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The maturation status of dendritic cells (DCs) determines whether they prime or tolerize T cells. We targeted ovalbumin peptide exclusively to DCs in situ using an antibody against DEC-205 and studied DC interaction with naïve CD4+ T cells under tolerizing or priming conditions. Two-photon microscopy was used to simultaneously track antigen-specific OT-II T cells, non-specific T cells and DCs in lymph nodes of living mice. In both tolerance and immunity, OT-II cells arrested on DCs near high endothelial venules beginning shortly after extravasation and regained their baseline speed by 18 h. Thus, early antigen-dependent T cell arrest on DCs is a shared feature of tolerance and priming associated with activation and proliferation.
Laser scanning microscopy allows imaging of live cells at a depth of up to 300 μm in lymph nodes removed from mice (explanted) 1–6 or exposed surgically in anesthetized mice (intravital)7–9. Both kinds of preparations have been used to study mature DCs actively participating in immune responses1,3–5,7, but lymph nodes imaged by intravital microscopy differed from the explanted preparations in preserving blood and lymph flow.
Mature DCs have been visualized by labeling with exogenous fluorescent dyes and were found to crawl slowly in random directions, rapidly extending and retracting long dendritic processes1,3–5,7. We have examined the behavior of steady state DCs in live anesthetized mice by intravital 2-photon microscopy CD11c-EYFP transgenic mice that express high amounts of enhanced yellow fluorescent protein (EYFP) in CD11c+ DCs10. DCs in these mice are indistinguishable from DCs of wild-type mice based on immunohistochemistry and flow cytometry. In the steady state, DCs actively probed passing T cells with dendritic processes, but showed little translational movement and were found in extensive interdigitating networks within the T cell zones. These DCs networks were present throughout the T cell area, including around High endothelial venules (HEVs), such that T cells exiting HEVs would encounter the DC network rapidly. In addition, we noted that transferred mature DCs displayed translational movement as they dispersed throughout the networks of endogenous DCs10.
The outcome of antigen presentation by DCs depends on the state of DC differentiation or maturation11–16. In the steady state, immature DCs capture, process, and present to T cells a variety of self and environmental antigens including proteins from serum, commensal microbes, and dead cells17–22. Presentation of agonistic peptides on major histocompatibility complex (MHC) by steady state DCs tolerizes T cells and prevents autoreactivity when the same antigens are subsequently presented under immunizing conditions16,23–27. In contrast, DCs activated to mature by inflammatory stimuli, including infection, Toll-like receptor ligands, and CD40 ligation, induce strong effector T cell responses to the same antigens11–16,23,24,28–30.
Intravital two-photon microscopy was used to image T cell priming in lymph nodes7, and showed that T cells interact with fully mature, antigen-loaded, DCs in 3 phases. For the first 8 h, T cells transiently contacted DCs, between 8–20 h they established stable contacts, and finally resumed dynamic movement. In contrast, when tolerance was examined in explanted lymph nodes6, T cells did not establish prolonged contacts with antigen-loaded DCs. These differences in early T cell migration suggests that the stability of T cell-DC interactions determines commitment to tolerance or immunity6. However, non-specific control T cells were not simultaneously visualized, and experiments based on classical immunological methods indicate that the early stages of T cell-DC interactions are similar in tolerance and priming23,24,27,31,32 and do not commit T cells to either 33.
To examine tolerance and immunity in live mice, we developed a method for visualizing endogenous DCs simultaneously interacting with antigen-specific and non-specific CD4+ T cells. This allowed us to control for the variability in T cell movement within different sub-compartments of the lymph node. Here we report a novel antigen-specific stop signal that acts within an hour of T cells leaving the HEVs in both immunity and tolerance.
