Visualization of mucosal homeostasis viasingle- and multiphoton intravital

fluorescence microscopyCassie Xu,* Yuelei Shen,† Dan R. Littman,†,‡ Michael L. Dustin,‡ and Peter Velázquez*,‡,§,�,1

*Department of Microbiology and Immunology, Indiana University School of Medicine, South Bend, Indiana, USA; †HowardHughes Medical Institute, New York University School of Medicine, New York, New York, USA; ‡The Kimmel Center for

Biology and Medicine of the Skirball Institute, New York, New York, USA; and §Department of Biological Sciences and �EckInstitute for Global Health, University of Notre Dame, Notre Dame, Indiana, USA

RECEIVED JULY 11, 2011; REVISED FEBRUARY 15, 2012; ACCEPTED FEBRUARY 16, 2012. DOI: 10.1189/jlb.0711344

ABSTRACTFIVM has provided many insights into the regulation ofimmunity. We report the validation of an approach forvisualizing murine small bowel via single- and multipho-ton FIVM. Tissue damage is limited to �200 �m, imme-diately adjacent to the incision, as confirmed by intravi-tal PI staining. Treatment with 10 KDa dextran-FITC and70 KDa dextran-TR confirms that perfusion is intact.Selective filtration of 10 KDa but not 70 KDa dextranfrom the blood indicated that kidney function is also in-tact. Interestingly, lamina propria vasculature is semi-permeable to 10 KDa dextran. Next, reporter mice ex-pressing egfp from the CX3CR1 locus, egfp from theFoxP3 locus, or RFP from the IL-17F locus were used totrack DC subsets, FoxP3� Tregs, or Th17f cells, respec-tively. Resident cx3cr1�/egfp cells were sessile but ac-tively probed the surrounding microenvironment. Both Tcell populations patrol the lamina propria, but the Th17fcells migrate more rapidly than Tregs. Together, thesedata demonstrate intact vascular perfusion, while intra-vitally visualizing the mucosal surface of the smallbowel. Lastly, the cx3cr1� DCs and T cells display activ-ity similar to that found in steady-state, secondary lym-phoid organs. J. Leukoc. Biol. 92: 000–000; 2012.

IntroductionThe technology of FIVM has provided significant advances inour understanding of immunobiology. Much has been learnedabout the regulation of immunity in secondary lymphoid or-gans [1]. Fewer studies have examined orchestration and regu-lation of immunity at effector sites, and studies that have care-fully applied FIVM to understand regulation of immunity inthe intestine are limited [2–5].

The intestine is unique in that it hosts �1014 microbes whilemaintaining relative immune quiescence [6]. The enteric flora

has long been known to play an important role in the develop-ment of the gut-associated lymphoid tissue, driving peristalsis,protecting from infectious disease, and assisting in food diges-tion [7–10]. It is further unique in that it contains immuno-logic priming and effector sites, such as organized lymphoidfollicles and lamina propria, respectively. As FIVM studies haveprovided valuable insight into the microanatomic organizationand orchestration of immunity in LN and spleen, we aimed toestablish a system that allows for intravital examination of theintestine while maintaining tissue viability and vascular perfu-sion.

We report here application of single- and multiphoton FIVMas a technique to study the regulation of immunity in the in-testinal mucosa. Following surgical exposure of the mucosalsurface of murine distal ileum, the tissue is mechanically stabi-lized and imaged. Using a combination of vital dyes and ge-netic reporters, the integrity of epithelial cells and underlyinglamina propria cells was confirmed to be intact. Fluorescent-conjugated dextrans, 70 KDa and 10 KDa, were used to con-firm blood perfusion. These dyes were cleared from the vascu-lature with predicted kinetics, indicating perfusion was intact.Surprisingly, cx3cr1�/egfp DCs in lamina propria were sessiledespite the constant exposure of this tissue to enteric mi-crobes. However, they actively extend and retract dendrities, aprocess called probing. The sessile, while probing behavior, issurprisingly akin to DC activity in a steady-state, secondary lym-phoid organ. Th17 Teff and FoxP3� Treg were motile, similarto T cell migration, reported at other organ sites [11–14]. To-gether, these data validate the ability to stably visualize the in-testinal mucosal effector site while maintaining tissue healthand demonstrate DC and T cell behavior surprisingly akin tothat in secondary lymphoid organs at steady-state, despite thepresence of enteric microbes.

