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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nat Immunol. Author manuscript; available in PMC Dec 18, 2007.
Published in final edited form as:
Published online Aug 14, 2005. doi:  10.1038/ni1238
PMCID: PMC2144916
NIHMSID: NIHMS5172

CC chemokine receptor 7 required for T lymphocyte exit from peripheral tissues

Abstract

Lymphocytes travel throughout the body to carry out immune surveillance and participate in inflammatory reactions. Their path takes them from blood through tissues into lymph and back to blood. Molecules that control lymphocyte recruitment into extralymphoid tissues are well characterized, but exit is assumed to be random. Here, we showed that lymphocyte emigration from the skin was regulated and pertussis toxin-sensitive. CD4+ lymphocytes emigrated more efficiently than CD8+ or B lymphocytes. T lymphocytes in the afferent lymph expressed functional CCR7, and CCR7 was required for T lymphocyte exit from the skin. The regulated expression of CCR7 by tissue T lymphocytes may control their exit, acting with recruitment mechanisms to regulate lymphocyte transit and accumulation during immune surveillance and inflammation.

INTRODUCTION

Naïve T cells preferentially recirculate between blood and secondary lymphoid tissues, entering lymph nodes from the blood by crossing high endothelial venules13. Memory and effector T cells, unlike naïve T cells, can migrate efficiently into non-lymphoid tissues and into sites of inflammation and infection (reviewed4), subsequently entering afferent lymphatic vessels and traveling to local lymph nodes in the afferent lymph3,5,6. In parabiotic mouse models, endogenous memory T cells in most peripheral tissues reach equilibrium with immigrating blood-borne donor T cells within a week, suggesting that there is rapid turnover of T cells in peripheral tissues7. Indeed, blood to lymph migration through peripheral tissues such as the skin is extraordinarily dynamic with a peak transit time for lymphocytes of approximately 24 h8. Classic studies of recirculation in the sheep demonstrated that 1/10 of the total lymphocytes9 and a major fraction of memory-effector lymphocytes3 entering a given lymph node do so through the afferent lymph. Collectively, these studies indicate that lymphocyte exit from tissues and migration via afferent lymph into the draining nodes is a critical homeostatic migration pathway for memory and effector T cells.

The afferent lymph from tissues such as the skin predominantly contains memory CD4+ T cells and few B cells5,6,10,11, which mirrors the preferential recruitment and accumulation of CD4+ T cells relative to B cells in peripheral tissues. This finding and the fact that erythrocytes can enter the afferent lymph when injected into tissues, led to the current notion that lymphocyte migration into the afferent lymph is a passive and random process, determined primarily by fluid flow12. This concept contrasts with the tight control of the various other steps of lymphocyte recirculation, from the multi-component combinatorial control of recruitment of circulating lymphocytes from the blood13 to the regulation of lymphocyte exit from lymph nodes by cell-cell interactions and sphingosine 1-phosphate and its receptors14,15.

To directly assess the selectivity of lymphcyte exit from non-lymphoid tissues, we have established an experimental system in which the migration of adoptively transferred lymphocytes from subcutaneous tissues into draining lymph nodes is monitored. Our studies show that lymphocyte exit into afferent lymphatics is a tightly regulated process under the control of the chemokine receptor CCR7.

RESULTS

Tissue exit is non-random

Earlier studies assumed that tissue exit is a random process, and that lymphocytes within the interstitium could passively enter afferent lymph12. To study lymphocyte exit from a peripheral site, we injected 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester- (CFSE) or PKH26 Red Fluorescent Cell Linker- (PKH26) labeled murine splenocytes subcutaneously into the footpads of recipient mice and evaluated the number and phenotype of cells that migrated into the draining popliteal lymph nodes (Fig. 1a). The frequency of migrated cells increased from 3 to 18 h; a 12-hour time point was selected for most subsequent experiments, because at this time after injection, the blood, spleen and contralateral lymph nodes remained essentially devoid of transferred cells (not shown), implying that cells that had migrated via the afferent lymph and reached the draining lymph node had not yet entered the circulation.

