Afferent lymph derived T cells and DCs use different chemokine receptor CCR7 dependent routes for entry into the lymph node and intranodal migration

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1 Afferent lymph derived T cells and DCs use different chemokine receptor CCR7 dependent routes for entry into the lymph node and intranodal migration Asolina Braun, Tim Worbs, G Leandros oschovakis, Stephan Halle, Katharina Hoffmann, Jasmin Bölter, Anika ünk & Reinhold Förster 211 Nature America, Inc. All rights reserved. Little is known about the molecular mechanisms that determine the entry into the lymph node and intranodal positioning of lymph-derived cells. By injecting cells directly into afferent lymph vessels of popliteal lymph nodes, we demonstrate that lymph-derived T cells entered lymph-node parenchyma mainly from peripheral medullary sinuses, whereas dendritic cells (DCs) transmigrated through the floor of the subcapsular sinus on the afferent side. Transmigrating DCs induced local changes that allowed the concomitant entry of T cells at these sites. Signals mediated by the chemokine receptor CCR7 were absolutely required for the directional migration of both DCs and T cells into the T cell zone but were dispensable for the parenchymal entry of lymph-derived T cells and dendrite probing of DCs. Our findings provide insight into the molecular and structural requirements for the entry into lymph nodes and intranodal migration of lymph-derived cells of the immune system. During the past two decades, many molecules have been identified that regulate the homing of blood-derived lymphocytes to lymph nodes via specialized high endothelial venules (HEVs). In contrast, little is known about homing routes and mechanisms that control the lymph-node entry of T cells and dendritic cells (DCs) that arrive with the afferent lymph. In peripheral tissues, DCs constitutively sample the environment for antigen and eventually travel via afferent lymphatic vessels toward draining lymph nodes (called primary lymph nodes here). This process requires chemotactic signals mediated by the chemokine receptor CCR7 (refs. 1,2). Whereas the first step of the migration of Langerhans cells from epidermis to dermis is assumed to be CCR7 independent 2,3, epidermal as well as dermal DCs are subsequently guided toward small initial afferent lymphatics originating in the dermis by the chemokine CCL21 expressed on the lymphatic endothelium 2,4. Entry into these initial lymphatic vessels might occur predominantly via special preformed portals, independently of integrin-mediated adhesion and pericellular proteolysis 5 7. Likewise, it has been suggested that the entry of recirculating T cells into the initial lymphatic vessels also relies on CCR7 expression 8,9. At least in mouse lymph nodes, the main producers for the CCR7 ligands CCL19 and CCL21 are fibroblastic reticular cells of the T cell zone (TCZ) stromal compartment Additionally, HEV-bound CCR7 ligands contribute to the transmigration of lymphocytes through HEVs 1,13, whereas CCL21 present on stromal cells accelerates the basal velocity of randomly migrating T cells in the lymph node paracortex 14. However, except for indirect indications from studies of plt/plt mice, a naturally occurring mutant that lacks expression of CCL19 and CCL21 in secondary lymphoid organs 15, the influence of CCR7 and its ligands on the entry into the lymph node and intranodal positioning of afferent lymph derived DCs and T cells has remained unclear. Naive circulating T cells enter a lymph node via HEVs of blood vessels and leave the organ by accessing efferent lymphatics. From there, the cells may directly re-enter blood circulation or immigrate into a lymph node (called the secondary lymph node here) located downstream of the primary lymph node. To systematically study the lymph node homing process of afferent lymph derived DCs to primary lymph nodes, as well as of naive T cells to secondary lymph nodes, we established a new technique of intralymphatic injection into mice that allows the efficient delivery of defined numbers of cells directly into the afferent lymph vessels of the popliteal lymph node. By combining that approach with twophoton microscopic imaging, we found that T cells, but not DCs, reached lymph nodes downstream of the primary draining popliteal lymph node after intralymphatic injection. After transmigrating through the afferent-side subcapsular sinus (SCS) floor of the popliteal lymph node, wild-type DCs showed highly directional migration toward the deep TCZ. In contrast, CCR7-deficient (Ccr7 / ) DCs exited the SCS with delayed kinetics and remained largely sessile in SCS-adjacent cortical regions while showing dendrite probing similar to that of wild-type DCs. In contrast, T cells were not retained in the SCS in larger numbers after intralymphatic delivery but instead entered the lymph-node parenchyma from peripheral medullary sinuses. Although wild-type as well as Ccr7 / T cells showed random-walk motility in peripheral medullary cords, Institute of Immunology, Hannover edical School, Hannover, Germany. Correspondence should be addressed to R.F. (foerster.reinhold@mh-hannover.de). Received 26 ay; accepted 7 July; published online 14 August 211; doi:1.138/ni.285 nature immunology VOLUE 12 NUBER 9 SEPTEBER

2 211 Nature America, Inc. All rights reserved. Figure 1 Ccr7 / BDCs do not reach the paracortical TCZ after intralymphatic injection. (a) Expression of CD11c and major histocompatibility complex class II (HCII) by DCs generated in vitro from the bone marrow of wild-type (WT) and Ccr7 / () mice. Numbers adjacent to outlined areas indicate percent CD11c + DCs positive for major histocompatibility complex class II. (b) Retrieval of wild-type and Ccr7 / BDCs labeled with DDAO (wild-type) or TARA (Ccr7 / ), mixed at a ratio of 1:1 and injected intralymphatically ( total cells); draining popliteal lymph nodes digested 2 h later were analyzed by flow cytometry. Numbers adjacent to outlined areas indicate percent DDAO + cells (wild-type) and TARA + cells (Ccr7 / ). (c) Quantification of BDCs retrieved from draining popliteal lymph nodes (n = 11) as described in b, presented as the ratio of wild-type cells to Ccr7 / cells. (d) icroscopy of popliteal lymph nodes (popln) obtained from mice 2 48 h (top right corners) after intralymphatic injection of egfp-expressing wild-type BDCs and TARA-labeled Ccr7 / BDCs (1:1 mixture; cells in 5 µl PBS). IgD, immunoglobulin D;, orientation of the central