We sought to determine how widely divergent endpoints of tolerance and immunity relate to the physical interaction between DCs and CD4+ T cells during early stages of antigen recognition. To deliver antigen to DCs in vivo, we injected mice with α–DEC-OVA, a recombinant monoclonal antibody against DEC-205 engineered23 to carry an in-frame C-terminal peptide (amino acids 323–339) from ovalbumin. CD4+ OT-II T cells recognize this peptide when it is presented by I-Ab34. To compare conditions of tolerance to priming we used α-CD40 as an adjuvant. α–CD40 is the only single-component adjuvant that we have examined that induces long-lived effector T cells with a single injection30. α-CD40 induced upregulation of CD80, CD86 and MHC class II on lymph node CD11c+ DCs between 6 and 12 h after injection (Supplementary Fig. 1 on-line). Accordingly we timed α-CD40 injection such that upregulation of CD80, CD86 and MHC class II were maximal in the time frame of our imaging experiments.
In the steady state (no α–CD40) OT-II cells responded to α–DEC-OVA by up-regulating CD69 and down-regulating CD62L surface expression within 3 h of exposure to antigen (Fig. 1a). By day 3 there was extensive T cell division, but most of the responding cells were deleted by day 7 (Fig. 1b,c). Whereas OT-II cells re-isolated after 3 days remained antigen responsive, the few OT-II cells remaining after 7 days were unresponsive to re-stimulation in vitro and therefore tolerant (Fig. 1d). When α-CD40 was given in conjunction with α–DEC-OVA, similar expression of CD69 and CD62L was found and similar amounts of OT-II cell division were seen at day 3 (Fig. 1a). However, OT-II cells responding to antigen in the presence of α-CD40 adjuvant persisted at day 7 and showed enhanced proliferation in response to antigen in vitro, indicating T cell priming (Fig. 1b,c,d). Thus, antigen presentation by DCs targeted with α–DEC-OVA resembles previously described α–DEC targeted antigens in that the outcome of antigen presentation in the steady state differs dramatically from presentation of the same antigen under immunizing conditions23,24,27,30. Therefore, despite the difference in outcome, T cells responding to antigen presented by DCs under tolerizing and immunizing conditions are initially (day 3) indistinguishable in terms of cell surface activation antigen expression and ability to respond to antigen in vitro.
To determine whether T cells were differentially distributed in lymph nodes under conditions of tolerance and immunity, we examined the fate of EGFP-expressing antigen-specific (CD4+ OT-II T cells) and non-specific T cells (naïve non-TCR transgenic CD4+ T cells) in lymph nodes at 3 or 24 h after exposure to antigen. At 3 h post-transfer, antigen-specific T cells were found in close proximity to HEVs (average distance of 59 μm), and only 13% of them were in the deep paracortex (Fig. 2). In contrast, the non-specific T cells were an average of 78 μm from HEVs (p<0.0001), and were more evenly distributed throughout the T cell area, with 32% in the deep paracortex. By 24 h, both specific and non-specific cells were uniformly distributed throughout the T cell area with 43–45% present in the deep paracortex, and were located on average 90 μm from the center of the nearest HEV (Fig. 2). CD40 ligation did not alter the distribution of either the antigen-specific or non-specific T cells at either time point (Fig. 2). Similar experiments simultaneously visualizing enhanced cyan fluorescent protein-expressing (ECFP) OT-II cells and EGFP-expressing non-specific T cells showed identical results (data not shown). We conclude that T cells that recognize specific antigen are restricted to the area around HEVs in the early phases of tolerogenic and immunogenic responses.
To visualize interactions between DCs and T cells, we injected CD11c-EYFP mice10 with α-DEC-OVA with or without α-CD40 (Supplementary Fig. 2 on-line); five h later, we adoptively transferred ~107 antigen-specific EGFP-OT-II cells and an equal number of naïve control non-TCR transgenic CD4+ ECFP-T cells. In some experiments blood vessels were identified by injection of Q-dots (quantum dots coated with polyethylene glycol, Supplementary Video 1 on-line). All fluorescent cell types were simultaneously imaged in anaesthetized mice, at multiple depths (10 to 200 μm) including the outer paracortex near HEVs to the deep paracortical T cell zones up to 18 h after T cell transfer (Fig. 3b). T cells extravasated in the first hour after injection, and few extravasation events were observed during the imaging period (Supplementary Video 2 on-line). Although other groups described depth as a critical determinant of T cell motility7,35, we found little correlation between depth and cell speed (Supplementary Fig. 3 on-line).
