C., Hintzen, G., Ohl, L. & Förster, R. Regulatory T cells interfere with the development of bronchus-associated lymphoid tissue. J. Exp. Med. 204, 723–734 (2007). Mueller, S. N. et al. Regulation of homeostatic chemokine expression and cell trafficking during immune responses. Science 317, 670–674 (2007). Kretschmer, K. et al. Inducing and expanding regulatory T cell populations by foreign antigen. Nature Immunol. 6, 1219–1227 (2005). Liang, S. et al. Conversion of CD4+ CD25-– cells into CD4+ CD25+ regulatory T cells in vivo requires B7 costimulation, but not the thymus. J. Exp. Med. 201, 127–137 (2005). Mann, M. K., Maresz, K., Shriver, L. P., Tan, Y. & Dittel, B. N. B cell regulation of CD4+CD25+ T regulatory cells and IL‑10 via B7 is essential for recovery from experimental autoimmune encephalomyelitis. J. Immunol. 178, 3447–3456 (2007). Huehn, J. et al. Developmental stage, phenotype, and migration distinguish naive- and effector/memory-like CD4+ regulatory T cells. J. Exp. Med. 199, 303–313 (2004). Lee, J. H., Kang, S. G. & Kim, C. H. FoxP3+ T cells undergo conventional first switch to lymphoid tissue homing receptors in thymus but accelerated second switch to nonlymphoid tissue homing receptors in secondary lymphoid tissues. J. Immunol. 178, 301–311 (2007). Davalos-Misslitz, A. C. et al. Generalized multi-organ autoimmunity in CCR7-deficient mice. Eur. J. Immunol. 37, 613–622 (2007). References 66–68 demonstrate that establishment of central tolerance is impaired in the absence of CCR7 signalling, and that CCR7-deficient mice develop autoimmunity. Kurobe, H. et al. CCR7-dependent cortex‑to‑medulla migration of positively selected thymocytes is essential for establishing central tolerance. Immunity 24, 165–177 (2006). Davalos-Misslitz, A. C., Worbs, T., Willenzon, S., Bernhardt, G. & Förster, R. Impaired responsiveness to TCR stimulation and defective negative selection of thymocytes in CCR7-deficient mice. Blood 110, 4351–4359 (2007). Hoepken, U. E. et al. CCR7 deficiency causes ectopic lymphoid neogenesis and disturbed mucosal tissue integrity. Blood 109, 886–895 (2006). Aloisi, F. & Pujol-Borrell, R. Lymphoid neogenesis in chronic inflammatory diseases. Nature Rev. Immunol. 6, 205–217 (2006). Fan, L., Reilly, C. R., Luo, Y., Dorf, M. E. & Lo, D. Cutting edge: ectopic expression of the chemokine TCA4/SLC is sufficient to trigger lymphoid neogenesis. J. Immunol. 164, 3955–3959 (2000). Luther, S. A. et al. Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis. J. Immunol. 169, 424–433 (2002).

nature reviews | immunology

73. Martin, A. P. et al. A novel model for lymphocytic infiltration of the thyroid gland generated by transgenic expression of the CC chemokine CCL21. J. Immunol. 173, 4791–4798 (2004). 74. Chen, S. C. et al. Ectopic expression of the murine chemokines CCL21a and CCL21b induces the formation of lymph node-like structures in pancreas, but not skin, of transgenic mice. J. Immunol. 168, 1001–1008 (2002). 75. Grant, A. J. et al. Hepatic expression of secondary lymphoid chemokine (CCL21) promotes the development of portal-associated lymphoid tissue in chronic inflammatory liver disease. Am. J. Pathol. 160, 1445–1455 (2002). 76. Christopherson, K. W., Hood, A. F., Travers, J. B., Ramsey, H. & Hromas, R. A. Endothelial induction of the T‑cell chemokine CCL21 in T‑cell autoimmune diseases. Blood 101, 801–806 (2003). 77. Weninger, W. et al. Naive T cell recruitment to nonlymphoid tissues: a role for endotheliumexpressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis. J. Immunol. 170, 4638–4648 (2003). 78. Marinkovic, T. et al. Interaction of mature CD3+CD4+ T cells with dendritic cells triggers the development of tertiary lymphoid structures in the thyroid. J. Clin. Invest. 116, 2622–2632 (2006). 79. Wengner, A. M. et al. CXCR5- and CCR7-dependent lymphoid neogenesis in a murine model of chronic antigen-induced arthritis. Arthritis Rheum. 56, 3271–3283 (2007). 80. Petrie, H. T. Cell migration and the control of postnatal T‑cell lymphopoiesis in the thymus. Nature Rev. Immunol. 3, 859–866 (2003). 81. Takahama, Y. Journey through the thymus: stromal guides for T‑cell development and selection. Nature Rev. Immunol. 6, 127–135 (2006). 82. Bleul, C. C. & Boehm, T. Chemokines define distinct microenvironments in the developing thymus. Eur. J. Immunol. 30, 3371–3379 (2000). 83. Liu, C. et al. The role of CCL21 in recruitment of T‑precursor cells to fetal thymi. Blood 105, 31–39 (2005). 84. Ueno, T. et al. CCR7 signals are essential for cortex– medulla migration of developing thymocytes. J. Exp. Med. 200, 493–505 (2004). 85. Ueno, T. et al. Role for CCR7 ligands in the emigration of newly generated T lymphocytes from the neonatal thymus. Immunity 16, 205–218 (2002). 86. Hogquist, K. A., Baldwin, T. A. & Jameson, S. C. Central tolerance: learning self-control in the thymus. Nature Rev. Immunol. 5, 772–782 (2005). 87. Baekkevold, E. S. et al. The CCR7 ligand elc (CCL19) is transcytosed in high endothelial venules and mediates T cell recruitment. J. Exp. Med. 193, 1105–1112 (2001). 88. Warnock, R. A. et al. The role of chemokines in the microenvironmental control of T versus B cell arrest in Peyer’s patch high endothelial venules. J. Exp. Med. 191, 77–88 (2000). 89. Ley, K., Laudanna, C., Cybulsky, M. I. & Nourshargh, S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nature Rev. Immunol. 7, 678–689 (2007). 90. Ohl, L. et al. Cooperating mechanisms of CXCR5 and CCR7 in development and organization of secondary lymphoid organs. J. Exp. Med. 197, 1199–1204 (2003).

Acknowledgements

We thank T. Worbs and G. Bernhardt for valuable suggestions on this manuscript. A.R. is supported by the EU 6th Framework Program collaborative grant INNOCHEM (LSHBCT‑2005‑518167). R.F. is supported by grants from the German Research Foundation (DFG SFB621‑A1, DFG SFB587‑B3, DFG SFB566‑A14, DFG SFB738‑B5). We regret that, owing to space limitations, we could not always adequately quote the work of our colleagues contributing to the field reviewed here.

DATABASES Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene CCL19 | CCL21 | CCR7

FURTHER INFORMATION Reinhold Förster’s homepage: http://www99.mh-hannover. de/institute/immunologie/ All links are active in the online pdf

volume 8 | may 2008 | 371 © 2008 Nature Publishing Group