1. Correspondence: Dept. of Microbiology and Immunology, Indiana Univer-sity School of Medicine, 1234 Notre Dame Ave., South Bend, IN 46617,USA. E-mail: pvelazqu@iupui.edu

The online version of this paper, found at www.jleukbio.org, includessupplemental information.

Abbreviations: 2D/3D�two/three-dimensional, BAC�bacterial artificialchromosome, FIVM�fluorescence intravital microscopy, FoxP3�forkheadbox P3, mRFP�monomeric RFP, p4E16�RP23-4E16, RFP�red fluores-cence protein, Teff�effector T cell, TR�Texas red, Treg�regulatory T cell

Article

0741-5400/12/0092-0001 © Society for Leukocyte Biology Volume 92, September 2012 Journal of Leukocyte Biology 1

Epub ahead of print March 27, 2012 - doi:10.1189/jlb.0711344

Copyright 2012 by The Society for Leukocyte Biology.

MATERIALS AND METHODS

MiceWT and cx3cr1egfp/egfp C57BL/6 animal breeders were purchased from Jack-son Laboratory (Bar Harbor, ME, USA) and maintained at the FreimannLife Science Center at the University of Notre Dame (Notre Dame, IN,USA). In some experiments, cx3cr1egfp/egfp animals were derived and main-tained in the Skirball Institute for Biomolecular Medicine (New York, NY,USA) [15]. WT or cx3cr1�/egfp F1 animals, 8–12 weeks of age, were used inall experiments. FoxP3-GFP knock-in mice were kindly provided by Drs.Vijay Kuchroo and Mohamed Oukka and have been described [16]. Ani-mals were age- and sex-matched as appropriate. All experiments were con-ducted as approved by the Institutional Animal Care and Use Committee.

Generation of Th17f-RFP FoxP3-EGFP miceScreening of rBAC and resolved BAC constructs has been described [17,18]. Briefly, the IL-17f gene carrying BAC p4E16 was purchased from LifeTechnologies (Carlsbad, CA, USA). Overlapping PCR was performed toamplify mRFP (with stop codon) [19], flanked with IL-17f genomic se-quences. The overlapping PCR product was cloned into shuttle vector [18]and transformed into competent p4E16 cells. After integration and resolu-tion, the rBAC carrying IL-17f-mRFP was cut with NotI to remove the vectorbackbone. The linearized rBAC DNA was purified by pulse-field gel electro-phoresis and injected into fertilized C57BL/6 oocytes. DNA injection wasdone in the transgenic facility of the Memorial Sloan Kettering CancerCenter (New York, NY, USA), and pups were screened by PCR. BAC trans-genic mice (p4E16-mRFP) were continually bred with C57BL/6 mice (pur-chased from Taconic, Hudson, NY, USA) for colony expansion. Animalswere crossed to Foxp3-ires-eGFP mice. All mice were bred and used in ourspecific pathogen-free animal facility, according to the New York UniversitySchool of Medicine Institutional Animal Care and Use Committee (NewYork, NY, USA). See Supplemental Data for further details about the Th-17phenotype.