medulla-hilus region. Repeat experiments with egfpexpressing Ccr7 / and TARA-labeled wild-type BDCs yielded similar results (data not shown). (e) icroscopic analysis of the positioning of wild-type and Ccr7 / BDCs in a popliteal lymph node explanted 12 h after intralymphatic injection of cells (as in d) and sectioned completely; composite images acquired by fluorescence microscopy were assembled for three-dimensional reconstruction (Supplementary Video 1). (f) icroscopy of intralymphatically injected BDCs in iliac lymph nodes (iln) from the mice in d. Scale bars, 1 µm (d,f) or 2 µm (e). Data are representative of at least three independent experiments (a c; mean and s.d. in c), two to five independent experiments with 4 12 lymph nodes per time point (d,f) or one experiment (e). only wild-type T cells proceeded into the deep TCZ, probably by haptotaxis (adhesive migration along concentration gradients of immobilized ligands 23 ) along a gradient of immobilized CCL21. Furthermore, mature DCs induced local changes in the SCS floor during transmigration that allowed the concomitant parenchymal entry of T cells at these sites. RESULTS Distinct distribution of wild-type and Ccr7 / DCs We developed an experimental protocol that allows the micromanipulator-guided microinjection of cells directly into the afferent lymphatic vessel of the popliteal lymph node in living mice, which ensures the synchronized arrival of injected cells into the SCS. After intralymphatic injection of ink, the dye almost immediately stained the efferent lymphatic vessel of the popliteal lymph node, followed by the ipsilateral medial iliac lymph node and the renal lymph node (Supplementary Fig. 1a c). These findings not only confirmed that lymphatic drainage occurs at least in part through lymph node chains in mice as in larger mammals but also indicated that downstream lymph nodes receive efferent lymph containing naive lymphocytes from more peripheral lymph nodes 16 (Supplementary Fig. 1d). Next we addressed the homing of bone marrow derived DCs (BDCs; Fig. 1). For this we labeled wild-type and Ccr7 / BDCs with the fluorescent cell tracers DDAO and TARA, respectively (Fig. 1a), or with the opposite labeling, then injected a 1:1 mixture of wild-type and Ccr7 / cells intralymphatically. Then, 2 h later, we prepared single-cell suspensions of popliteal, iliac and renal lymph nodes and analyzed these by flow cytometry (Fig. 1b). Although we were able to reisolate wild-type and Ccr7 / BDCs at a ratio of 1:1 from primary draining popliteal lymph nodes (Fig. 1c), we consistently failed to detect any BDCs in iliac or renal lymph nodes a CD11c d e WT HCII TARA f (Fig. 1f and data not shown). These data indicated that afferent lymph derived DCs were quantitatively retained in the primary draining lymph node and that this retention was independent of CCR7. We further investigated the subsequent intranodal fate of these DCs by intralymphatic injection of mice with a 1:1 mixture of TARA-labeled BDCs together with BDCs from mice expressing a transgene encoding enhanced green fluorescent protein (egfp) under control of the promoter of the chicken gene encoding β-actin. As shown by histology, wild-type BDCs rapidly entered the parenchyma of the lymph node cortex by exiting mainly from parts of the SCS that overlaid interfollicular areas while sparing cortical B cell follicles. The wild-type BDCs began to reach the outer paracortex within 2 h of intralymphatic injection and populated the entire TCZ 12 h and 24 h after delivery while concentrating in particular in deeper paracortical and medullary areas at 48 h (Fig. 1d). Notably, further histological analysis using the application of monoclonal antibody to the lymphatic endothelial cell marker LYVE-1 to popliteal lymph node sections collected 48 h after cell transfer indicated that few of the transferred DCs had actually entered the medullary sinuses and thus acquired the potential to disseminate to other organs (Supplementary Fig. 2). In contrast, Ccr7 / BDCs injected intralymphatically entered the lymph-node parenchyma with delayed kinetics and did not reach the paracortical TCZ throughout the observation period (Fig. 1d). Three-dimensional reconstruction of a complete popliteal lymph node collected 12 h after the intralymphatic transfer of DCs further substantiated the massive intranodal migration and positioning defect of Ccr7 / DCs (Fig. 1e and Supplementary Video 1). Notably, early after intralymphatic injection, DCs were efficiently retained in the afferent hemisphere of the popliteal lymph node; that is, proximal to those sites at which afferent lymph vessels merged with the SCS (Fig. 1d and Supplementary Video 1). b BDCs WT BDCs IgD popln 2 h popln 24 h DDAO illn 2 h c BDCs (WT/) popln 12 h i 2 1 popln 48 h illn 48 h 88 VOLUE 12 NUBER 9 SEPTEBER 211 nature immunology

3 a 1 h 4 h 8 h d WT e min 25 min min 25 min f s 3 s 6 s 9 s WT b min 4 min g min 75 min 211 Nature America, Inc. All rights reserved. c min 4 min Figure 2 Intranodal migratory activity of intralymphatically injected DCs in popliteal lymph nodes, as visualized by two-photon microscopy. (a) icrocopy of intranodal DC positioning in draining popliteal lymph nodes explanted 4 min after intralymphatic injection of TARA-labeled h Speed (µm/min) egfp WT BDCs ex vivo 2-P- egfp WT BDCs ex vivo 2-P- Coeff (µm 3 /min) min CD11c-YFP skin DCs in vivo 2-P- CD11c-YFP skin DCs ex vivo 2-P- wild-type BDCs (red) and egfp-expressing Ccr7 / BDCs (green), imaged ex vivo at 1 h, ~4 h and ~8 h after cell transfer. Blue indicates the secondharmonics generation (SHG) signal of collagen fibers of the lymph-node capsule. (b) Ex vivo time-lapse imaging of a lymph node ( min, top left) beginning 3 h after intralymphatic injection of egfp-expressing wild-type BDCs (green). Colors of scale bars (bottom right corners) indicate time scale of DC tracks (right), from the start (blue) to the end (yellow) of imaging. (c) Ex vivo time-lapse imaging of a lymph node (as in b) beginning 4 h 2 min after intralymphatic injection of egfp-expressing Ccr7 / BDCs (green). (d,e) Ex vivo time-lapse imaging of a lymph node (as in b) beginning 1.5 h (d) or 3 h 45 min (e) after intralymphatic injection of wild-type BDCs (d) or Ccr7 / BDCs (e), showing the cellular morphology of egfp-expressing BDCs (green) during intranodal migration. SHG signal (blue) at top indicates SCS position. (f) Ex vivo time-lapse imaging of a lymph node (as in b) beginning 6 h 3 min (wild-type) or 6 h (Ccr7 / ) after intralymphatic injection of to wild-type or Ccr7 / BDCs, analyzed after directional migration of wild-type BDCs had ceased (additional images, Supplementary Video 5). (g) Ex vivo time-lapse imaging of a lymph node (as in b) beginning 2 h after intralymphatic injection of CD11c-YFP DCs (green) that emigrated from the skin (additional images, Supplementary Video 6). SHG signal (blue) as in a. (h) Statistical analysis of intranodal DC migration. For track speed and straightness, each symbol represents an individual cell track and red horizontal bars indicate the median; for the motility coefficient (Coeff), each symbol represents a single time-lapse recording of a different imaging day and red horizontal bars indicate the mean (colors in key match colors in plots). ean displacement plots were calculated with one time-lapse recording per group. 