Figure 1
Tissue exit is non-random

If lymphocyte exit were random, then the cells recovered from the draining lymph node would have the same cellular composition as the injected population. Instead, CD4+ T cells were dramatically enriched among cells recovered from the lymph node compared with the injected population (Fig. 1a). Naïve (CD44lo CD45RBhi) CD4+ T cells migrated somewhat better than memory (CD44hi CD45RBlo) CD4+ T cells as indicated by a slight enrichment of naïve CD4+ T cells in the migrated fraction compared with the injected cells (Fig. 1b). Although there was substantial variability in the overall numbers of migrated cells recovered from the draining lymph nodes of individual mice, CD4+ T cells consistently exited the footpads more efficiently than CD8+ T cells or B cells (Fig. 1c, left panel). CD4+ T cells migrated, on average, ~12-fold and ~3-fold more efficiently than CD8+ T cells and B cells, respectively (Fig. 1c, right panel). We conclude that exit from peripheral sites is a selective process.

Tissue exit requires Gαi protein-coupled receptors

To ask whether T cell exit from peripheral tissues and migration into the draining lymph node through the afferent lymph is an active process that involves chemoattractant receptor signaling, we treated mononuclear cells from spleen with pertussis toxin (PTX), which irreversibly modifies Gαi proteins and prevents most chemotactic responses. PTX-treated splenocytes were labeled with CFSE and injected into the footpads of recipient mice. Concurrently, equal numbers of mock-treated PKH26-labeled splenocytes were injected into the contralateral footpads, and migration to the draining lymph nodes was assessed 12 h later. PTX pre-treatment almost completely inhibited lymphocyte migration to the draining lymph node (Fig. 2). The same result was obtained when the dyes were reversed (data not shown). At later time points after transfer (18–24 h), infrequent untreated cells, but no PTX-treated cells, could be detected in the recipients’ blood (data not shown). Cells re-isolated from the injected footpad (whether PTX-treated or not) excluded propidium iodide and failed to bind annexin V, excluding a general effect on cell viability. We conclude that tissue exit is a regulated, non-random, Gαi-coupled receptor signaling-dependent process that likely involves chemoattractant receptors.

Figure 2
Tissue exit involves Gαi protein-coupled receptor signaling

CCR7-deficient lymphocytes show impaired tissue exit

Having established a role for G protein signaling in tissue exit, we next tested whether CCR7 expression controls lymphocyte exit from peripheral tissues. To evaluate the migration of CCR7-deficient (Ccr7−/−) cells, a mixture of CFSE-labeled splenic lymphocytes from Ccr7−/− mice16 and PKH26+ wild-type lymphocytes or a control mixture of CFSE- and PKH26-labeled lymphocytes from wild-type mice were injected into the footpads of recipient mice. At 12 h after transfer, equal numbers of CFSE+ and PKH26+ wild-type cells reached the draining lymph nodes (Fig. 3a, left panel). In contrast, CFSE+ Ccr7−/− cells were much less frequent in the draining lymph node (Fig. 3a, right panel): the migration of Ccr7−/− CD4+ T cells was reduced by 90 % (Fig. 3b), and CCR7 deficiency inhibited the migration of CD45RBhi naïve and CD45RBlo memory CD4+ cells to the same extent (Fig. 3b). The relatively inefficient exit of CD8+ T cells and B cells from the skin (Fig. 1) was also largely CCR7-dependent (Fig. 3b). These differences in migration between Ccr7−/− and wild-type lymphocyte subsets were apparent at 3 h, 6 h, and 9 h after injection as well, although fewer cells had reached the lymph nodes at these earlier time points (data not shown). As observed for wild-type lymphocytes, at these time points, and in most experiments at 12 h after injection, no Ccr7−/− cells were detected in the spleen, the site of preferential localization of Ccr7−/− lymphocytes16 or in the contralateral lymph nodes; and no cells were seen in blood (data not shown). These observations implicate CCR7 as a key regulator of T lymphocyte exit from the skin and subsequent migration into the draining lymph node.