Surgery and anesthesiaAnimals were anesthetized and maintained under anesthesia, as describedpreviously [20]. Animals were anesthetized with a rodent cocktail of ket-amine (50 mg/Kg), xylazine (10 mg/Kg), and acepromazine (1.7 mg/Kg),injected into the peritoneum. Anesthesia was maintained during image ac-quisition with one-half dose s.c. boosting every 45 min for up to 2.5 h. Ani-mals can be maintained under anesthesia for up to 4 h. However, in someanimals, respiration is affected at later time-points (�3 h), as indicated bylabored, slowed, and deep breathing. For surgery, the anesthetized animalwas placed on a heating pad set at 37°C, and abdominal fur was trimmed.To access the peritoneal cavity, a 1- to 1.5-cm incision was made throughthe skin along the abdominal midline to expose the peritoneal wall. Anadditional incision was made through the peritoneal wall to expose the ab-dominal cavity. Next, a 3- to 4-cm loop of the distal ileum was externalized.A segment of the mucosal surface was exposed by making two partial trans-verse incisions along the gut wall, �0.5 cm apart. A longitudinal incisionwas next made to expose the mucosal surface, and blood loss was limitedby cauterizing at the edges of the incisions. The intestinal contents at theexposed area and up to 1 cm of the flanking region were removed withcotton swabs. Care is taken to avoid disrupting the mucosal layer. At nopoint was mesentery or mesenteric vasculature disrupted. Upon completionof surgery, the entire animal was then placed on an inverted microscopestage insert, with the mucosal surface of the intestine resting on the cover-slip, which itself has glued to it single, 1.5 � 1.5-cm nonmoisturized, 20-lbprinting paper with a 0.5 � 0.5-cm slit, on center, through which the intes-tine is aligned with the objective. Consequently, the externalized intestineis sandwiched between the paper glued to the coverslip and sterile gauzeplaced on the abdomen, upon which, the animal is now resting. Therefore,the entire exposed tissue is covered by the animal, which protects the tis-sue from dehydration. The animal is maintained at 37°C in a black-out en-

vironmental chamber with supplemental medical-grade oxygen supplied viaa nose cone.

Rodent chowAnimals were maintained on Harlan Teklad Irradiated Global 19% ProteinExtruded Rodent Diet 2919 (Indianapolis, IN, USA). Rodent chow did notcause any significant autofluorescence in the intestinal mucosa.

Intravital imagingAnimals were placed with the abdominal side down on a custom-madestage insert (Ludl Electronic Produces, Hawthorne, NY, USA), which con-tains a hole with coverslip (No. 1.5: 0.16–0.19 mm thick) to allow for ac-cess of an inverted objective. The stage insert with the animal is thenplaced on an inverted microscope stage of an Olympus FV1000 (CenterValley, PA, USA), equipped with a fempto-second-pulsed Ti:Sapphire laserwith dispersion compensation (Newport Mai Tai DeepSee HP, Irvine, CA,USA) and 25�/1.05NA XL PlanN objective with 37°C objective heater.The microscope is equipped with customized filters mounted on four ex-ternal nondescanned detectors. This microscope stage and head unit areenclosed in a blacked-out environmental chamber, which is maintained ata constant 37°C. In some studies, image acquisition was conducted on aZeiss LSM 510 confocal with Plan-Apochromat 20�/0.75NA objective(Zeiss, Thornwood, NY, USA). Animals were euthanized at the terminationof each experiment. For time-lapse image acquisition, 1 vol was collectedevery 30 s, with z-slices acquired every 5 �m. The maximum depth ac-quired over a 30-s interval by multiphoton was 75 �m with a 640 � 640pixel size and 2 �s dwell time. The maximum depth acquired with a single-photon light source was 20 �m. The appearance of labeled blood vessels orcx3cr1�/egfp cells indicated that the focal plane was past the epitheliumand just penetrating into the lamina propria. Therefore, the zero point wasdefined at 10 �m lower (on an inverted platform) than the plane contain-ing the first signals on blood vessels or cx3cr1�/egfp cells. An area of auto-fluorescence was observed 15–20 �m lower than the first signals of bloodvessels, which was interpreted as autofluorescence in the mucosal layer. Al-though autofluorescence was present, it was limited to the mucosal layerand did not pose a challenge to imaging epithelia or lampina propria. Thedepth routinely chosen for multiphoton image acquisition was 0–75 �m.Resolution at depths as much as 120 �m can be achieved with multiphotonexcitation.

Treatment with vital dyesImmediately prior to image acquisition, animals were retro-orbitally in-jected with fluorescently conjugated dyes, as indicated, in a 50- to 100-�lvol in saline at the following doses: 20 �g propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO, USA), 20 pmol Qtracker 655 (Life Technologies), 2mg 10 kD-FITC (TdB Consultancy, Uppsala, Sweden), 0.5 mg 70 kD TR(Life Technologies), and 0.25 mg Hoechst 33342 (Life Technologies).

