The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.
The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.
The effector function of CD4 T cells is critically dependent on their ability to rapidly enter and traverse a wide variety of peripheral tissues to survey for damage, locate foci of infection, or cause pathology from chronic infection or autoimmunity. While the processes of homing to inflamed sites1-4 and extravasation5-7 from the vasculature into tissues have been well-characterized, the factors that drive and regulate the interstitial motility of T cells remain undefined. The migration of T cells in complex 3D environments has been studied in vitro through the use of artificial matrices8-10 or microfluidic devices11,12, but these fail to recapitulate the complex and dynamic environment of an in vivo system. It is only recently, with the advent of high-resolution multi-color intravital imaging that it has become possible to study the dynamic behavior of immune cells in situ, allowing for a better understanding of intact immune responses.
Over a decade ago, several influential studies were published that first utilized multiphoton microscopy to address immunological questions. Early studies focused on the behavior of immune cells within explanted lymphoid organs13-16, which were soon followed by techniques to image exposed lymph nodes in anesthetized mice17. Imaging allowed for new fundamental observations about the stages of lymph node priming of T cells18, the mechanisms by which T cells migrate in secondary lymphoid organs19, T cell interactions with other immune cells20,21, and dynamic T cell positioning within the lymph node22. Although many early studies focused on lymph node dynamics, intravital imaging has been since been utilized to image the immune response in many peripheral tissues, including the brain23-25, liver26, lung27, and skin28-30.
The mouse ear dermis is particularly well poised for imaging, due to the thinness of ear skin, a relative lack of hair, and the ease with which it can be isolated from respiratory movements31. Indeed, the ear dermis has been used to image the interstitial behavior of dendritic cells32,33, T cells28,29,34,35, and neutrophils36,37, and is a well-established site for studying dermal inflammation. Increasingly, non-invasive procedures have been replacing surgical preparations of the skin, including split dermis38,39, flank39,40, or dorsal skin flap window39,41 models, that can induce changes to the local inflammatory milieu. The use of transferred, in vitro-primed, antigen-specific CD4 effector T cells allows for the study of a homogenous population of cells in the context of a dermal inflammatory response30. Here we describe a non-invasive imaging procedure that allows for the visualization of antigen-specific effector CD4 T cells in the dermal interstitium of the inflamed mouse ear, and the ability to manipulate these cells in real-time by introducing blocking antibodies through a venous catheter. We show that this model is effective for tracking the movement of CD4 T cells in the dermis and for querying the mechanisms that govern this motility.
All procedures involving mice were approved by the Institutional Animal Care and Use Committee of the University of Rochester, and carried out in strict accordance with the Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals administered by the National Institutes of Health, Office of Laboratory Animal Welfare.
1. Preparation of Effector CD4 T Cells
NOTE: BALB/c TCR-transgenic DO11.10 mice that specifically recognize a peptide from chicken egg ovalbumin (pOVA: ISQAVHAAHAEINEAGR). Other TCR-transgenic systems can be substituted, using the appropriate cognate peptide in place of pOVA where indicated.
2. Transfer of Cells and Induction of Inflammation
NOTE: For optimal cell numbers for imaging, 5×106 fluorescently labeled Th1 cells should be transferred to each mouse in a total volume of 200 µl PBS. Cells here are labeled with the green dye CFSE or the near-red dye CMTMR, although other cell tracker dyes can be used. CFSE and CMTMR-labeled cells can be co-transferred to allow for tracking of two distinct effector CD4 populations.
3. Preparing the Mouse for Imaging
4. In Vivo Time-lapse Imaging and Intravenous Antibody Administration
NOTE: This protocol requires the use of a multiphoton microscope equipped with a Ti:Sa laser system. The objective used is a 25x magnification lens with 1.05 N.A., affixed with an objective heater set to 40 °C. The optimal temperature for this heater was determined empirically to be the appropriate temperature to maintain the ear dermis at 37 °C, and may need to be adjusted for use in other imaging systems. The acquisition software used may vary between instruments and adjustments to the protocol may have to be made to work on differently configured systems. Ensure that images can be saved in a format that is compatible with any desired analysis software.
The ability to study immune responses in situ without altering the immune environment is essential in studying real-time interactions of T effector cells with an inflamed tissue. Imaging of the intact ear dermis by this protocol, outlined in Figure 1A and B, allows for the visualization of transferred fluorescently labeled T effector cells in the dermal interstitium. This permits both high-resolution (Figure 1C) and time-lapse (Figure 1D, Movie 1) images of effector T cell dynamics in the inflamed dermis.
