Mice

C57BL/6J (B6) mice were purchased from Jackson Labs (Bar Harbor, ME) and used from 8–12 weeks of age. A colony of the OT-I transgenic mouse strain that expresses a TCR specific for the OVA SIINFEKL (OVA257–264) peptide presented in the context of H-2Kb (14) was crossed with a transgenic mouse expressing GFP under a chicken beta-actin promoter [23]. Both lines of mice were maintained at the University of Rochester Vivarium. All animals were housed in the University of Rochester Vivarium facilities under specific pathogen-free conditions using microisolator technology.

Ethics statement

All experimental protocols were performed in accordance with the standards established by the United States Animal Welfare Act, as set forth by the National Institutes of Health guidelines. The experimental protocols were reviewed and approved by the University Committee for Animal Resources, number 101431-UCAR-2006-029R, and conducted in Association for Assessment and Accreditation of Laboratory Animal Care International accredited facilities.

Influenza viruses and infection

The influenza H3N2 A/Hong Kong/X31 (X31) virus, and A/X31-OVA-I influenza virus that expresses the ovalbumin (OVA) 257–264 (siinfekl) peptide in the hemagglutinin viral protein [24] were grown and titered in embryonated chicken eggs and harvested as allantoic fluid preparations (Allan et al., 1990). Mice were sedated with avertin (2,2,2-tribromoethanol) prior to intranasal (i.n.) challenge with 10⁵ EID₅₀ of X31 or 3 x 10³ EID₅₀ X31-OVA-I in 30 μl of PBS.

Cell Isolation and flow cytometry

Following cardiac perfusion of the mice with PBS, the trachea was canulated to just above the tracheal carina (first bifurcation of the airways) and bronchoalveolar lavage cells were collected by lavage with 1 ml HBSS three times. These were then resuspended in complete (+10% fetal bovine serum) minimal essential medium (cMEM) (Gibco, Grand Island, NY). Single-cell suspensions were prepared from spleen and lymph nodes by disruption in cMEM by Dounce homogenization, washed, and resuspended in cMEM. Trachea and lung were collected and disrupted using a tissue chopper, then digested using Collagenase Type II (Worthington). Lymphocytes were isolated on a Percoll (Sigma, St. Louis, MO) gradient. Viable cell counts were obtained by trypan blue exclusion.

Cells collected from trachea were pooled in like groups (3–5 trachea per group) and stained with various combinations of mAbs to CD8a (53–6.7), CD4 (RM4-5), CD49a (Ha31/8), CD3 (145-2C11), Live/Dead Aqua (Invitrogen), and CD19 (1D3). The conjugated mAbs were purchased from BD Pharmingen, eBioscience or BioLegend and are referenced in their current catalogs. Tetrameric complexes of H-2Db/influenza polymerase (PA)224–233 (DbPA), and H-2Db/influenza nucleoprotein (NP)366–374 (DbNP), were prepared by the NIH Tetramer Facility (Atlanta, GA). Data was collected using an 18-color LSR-II, and analyzed using FlowJo software (Tree Star).

Tissue staining and preparation

To obtain tissue for immunohistology, mice were infected intranasally with HK/X31. At time of harvest, mice were injected with Avertin and placed in a surgical plane. The renal artery was severed and the mouse was euthanized via exsanguination. The chest cavity was opened and the lungs were perfused with PBS. The trachea was exposed and a canula was inserted and fixed in place with a suture. Using a 1ml syringe, 0.8ml of warmed OCT was instilled to inflate the lungs, and a suture was placed at the base of the trachea to maintain the inflation. The lungs were excised and placed in OCT, then immediately frozen by floating on top of liquid Nitrogen. The trachea was also excised, inserted into OCT and frozen as for the lungs. Tissues were wrapped in aluminum foil and stored in a -80°C freezer.

Sections from 5–10 μm were cut transversally using a Shandon LSE Cryotome Cryostat. They were mounted on pre-cleaned superfrost plus slides from VWR. Slides were immediately placed in ice-cold acetone for 10 minutes for fixation, removed and allowed to dry for 10 minutes. They were then placed in a slide box, wrapped in aluminum foil, and stored at -80°C until ready for staining.