Analysis of perfusion and permeabilityAll images were analyzed using Imaris software (Bitplane Scientific Soft-ware, South Windsor, CT, USA). Blood vessels were delineated by nonper-meable Qtracker 655. For image analysis at select time intervals, 10 vol ofinterest (5�5�1 �m) was chosen inside and outside, immediately adjacentto the blood vessel. The relative fluorescence intensities of each volume of10 KDa-FITC and 70 KDa-TR dextran were determined. A total of threemice was examined. Therefore, each time-point is represented by 30 mea-surements for each dye. To quantify perfusion, the intravascular relativefluorescence intensity of 10 kD-FITC and 70 kD-TR was plotted over time.Percent fluorescence was calculated by normalizing the relative fluores-cence intensity at 1 min postinjection. To quantify vascular permeability,the relative fluorescence intensities of each dye were calculated ratio-metri-cally and plotted as extra- versus intravascular, at 1 min postinjection.

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Adoptive transfer of primed CD4 T cells in smallintestineSplenocytes from C57BL/6 mice were sorted for CD4�CD62 ligand� cellsusing a MACS separator kit (Miltenyi Biotec, Auburn, CA, USA). The cellswere activated in 96-well polystyrene flat-bottom plates (Costar, Corning,NY, USA) in the presence of 3 �g/ml immobilized anti-CD3, 1 �g/ml solu-ble anti-CD28, 50 U/ml IL-2, and 1 �M retinoic acid for 4 days and thenallowed to rest in the presence of IL-2 only, for an additional 2 days. Subse-quently, the cells were labeled with 1 �M CFSE and retro-orbitally injectedinto syngeneic recipient C57BL/6 mice. Recipient animals were imagedintravitally using multiphoton microscopy at 1 day post-transfer.

Leukocyte migration analysisLeukocyte migration was quantified using Volocity (Improvision, nowPerkinElmer, Waltham, MA, USA) and Imaris (Bitplane Scientific Soft-ware). Each cell was tracked semiautomatically and confirmed visually dur-ing each frame collected. For each analysis, every cell is examined duringeach acquisition volume (once every 30 s).

StatisticsStatistical analysis was conducted using GraphPad Prism (GraphPad Soft-ware, La Jolla, CA, USA). All data sets were examined for Gaussian distribu-tion via a D’Agostino and Pearson normality test. For determination of sig-nificance of differences between two groups, a two-tailed nonparametrict-test (Gaussian) or Mann-Whitney test (non-Gaussian), as appropriate, witha 95% confidence interval, was conducted. Significance is defined as P �

0.05.

RESULTS AND DISCUSSION

The technology of multiphoton FIVM has provided many ex-citing and sometimes unexpected insights in immunology [1].The application of this technology to mucosal immunobiologyhas the potential to provide many novel insights into the fun-damental cellular and molecular regulation of homeostasis inthis unique organ. A major challenge to microscopically visual-izing internal organs via FIVM is to develop surgical proce-dures that minimize the impact on tissue health and move-

ment. This study demonstrates a careful evaluation of tissuehealth and viability, while overcoming the additional challengeof peristaltic movement. Additionally, we describe homeostaticleukocyte behavior, surprisingly similar to that found in steady-state, secondary lymphoid organs.

Following anesthesia, a distal loop of the small bowel is ex-ternalized by making an incision along the midline to mini-mize rupturing vasculature along the parietal peritoneum. Aloop of the distal ileum is then externalized and placed onsterile gauze, which is resting on the abdomen of the animal.This prevents contact of the intestinal wall with abdominal fur.Following externalization of the distal ileum, an incision wasmade along the intestinal wall to access the mucosal surfacefor visualization. The incision was cauterized to prevent bloodloss (Fig. 1). The entire animal was then placed on an in-verted microscope stage insert as described in Materials andMethods.

Visualization on inverted and upright microscope platformswas attempted, but maximal success with mechanical tissuestabilization was observed on an inverted platform. We believethe combination of the weight of the animal and tactile resis-tance provided by paper and gauze are what provide for imagestability. Nonetheless, there is variability of peristaltic move-ment along the intestine and even within a viewing field.