EYFP+ DCs present in the imaging fields sensitively indicated the viability of the preparation. By simultaneously visualizing OVA-specific EGFP-OT-II cells, non-specific ECFP-T cells and EYFP DCs in each field, we could identify immobility caused by phototoxicity, suboptimal temperature, inadequate blood supply or surgical trauma. Fields in which T cells were globally arrested and DCs stopped probing were excluded from analysis (Supplementary Video 3 on-line). Cells were tracked in three dimensions over the course of each imaging experiment, and T cell motility was quantified with regard to velocity (Fig. 4, Supplementary Figs. 4 and and55 on-line), immobility (Fig. 5), and directionality (Fig. 6). Overall, 2100 cell tracks from 62 imaging fields in 29 mice were included in our final analysis.
We found that tolerance was associated with early T cell immobility followed by recovery over a period of 6–12 h. One to 6 h after injection with α-DEC-OVA (Figs. 3, ,4,4, Supplementary Figs. 4, ,55 on-line, Supplementary Video 4 on-line), antigen-specific EGFP-OT-II T cells moved significantly more slowly than non-specific ECFP-T cells (mean velocity, 4.41 vs. 9.41 μm/min, p< 0.0005, Fig. 4). A large proportion of the specific T cells were immobile (slower than 2 μm/min) in this interval (35% vs. 8.7% specific vs. non-specific T cells p<0.0001, Fig. 5a), and many sessile antigen-specific cells were found just outside HEVs (Fig. 2d, Supplementary Video 5 on-line). In addition to immobility we measured the arrest coefficient6, which is the proportion of time that a T cell is arrested (instantaneous velocity < 2 μm/min). Antigen-specific T cells spent more time arrested than did control cells (47% vs. 20%, p<0.0001, Fig. 5b). To determine the directionality of T cell movement, we measured the turning angle7 and confinement index6 (see Methods). Consistent with their relative immobility, we found that antigen-specific T cells moved less linearly than non-specific cells, as evidenced by a higher turning angle (82° vs. 61°, p<0.0001, Fig. 6a) and a lower confinement index (41% vs. 56%, p<0.0001, Fig. 6b). In the 6–12 h interval (Supplementary Video 6 on-line), the specific T cells remained significantly slower than the non-specific T cells (5.94 vs. 7.46 μm/min, p<0.0001, Fig. 4), but the specific T cells were moving faster than in the 1–6 h interval (5.94 vs. 4.41 μm/min, p<0.001, Fig. 4). Between 6 and 12 h, antigen-specific T cells were more immobile (12.2% vs. 6.0%, p<0.05, Fig. 5a), had larger arrest coefficients (32% vs. 13%, p<0.0001, Fig. 5b) and smaller confinement index (53% vs. 66%, p<0.0001, Fig. 6b) than the non-specific T cells. Finally, at 12–18 h (Supplementary Video 7 on-line), the antigen-specific T cells were still significantly slower (7.05 vs. 8.83 μm/min, p<0.0001, Fig. 4), more immobile (12% vs. 2.8%, p<0.002, Fig. 5a) and had higher arrest coefficients (32% vs. 22.3%, p<0.001, Fig. 5b) than control T cells, but differences in the turning angle and confinement index were not significant (Fig. 6). Two-way analysis of variance (ANOVA) showed that the speed difference between antigen-specific and non-specific cells was significantly greater at 1–6 h than at either 6–12 or 12–18 h (p<0.0001 for both), whereas the difference at 6–12 h was statistically indistinguishable from the difference at 12–18 h. This suggests that antigen-specific cells were regaining motility at 6–12 h. We conclude that T cells exposed to antigen under tolerizing conditions arrest near HEVs, and that mobility recovers significantly by 6–12 h after antigen exposure.