2-P-, two-photon microscopy. Time-lapse recordings are in Supplementary Video 2 (for a), Supplementary Video 3 (for b,c) or Supplementary Video 4 (for d,e). Scale bars, 5 µm (a c,g), 1 µm (d,e) or 5 µm (f). Data are representative of four or more independent experiments with 7 19 lymph nodes (a e), six independent experiments with six to eight lymph nodes (f), three independent experiments with six lymph nodes (g) or three independent experiments (h). Straightness (Au) 1 ean displacement (µm) Entry into the lymph node and intranodal dynamics of DCs We next applied two-photon imaging to characterize the dynamic activity of intralymphatically injected DCs either in vivo 14 or ex vivo by superfusion of the explanted popliteal lymph node with oxygenated medium at 37 C in a custom-built imaging chamber 17. We injected TARA-labeled wild-type BDCs and egfp-expressing Ccr7 / BDCs (or cells with opposite labeling) together intralymphatically at a ratio of 1:1, explanted the popliteal lymph nodes 4 min later and repeatedly imaged the same lymph node area over the ensuing 8 h. Between 1 h and 4 h after cell transfer, wild-type DCs rapidly transmigrated through the floor of the afferent side SCS and showed highly directional migration toward the central TCZ. Thus, at approximately 8 h after injection, most wild-type DCs had left the SCS as well as adjacent areas of the lymph node cortex. In contrast, most Ccr7 / BDCs remained inside the SCS at 1 h. After 4 h and 8 h, only part of the Ccr7 / cells had entered the lymphnode parenchyma, generally residing in SCS-adjacent cortical areas (Fig. 2a and Supplementary Video 2). Some of the cells still residing in the SCS after 8 h might have undergone apoptosis, as cell fragments were distinctly visible at this point of time (Supplementary Video 2). However, as we observed this more frequently with TARA-labeled BDCs than with egfp-expressing BDCs, we assume that this effect was caused mainly by the cell-labeling procedure. The intranodal migratory activity of wild-type egfp-expressing BDCs was characterized by extensive cell polarization that resulted in the formation of a prominent leading edge as well as a pronounced long uropod, both characteristic features of directionally migrating cells (Fig. 2b and Supplementary Videos 3 and 4). Although Ccr7 / egfp-expressing BDCs also showed dynamic changes of their cellular morphology, they showed almost no prolonged directional cell polarization (Fig. 2c and Supplementary Videos 3 and 4) and consequently failed to translocate into the deeper lymph node paracortex. ore detailed visualization of migrating DCs confirmed those observations (Fig. 2d,e). To specifically compare the intranodal probing activity of wildtype and Ccr7 / BDCs (that is, their ability to protrude and retract dendrites), we imaged egfp-expressing BDCs 6 h after transfer. The dendrite movement characteristics of these sedentary wild-type BDCs were essentially indistinguishable from those of Ccr7 / BDCs residing at similar locations in the lymph node nature immunology VOLUE 12 NUBER 9 SEPTEBER

4 a b c Figure 3 Intranodal positioning of afferent lymph derived DCs contributes WT ** 4 to T cell proliferation but not lymph-node shutdown. (a) Total living cells WT * * in the draining popliteal lymph node 72 h after intralymphatic (IL) or 1 Ctrl subcutaneous (SQ) injection of wild-type or Ccr7 / BDCs ( cells per afferent lymph vessel or ** cells per footpad) or PBS NS ** (control (Ctrl)). NS, not significant; *P <.1 and **P <.1 (unpaired 1 2 two-tailed Student s t-test). (b) CFSE profiles of CD8 + Ly5.1 + V α 2 + V β * lymphocytes isolated from draining popliteal lymph nodes (black lines) from bm1 recipients given adoptive transfer of CSFElabeled 1 OT-I Ly5.1 + T cells by intravenous injection and, 1 d later, given intralymphatic injection of SIINFEKL-pulsed wild-type BDCs (top) or Ccr7 / IL SQ BDCs (bottom), followed by flow cytometry 65 h later. CFSE Shaded curves, CFSE profile of the brachial lymph node of the same WT Ctrl mouse (control). (c) Proliferation index of the cells in b; cell cycle number was calculated with FlowJo software. Each symbol represents an individual node; small horizontal bars indicate the mean. *P <.1 (unpaired two-tailed Student s t-test). Data are from three to four independent experiments with 8 15 mice (a; mean and s.d.) or three independent experiments with 12 mice per group (b,c). Cells ( 1 6 ) Frequency (relative) Proliferation index 211 Nature America, Inc. All rights reserved. cortex at 6 h (Fig. 2f and Supplementary Video 5). These findings suggest that signals mediated via CCR7 are not required for efficient intranodal DC probing in vivo, in contrast to published in vitro observations suggesting that CCL19 contributes to the dendrite extension activity of DCs 18. We then intralymphatically injected DCs that had crawled out of the split ear skin of mice expressing yellow fluorescent protein (YFP) under control of the promoter of the gene encoding the common DC marker CD11c (CD11c-YFP mice). These cells also had high expression of CCR7 (Supplementary Fig. 3a,b). Two-photon microscopy showed that skin-derived DCs also entered the parenchyma of the popliteal lymph node by transmigration through the floor of the SCS and showed cell polarization, uropod formation and inward directional migration similar to that of egfp-expressing wild-type BDCs (Fig. 2g and Supplementary Video 6). Of note, skin-derived DCs characteristically showed a more frequent branching of the leading edge during intranodal migration than did wild-type BDCs (Supplementary Video 6). Quantitative migration analysis showed that wild-type