We varied on the use of confocal (single-photon) versusmultiphoton application in this study as a result of availabilityof the technology. Early in the study, we aimed to establishedparameters for acquiring stable images. As described above,minimizing peristaltic movement, while visualizing the intesti-nal mucosa, was best achieved on an inverted microscopystage. However, in the early phase of this study, a multiphotonlight source was not available on an inverted stage. Although,we later re-examined T cell trafficking of endogenous Th17-RFP animals (described below), the signal-to-noise ratio wasnot sufficient to visualize these cells via multiphoton.

Mucosa

Mesenteric blood vessel

Cauterized wall

Figure 1. Schematic of surgery. A 1-cm cut is made along the abdomi-nal midline (dashed line) of anesthetized animals following hair re-moval. A loop of the distal small bowel is then externalized. A small,0.5-cm incision is made along the intestinal wall, and luminal contentsare removed carefully to expose the mucosal surface (inset, with dots).Bleeding is stopped by cauterizing the incision (inset, bold lines).

Figure 2. Tissue damage limited to incision region. Animals weretreated i.v. with 20 �g PI (yellow/orange) in saline, while intravitallyimaging distal ileum via multiphoton. In some experiment, animalswere coinjected with 250 �g Hoechst 33342 (green; n�4).

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To evaluate the effect of surgery on tissue viability, animalswere treated with 20 �g PI, and 250 �g Hoechst counterstainvia i.v. injection. PI-positive cells were limited to �200 �mfrom the incision (Fig. 2). Therefore, cell death as a result ofthe surgical technique was limited to the region immediatelyadjacent to the incision. For all subsequent experiments, im-ages were acquired in regions of normal tissue health.

To evaluate tissue perfusion, 10 KDa-FITC and 70 KDa-TRwere injected i.v. into WT C57BL/6 mice while imaging. Bothdyes were observed in the lamina propria blood vessels in �30s, indicating that perfusion is intact (Fig. 3 and SupplementalVideo 1). The loss of intravascular fluorescence intensity overtime was more readily apparent with 10 KDa-FITC than 70KDa-TR dextran (Fig. 3A). Quantitative analysis of intravascu-lar fluorescence intensity revealed that �50% of the intravas-cular 10 KDa-FITC dextran is lost within 10 min. Conversely,�90% of the intravascular 70 -Da dextran is retained as late as20 min postinjection (Fig. 3B). These finding are consistentwith the observation that kidneys filter 3–10 KDa dextranswithin minutes, whereas larger molecular weight dextrans,

�40 KDa, persist for an extended period in blood vessels [21].The presence of dextrans in the bladder was confirmedgrossly. Therefore, whereas it is likely that anesthesia has ef-fects on the cardiovascular system, the intestinal mucosa andkidneys remain perfused using this surgical procedure.

To examine vascular permeability, a ratiometric analysis ofextra- versus intravascular dextran was conducted. At 1 minpostinjection, the relative fluorescence of 10 KDa dextran out-side of the blood vessel is almost equal to that inside of theblood vessel, with a ratio of 0.80 � 0.02 (Fig. 3C). Conversely,70 KDa dextran is mostly intravascular, with a ratio of 0.18 �0.02 (Fig. 3C). Therefore, 10 KDa dextran is 4.4-fold morepermeable than 70 KDa dextran in distal ileum. This tech-nique allows for the intravital analysis of the mucosal vascularintegrity in almost real time. Such measurements are impor-tant for target validation and delivery of therapeutics aimed attargeting the intestine.

Animals expressing EGFP under control of a single allele ofthe endogenous cx3cr1 promoter, cx3cr1�/egfp, allow for thevisualization of DCs and were examined by IVM [15, 22]. Im-

Figure 3. Perfusion and vascular permeability.(A) The lamina propria of distal ileum was visu-alized via multiphoton, immediately followingi.v. coinjection of 2 mg 10 kDa dextran-FITC(green) and 0.5 mg 70 kDa dextran-TR (or-ange) in saline. Time-lapse images were ac-quired every 30 s for up to 20 min following

treatment of dextran dyes (Supplemental Video 1). (B) Quantitative kinetic analysis of the relative intravascular fluorescence up to 20 min, nor-malized to total fluorescence of each dye at 1 min postinjection. (C) Ratiometric analysis of relative fluorescence intensity of extra- versus intravas-cular dextran at 1 min postinjection; n � 3.