Figure 1. High-resolution and 4D imaging of T effector cells in the intact dermal interstitium. (A) Experimental outline. (B) Photograph of an ear prepared for imaging with the site of the emulsion (black dashed line) and optimal area for imaging (red line) indicated. (C) Maximal 3D projection of a high-resolution stack showing CFSE-labeled Th1 cells (green), second harmonic signal from fibrillar collagen (blue) and Texas Red-Dextran labeled vasculature (red). The white arrow indicates one of several autofluorescent hair follicles. Scale bars represent 50 µm (D) Migratory paths of Th1 cells tracked for 30 min in the CFA-inflamed dermis. Please click here to view a larger version of this figure.
This imaging protocol requires the use of a specialized imaging platform that was constructed in-house, as well as an adapted nosecone for the delivery of inhaled anesthesia while imaging. The imaging platform (Figure 2A-B) consists of an aluminum base plate with a raised central section. This raised section has an inset portion lined with acrylic felt to provide support to the ear without compressing and potentially damaging the thin tissue. The geometry of our setup required the construction of a flexible and adjustable nosecone to deliver inhaled anesthesia during imaging. This nosecone, consisting of a modified microcentrifuge tube connected to a 50 mm section of flexible tubing (Figure 2C), is secured in place with a holder composed of modified microcentrifuge tubes (Figure 2D) and affixed to the imaging platform via hook and loop fastener. The use of hook and loop fastener allows for the repositioning of the nosecone assembly so that either ear of a mouse can be imaged, and can be optimally positioned for individual mice.
Figure 2. Equipment for intravital imaging. Custom-built imaging platform in top (A) and side (B) views. Nosecone for isoflurane administration (C) and nosecone holder (D). Please click here to view a larger version of this figure.
Catheterization of the tail vein allows for continuous access to the circulation to administer antibodies and other small molecules that can diffuse out of the vasculature, or larger fluorescent molecules such as high molecular weight dextran to label blood vessels. After the administration of 100 µg of anti-β1 and anti-β3 integrin blocking antibodies through the catheter, previously motile cells arrest within the dermis (Movie 2). These cells have a decreased average velocity after antibody administration (Figure 3A), as well as a significant decrease in meandering index, the ratio of the total displacement to the total track length (Figure 3B).
Figure 3.Administration of anti-β1 and anti-β3 antibodies inhibits Th1 cells migration in CFA-inflamed skin. (A) Average velocity of Th1 cells before and after administration of 100 µg anti-β1 and anti-β3 integrin blocking antibodies. (B) Meandering index of Th1 cells before and after antibody blockade. Approximately 100 tracked cells are from images before and after antibody blockade from a single mouse in one representative experiment. Statistics by Mann Whitney. Please click here to view a larger version of this figure.
Because hair is not removed from the surface of the ear, imaging artifacts from the hair are common. Autofluorescence from hair follicles (Figure 4A) and shadowing and autofluorescence from overlying hair (Figure 4B) should be avoided where possible as they can obscure T cells and interfere with automated image analysis software. Similarly, air bubbles trapped between the ear surface and the coverslip can lead to imaging artifacts (Figure 4C). Proper preparation of the ear should minimize the number and size of any remaining bubbles.
Figure 4. Common artifacts from hair autofluorescence and poor ear preparation. (A) Autofluorescent hair follicles. (B) Autofluorescent hair and hair follicles (green) and collagen (white) with overlying hair shadows, causing dark lines in the image. (C) Artifact from an air bubble, showing the edge of the displaced, autofluorescent keratinized epidermis (dotted line). Scale bars represent 50 µm. Please click here to view a larger version of this figure.
Additionally, the use of multiple sources of heat can lead to issues with stability of the tissue due to thermal expansion and contraction. Incorrect thermostat settings, for instance, can lead to large oscillations during imaging that can make interpretation of results difficult (Movie 3). It is essential to determine the optimal settings for any system that provides a constant temperature and maximizes stability. Ultimately, a temperature controlled imaging chamber is the best way to remove variability from changes in temperature.
Movie 1. Th1 cells migrating in the CFA-inflamed dermis (Right click to download). 30 min time-lapse image showing Th1 cells (green) migrating in the dermal collagen network (second harmonic generation, blue).
Movie 2. Th1 cells arrest upon blockade of β1 and β3 integrins (Right click to download). Cells (green) were imaged for 30 min before the administration of blocking antibodies, and then imaged for another 20 min in the same location.
Movie 3. Oscillations from a poorly controlled heating plate (Right click to download). 30 min time-lapse image of Th1 cells (green) and second harmonic generation (blue). After this image was collected, the heating plate used was found to have a faulty thermostat, causing oscillations in the temperature of the imaging platform and subsequent thermal expansion and contraction.