For immunofluorescent staining, sections were removed from -80°C and thawed on ice for 10 minutes. They were then placed at room temperature and allowed to dry for 10 minutes. Residual OCT was removed and rehydration of tissues was accomplished by incubating slides for 10 minutes with 1ml of PBS. The Fc receptor was blocked with PBS-Tween20 (0.05%) containing anti-CD16/32 (BD Pharmingen 1:200), normal rat serum and normal goat serum (Jackson, 1:100). Sections were incubated for 15 minutes at room temperature in a humidified chamber. From this point, all washing and staining was done using PBS-Tween20.

Sections were washed for 5 minutes and stained with the following antibodies: Alexa Fluor 647 labeled rat anti-mouse CD8a (BioLegend; 1:200), FITC labeled Monotope–Influenza A Virus (Virostat 1:100) and unlabeled goat anti-type IV collagen (Southern Biotechnology Associates; 1:400) for 60 min. Sections were then washed and incubated with a secondary donkey anti-goat Cy-3 for 30 min. The sections were then washed and mounted using a cover slip and DAPI Fluoromount-G (Southern Biotech). Fluorescence microscopy was performed at 20X using a Nikon Eclipse TE2000E fluorescence microscope equipped with an X-Cite Series 120 fluorescence Illuminator and a Cool-SNAP HQ charge-coupled device camera (Roper Scientific) with NIS-Elements software. All imaging filters were from Chroma Technology Corp. (Bellows Falls, VT).

For whole mount, intact tracheas were excised from infected mice. The tracheas were cut in half longitudinally and placed in PBS/BSA 1% (PBA). Fc receptors were blocked with CD16/32 (BD Pharmingen, 1:100). Primary antibodies were added in various combinations in excess (1:50): CD31, CD8, CD19, CD4 PE, Class II, ICAM, LYVE-1, and CD45 conjugated to FITC, PE, Alexa Fluor 647, Cy3, PerCP and APC. The conjugated mAbs were purchased from BD Pharmingen, eBioscience or BioLegend and are referenced in their catalogs. Tissue sections were incubated in a cold room at 4°C, on a rocker, for 4 hours. They were washed with 4 ml of PBA for 5 minutes and then mounted on a clean glass slide in 100 μl of PBA under a cover slip.

Imaging was performed with an Olympus BX40 with fluorescent attachment, a Retiga 1300 Monochrome cooled 12 bit camera from Q-Imaging systems, and Image Pro Plus Version 5.0 from Media Cybernetics.

Multiphoton imaging

Imaging was performed using an Olympus FV1000AOM-Multiphoton imaging system in combination with a Spectra-Physics MaiTai-HP Deep See fs Ti:Sa laser system. Fluorescence was collected with a water immersion objective Olympus XLMUMPlanFI 25x NA1.05 for high-resolution imaging or a Zeiss C-Apochromat 10x NA0.45 water immersion objective for imaging large fields of view (~1200x1200um2). Fluorescence was separated from the excitation light using a dichroic mirror / blocking filter combination (Dichroic Olympus 650LP; Blocking Filters FF01-680/SP, Semrock, Rochester, NY). The fluorescence was divided into two channels using an appropriate dichroic mirror (505DCXRU, Chroma). The laser-power and the laser-pulse-width were continuously monitored using a Pulse-Scout-Autocorrelator (Spectra-Physics) and a wavelength-calibrated laser-power power meter (1918-C. Spectra-Physics). All imaging data was taken at 12bit-depth and analyzed and visualized using Volocity (Perkin Elmer) and Imaris (Bitplane) software, respectively.

Cell transfer and infection

A line of GFP+/OT-1+ animals was maintained and used as donors for adoptive transfer of C57BL/6 naïve animals prior to infection. Whole splenocytes were isolated, and 1x10⁶ cells contained in 200ul of DPBS were transferred via tail vein. 24–48 hours post transfer, the animals were sedated with 120 mg/kg of Avertin. Once animals were sedated and exhibited no pedal reflex, 30 μl of X31-OVA-1 (2.7x10³ EID₅₀/ml) was administered intra-nasally. Animals were monitored until fully recovered from anesthesia, and then daily for weight-loss.