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mediately prior to image acquisition, animals were i.v.-treatedwith 10 KDa dextran-TR to ensure perfusion. Analysis of DCactivity in lamina propria reveals a sessile but probing activityof endogenous CX3CR1 cells (Fig. 4 and Supplemental Video2). Interestingly, this activity is akin to that of DCs in second-ary lymphoid organs [11]. cx3cr1�/egfp cell migration was notobserved in a total of three animals, six villi/animal, and10–40 cells/villus, examined over a period of 20 min each.Although the current paradigm is that a subset of cx3cr1�/egfp

DCs sample mucosal antigen and traffic to the mesenteric LNto present antigen to T cells [2, 23, 24], the relative frequencyof this behavior may be rare during homeostasis. Previousstudies have demonstrated that cx3cr1�/egfp cells include a sub-set of DCs, which reach through epithelial cell tight junctionsand play an important role in protection from salmonella[22]. Whereas these cells may play a sentinel role, their mobi-lization may require a pathogenic signal, such as that derivedfrom Salmonella. The probing action of cx3cr1�/egfp cells indi-cated that the relative lack of cell migration in this study is notsecondary to a lack of cellular metabolic activity.

To further ensure that the lack of cx3cr1�/egfp cell migrationwas not a result of a lack of tissue health, endogenous T cell

migration was examined. Migration of Teffs and Tregs in tis-sue is a highly controlled process, dependent on vascular per-fusion, gas exchange (normoxia), and temperature regulation[12, 13, 25]. We examined the behavior of endogenous Th17Teffs and FoxP3 Tregs in vivo via intravital confocal laser-scan-ning fluorescence microscopy, using a BAC transgenic-express-ing RFP under the IL-17f promoter (Th17-RFP), crossed totransgenic animals expressing EGFP under the FoxP3 pro-moter (FoxP3-EGFP). Visualization of Th17-RFP required con-focal (single-photon) microscopy as a result of a relativelyweak signal of RFP in Th17 cells. We carefully discriminatedbetween autofluorescence and Th17f-RFP cells by excludingautofluorescent signals found in multiple detectors (Fig. 5Aand B, and Supplemental Video 3). FoxP3� T cells (green)can be seen migrating in situ, in and out of the focal plane,and can be readily identified by dynamic, morphologicchanges, as they traffic in villous lamina propria. Th17f cells(red) can also be identified as a result of their dynamic behav-ior, including a discernable leading edge, and they migrate insitu (Fig. 5A and B, and Supplemental Video 3). Th17f T cellsmigrated at a mean speed of 7 �m/min, whereas FoxP3 Tcells migrated at a mean speed of 5 �m/min; P � 0.004 (Fig.

2 min 5 min 12 min

50 um

Figure 4. Monocyte and DC activity inlamina propria. cx3cr1�/egfp mice were im-aged via intravital laser-scanning confocalmicroscopy over a 20-min period, with aframe rate of 1/30 s. Each mucosal villusis abundant with DCs, which are extend-ing and retracting dendrites, but remainsessile over time (highlighted by whitecircles); n � 3.

C

Th17f FoxP30

5

10

15p=0.004

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Figure 5. Migration of endogenous T cells in intestinal mucosa. (A) Th17f Teff (red) and FoxP3 Treg(green) in lamina propria were visualized intravitally via laser-scanning confocal microscopy. (B) Insetof A. Th17f Teff (arrows) and FoxP3 Tregs (arrowheads). Original scale bars � 100 �m; n � 3. (C)FoxP Tregs (open circles) migrate slower than Th17f Teffs (closed circles); P � 0.004. Each dot repre-sents the average 2D speed of an individual cell. (D) FoxP cells (open squares) show greater arrest co-efficient than Th17f cells (closed squares); P � 0.01. (E) No difference is observed in meandering be-tween Th17f (closed triangles) and FoxP3 cells (open triangles); P � 0.15. The horizontal line on eachgraph indicates the mean of all of the cells depicted, which are pooled together from three mice.