To determine whether the difference in T cell speeds reflects the induction of antigen-specific tolerance or inherent differences between EGFP-OT-II cells and ECFP-T cells, we examined the behavior of EGFP-OT-II cells under control conditions. Mice were injected with an irrelevant peptide targeted to DEC-205 (α-DEC-CSP) or with OVA peptide fused to an isotype-control antibody (Iso-OVA). Under both conditions, EGFP-OT-II cells moved at around 9 μm/min, a speed comparable to those of the non-specific ECFP-T cells in α-DEC-OVA treated mice (data not shown) and to those reported previously2–5,7,8. Thus, the arrest of EGFP-OT-II cells in mice treated with α-DEC-OVA was antigen-specific.
As in tolerance, immunity was associated with an early antigen-specific arrest of T cells, but recovery to normal movement was somewhat slower (Fig 4, Supplementary Figs. 4, ,55 online). In mice treated with α-DEC-OVA and α-CD40 (Supplementary Video 8 on-line), specific EGFP-OT-II T cells moved significantly slower than non-specific ECFP-T cells in the 1–6 h interval (mean velocity, 5.20 vs. 8.78 μm/min, p<0.0001, Fig. 4). In this interval the specific T cells were more immobile (31% vs. 9.6%, p<0.0001, Fig. 5a), had a higher arrest coefficient (40% vs. 21%, p<0.0001, Fig. 5b), a sharper turning angle (71% vs. 61%, p<0.05, Fig. 6a) and a smaller confinement index (41% vs. 49%, p<0.02, Fig. 6b) than non-specific T cells. In the 6–12 h interval (Supplementary Video 9 on-line), specific T cells remained slower than non-specific cells (4.35 vs. 8.05 μm/min, p<0.0001, Fig. 4), had a higher arrest coefficient (46% vs. 27%, p<0.0001, Fig. 5b), but were no longer significantly more immobile (Fig. 5a). They displayed a smaller confinement index (40% vs. 49%, p<0.02, Fig. 6b), but showed no significant difference in turning angle (Fig. 6a). Finally, in the 12–18 h interval (Supplementary Video 10 on-line), specific T cells moved slightly slower (6.08 vs. 7.54 μm/min, p<0.005, Fig. 4), and had slightly higher arrest coefficient (35% vs. 28%, p <0.05, Fig. 5b) than non-specific cells, but were otherwise indistinguishable. Two-way ANOVA showed that the speed difference between antigen-specific and non-specific cells was significantly smaller at 12–18 h than at 1–6 h (p<0.003) and 6–12 h (p<0.006), while the difference at 1–6 h was statistically indistinguishable from the difference at 6–12 h. We conclude that under conditions of immunity, specific cells arrest initially and then recover mobility significantly 12–18 h after antigen exposure. Occasionally, we observed discrete T-DC clusters forming around DCs (Supplementary Video 11 on-line), but T cells were not always in clear contact with CD11chi DCs (Supplementary Video 12 on-line), perhaps because not all DEC-205+ cells express high amounts of EYFP in CD11c-EYFP transgenic mice10.
Tolerance and immunity were similarly characterized by an early arrest followed by restoration of mobility. This was also seen when the speed of antigen-specific T cells was normalized to that of in-field non-specific control cells (Supplementary Fig. 5 on-line). Overall, there was little evidence for perturbation of control T cells, which maintained their mobility for at least 18 h. The most revealing parameter for comparison of specific cells under conditions of tolerance and immunity was the arrest coefficient6, a continuous parameter reflecting the percentage of time T cells were arrested (Fig. 5b). At 1–6 h the arrest coefficient for specific T cells was slightly higher under conditions of tolerance than immunity (47% vs. 40%, p<0.02). In contrast, at 6–12 the arrest coefficient was higher in immunity (32% vs. 46%, p<0.005). Finally, at 18 h the arrest coefficients were uniform (32% vs. 35%, n.s.). Over the entire 1–18 h period the average arrest coefficient for antigen-specific cells was similar for both conditions (~38%), as was the average cell speed (5.7 vs. 5.4 μm/min, n.s.). We conclude that while there were small differences in the dynamics of specific T cell DC interactions in tolerance and immunity, the trend of early arrest and slow recovery over 18 h was similar.
Our results show that OVA-specific T cells slowed down and exhibited confined motion upon encountering antigen, many of them arresting in the vicinity of HEVs. Meanwhile, the non-specific cells in the same field maintained their baseline speed and directionality. The early arrest near HEVs was identical in conditions of tolerance and priming, but antigen-specific T cells regained mobility more rapidly under conditions of tolerance than priming.