Figure 3
T cells require expression of CCR7 to exit the skin and reach the draining lymph node

Transduced CCR7 rescues the exit of Ccr7−/− T cells

The impaired migration of Ccr7−/− lymphocytes from the skin to the draining lymph node strongly suggested that CCR7 mediates T cell exit from peripheral sites. However, T cells from Ccr7−/− mice could be abnormal due to their development in an organism with disrupted lymphoid architecture. Therefore, we asked if reintroducing CCR7 could restore the tissue exit of Ccr7−/− T cells. We transduced Ccr7−/− CD4+ T cells with a retroviral expression vector encoding CCR7 and an internal ribosomal entry site-green fluorescent protein (IRES-GFP) (‘CCR7’) or with a control vector encoding only GFP (‘control vector’). To confirm expression of surface CCR7, we tested cell binding to a fusion protein consisting of the murine CCR7 ligand, CCL19, fused to human immunoglobulin G17 (CCL19-Ig). Wild-type T cells expressed high amounts of surface CCR7 (Fig. 4a, left panel), whereas Ccr7−/− T cells, whether transduced with the control vector or not, failed to bind CCL19-Ig (Fig. 4a, middle). Ccr7−/− T cells transduced with CCR7 (GFP+) bound CCL19-Ig (Fig. 4a, right panel), although less efficiently than wild-type cells. Consistent with the CCR7 surface expression, wild-type T cells migrated efficiently toward the CCR7 ligand, CCL21, in an in vitro chemotaxis assay, whereas Ccr7−/− T cells responded only when transduced with CCR7 (Fig. 4b). Transducing cells with the control vector had no influence on the capacity of wild-type or Ccr7−/− T cells to exit the skin of congenic Thy1.1 mice (Fig. 4c, left and middle panel). In contrast, transducing Ccr7−/− CD4+ T cells with CCR7 rescued their ability to migrate to the draining lymph node (Fig. 4c, right panel). We conclude that the migratory defect of Ccr7−/− T cells is due to their lack of CCR7.

Figure 4
Transduction of CCR7 rescues Ccr7−/− CD4+ T cell exit from the skin and migration to the draining lymph node

T cells in the afferent lymph express functional CCR7

If physiologically recirculating T cells use CCR7 to exit from peripheral tissue sites, then T cells in the draining afferent lymph should express functional CCR7. To test this prediction, we cannulated the skin draining afferent lymph vessels (proximal to the draining lymph node) of sheep, an animal used in classical recirculation studies. We analyzed CCR7 (CCL19-Ig binding) expression by memory (CD45R) and naïve (CD45R+) T cell subsets. Consistent with prior reports3, almost all CD4+ T cells in afferent lymph draining the skin were of memory phenotype (Fig. 5a). Most of these afferent lymph CD4+ T cells bound CCL19-Ig (Fig. 5a). Memory CD4+ T cells in the blood were divided into major CCL19-Ig binding and non-binding populations, as expected18,19. Almost all afferent lymph CD8+ T cells were also of memory phenotype (CD45R), and the majority of these bound CCL19 as well (Fig. 5a; range: 50–89 %; mean: 69.8 %, from 6 different experiments). Moreover, in Transwell chemotaxis assays, memory T cells from afferent lymph and from blood migrated efficiently toward the CCR7 ligands CCL21 and CCL19 (Fig. 5b). Lymph memory T cells exhibited equal or greater sensitivity toward CCR7 ligands relative to blood memory T cells as indicated by half-maximal responses at lower chemokine concentrations (Fig. 5b). These data demonstrate that shortly after tissue exit, and prior to arrival at the draining lymph node, memory T cells that recirculate through the skin express CCR7 and are enriched in responsiveness to CCR7 ligands.

Figure 5
Ovine T cells in the afferent lymph express functional CCR7

DISCUSSION

Lymphocyte recirculation is a highly regulated process, controlling the distribution and targeting of immune cells throughout the body. Migration from the blood into lymphoid as well as peripheral tissues is determined by complex, combinatorially determined events involving multiple molecules that together regulate the tissue access of specialized lymphocyte subsets. Exit of lymphocytes from organized lymphoid tissues into the efferent lymphatics is controlled by distinct mechanisms, involving sphingosine 1-phosphate and its receptors14,15. The exit of lymphocytes from peripheral tissue such as the skin, however, has long been assumed to be random12. The goal of our study was to evaluate the selectivity and mechanisms of lymphocyte exit from peripheral tissues.