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5B). The slower speed of FoxP3 T cells compared with Th17 Tcells was a result of a significant increase in arrest coefficient,31% and 19%, respectively; P � 0.01 (Fig. 5C). These differ-ences were not a result of global variations in T cell migrationof each subset, as no differences were seen in meandering in-dex; P � 0.15 (Fig. 5E). The mean speed of Th17 Teffs isslightly slower to that of CD4 T cells reported previously insitu [11–14]. Nonetheless, the combination of perfusion andthe dynamic behavior of T cells and DCs together indicatethat cell and tissue health are intact in this system. The signifi-cance of the distinct behavior of Teff and Treg subsets duringhomeostasis is unclear. However, the increased arrest of FoxPT cells suggests that Treg interactions with resident DCsand/or other cells are more prolonged compared with that ofTeffs. These data further suggest that Th17 Teffs survey moretissue/unit time and therefore, more APCs than FoxP T cellsduring homeostasis.

As the studies with endogenously primed T cells were lim-ited to confocal microscopy, it is possible that the slower speedobserved was a result of the lack of 3D resolution. Therefore,we used an adoptive transfer system, where naïve donor T cellswere differentiated into gut homing T cells, loaded with aCFSE tracer, and visualized in recipient animals via multipho-ton FIVM (Fig. 6, Supplemental Video 4). A comparative anal-ysis of 2D versus 3D migratory behavior revealed that 2D analy-sis shows a slower speed compared with 3D analysis (10 �m/min vs. 12 �m/min, respectively) of the same cells (Fig. 6B).Cells that move along the z-plane are not quantified in a 2Danalysis, and thus, speeds are likely biased compared with 3Danalysis. No significant difference in arrest coefficient or me-andering index was observed (Fig. 6C and D).

Together, these data validate procedures to quantitativelyevaluate mucosal perfusion, vascular permeability, and leuko-cyte behavior at single-cell resolution in vivo and provide animportant tool for more closely examining the role of DC mi-gration in maintaining homeostasis in the gastrointestinaltract. This system also provides an important tool for under-standing the microanatomic locale of T cell-DC interactionsand the genetic requirements for T cell-APC interactions anddefining the relative contribution of APC subsets in spatial-temporal regulation of T cell activation in vivo.

AUTHORSHIP

P.V. and M.L.D. designed experiments. Y.S. and D.R.L. de-signed and developed BAC transgenic animals. C.X. and P.V.conducted experiments and prepared the manuscript.

ACKNOWLEDGMENTS

This work was supported in part by National Institute of Dia-betes and Digestive and Kidney Diseases DK078153 (P.V.), In-diana University School of Medicine-South Bend (P.V.), andNIH R01055037 (M.L.D.). We thank M. Oukka and V.Kuchroo for the FoxP3-ires-eGFP mice. We also thank SkirballAnimal Facilities at New York University Langone School ofMedicine and Freimann Life Science Center at University ofNotre Dame.

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Figure 6. Migration of primed Tcells. (A) In vitro-primed CD4 cellswere adoptively transferred into syn-geneic WT C57BL/6 mice and visu-alized intravitally via multiphotonmicroscopy. Qtracker 655 (LifeTechnologies) was inject i.v. tohighlight vasculature (red). CFSE-loaded T cells (green) were tracked(white line) for a 15-min time pe-riod at 16 h post-transfer. Originalscale bar � 50 �m; n � 3. (B–D) T

cell migration was quantified using a 3D volume and 2D slice from a total of three animals, and data were pooled. Each symbol represents an in-dividual cell, and the horizontal line on each graph indicates the mean. (B) Average speed of T cells from a 2D image (open circles) was slowerthan as the same cells analyzed in 3D (12 �m/min and 10 �m/min, respectively; P�0.03). (C) No significant difference was seen in arrest coeffi-cient in 3D (3 �m/min) versus 2D (10 �m/min). (D) No significant difference in meandering index was observed in 3D versus 2D analysis (0.48vs. 0.44, respectively).

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KEY WORDS:T cells � dendritic cells � intestine

Xu et al. Visualizing intestine via intravital microscopy

www.jleukbio.org Volume 92, September 2012 Journal of Leukocyte Biology 7