Intravital imaging of lymph nodes in mice injected with mature antigen-loaded DCs, showed that naïve T cells interacted with DCs in three stages: during the first 8 h, or phase 1, T cells interact with antigen-bearing DCs dynamically with no prolonged stopping, T cell immobility was seen between 8–20 h, or phase 2, and motility recovered thereafter in phase 37. Similar kinetics were observed in explanted lymph nodes when antigen was delivered by migrating mature DCs5. It was suggested that transient T-DC interactions in phase 1 changed the T cell activation state so that it could undergo stable interactions in phase 27,36. Although our finding that T cells stop shortly after leaving the HEVs was surprising in this light, it was independently supported by histological evidence showing preferential retention of antigen-specific T cells around HEVs, which indicated constrained movement. Our results agree with previous work showing histologically that immunization with complete Freund’s adjuvant resulted in early antigen-dependent retention of T cells near HEVs37. Furthermore, in vitro experiments show that T cells stop migrating immediately upon exposure to their cognate antigen38,39. Steady-state lymph node DCs express high amounts of MHC class II and intermediate amounts of CD80 and CD8610,40. Therefore, the rapid arrest we observe under tolerogenic conditions in vivo is not inconsistent with in vitro observations of poor immunological synapse formation in the absence of costimulation41,42. We conclude that a prolonged period of transient interactions is not necessary to form stable conjugates between T cells and DCs. Potential explanations for the differences in the length of phase 1 between ours and previous studies include their use of exogenously administered DCs that must migrate to the lymph node, and enter the T cell area before presenting antigen7. Another possibility is that T cells integrate signals during phase 1 encounters with DC. In this model, antigen presented by many DCs after DEC-205 targeting43,44 might produce an integrated signal of the same magnitude as that obtained by T cells interacting with much less frequent immigrant DCs for 8 h. While further experimentation is needed to test these possibilities, it is likely a priori that the number of antigen-positive DCs will vary depending upon how antigen reaches the lymph node. For example, soluble antigen rapidly drains to the lymph nodes and is widely accessible to resident lymph node DCs45. In addition, self-antigens from dead cells and serum proteins are continually picked up, processed and presented by large numbers of resident DCs in the steady state. Thus, it seems likely that under conditions of immunity or tolerance, T cells can encounter varying numbers of antigen-bearing DCs, and that the kinetics of the initial T cell-DC interaction will depend on the relative frequency of antigen-positive DCs.
Imaging of T cell priming in explanted lymph nodes showed antigen-specific T cell clustering around DCs in the deep paracortex3,6. We rarely observed formation of discrete T-DC clusters but this was expected, as large numbers of DEC-205+ DCs in the lymph node present antigen after targeting with the α-DEC-OVA antibody23,30, and the number of T cells in each cluster is inversely proportional to the number of antigen presenting DCs3. In our experiments, arrested antigen-specific T cells were not always in clear contact with CD11chi DCs, which is consistent with the partial overlap between EYFP and DEC-205 expression in CD11c-EYFP Tg mice10.
We found that the early stages of tolerance and immunity were similar in terms of T cell arrest near HEVs at 1–5 h followed by resumed movement and dispersion in the deep paracortex by 18 and 24 h, respectively. The only difference observed was that return to rapid migration was slower under conditions of priming than tolerance. Our imaging experiments are consistent with our finding that T cell activation markers are identical and proliferation and responses to antigen in vitro are similar in the first 3 days of priming and tolerance induction. These experiments are also in agreement with physiologic studies showing that T cells activated by soluble peptide in vivo are not committed to tolerance after 3 days31–33. Our results differ from an earlier two-photon study of tolerogenic T-DC interactions which used the same antigen delivery method (α–DEC-205-OVA)6. This study reported that CD8+ T cell arrest exclusively in priming and not in tolerance. However, these experiments were performed in explanted lymph nodes where DCs were labelled by direct lymph node injection of anti-CD11c, and non-specific T cells were not simultaneously imaged6. Importantly, our use of intravital microscopy preserves blood and lymph flow whereas these physiological processes were lacking in the explanted lymph nodes used previously6.