Sedation and Anti-convulsives

Prior to surgery, the animal was anesthetized with pentobarbital (65 mg/kg). During imaging the level of anesthesia was maintained with isofluorane, administered at 0.5–2% as necessary based on heart rate. Pancuronium bromide (0.4mg/kg) was administered prior to imaging, to prevent movement of the imaging area.

Surgical set-up

The hair was removed with a shaver from one hind leg, thigh to groin, exposing the skin for the MouseOX Plus sensor (Starr Life Sciences). The hair was removed from the thoracic area with scissors and/or a shaver. The animal was placed in a supine position, on a warming blanket, once surgical plane of anesthesia was determined by lack of both pedal and palpebral reflex. The coat was opened from below the chin to the top of the ribcage. The salivary glands were separated to reveal the muscles covering the trachea. Using round forceps, the muscles were separated to expose the trachea. The forceps were then inserted beneath the trachea to lift and separate it from the muscle and mouse body. A small flexible plastic support was placed in the space created by the forceps to permanently hold the trachea above and separated from the muscle, surrounding tissue and coat.

The animal was moved to the previously warmed stage. A small incision was made between the cartilage rings below the larynx. The steel cannula was inserted into the opening in the trachea until it reached just below the sternum. The cannula provided physical immobilization of the trachea. The cannula was secured on the stage with a support that holds it in position so it is correctly aligned with the trachea. It was held in place with 2 screws that prevent it from moving in transport, or during the attachment of the respirator. The forepaws were secured to the stage with surgical tape to maintain position of the mouse body on the stage. A few drops of saline were placed on the exposed tracheal tissue to prevent drying.

Maintaining blood O₂ saturation and temperature

The cannula was quickly attached to the Harvard Inspira ASV ventilator, and both 100% O₂ and 0.5% isofluorane flow was started, according to mouse weight. The MouseOX Plus thigh sensor was attached to the exposed thigh and monitoring was started immediately. Oxygenation levels were maintained at >95% and heart rate ranged between 250 and 600 beats per minute. The rectal body-temperature was continuously monitored and maintained using a small animal temperature controller that is connected to a rectal probe and a feedback-regulated rodent heating pad. After achieving stable physiology, and verification of lack of both pedal and palpebral reflex, the pancuronium bromide (0.4 mg/kg) was administered, based on body weight. The saline covering the exposed trachea was blotted away and replaced with 0.05% agarose to seal the exposed area. Once the agarose was solidified, a support ring was placed over the imaging area and covered with a piece of plastic wrap. Approximately 5 ml of water was pooled over the imaging area to submerse the objective.

OVA peptide-MHC (Kb/siinfekl) blocking studies

Prior to the start of surgery, 100μg of anti-H2 Kb/ siinfekl (25-D1.16) antibody [25] was injected via tail vein. Animals were then prepared for imaging, which took place either one or two hours after administering the antibody. For the imaging, the animals were maintained either at 37°C or 35°C as described in the results.

Kinetics of influenza virus replication in the trachea

The well-established mouse model of non-lethal influenza infection has defined kinetics of viral replication in the lung, with clearance of the virus by 9 to 10 days after infection [26, 27], while T cell infiltration of the airways sampled by broncho-alveolar lavage (BAL) becomes easily detectable by day 6 [26, 28]. For the trachea, our initial goal was to determine if the kinetics of the virus and cellular immune responses were comparable to the BAL or lung tissue. To establish a baseline, mice were infected with a standard laboratory strain of H3N2 influenza A virus (HKx31) using an intranasal route of administration to sedated animals. Each mouse received 10⁵ EID₅₀ of virus diluted in 30μl PBS [27, 29]. To determine the localization of infected cells and lymphocyte infiltrates, frozen sections were prepared for immunohistology and stained for CD4 and CD8 T cells, collagen IV, and influenza nucleoprotein (NP). On day 2 in mice infected with HKx31, the cells of the epithelium were uniformly positive for NP, with few T cells visible (Fig 1A). At day 8, the epithelium now demonstrated substantial T cell infiltrates in and below the epithelial surface (Fig 1B). This data suggests that HKx31 virus replicates efficiently in the tracheal epithelium.