BDCs and skin-derived CD11c-YFP DCs had a very similar average cellular velocity, straightness of migration and mean displacement plot, as well as motility coefficient (Fig. 2h). Skin-derived CD11c-YFP DCs had slightly higher average cell velocities during intravital imaging than during ex vivo imaging, with essentially no difference in migration straightness, mean displacement or motility coefficient (Fig. 2h). Thus, quantitative motility analysis confirmed our qualitative assessment that the migratory activity of wild-type BDCs in explanted lymph node mirrored the physiological situation. Notably, Ccr7 / BDCs had a lower average cell velocity, less migration straightness and less mean displacement over time than did all wild-type DC groups analyzed, which resulted in a fourfold higher motility coefficient in wild-type mice than in Ccr7 / mice (Fig. 2h). Ccr7 / DCs are potent inducers of lymph-node shutdown During the so-called lymph-node shutdown reaction, the homing of inflammatory DCs is thought to induce the intranodal accumulation of lymphocytes, which results in lymph node swelling. At 72 h after intralymphatic injection of wild-type or Ccr7 / BDCs, we observed a similarly greater number lymphocyte numbers regardless of the BDC genotype (Fig. 3a). It is well established that after subcutaneous injection of DCs, only a small fraction of wild-type DCs actually enters the afferent lymphatic vessels and subsequently the connecting lymph node 19. Therefore, we used many more BDCs (1 1 5 ) for subcutaneous injection into the hind footpad. Although the injection of wild-type BDCs via this route led to ~11-fold more cells in the draining popliteal a b c Figure 4 CD4 + T cells enter lymph-node CD4 + WT CD4 + lgd CD4 + WT CD4 + LYVE-1 parenchyma in peripheral medullary areas after intralymphatic injection and require CCR7 signals for migration into the paracortical TCZ. (a,b) Immunohistological analysis of draining popliteal lymph nodes after intralymphatic injection of TARA-labeled wild-type (red) and egfp-expressing Ccr7 / Beads LYVE-1 (green) polyclonal CD4 + T cells. Similar results were obtained with opposite labeling (data not 2 h 4 h shown). (a) Overview composite images at 2 h and 4 h after injection. Immunoglobulin D staining (blue) marks B cell follicles as well as medullary B cells; indicates the orientation of the central medulla-hilus region. (b) LYVE-1 staining (blue) of lymphatic endothelium d LYVE-1 Dextran CD4 + indicates sinus structures. White arrow indicates peripheral medullary sinus lumen; white arrowheads indicate medullary cords; 4 h white, DAPI nuclear staining. (c) icroscopy of LYVE-1 + peripheral medullay sinus structures (red) 15 min after intralymphatic injection of inert 6-µm latex beads (green). (d) icroscopy of draining popliteal lymph nodes after intralymphatic injection of TARA-labeled Ccr7 / CD4 + T cells (red), followed by injection of 5 µl fixable fluorescein isothiocyanate conjugated 5-kilodalton dextran (green) into the same afferent lymph vessel 15 min later and isolation and fixation with paraformaldehyde 5 min after that. LYVE-1 staining (blue) of lymphatic endothelium indicates sinus structures. Scale bars, 1 µm (a), 2 µm (b) or 5 µm (c,d). Data are representative of five (a,b) or two (c,d) independent experiments (n = 15 lymph nodes per time point (a,b); n = 6 (c); n = 12 (d)). 882 VOLUE 12 NUBER 9 SEPTEBER 211 nature immunology

5 211 Nature America, Inc. All rights reserved. Figure 5 Two-photon imaging of the entry into the lymph node of intralymphatically injected CD4 + T cells in peripheral medullary areas. (a,b) Ex vivo time-lapse imaging of a lymph node beginning 1 h 2 min (a) or 35 min (b) after intralymphatic injection of to polyclonal egfp-expressing wild-type CD4 + T cells, showing entry into peripheral medullary cords by transmigration through LYVE-1 + endothelium (red, cyan outline) of the capsule-lining sinus floor (additional images, Supplementary Video 7). Blue indicates SHG signal of the lymph-node capsule; lines indicate tracks of individual cells. (c) Ex vivo timelapse imaging of a lymph node beginning 2 h after intralymphatic injection of wild-type CD4 + T cells (green), showing entry of cells into peripheral medullary cords by transmigration through LYVE-1 + endothelium (red) after extended migration in the lumen of the peripheral medullary sinus system (additional images, Supplementary Video 8). (d) Ex vivo time-lapse imaging of a lymph node beginning 1 h after intralymphatic injection of polyclonal wild-type CD4 + T cells, showing intranodal migratory activity (tracks in red) in peripheral medullary areas directly after exit from the capsule-lining sinus lumen (white arrowhead). Grey three-dimensional structure represents the LYVE-1 + lymphatic endothelium and delineates the border between the peripheral medullary sinus lumen (S) and the parenchyma of peripheral medullary cords (P). Additional images, Supplementary Video 9. (e g) Statistical analysis of the migration of wild-type CD4 + T cells (n = 22) in peripheral medullary areas.5 2 h after intralymphatic injection (three time lapse recordings; duration, 4 6 min). (e) Dwell index in the parenchyma versus sinus lumen (P/S), relative to compartment volume. (f) Transmigration events (compartment changes by migration through a LYVE-1 + lymphatic endothelial layer) per cell track. (g) Localization and direction of transmigration, presented as the frequency of cell tracks that remained in the lymphnode parenchyma (P) or the lumen of the peripheral medullary sinus (S) without transmigration or started in the lumen of the peripheral medullary sinus and ended in the peripheral medullary cords ( P) or vice versa ( S). Scale bars, 1 µm (a c) or 2 µm (d). Data are representative of three independent experiments (a d) or are from three independent experiments (e g; mean and s.d.). lymph node, the injection of the same number of Ccr7 / BDCs resulted in only fourfold more cells (Fig. 3a). Thus, the efficiency of homing to the lymph node (that is, arrival in the SCS of the draining lymph node) contributed to the degree of lymph-node shutdown induced by migratory DCs, whereas intranodal positioning had no major influence on this process, which substantiated published findings that locally produced soluble mediators rather than direct cell-cell interactions transiently inhibit lymphocyte egress 2,21. Less T cell proliferation in vivo by Ccr7 / DCs We next used T cell antigen receptor transgenic OT-I T cells specific