Our findings reveal that lymphocyte exit into afferent lymphatics, far from being random, is controlled by specific chemotactic mechanisms, and that CCR7 is an essential mediator of this process. Memory as well as naïve CD4+ T cells exit more efficiently compared to CD8+ T cells or B cells, but CCR7 is important in each case. The enhanced efficiency of exit into lymph by CD4+ T cell relative to CD8+ T cells or B cells is mirrored in the predominance of CD4+ T cells in afferent lymph5,6,10,11. The differential emigration also suggests that additional signals may be required for tissue exit, because CD4+ and CD8+ T cells from murine spleen have similar CCR7 expression17. Additional control mechanisms might include interactions with adhesion molecules expressed by lymphatic endothelium such as Clever-120. The results are in line with previous reports of a higher capacity of CD4+ relative to CD8+ T cells to recirculate from blood through tissues such as the peripheral and mesenteric lymph nodes21, intestine, and skin22. The reason for these T cell subset-specific differences in recirculation remains to be elucidated.

The critical role of CCR7 in T cell recirculation at the stage of exit from peripheral tissues may be considered surprising in light of concepts of CCR7 as a marker for “central memory T cells” that circulate exclusively through secondary lymphoid organs18,23. Recent studies, however, have shown that CCR7 is expressed by T cells that exert immediate effector functions17,24,25, and by many memory-effector T cells that infiltrate extralymphoid tissues, including the skin, synovium, and lung19,24,26.

Moreover, the CCR7 ligand, CCL21, is constitutively expressed by lymphatic endothelial cells within extralymphoid tissues2729, where it is known to mediate the CCR7-dependent migration of mature antigen-presenting dendritic cells into the afferent lymph16,28,30,31. CCL21-expressing lymphatics can be visualized immunohistochemically within skin, primarily adjacent to venules involved in lymphocyte recruitment or immediately underlying the epithelium32,33; thus, they are well positioned to mediate the CCR7-dependent exit of T cells from physiologic sites of lymphocyte infiltration. In this context, the efficiency of short term exit of footpad-injected cells is expected to depend not only on their expression of CCR7, but also on their proximity to these lymphatic-rich microenvironments; the distribution of injected cells to the deep connective tissues versus the superficial dermis may thus underlie the relatively low overall efficiency and may also explain the animal to animal variability of T cell exit in our experiments. In addition, in normal skin, in the absence of inflammatory stimuli, CCL21 expression by lymphatic endothelial cells is relatively low32,33, perhaps paralleling the low rate of homeostatic lymphocyte recirculation through this site. Interestingly, the efficiency of CD4+ T cell migration we observed (range, 0.3 to 1.7 percent of injected cells; mean of 5 experiments = 0.81) is similar to that reported for dendritic cells in similar studies (0.01 to 3% migration, 48 h after injection32). CCR7-CCL21 interaction also controls lymphocyte migration from the blood into secondary lymphoid tissues through high endothelial venules16,34,35. Our results thus extend our understanding of the roles of CCR7, suggesting a unifying mechanism for the control of lymphocyte access to secondary lymphoid tissues, whether via high endothelial venules from the blood or via lymphatic endothelium and afferent lymph from peripheral tissues.

Exit is likely to be as important as entry in controlling the accumulation of lymphocytes at peripheral sites. In this regard, the importance of CCR7 in lymphocyte transit may help explain observations of lymphocyte accumulation in the gastrointestinal tract, lung, and body cavities of Ccr7−/− mice (Uta E. Höpken and M.L., unpublished data). In a mouse model of influenza, most influenza virus-specific interferon-γ-positive (IFN-γ+) CD4+ T cells in the lungs of actively infected mice are CCR7-negative, but the majority of bystander effector T cells (which produce IFN-γ in response to polyclonal stimuli) are CCR7-positive26. Moreover, antigen recognition transiently down-regulates CCR7 function on T cells, at least in lymph nodes36. Together, these data suggest that down-regulation of CCR7 on actively responding effector T cells may help antigen-specific T cells accumulate at the peripheral sites of antigen-recognition37, while allowing bystander memory and effector T cells to exit rapidly and continue their immune surveillance elsewhere. Consistent with this concept, in vitro activated CCR7-transgenic effector T cells show a deficit in pulmonary accumulation in asthma models associated with enhanced exit from the lung and migration into the draining mediastinal lymph nodes compared to control (CCR7-negative) Th2 cells (Shannon Bromley and Andrew Luster, personal communication).