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Fig 1. Influenza infection of the mouse trachea.

C57BL/6 mice at 8 weeks of age were infected with 10⁵ EID₅₀ of H3N2 influenza A/HK/X31 by an intranasal route. On days 2 and 8 the trachea was excised, snap frozen, and later sectioned and stained with antibodies to influenza nucleoprotein (NP, blue), collagen IV (Col IV, green), and CD8 (red); A = day 2, B = day 8. Separate cohorts of mice (n = 3–4) were similarly infected and virus titers in the trachea and lung were determined after homogenization of the tissues followed by dilution and inoculation of embryonated hen eggs (Methods). Titers of influenza virus in lung and trachea expressed as EID₅₀ per ml of tissue homogenate (C) or per gram of tissue weighed prior to homogenization in 1 ml serum-free media (D). Closed circles = lung, open circles = trachea.

https://doi.org/10.1371/journal.ppat.1005881.g001

To determine the kinetics of virus replication and production of infectious virus, the whole lung and a section of trachea (~3mm) were individually dissected from the animals, weighed, and snap frozen. When homogenized in an equal volume of media, the concentration of recovered virus in the trachea was lower than the lung at all time points, but followed a similar time course (Fig 1C). However, the amount of virus being produced per gram of tissue was equivalent to the lung (Fig 1D), despite the larger epithelial surface area. In the experiment shown, infectious virus was cleared from the trachea by day 9, and only one of three mice cleared virus from the lung by day 10, though the residual amount (~100 EID₅₀) was low. Overall the kinetics of HKx31 are similar in trachea and lung tissue.

Host cellular immune response in the trachea compared to lung tissue and airway

Since the virus replication and clearance kinetics were similar in lung and trachea, we wanted to also compare T cell responses in the trachea to the airways sampled by BAL, and to lung tissue. Although the total cell numbers were small given the size of the tissue section (~10mg) and relative surface areas being sampled, CD4+ and CD8+, CD3+ T cells were easily detectable by flow cytometry on day 8 (Fig 2A, 2C and 2D), including Db/NP (8–11% of CD8+) and Db/PA (7–17% of CD8+) tetramer positive cells (Fig 2B and 2E). The trachea also contained higher proportions of CD19+ cells (5–10%) than the BAL (>1%), but less than the lung (~25%). Total CD3+ cells peaked 8 days after infection, and then declined. The peak of the response was less sharp than in the BAL or lung tissue (Fig 2), though it should be noted that the difference in cell numbers in the trachea from days 6, 8 or 10 are statistically insignificant, reflecting variability in cell recovery and a smaller denominator in terms of total cell counts. Collectively, these data show that the adaptive cellular immune response to influenza infection in the trachea is robust, with a rise and fall that corresponds with clearance of the virus as it does in the BAL and lung tissue.

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Fig 2. Cellular composition of influenza infected trachea, lung, and BAL.

Mice were infected with influenza (as in Fig 1) and tissues harvested on different days after infection. Lung tissue was taken after broncho-alveolar lavage and perfusion of the blood vessels. Experiments consisted of pooled groups of 3 mice per time point, and 3–4 experiments per time point. Lung and trachea were enzyme digested and gently homogenized to make a single cell suspension and counted. Cells were stained with a panel of antibodies against T- and B-cell markers and MHC-Class I tetramers corresponding to immunodominant CD8+ T cell epitopes and analyzed by flow cytometry (Methods). Trachea = A-E, BAL = F-J, Lung = K-O. Each point represents the average of 3–4 experiments ± standard error of the mean.