for the ovalbumin-derived SIINFEKL peptide as reporter cells to determine whether misplaced DCs are impaired in their activation of T cells. We labeled OT-I T cells with the cytosolic dye CSFE and adoptively transferred them by intravenous injection into bm1 recipient mice, which are unable to cross-present the SIINFEKL peptide. Subsequently, we delivered BDCs intralymphatically and analyzed T cell proliferation 65 h later. Ccr7 / DCs were less efficient than their wild-type counterparts in inducing the antigen-specific proliferation of naive T cells (Fig. 3b,c), which indicated that the CCR7-dependent inward migration of DCs into the paracortex indeed contributed to T cell activation efficiency. Entry of T cells into lymph nodes via afferent lymphatics Lymph nodes are often organized in chains, which indicates that lymphocytes exiting a peripheral primary lymph node via efferent lymphatics would arrive in the SCS of a more central secondary lymph node via an afferent lymph vessel (Supplementary Fig. 1). To study the homing of lymph-derived T cells, we injected TARA-labeled CD4 + a SHG d e f S P b LYVE-1 WT CD4 + SHG LYVE-1 WT CD4 + LYVE-1 WT CD4 + Dwell index P/S Frequency (%) >3 Transmigration events wild-type T cells intralymphatically together with egfpexpressing Ccr7 / CD4 + T cells (or cells with opposite labeling). In contrast to the results obtained with DCs, wild-type as well as Ccr7 / T cells localized predominantly to outer parts of the peripheral medullary sinus system, which includes the capsule-lining sinus of the medullary lymph node hemisphere (Fig. 4a; topological details, Supplementary Fig. 4). Furthermore, whereas DCs homed exclusively into the primary draining popliteal lymph node, many T cells were present in the downstream second-level iliac lymph node at 2 h and 24 h after intralymphatic injection (Supplementary Fig. 5). At 2 h after intralymphatic injection, some wild-type T cells had already reached the outer paracortex of the primary draining popliteal lymph node, whereas at 4 h, wild-type T cells had populated the entire TCZ. In contrast, most intralymphatically injected Ccr7 / T cells remained in outer peripheral medullary areas at all time points analyzed (Fig. 4a). We further analyzed lymph node entry routes through the use of monoclonal antibody to LYVE-1 to delineate the peripheral medullary sinus system from the parenchymal compartment of the peripheral medullary cords (Fig. 4b; orientation, Supplementary Fig. 4). Ccr7 / T cells were able to leave the lumen of the peripheral medullary sinus system, thus efficiently entering the parenchymal compartment of the lymph node. At 4 h after intralymphatic injection, Ccr7 / T cells showed considerable accumulation in peripheral medullary cords characterized by a more densely packed cellular environment, as shown by the staining of nuclei with the DNA-intercalating dye DAPI (Fig. 4b). As we found T cells initially concentrated in the peripheral medullary sinus system after intralymphatic delivery, we checked whether passive transport and deposition due to the fluid dynamics of lymph flow resulted in such preferential accumulation c g Frequency (%) P S P S nature immunology VOLUE 12 NUBER 9 SEPTEBER

6 211 Nature America, Inc. All rights reserved. Figure 6 CCR7-mediated signals are required for the progression of T cells from peripheral medullary areas into the paracortical TCZ. (a) Ex vivo time-lapse imaging ( min, far left) of a lymph node beginning 45 min after intralymphatic injection of total CD4 + T cells (1:1 mixture of wild-type cells (red) and Ccr7 / cells (green)), showing intranodal migration in peripheral medullary areas (additional images, Supplementary Video 1). Far right, cell track overlay; blue, SHG signal of the lymph-node capsule. (b) Statistical analysis of intranodal T cell migration, derived from time-lapse recordings starting 45 min to 3.5 h after intralymphatic injection of cells as in a (presented as in Fig. 2h). *P <.5 (unpaired two-tailed Student s t-test). (c) Immunohistological analysis of CCL21 expression (red) in peripheral medullary areas. Blue, LYVE-1 + lymphatic endothelium; white dots outline a B cell follicle (B) devoid of CCL21 signal. Right, topological quantification of the LYVE-1 signals (blue) and CCL21 a b Speed (µm/min) min CD4 + WT CD4 + SHG 3 1. signals (red) in the area demarcated by the yellow lines at left. Scale bars, 5 µm (a,c). Data are representative of three experiments (a,c) or are from three independent experiments (b). of lymphocytes. For this we used the intralymphatic injection setup to inject fluorescent 6-µm latex microspheres as inert model particles. Notably, these particles also deposited mainly in outer parts of the peripheral medullary sinus system (Fig. 4c). We further analyzed the distribution of lymph fluid and lymph-borne T cells in the lymph node sinus system by injecting TARA-labeled Ccr7 / CD4 + T cells intralymphatically, followed by the injection of a soluble fluorescent tracer (fixable fluorescent isothiocyanate conjugated high molecular-weight dextran) via the same route 15 min later. After an additional 5 min, we killed the recipient mice and analyzed the draining popliteal lymph nodes by histology. We found that Ccr7 / T cells injected intralymphatically resided mainly in superficial parts of the peripheral medullary sinus system lumen that had also been reached by the soluble fluorescent tracer (Fig. 4d). Together these observations suggest that to a large extent, afferent lymph derived T cells are passively transported by lymph flow into the peripheral medullary sinus system. Subsequently, they can cross the LYVE-1 + endothelium and thereby enter the lymphnode parenchyma independently of CCR7 signals by migrating into adjacent peripheral medullary cords. igration dynamics of afferent lymph derived T cells We next aimed to delineate the boundaries between medullary sinuses and cords during two-photon microscopy by visualizing the LYVE-1 + lymphatic endothelium in vivo. For this we applied labeled monoclonal antibody to LYVE-1 intralymphatically 3 min before delivering egfp-expressing CD4 + wild-type T cells via the same route. We found that many wild-type T cells entered peripheral medullary cords by directly transmigrating through the floor of the capsule-lining medullary sinus (Fig. 5a,b and Supplementary Video 7). Notably, in some cases, several wild-type T cells crossed the LYVE-1 + lymphatic endothelial layer of the sinus floor at the same place within a short