Significance
Here we present a complete protocol for the 4D visualization of transferred, antigen-specific effector Th1 cells in the intact mouse ear dermis. This method provides advantages over some current imaging modalities for several reasons. By imaging the ventral ear dermis, we are able to forego hair removal that is required for imaging protocols involving other skin sites. Although depilatories are generally mild, they have been shown to cause disruption to the skin barrier 42, a process that can stimulate an immune response 43,44. By also avoiding invasive surgical procedures to expose the dermis or hypodermis, this protocol prevents damage-induced inflammation and the rapid recruitment of neutrophils37 and other immune cells into the dermis. The use of a venous catheter in this system to deliver blocking antibodies against key molecules allows for real time interrogation of the dynamic behavior of CD4 T cells. The use of this imaging protocol has revealed critical requirements for CD4 T cell interstitial motility30 that were not detected in in vitro systems8.
Critical steps in the procedure
An essential step in any imaging protocol is ensuring a stable tissue preparation for imaging. It is important to ensure that there is enough PBS between the ear and the coverslip that there are no air bubbles and the ear is in contact with the glass, but not so much as to cause the tape to become de-adhered from the glass. Similarly, avoiding contact between the tape holding the coverslip to the platform and any vacuum grease will prevent the tape from loosening over time. Temperature must also be kept constant to avoid oscillations and drift from thermal contraction or expansion of the platform materials.
Limitations and Modifications
Imaging by this non-invasive protocol is limited to skin areas that are thin enough to allow for effective visualization of fluorescence through multiphoton excitation. The ear is advantageous due to ease of preparation and ability to be isolated from respiratory movements, and has been used as a model for intravital time-lapse imaging since the 1980s45. However, the resident immune population of the ear is distinct from skin on the flank or footpad46, and the mouse ear has distinct vascular properties when compared to other sites47. Thus, for some applications, comparison of ear imaging data to other skin sites may be difficult.
This protocol also requires the effective migration of transferred T effector cells out of the blood stream and into the dermis. This limits the ability to use cells that have defects in homing or extravasation as they will not be able to enter the interstitial space. Extravasation can be bypassed by injecting cells directly into the ear dermis37,48, although this will cause some mechanical damage and delivery of cells in this way may not recapitulate the localization or behavior of cells that undergo in vivo extravasation.
It is also critical to consider using non-pigmented recipient mice for imaging experiments, such as the BALB/c strain used here or Albino C57BL/6-Tyrc-2J mice. The melanin in pigmented mice, in addition to being highly autofluorescent, heats up under even relatively low-power excitation from a multiphoton laser37. This can cause thermal damage to the skin and subsequent inflammation36 or fluorescent speckling31, complicating results. This may limit fluorophores that can be used in a multi-parameter imaging experiment. However, some very bright or highly expressed fluorescent molecules can be effectively excited at low laser power, allowing for effective visualization in pigmented mice.
Future applications
Because this is a non-invasive procedure, it could be easily adapted for longitudinal studies on mice with sequential imaging over extended time periods. While fluorescent labeling as described here would fade over time periods greater than 3-4 days, use of endogenously fluorescent cells or fluorescent reporter cells eliminates this problem. Indeed, we have previously used this protocol to track in vivo-generated antigen-specific effectors bearing cytokine reporters, including the IFNγ reporter Yeti30,49 and IL-4 reporter 4get50. We have additionally visualized endogenous CD4 cells in CD4-Cre ROSA26-stop-floxed eYFP fluorescent reporter mice30. As the fluorescent properties of eYFP and other fluorescent proteins differ from chemical dyes such as CFSE, modifications to the imaging parameters may be needed for efficient visualization. Changes such as increasing the pixel dwell time can enhance fluorescent signal of some dim fluorophores, and decreasing the length of time lapse images can mitigate any photobleaching that may be observed. At 900 nm excitation, we have not previously observed significant photobleaching of CFSE, CMTMR, or eYFP over short imaging intervals.
Although this procedure focuses on measuring the dynamics of CD4 effector T cell motility, it is not limited to this application. Work is currently ongoing to measure the dynamic interactions of effector T cells with antigen presenting cells through the use of fluorescent reporter mice and injection of fluorescently conjugated antibodies into the dermis to label cells or tissue structures prior to imaging51,52. Additionally, while this protocol demonstrates the use of the catheter to deliver blocking antibodies while imaging, other compounds, including small molecule inhibitors, can be administered. Ultimately, this protocol provides a flexible platform to measure immune dynamics over time, in vivo, in a non-invasive manner.
The authors have nothing to disclose.
The authors thank the University of Rochester Multiphoton Microscope Core facility for help with live imaging. Supported by NIH AI072690 and AI02851 to DJF; AI114036 to AG and AI089079 to MGO.