https://doi.org/10.1371/journal.ppat.1005881.g002

The trachea imaging site

Having established that the trachea is a suitable site of infection with HKx31 and X31-OVA, we proceeded to develop it as a site for imaging. To give some perspective on where in the tissue the imaging is performed, the trachea was surgically exposed in a live animal, showing it has a translucent appearance between the cartilage rings (Fig 3A). Imaging was performed on the tissue between the rings. For further perspective, explanted trachea was split longitudinally, opened and placed between a cover slip and glass slide for whole mount imaging. The tissue was stained for CD4 (blue) and CD8 (red) to image the T cells, and collagen IV (green) to visualize the lamina densa of the basement membrane, just underlying the epithelial surface (Fig 3B). Next, because of its ability to optically penetrate intact tissues to a greater depth, we used multiphoton imaging of the infected trachea to further reveal structural features and T cell localization. Dextran (red) was injected intravenously to highlight the blood vessels. Peering through the tissue, the blood vessels, T cells (green) and outer collagen fibers (white) can be visualized (Fig 3C–3E and S1 Movie). CD8+ cells are close to the blood vessels, in the parenchymal space below the epithelial surface. Unfortunately, the collagen bundles directly underlying the epithelium do not produce a detectable second harmonic signal. Using explanted trachea stained with antibodies to collagen IV, the T cells can be seen above and below the Col-IV layer corresponding to the lamina densa at the base of the epithelial surface, suggesting some of the T cells are in the epithelium itself (S1 Fig). This is consistent with conventional immunohistology using frozen sections [30]. The ability to optically penetrate the trachea from the outside in opens the possibility of imaging the infected trachea in situ in an intact live animal.

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Fig 3. Intravital imaging site of the mouse trachea.

B6 mice were infected as in Fig 1 or T cell receptor transgenic OT-I x GFP spleen cells (10⁵) were adoptively transferred into recipient B6 mice one day prior to infection with 3x10³ EID₅₀ recombinant influenza A/HK/X31-OVA expressing the ovalbumin peptide siinfekl recognized by the OT-I T cells. (A) Exposed trachea just prior to imaging reveals its translucent appearance. (B) Whole mount imaging of excised trachea viewed from the inside. Tissue was stained with antibodies to CD8 (red) and Col IV (green). (C-E) Intact trachea from mice that received adoptive transfer of OT-I GFP+ cells, intravenous dextran (red) to label the blood vessels followed by imaging by multiphoton microscopy. Images C and D show 0 and 90 degree points of view through the trachea, with the SHG signal towards the outer wall of the trachea appearing in the distal layer of C and bottom of D.

https://doi.org/10.1371/journal.ppat.1005881.g003

Intravital multiphoton microscopy (IV-MPM) of lymphocyte migration in an intact, influenza infected trachea

Migration into the infected epithelium is critical for CTL mediated control of infected cells. Mechanisms that regulate effector CD8+ T cell motility in the infected tissue are largely undefined, and studies need to be done in the intact tissue to begin interrogating the mechanisms. Live imaging of the lung in live animals is limited by the need for the lung to continue functioning in breathing, with movement of the tissue and the challenge of maintaining intrathoracic pressure during respiration as significant technical hurdles [31]. Mice infected with influenza have lower body temperatures and low blood oxygen saturation [32]. Anesthetization can further lower body temperature [33, 34] and suppress breathing, affecting blood oxygen levels [34]. In our experiments, rectal temperatures of infected mice after sedation for imaging were variable, and lowest at days 5–6 (Fig 4A), a time when virus titers are still relatively high and the T cell infiltrates are just becoming measurable. As the animals recover and virus titers were reduced, pre-imaging body temperatures rise, though were still lower than reported for unsedated animals (Fig 4A) [32]. Mice being imaged also exhibited low blood oxygen saturation and erratic heart rates (Fig 4B). Because of concerns about the effects of physiological variability on cell motility, we elected to provide respiratory and temperature support to maintain normal physiology since cell movement is affected by both temperature and oxygenation [35]. Body temperature was controlled via a heat block on the stage, a warming blanket with feedback from the animal using a rectal probe, as well as a warmed objective lens. A respirator maintained breathing rate and oxygenation, with pulse oximetry. This setup allows longer-term (2h) imaging. Tracheostomy and cannulation of the trachea both stabilized the tissue from respiratory movement and facilitated mechanical ventilation.