What is the significance of the prolonged T cell-DC interactions early in immune responses? Our experiments suggest that these conjugates are antigen-specific, but not associated with commitment to immunity or tolerance and independent of the state of DC maturation. We speculate that stable contacts between DCs and T cells are an integral part of initial T cell activation in vivo, and that commitment to immunity or tolerance occurs later as T cells dynamically contact DCs.
EYFP-CD11c mice, described previously10 express EYFP-Venus under the control of the CD11c promoter, and exhibit bright fluorescence specifically in dendritic cells. EGFP-OT-II transgenic mice were products of a cross between OT-II mice and mice ubiquitously expressing EGFP on the β-actin promoter46,47; ECFP-OT-II mice are a cross between mice expressing ECFP on the β-actin promoter from Jackson Labs48 and OT-II mice. All strains used are fully backcrossed to a C57Bl/6 background. Mice were used for experiments at the age of 4–6 weeks; all mice were housed under SPF conditions and treated in accordance with IACUC protocols of Rockefeller and New York universities.
Lymph nodes were fixed in PBS with 4% paraformaldehyde + 10% sucrose and cryoprotected in PBS + 30% sucrose before embedding in OCT and freezing. Frozen tissue was sectioned on a microtome and fixed in acetone. All incubations were performed in a humidified chamber. Sections were blocked in 5% BSA, 10% serum in PBS in the presence of α-CD16/CD32 (BD Biosciences), and sequentially blocked with excess streptavidin and biotin (Vector Labs). The antibodies used were: directly labeled α-CD3-Alexa 647, α-B220-Alexa 647 (CalTag), biotinylated α-rat IgM, and unconjugated MECA-79 (BD Biosciences). Streptavidin-Cy3 was from Jackson ImmunoResearch. Sections were mounted in Fluoromount-G (Southern Biotech) and stored at 4 °C.
Confocal images were acquired on a Zeiss LSM 510 system with 405, 488, 543, and 633 nm excitation lines; EGFP and ECFP fluorescence were visualized directly in all images. An array of 40x images was taken with a motorized stage to capture the entire cut surface of the lymph node, and images were exported into Adobe Photoshop for final processing. Positions of T cells, HEVs, and the deep paracortex were measured in Volocity software. Cells were scored as being in the deep paracortex only if the entire cell was within the boundary; cells touching the boundary were excluded. The minimum distance from T cells to HEVs were calculated as described elsewhere37 with the following equation: , where i and j are indices from 1…n for T cells and HEVs, respectively, and min is the minimum function. It should be noted that this yields the distance to the center of the HEV, which is greater than the distance to the basal side of the HEV endothelium.
α–DEC-OVA, and α-DEC-CSP (anti-DEC-205 fused to the irrelevant peptide SYVPSAEQI) were produced by replacing the HEL peptide-encoding sequence in α-DEC-HEL23 by molecular cloning. Iso-OVA was prepared as described previously23. Recombinant antibodies were produced by transient transfection and purified as previously described23.
CD11c-EYFP mice were injected i.p. with 2 μg of α-DEC-OVA, Iso-OVA or α-DEC-CLS either alone or together with α-CD40 (clone 1C10, 50 μg). Five h later mice were injected i.v. with 1–2 × 107 purified EGFP-OT-II cells and naïve CD4+ ECFP-T cells (>98% purity) prepared by magnetic depletion of non-CD4+ T cells (MACS CD4+ T cell Kit, Miltenyi Biotec). Lymph nodes were imaged 2–18 h after T cell transfer (Fig. 3a). To examine HEVs in vivo, we injected 10 μg of Alexa 594-labeled MECA-79 antibody i.v.