Of course, a substantial fraction of normal circulating memory and effector T cells lack detectable CCR7 expression even in the blood; these cells may be destined to enter but not leave tissue sites, or may be able to up-regulate the CCR7 ‘exit pass’ after tissue entry. It will be important in future studies to characterize the regulation of CCR7 expression on different lymphocyte subsets during peripheral immune responses, and to evaluate the relevance of CCR7-regulated lymphocyte exit to the course of inflammation and the defense against pathogens.

In conclusion, we have shown that lymphocyte exit from tissues into lymph is selective and active, and that CCR7 is key in this process. These findings fill a major gap in our understanding of the cycle of lymphocyte recirculation, and reveal tissue exit as a novel potential control point for the modulation of local inflammatory and immune responses.

METHODS

Animals, surgery, and lymph cannulation

For cell transfers, sex and age-matched BALB/c Ccr7−/− and wild-type mice were used. Donor mice for experiments comparing memory T cell exit were 6–9 months of age; those used for transduction were 6 weeks old. As recipients, 6–12 week-old recipient BALB/c mice (Jackson Laboratories) or Thy1.1 congenic BALB/c mice (a gift from I. Weissman, Stanford University) were used. 8–10 month old randomly bred sheep (Central Animal Facility, University of Guelph, Ontario, Canada) or Cheviot wethers (Ovis, South Dakota) were used. For the collection of pseudoafferent lymph38, subiliac lymph nodes were surgically removed >4 weeks before lymph cannulation as described38. Afferent (popliteal) and pseudoafferent lymph vessels were cannulated, and lymph was collected over 12-h periods as described38. The cannulated lymph vessels drained the skin, muscles, and bones of the hind leg or the rear flank and abdomen39. All animal experiments were approved by local review boards (Veterans Affairs Palo Alto Health Care System Institutional Animal Care and Use Committee (IACUC), Stanford University’s Administrative Panel on Laboratory Animal Care, South Dakota State University IACUC, and the University of Toronto Animal Care Committee), and performed according to institutional, federal, and state guidelines.

Cell isolation, PTX treatment, cell labeling, and transfer

Cells were isolated from murine lymph nodes and spleens as described26. Ovine lymphocytes from lymph were washed with RPMI 1640 containing 5% FCS and 25 mM HEPES, pKa 7.55 (cRPMI). Ovine blood was collected from the jugular vein and mixed with anti-coagulant. Mononuclear cells were isolated from murine spleen and ovine blood by gradient centrifugation using Histopaque (Sigma). In some experiments, CD8+ T cells were enriched 5-fold in the injected population by negative selection using CD19 microbeads (Miltenyi Biotec). For PTX treatment, splenocytes were incubated for 2 h at 37°C with 200 ng/ml PTX (Sigma) in cRPMI. Cell labeling with PKH26 was performed according to the manufacturer’s instructions (Sigma). For CFSE (Molecular Probes) labeling, cells at 5×106/ml were incubated in 0.2 μM CFSE in HBSS containing 25 mM HEPES for 5 min at 37°C. PKH26 and CFSE labeling reactions were stopped by adding serum. Cells were subsequently washed three times using cRPMI followed by one PBS wash. 1-5×106 cells in 10 μl PBS were injected into the footpads of recipient mice. 12 h after transfer, single cell suspensions of the draining popliteal lymph nodes were analyzed for transferred cells (identified by fluorescent labels or Thy1.2 congenic marker), and total cell numbers were enumerated by flow cytometry using a fixed number of polystyrene beads (Polybead, Polysciences). In experiments comparing the relative migration efficiency of Ccr7−/− to wild-type cells of specific lymphocyte subsets (total lymphocytes, total CD4+ T cells, naïve (CD45RBhi) and memory (CD45RBlo) CD4+ T cells, CD8+ T cells, and B cells), the ratio of migrated CFSE+ (Ccr7−/− in test mice; wild-type in control mice) to PKH26+ cells (admixed wild-type cells as a common internal standard) of the indicated subset was determined by flow cytometry. The relative migration of Ccr7−/− to wild-type cells was determined by calculating the ratios of migrated CFSE/PKH26 to injected CFSE/PKH26 cells within each lymphocyte subset. To control for variability in overall recovery, results were normalized to the mean ratio of CFSE+ wild-type to PKH26+ wild-type cells for each subset (set as 100%). In different experiments, the injected wild-type and Ccr7−/− populations consisted of 18–25 % CD4+ with 22–40 % of these being CD45RBlo, 48–67 % CD19+ B cells, and 10 % CD8+ (after enrichment).