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Fig 4. Physiological and motility parameters measured during intravital imaging of influenza infected mouse tracheas by multiphoton microscopy.

Mice that received adoptively transferred GFP+ OT-I spleen cells and subsequently infected with X31-OVA-I virus as in Fig 3 were prepared and imaged on sequential days after infection by intravital multiphoton microscopy (Methods). (A) Rectal body temperatures of infected mice measured after sedation just prior to imaging. Each point is the average of 3–5 mice ± standard deviation. (B) Pulse-oximetry measurements of heart rate (HR) and percent blood oxygenation with and without respiratory support. Green and blue symbols represent blood O₂ and HR, respectively, in a mouse on respiratory support. Black and red symbols represent O₂ and HR, respectively from a mouse not receiving respiratory support. (C-F) Cell motility parameters calculated using Volocity software (Perkin-Elmer). Each symbol represents a single cell, and the plots contain 3–5 replicate experiments on individual mice. Horizontal gray bars denote the mean. C = velocity; D = Meandering index (also called confinement ratio); E = Displacement (distance form origin) for an entire cell track; F = average turning angle for each imaging step in a track. (G-H) Spider plots showing individual cell tracks plotted from a common origin using data from days 7 and 9 after infection.

https://doi.org/10.1371/journal.ppat.1005881.g004

CD8+ T cell motility varies over the course of infection

First, we wanted to know how variable T cell motility was over the course of an acute infection. The number of studies measuring CD8+ T cell motility during respiratory infection is limited. CD8+ T cell velocity during a model of influenza infection has been reported to be similar on days 6 and 8, with significantly higher velocities on day 10 [36]. These experiments were performed using an excised lung slice and combined imaging of lung tissue and airway versus airway alone. To get a more comprehensive set of T cell motility parameters, and compare motility in primarily an airway to a more heterogeneous tissue, we adoptively transferred OT-I TCR transgenic CD8+ T cells crossed to a mouse that expresses GFP under an actin promoter, and then infected mice with X31-OVA-I at a sublethal dose. Note that this dose was lower than for wild-type X31 because mice infected with higher doses were too fragile for long-term imaging. Animals were imaged between days 5 and 14.

Events in the lymph node and spleen related to T cell activation and differentiation initially occur between one and 3 days after infection [22, 28, 29, 37, 38], well before virus-specific T cells become detectable in the respiratory tract. Few cells were visible prior to day 5, so imaging was done on days 5, 6, 7, 8, 9, 10, and 14 (S1–S7 Movies show an extended focus, while S8–S12 Movies are animated to visualize the cells and tissue from different angles in 3D). Visible cells were still variable on day 5 in some experiments, so we include only motility parameters from day 5 experiments that had substantial T cell infiltrates. Each imaging time point was repeated 3–5 times in replicate experiments using separate mice conducted over a period of more than a year.

CD8+ T cell motility was not uniform throughout the infection. Cell velocity began relatively high at days 5 and 6 and decreased steadily, reaching a nadir (p < 0.001) at day 7–8, presumably as effector CD8+ T cells accumulate in the respiratory epithelium where the infected cells are located (Fig 4C, S4b and S5b Movies). Remarkably, at day 9 there was a significant (p < 0.001) increase in velocity that then returned to slower speeds by day 10 and beyond (p < 0.001) (Fig 4C, S4a & S4b, S5a & S5b, S6a & S6b Movies). The meandering index, a measure of the straightness or confinement of cell tracks that is the ratio of the displacement of a cell to the total length of the path that the cell has travelled [39], also changed dramatically (p < 0.001) from day 8 to 9 and 9 to 10, being lowest at day 9, suggesting the cells were more restricted in their movement (Fig 4D and S12–S16 Movies). Displacement was highest at days 5 and 6 (the only time points significantly different than the others), suggesting rapid infiltration of the infected tissue (Fig 4E). Differences in displacement and cell tracks are depicted in spider plots for days 7 and 9 (Fig 4G and 4H).