BALB/c mice | Jackson Laboratories | 000651 | Mice used were bred in-house |
DO11.10 mice | Jackson Laboratories | 003303 | Mice used were bred in-house |
HBSS | Fisher | 10-013-CV | Multiple Equivalent |
Newborn Calf Serum (NCS) | Thermo/HyClone | SH30118.03 | Heat inactivated at 56 °C for 30 minutes |
Guinea Pig Complement | Cedarlane | CL-5000 | |
anti-CD8 antibody | ATCC | 3.155 (ATCC TIB-211) | Antibodies derived from this hybridoma |
anti-MHC Class II antibody | ATCC | M5/114.15.2 (ATCC TIB-120) | Antibodies derived from this hybridoma |
anti-CD24 antibody | ATCC | J11d.2 (ATCC TIB-183) | Antibodies derived from this hybridoma |
anti-Thy1.2 antibody | ATCC | J1j.10 (ATCC TIB-184) | Antibodies derived from this hybridoma |
Ficoll (Fico/Lite-LM) | Atlanta Biologicals | I40650 | |
PBS | Fisher | 21-040-CV | Multiple Equivalent |
EDTA | Fisher | 15323591 | |
biotinylated anti-CD62L antibody (clone MEL-14) | BD | 553149 | |
streptavidin magnetic separation beads | Miltenyi | 130-048-101 | |
MACS LS Separation Column | Miltenyi | 130-042-401 | |
recombinant human IL-2 | Peprotech | 200-02 | |
recombinant mouse IL-4 | Peprotech | 214-14 | |
recombinant mouse IL-12 | Peprotech | 210-12 | |
anti-IFNg antibody (clone XMG 1.2) | eBioscience | 16-7311-85 | |
anti-IL-4 antibody (clone 11b11) | eBioscience | 16-7041-85 | |
RPMI | VWR | 45000-412 | |
Penicillin/Streptomycin | Fisher | 15303641 | |
L-glutamine | Fisher | 15323671 | |
2-mercaptoethanol | Bio-Rad | 161-0710 | |
ovalbumin peptide | Biopeptide | ISQAVHAAHAEINEAGR-OH peptide | |
Fetal Calf Serum (FCS) | Thermo/HyClone | SV30014.03 | Heat inactivated at 56 °C for 30 minutes |
24-well culture plate | LPS | 3526 | Multiple Equivalent |
CFSE | Life Technologies | C34554 | |
CMTMR | Life Technologies | C2927 | |
28 G1/2 insulin syringes, 1ml | BD | 329420 | |
28 G1/2 insulin syringes, 300μl | BD | 309301 | |
27 G1/2 TB syringes, 1ml | BD | 309623 | |
30 G1/2 needles | BD | 305106 | |
PE-10 medical tubing | BD | 427400 | |
cyanoacrylate veterinary adhesive (Vetbond) | 3M | 1469SB | |
heating plate | WPI | 61830 | |
Heating plate controller | WPI | ATC-2000 | |
Water blanket controller | Gaymar | TP500 | No longer in production, newer equivalent available |
water blanket | Kent Scientific | TP3E | |
Isoflurane vaporizer | LEI Medical | Isotec 4 | No longer in production, newer equivalent available |
isoflurane | Henry Schein | Ordered through Veterinary staff | |
microcentrifuge tubes | VWR | 20170-038 | Multiple Equivalent |
medical tape | 3M | 1538-0 | |
isoflurane nosecone | Built In-house, see Fig 2 | ||
imaging platform | Built In-house, see Fig 2 | ||
curved forceps | WPI | 15915-G | Multiple Equivalent |
scissors | Roboz | RS-6802 | Multiple Equivalent |
glass coverslips | VWR | Multiple Equivalent | |
high vacuum grease | Fisher | 146355D | |
cotton swabs | Multiple Equivalent | ||
delicate task wipes | Fisher | 34155 | Multiple Equivalent |
Olympus Fluoview 1000 AOM-MPM upright microscope with Spectra-Physics MaiTai HP DeepSee Ti:Sa laser | Olympus | call for quote | |
optical table with vibration control | Newport | call for quote | |
25x NA 1.05 water immersion objective for multiphoton imaging | Olympus | XLPLN25XWMP2 | |
objective heater | Bioptechs | PN 150815 | |
Detection filter cube | Olympus | FV10-MRVGR/XR | Proprietary cube, can be approximated from individual filters/dichroics |
anti-integrin β1 antibody (clone hMb1-1) | eBioscience | 16-0291-85 | Azide free, low endotoxin |
anti-integrin β3 antibody (clone 2C9.G3) | eBioscience | 16-0611-82 | Azide free, low endotoxin |
Texas Red Dextran (70,000 MW) | Life Technologies | D-1830 |