Retroviral transduction

Human CCR7 upstream of an internal ribosomal entry site-green fluorescent protein (IRES-GFP) construct in MSCV 2.2β retroviral vector40 was provided by D. Campbell, University of Washington. Virus was produced as described41. Thy1.2+ CD4+ T cells were isolated from spleens of wild-type and Ccr7−/− mice using CD4 microbeads according to the manufacturer’s instructions (Miltenyi Biotec) and stimulated with plate-bound Abs to CD3 and CD28. 24 h after stimulation, the cells were infected with retrovirus containing either CCR7 and IRES-GFP or only IRES-GFP (control vector), as described41. On day 4 or 5 of culture, the cells were used for experiments.

Flow cytometry

Murine and ovine cells were pre-incubated in rat IgG plus anti-CD16/CD32 (2.4G2, BD Pharmingen) or sheep IgG plus donkey IgG, respectively. Purified IgGs were purchased from Jackson Immunoresearch. The following biotin- or fluorochrome- (phycoerythrin, peridinin chlorophyll-protein-cyanine 5.5, allophycocyanin, Alexa Fluor 647, phycoerythrin-cyanine 7, or fluorescein isothiocyanate) conjugated rat monoclonal antibodies recognizing murine surface markers, obtained from BD Pharmingen, were used: CD4 (RM4-5), CD45RB (16A), CD44 (IM7), CD19 (1D3), CD8 (53–6.7), Thy1.2 (30-H12). Alexa Fluor 405-labeled streptavidin (Molecular Probes) was used as a second step reagent. The following fluorochrome-conjugated mouse monoclonal antibodies recognizing ovine surface markers, obtained from Serotec, were used: CD4 (44.38), CD8 (CC63), CD45R (20.96). Where indicated, unlabeled monoclonal antibodies were labeled with Zenon mouse antibody labeling kits according to the manufacturer’s instructions (Molecular Probes). CCL19-Ig17 binding26 was detected using biotinylated multi-species adsorbed F(ab′)2 donkey anti-human IgG (Jackson Immunoresearch) followed by phycoerythrin-labeled streptavidin (BD Pharmingen). Specificity of the staining was shown by blocking with excess unlabeled recombinant murine CCL19 (R&D Systems) or staining with control Ig chimera (not shown).

Chemotaxis assay

Single cell suspensions were incubated for 1 h at 37°C prior to the assay. Recombinant murine CCL21 and human CCL19, obtained from R&D Systems, were titrated in triplicate between 1 and 300 nM. The assay was performed using 5 μm pore-size, 24-well Transwell plates (Corning Costar, obtained through Fisher Scientific) and analyzed as described26.

Acknowledgments

The authors thank D. Campbell (University of Washington) for comments on the manuscript and M. Zasio for sheep blood. This work was supported by grants from the NIH and a Merit Award from the Department of Veterans Affairs to ECB, the FACS Core of the Stanford Digestive Disease Center under NIH P30 DK56339 and a fellowship from the Howard Hughes Medical Institute to CNA. GFD is a recipient of an Arthritis Foundation Postdoctoral Fellowship and was supported by a Deutsche Forschungsgemeinschaft fellowship.

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