To reinforce our interpretation of the motility data, we used an approach that provides a better visual representation of the changes in cell behavior. Since the cell motility parameters for each cell are linked, average cell velocities can be plotted against the meandering index to give an assessment of migratory properties over the course of the infection. Mrass et al. used this approach to describe four quadrants in the plot into which the cell populations fall (see inset Fig 5) [39, 40]. Quadrant 1 contains cells with high velocity and high apparent confinement (low meandering index); these are the cells exhibiting active directional migration, but that do not cover much distance from the origin. Quadrant 2 contains the cells with high velocity and low confinement, which are the cells showing sustained motility, with limited stopping. Quadrant 3 contains the cells of low velocity and high confinement, the so-called low motility or stopped cells. The fourth quadrant contains the cells with low velocity and low confinement, indicating cells that actively move only during certain periods, consistent with non-sustained motility. Histograms along the top and right side of each plot show the cell distribution. Similar to a flow cytometry plot, the locations of the crosshairs are based on the cell densities most easily seen in Fig 5D, but are otherwise arbitrary. The plots of velocity versus meandering index change rather dramatically over days 6–10 and day 14 (Fig 5). On day 6, cells are found distributed in all four quadrants, with many in quadrants 1 and 2, suggesting active directional and sustained motility (Fig 5A and S2 Movie). Fewer cells are in quadrant 3, exhibiting low motility. This is consistent with rapidly infiltrating cells that need to cover a lot of ground as they seek infected and antigen-bearing cells. On days 7–8 (Fig 5B and 5C), the majority of the cells move out of quadrants 1 and 2 (reduced velocity), and by day 8 most are in quadrant 4, exhibiting the non-sustained motility of cells that are not confined and actively moving during short periods (Fig 5C, S3 and S4 Movies). This behavior is consistent with cells that have reached their destination and are perhaps sequentially encountering antigen bearing and infected cells. On day 9 (Fig 5D), there is a major shift towards quadrant 1 and 3 as the velocities rise and the cells are moderately confined, consistent with low motility and displacement, showing that the cells are very active but not going very far. Closer examination of the d9 images shows many cells arrested and rounded in appearance compared to d8, while others exhibit rapid local migration (S13–S16 Movies). The high apparent velocities and increased confinement are consistent with rapid surveillance, perhaps sampling their immediate cellular environment for infection. On day 10 (Fig 5E) the cells shift again down to quadrants 3 and 4, which remains consistent on day 14, though with fewer cells (Fig 5F, S6 and S7 Movies). These are cells with low motility and high apparent confinement, possibly because they are entering a more sessile state. Calculation of the arrest coefficients supports these conclusions. On day 6, few T cells are arrested (Fig 6A). On days 7 & 8, the values are broadly distributed, with an increase in cells exhibiting arrest (Fig 6B and 6C), again perhaps as they accumulate at the epithelium. On day 9 few cells are arrested though their displacement values are low (Fig 6D), indicating that they are very actively moving, but not traveling far. On day 10, the majority of cells are close to arrest, while by day 14 the majority of cells are fully arrested (Fig 6E and 6F), which is not surprising given the absence of active infection at these time points.

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Fig 5. Meandering index versus velocity plots on different days after infection.

The values for individual cells of meandering index and velocity are graphed to depict changing cell population behavior over the course of infection. Each symbol is data from a single cell. Histograms along the top and right axes indicate the cell densities. Crosshairs defining the quadrants are based on the densities in (D). A = day 6; B = day 7, C = day 8; D = day 9; E = day 10, and F = day 14.

https://doi.org/10.1371/journal.ppat.1005881.g005

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Fig 6. Frequency distributions of the arrest coefficients for each day after infection.