period of time (Supplementary Video 7). These hot spots might represent preformed or temporarily induced transmigration portals in the capsule-lining medullary sinus floor. Alternatively, intralymphatically injected wild-type T cells entered the lymph-node parenchyma by transmigrating through the LYVE-1 + boundary of Straightness WT WT c ean displacement (µm) 3 min 3 min min LYVE-1 CCL21 inner peripheral medullary sinus system branches after migration in the sinus lumen (Fig. 5c and Supplementary Video 8). To comprehensively visualize the three-dimensional topology of LYVE-1 + sinus boundaries, we used the image-processing and analysis software Imaris to generate three-dimensional isosurface models that allowed us to plainly differentiate between sinus lumen and lymph-node parenchyma (Fig. 5d and Supplementary Video 9). We analyzed the compartmental distribution and intranodal migratory activity of wild-type T cells during the first.5 2 h after intralymphatic delivery by semiautomated tracking of T cells that had already left the capsule-lining medullary sinus at the beginning of track analysis. To determine their subsequent distribution between peripheral medullary cords and peripheral medullary sinus system, we calculated the dwell index as the ratio of the accumulated dwell time (track durations) in the parenchyma to that in the sinus lumen for all wild-type T cells tracked in each compartment. The resultant dwell index of ~2.2 (Fig. 5e) indicated that early after intralymphatic injection, wild-type T cells had a more than twofold likelihood of migrating in peripheral medullary cords (that is, within lymph-node parenchyma). Once located in a compartment, wild-type T cells largely respected compartmental boundaries during migration in the peripheral medulla, as ~75% of all cell tracks did not cross the LYVE-1 + endothelium of peripheral medullary sinuses. ost of those cells that transmigrated did so only once, and no T cell transmigrated more than three times during the imaging period of 4 6 min (Fig. 5f). Finally, after we classified all cell tracks according to the compartment in which the cell resided at the end of the imaging period (Fig. 5g), the preferential migration of wild-type T cells into and within peripheral medullary cords became obvious: although more than 7% of all cell tracks were located completely within peripheral medullary cords, less B Coeff (µm 3 /min) Radius WT * HEV 884 VOLUE 12 NUBER 9 SEPTEBER 211 nature immunology

7 211 Nature America, Inc. All rights reserved. Figure 7 Transmigration of activated DCs changes the morphology of the SCS floor on the afferent side and allows direct entry of coinjected T lymphocytes into lymph-node parenchyma. (a) Ex vivo time-lapse imaging ( min, left) of a lymph node beginning 3 min after intralymphatic injection of wild-type CD4 + cells. Blue, SHG signal of the lymph-node capsule. (b) Ex vivo min SHG WT CD4 + 4 min min SHG WT CD4 + WT BDC 4 min time-lapse imaging ( min, left) of a lymph node after intralymphatic injection of wild-type CD4 + cells and TARA-labeled wild-type BDCs (red) 4 min before injection of wild-type CD4 + T cells (green) into the same afferent lymph vessel, followed by imaging 1 h 2 min later. SHG signal (blue) as in a. Right, overlay of wild-type CD4 + T cell tracks (green). (c,d) Cellular composition and morphology of the SCS after intralymphatic injection of 6-µm beads (green; c) or wild-type CD4 + T cells (green; d). Red, LYVE-1 + lymphatic endothelial cells; blue, CD169 + SCS-lining macrophages. (e) icroscopy of the transmigration of intralymphatically injected activated DCs (egfp + wild-type BDCs; green) in the SCS. Blue, SCS-lining CD169 + macrophages (blue); red, LYVE-1-staining (red). Below (c e), enlargement of areas c Beads CD169 LYVE-1 d CD4 + CD169 LYVE-1 e CD169 LYVE-1 WT BDC outlined in main images above. Scale bars, 5 µm (a,b) or 1 µm (c e); inset size, 2 µm 2 µm. Data are representative of three independent experiments with four to six lymph nodes (a,b) or two to four independent experiments with six to thirteen lymph nodes (c e). than 5% of the T cells stayed in the lumen of peripheral medullary sinuses throughout the imaging. Among the ~25% of wild-type T cells that had changed compartments (at least once), fourfold more T cells were located in the peripheral medullary cords than in the peripheral medullary sinuses at the end of imaging (~2% versus ~5%). In summary, T cells preferentially migrated in peripheral medullary cords rather than returning into the peripheral medullary sinus system early after arrival via afferent lymphatics. We also observed rapid and preferential localization into the parenchymal compartment after intralymphatic injection of Ccr7 / CD4 + T cells during two-photon imaging (Supplementary Video 1). However, whereas wild-type T cells showed considerable inward migration toward the paracortex with high directionality and displacement over time, most Ccr7 / T cells showed an apparently random-walk migration restricted mainly to peripheral medullary cords (Fig. 6a and Supplementary Video 1). Although the average migration speed of Ccr7 / CD4 + T cells was only slightly lower than that of wild-type cells (Fig. 6b), their movement was much more confined than that of their wild-type counterparts, as shown by their lower motility parameters of directionality (track straightness, Fig. 6b) and displacement over time (mean displacement plot and motility coefficient, Fig. 6b). Hypothesizing that afferent lymph derived T cells might use CCR7-derived signals as guidance cues to successfully navigate from peripheral medullary cords to the paracortical TCZ, we analyzed the distribution of the CCR7 ligand CCL21 in peripheral medullary areas by immunohistology and found higher CCL21 expression toward the lymph node center (Fig. 6c). Altered homing of T cells in the presence of mature DCs When injected alone, afferent lymph derived T cells entered the lymphnode parenchymal compartment from the lumen of the peripheral a b medullary sinus system, whereas DCs injected intralymphatically transmigrated directly through the floor of the SCS on the afferent side. This raised the question of whether, apart from flow dynamics, morphological properties of the SCS floor might prevent the transmigration of T cells at this site under steady-state conditions. As shown by two-photon imaging of the SCS on the afferent side 3 min after intralymphatic injection, CD4 + wild-type T cells remained almost completely restricted