OT-II T cells (CD45.2) were labeled with CFSE, transferred into 4 to 6 week-old B6/SJL mice and challenged i.p. 24 h later with 2 μg of α-DEC-OVA, 2 μg Iso-OVA, PBS, or 2 μg α-DEC-OVA plus 90 μg α–CD40. T cells were purified from peripheral lymph nodes 3 d or 7 d later and either cultured with irradiated CD11c+ cells and OVA peptide to measure proliferation or analyzed by flow cytometry on a FACSCalibur (BD Pharmingen) to measure CFSE dye dilution (gated on Vα2 and CD45.2). To monitor early T cell activation, CD45.2 OT-II T cells were transferred into CD45.1 B6/SJL mice injected with α-DEC-OVA (with or without α-CD40) five h previously; T cells were purified from lymph nodes at 3, 12, or 24 h after T cell transfer and analyzed by flow cytometry to measure surface expression of CD69 and CD62L (gated on Vα2 and CD45.2). Antibodies were from BD Pharmingen.
Mice were anaesthetized with 100 mg ketamine, 15 mg xylazine and 2.5 mg acepromazine per kg, and kept anaesthetized with hourly injections of half this dose. Mice were restrained on a stage warmer at 37 ºC (BioTherm Micro S37, Biogenics) and the abdominal skin was incised from the edge of the rib cage through the midline to the thigh. A skin flap was separated from the abdominal muscle to expose the inguinal lymph node..
To stabilize, moisturize and maintain the lymph node at 37 ºC, we placed the skin flap on a thermoconductive base made of silicone elastomer (Sylgard 184, Dow Corning) surrounding a core of thermoconductive putty (T-putty 502, Thermagon). The connective and adipose tissue covering the lymph node was removed by microsurgery keeping blood and lymph vessels intact, and a saline filled chamber consisting of a cover-slip glued to a nylon washer was mounted on the skin flap over the lymph node. The chamber was laterally secured by 3 insect pins piercing the skin and lodged in the silicone base. The temperature was monitored using a probe (Bioptechs) placed near the lymph node to ensure that it was at 37±1 ºC. To compensate for anesthesia-induced respiratory depression, we provided mice with 100% oxygen by mask throughput the imaging session.
The lymph node was imaged using a Bio-Rad Radiance multiphoton microscope fitted with a Tsunami pulsed laser (Spectraphysics) and controlled by Lasersharp2000 software (Bio-Rad) We used a Nikon 40× objective (water immersed, NA 0.8). To image EYFP, EGFP and ECFP, the excitation wavelength was set to 910 nm. Band-pass filters optimized for detecting EYFP (550/30) and ECFP (510/30) and a 530 long-pass dichroic mirror were used to detect fluorescent proteins, with EGFP bright in both channels. Polyethylene glycol-coated quantum dots (Qtracker 655, Quantum Dot Corporation, Hayward, CA) were injected with the same excitation to visualize blood flow. To create time-lapse sequences, we typically scanned 50×400×400 μm volumes of tissue at 3 μm Z-steps and 30 second intervals.
Cell movement was analyzed with Volocity software (Improvision). To calculate cell speeds, the coordinates of each cell were calculated and tracked over time, and motility parameters were calculated. Average cell velocity was calculated as described previously10. Cells were characterized as immobile if their average velocity was less than 2 μm/min. The arrest coefficient was calculated as the percentage of each track in which the cell moved more slowly than 2 μm/min6. The turning angle is the absolute angle between the velocity vectors before and after a measurement time point7. The confinement index is calculated as the ratio of maximum displacement to path length6. Multivariate ANOVA was conducted to assess statistical significance, and planned contrasts were used to assess differences between individual conditions when appropriate. p values smaller than 0.05 were considered significant.
We thank R. Steinman for discussions, E. Besmer for help with the manuscript, W. Gan for guidance on two-photon microscopy, S. Boscardin for α-DEC-CSP and R. Masalimani for α-DEC-OVA plasmid. Supported by NIH grants AI055037 (M.L.D.) and AI051573 (M.C.N.), the Irene Diamond Foundation (M.L.D.), and the Howard Hughes Medical Institute (M.C.N.). G.S. was supported in part by a post-doctoral fellowship from the Rothschild Foundation; R.L.L. was supported by MSTP grant GM07739; D.D. was supported by the German Research Foundation DU 548/1-1.
Competing interests statement The authors declare that they have no competing financial interests.