For each day after infection measured, the arrest coefficients (defined as the fraction of time that a cell has a speed less than 2μm per minute) were calculated for individual cell tracks and the values of the population were binned into 0.1 increments. A value of 1 indicates full arrest. Y-axis indicates the number of coefficient values in each bin. A = day 6; B = day 7, C = day 8; D = day 9; E = day 10, and F = day 14.

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The effects of replicating virus and antigen availability on CD8+ T cell motility

A substantial infiltrate of OT-I T cells in mice infected with influenza encoding the cognate siinfekl OVA H2Kb restricted peptide is present by day 5 post infection [24, 27]. To determine whether there were differences in virus clearance compared to non-OVA HKx31, we performed virus titers of lung and trachea at several time points after infection using an indirect immunofluorescence assay. Possibly due to the more rapid T cell response and lower dose of virus, virus titers were lower in the lung and below detection in the tracheas of two out of three mice at days 6, 7, and 8. However, one mouse was positive on all three of those days. The incomplete control may relate to a requirement for neutralizing antibodies, which are not typically detectable until day 7 [41]. We conclude that while virus was below the limit of detection in some mice, the data suggest that it may persist as far as day 8, which is similar to the wild-type kinetics. (Fig 7A)

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Fig 7. Virus clearance in mice infected with X31-OVA and the effect of OVA-peptide-MHC blocking antibodies on cell velocities.

As described in Methods, OT-1-GFP CD8+ T cell receptor transgenic T cells were adoptively transferred into mice prior to infection with X31-OVA virus expressing the siinfekl OVA peptide. (A) Lungs and trachea were excised and titered by indirect immunofluorescence assay (B-D) The day 7 velocities of OT-I-GFP T cells are plotted over time during a 25-minute long imaging session. Control mice (B) were untreated. Experimental mice were treated with monoclonal antibody 25-D1.16 against Kb-siinfekl peptide-MHC complex, and imaged 2h later at 37°C (C) or at 35°C (D) with the warming objective turned off. The red lines indicate the average velocities at each increment over time, while the black horizontal line in (C) and (D) indicates the overall average of the day 7 control mice.

https://doi.org/10.1371/journal.ppat.1005881.g007

Collectively, the data show that, early in the response, the cells travel at higher speed over more distance. As the cells accumulate along the epithelium, the displacement decreases and confinement increases, presumably due to physical restrictions to movement at the epithelial surface. The data also indicate an abrupt shift in motility that occurs between days 8, 9, and 10. The increase in velocity, decrease in angle and meandering index would be consistent with the cells becoming established in the epithelium, and exhibiting behavior associated with surveillance for residual antigen [42, 43]. To test whether this could be the case, mice were treated on day 7 with a monoclonal antibody that specifically blocks Kb/siinfekl peptide-MHC complex, while leaving other Kb class I complexes intact [44]. CD8+ T cell motility was measured 1 and 2 hours later. On day 7 in control mice, average CD8+ T cell velocities hover close to 2 μm/min (Fig 7B). In the antibody treated mice however, initial cell velocities at the start of the imaging session were between 3 and 4 μm/min, suggesting the antibody was having an effect (Fig 7C). However, over the 25-minute imaging session, the velocities returned to the lower values more closely resembling day 7–8 in untreated mice. We wondered whether the imaging conditions that include warming both the mouse body and the imaging site to 37°C with a warming objective was having an effect on the peptide MHC blockade. To test, we performed the imaging at 35°C, and turned off the objective lens warmer. Under the lower temperatures, cell velocities remained higher at between 3–4μm/min (Fig 7D). Both the slow decline at 37°C, and the stable cell behaviors at 35°C were reproduced in multiple mice on multiple imaging sessions. The data suggests that the low velocities and increased confinement on day 7, similar to day 8, is a result of cells arresting through interaction with antigen-bearing cells. The increased velocity on day 9 is most likely due to the elimination of the majority of the antigen-bearing cells (and virus). After day 10, the cells return to slower velocities, are less confined, with many cells arrested (velocities < 2μm/min). Given that the virus is absent at this and later time points, it is most likely a reflection of a less activated phenotype.