to the SCS lumen, showing only very limited motility (Fig. 7a and Supplementary Video 11). In contrast, when wild-type BDCs were injected 4 min before T cells were injected, afferent lymph derived wild-type T cells transmigrated through the SCS floor and showed avid inward migration at sites of DC transmigration (Fig. 7b and Supplementary Video 11). To assess the role of lipopolysaccharide that potentially might have also been transported into the SCS in trace amounts by in vitro matured DCs, we incubated T cells with the same concentrations of lipopolysaccharide used for DC maturation and delivered them intralymphatically. As under these experimental conditions T cells were still excluded from efficient homing via the SCS (Supplementary Video 12), our findings suggest that DCs have an important role in the induced homing of T cells via this route. Hypothesizing that altered penetrability might correlate with morphological changes of the SCS floor, we used immunohistology to analyze in greater detail the configuration of the sinus-parenchyma border in various lymph-node regions. After intralymphatic injection of inert fluorescent microspheres or CD4 + wild-type T cells alone, the cellular composition and morphology of the SCS remained almost completely unchanged relative to those properties under steadystate conditions (Fig. 7c,d and data not shown). The SCS floor of the afferent popliteal lymph node hemisphere was composed of a thin layer of LYVE-1 + lymphatic endothelial cells as well as many nature immunology VOLUE 12 NUBER 9 SEPTEBER

8 211 Nature America, Inc. All rights reserved. sialoadhesin-positive (CD169 + ) SCS-lining macrophages that had an outspread cellular morphology, extending into the SCS lumen (Fig. 7c,d). In contrast, medullary sinuses were characterized by much higher endothelial LYVE-1 expression, with only few solitary CD169 + macrophages lining the parenchymal surface of these sinus structures. Instead, CD169 + macrophages were interspersed in the lumen of medullary sinuses, concentrating in particular toward the central hilar region of the medulla (Fig. 7c e and Supplementary Fig. 4). Notably, the transmigration of wild-type BDCs injected intralymphatically induced profound morphological changes in the SCS floor on the afferent side, in that SCS-lining CD169 + macrophages seemed elongated and aligned in parallel to immigrating DCs, whereas the LYVE-1 signal in the SCS floor was considerably diminished (Fig. 7e). These effects were restricted to parts of the SCS that had many immigrating DCs. DISCUSSION Little is known about mechanisms that regulate the entry into the lymph node of cells that arrive via afferent lymphatics. This is due mainly to the generally low frequency of subcutaneously injected cells that actually enter afferent lymphatic vessels and thus reach the draining lymph node. To overcome that fundamental limitation, we established a new microinjection technique to deliver defined populations of cells of the immune system directly into the afferent lymph vessel of the popliteal lymph node in mice. Although we consider intralymphatic injection a major technical advance, this procedure does not necessarily reflect the naturally occurring scenario of the entry of cells, which are unlikely to arrive as a bolus via afferent lymphatics during steady-state or inflammatory conditions. By intralymphatic infusion of cells, we found the following four fundamental differences in the lymph node homing activity of afferent lymph derived DCs and T cells: initial positioning after intralymphatic injection; location and degree of CCR7 dependence of parenchymal entry; route and character of intranodal migration; and degree of confinement to the primary draining lymph node. Directly after intralymphatic injection, T cells accumulated in the peripheral medullary sinus system where the capsule-lining medullary sinus (as a direct extension of the SCS on the afferent side) connected to the first branches of peripheral medullary sinuses. The labyrinthine branching of the peripheral medullary sinus system probably causes a relatively abrupt decrease in the fluid velocity of lymph flow, which in turn might result in the observed preferential deposition of lymphocytes and latex beads in the peripheral medullary sinus system. In contrast, DCs injected intralymphatically were present almost exclusively in the SCS lumen of the afferent lymph-node hemisphere. This might have been the consequence of differences in cell size and shape, which would lead to faster passive deposition of the larger DCs, or might have resulted from the active retention of DCs. Notably, the initial distribution of DCs in the SCS not only influences the site of parenchymal entry but probably contributes to the strict confinement of afferent lymph derived DCs homing to the primary draining lymph node as well. This in turn might favor the regional compartmentalization or, alternatively, the functional specialization of adaptive immune responses elicited by migratory DCs, or might represent a general protective mechanism to prevent the systemic spread of cell-bound antigen (for example, early during a local infection). In this context, it is noteworthy that the presentation of cell-bound antigen has been shown to efficiently induce mechanisms of peripheral tolerance, but soluble antigen has not been shown to do so 22. Notably, wild-type and Ccr7 / T cells entered the lymph-node parenchymal compartment with similar efficiency, whereas the exit of afferent lymph derived DCs from the SCS was impaired in the absence of CCR7 signals. This probably reflected, at least in part, the high intrinsic motility of T lymphocytes, which supports a basic random-walk migration even in the absence of functional CCR7 interactions 14. DCs, in contrast, seem to rely heavily on chemotactic or haptotactic CCR7 cues 23 to achieve cell polarization, cytoskeletal reorganization and, consequently, directional migration into the lymph-node parenchyma. Once inside the lymph-node parenchyma, both DCs and T cells strictly required CCR7 signals to translocate into the deeper paracortical TCZ, but they had different characteristics during intranodal migration. Although DCs followed straight pathways through interfollicular cortical regions, indicative of strong chemotactic or haptotactic guidance, as described before 23, T cells seemed to progress from peripheral medullary cords into the TCZ by means of a skewed random walk. As it has been proposed for naive T cells migrating in the paracortical TCZ after entry via HEVs 24,25, afferent lymph derived T cells might use the stromal cell network as a scaffold for basic random-walk migration. Increasing concentrations of immobilized CCL21 might then bias the migration of wild-type T cells toward the lymph node center, resulting in the observed skewed random walk into the TCZ. After arriving via afferent lymph, wild-type as well as Ccr7 / T cells largely respected compartmental boundaries while navigating the complex topology of peripheral medullary areas and preferentially migrated within peripheral medullary cords. This might have reflected a certain structural barrier function of the LYVE-1 + endothelial cell layer that makes up the wall of medullary sinuses or, more likely, resulted from preferential adherence of migrating T cells to the surface of parenchymal stroma cells. Notably, medullary sinuses are directly connected to the efferent lymph vessel and have been regarded, in addition to cortical sinuses 26 28, as important exit structures for the egress of lymphocytes from lymph nodes. Thus, the question arises of how medullary sinuses can at the same time provide a suitable environment for both lymphocyte homing and egress. Fundamental work has identified sphingosine 1-phosphate receptor type 1 (S1P 1 ) as the most important egress-promoting receptor on T lymphocytes 29,3. Although it is largely absent from lymph-node parenchyma, sphingosine 1-phosphate (S1P), a ligand for S1P 1, is present at high concentrations in lymph fluid. This gradient guides lymphocytes from lymph-node parenchyma into medullary and cortical sinuses, thus facilitating their egress. Notably, lymphocytes in lymph lack surface S1P 1 (ref. 31), and ligand binding has been shown to induce transient internalization of the receptor, thus rendering cells unresponsive to S1P 32. Together these data suggest a scenario in which lymph-derived wild-type T cells as well as Ccr7 / T cells would be temporarily unresponsive to a S1P signal intended to retain them in the sinus lumen and would thus efficiently enter peripheral medullary cords. In contrast, CCR7 ligands expressed on stromal cells in lymph-node parenchyma have been shown to provide a functional counterpart that helps to retain T cells in this compartment 26. Wild-type T cells, but not Ccr7 / T cells, could then follow an immobilized CCL21 gradient to progress into the paracortical TCZ. Because of low intranodal concentrations of S1P, afferent lymph derived T cells should regain their surface expression of S1P 1 with increasing dwell time in the lymph node, similar to T cells that have homed via HEVs. Consequently, medullary sinuses most probably also function as exit sites for lymph-derived T cells at later time points. Our preliminary analysis of the homing efficiency of lymph-derived naive wild-type T cells showed that 2 h after intralymphatic delivery, approximately 15% of the total injected T cells were present in the 886 VOLUE 12 NUBER 9 SEPTEBER 211 nature immunology

9 211 Nature America, Inc. All rights reserved. popliteal lymph node and 5% were present in the respective secondlevel iliac lymph node (data not shown). These observations suggest that homing via afferent lymphatics can contribute substantially to the homing of naive T cells to lymph nodes, which is probably most relevant for downstream lymph nodes that receive afferent lymph derived naive T cells that have previously egressed from a more peripheral lymph node. Furthermore, mature DCs actively transmigrating through the SCS induce changes in the SCS floor morphology that additionally allow the direct entry of afferent lymph derived T cells. This might represent a strategy for increasing not only the lymph node homing frequency of lymph-derived T cells but also the likelihood of DC T cell interactions in interfollicular areas, potentially facilitating the reactivation of tissue-derived memory T cells as well as the priming of naive T cells under conditions of acute inflammation. The relatively efficient T cell proliferation induced by Ccr7 / DCs in these lymph node regions further supports the idea proposed above. These results also emphasize the obvious potential of naive T lymphocytes to scan large volumes of lymph-node parenchyma and indicate that in addition to the paracortical TCZ, interfollicular cortical areas can provide a suitable environment for productive T cell DC encounters, as suggested before 33. As different subpopulations of migratory DCs have been described to pass through and accumulate within distinct subcompartments of the lymph node 33,34, it seems possible that functionally specialized T cell immune responses could be primed at different intranodal locations. The intralymphatic injection technique described here represents an efficient approach for the targeting of defined cell populations to peripheral lymph nodes and will allow further elucidation of many aspects of lymph node development, function and homeostasis. Furthermore, it might also help delineate the series of events that occur during lymph-node invasion and the spread of tumors that metastasize via lymphatics. ethods ethods and any associated references are available in the online version of the paper at Note: Supplementary information is available on the Nature Immunology website. Acknowledgments We thank. Herberg for animal care; E. Kremmer (Helmholtz Zentrum ünchen) for antibodies; and G. Bernhardt for discussions and comments on the manuscript. Supported by Deutsche Forschungsgemeinschaft (SFB566-A14, SFB587-B3 and EXC62, Rebirth, to R.F.) and Boehringer Ingelheim Fonds (A.B.). AUTHOR CONTRIBUTIONS R.F., A.B. and T.W. designed experiments, analyzed data and wrote the paper; A.B. did experiments, including intralymphatic injection; T.W. contributed to Figure 1, two-photon imaging and data analysis; S.H. contributed to Figure 7 and read and commented on the manuscript; G.L.. contributed to Supplementary Figure 2 and read and commented on manuscript; and A.., K.H. and J.B. did histology, flow cytometry staining and DC cultures. COPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at Reprints and permissions information is available online at reprints/index.html. 1. Förster, R. et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99, (1999). 2. Ohl, L. et al. CCR7 governs skin dendritic cell migration under inflammatory and steady-state conditions. Immunity 21, (24). 3. Ouwehand, K. et al. 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