Method Article

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

DOI:

10.3791/58704

⸱

January 7th, 2019

 ,  ,  ,  ,  , 
1Department of Cell, Developmental, & Cancer Biology, Oregon Health and Science University, 2Department of Molecular Microbiology & Immunology, Oregon Health and Science University, 3Department of Dermatology, Oregon Health and Science University, 4Knight Cancer Institute, Oregon Health and Science University

In This Article

Summary

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Here, we demonstrate the methods for in vivo quantification of leukocyte egress from naïve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Abstract

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Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Introduction

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Peripheral tissue immune responses are shaped not only by leukocyte recruitment to the sites of inflammation but also by mechanisms that regulate their subsequent retention. Thus, protective immunity is dictated by cumulative cellular and molecular mechanisms that determine whether a leukocyte enters, stays within, or rather migrates out of peripheral tissue via lymphatic vessels. Importantly, the propensity for leukocytes to exit tissue through lymphatic vessels (termed egress) is linked to their specialized functions. Dendritic cells (DC) acquire migratory behavior in response to maturation signals leading to antigen transport and presentation in draining lymph nodes (dLN), a process that is necessary for adaptive immunity1. Scavenging myeloid cells, such as macrophages and neutrophils, serve to clear apoptotic debris through phagocytosis. During bacterial infection, neutrophils egress tissue and ultimately undergo apoptosis in dLNs2 and in a model of DSS-induced colitis, data supports the hypothesis that macrophage egress is necessary to resolve local inflammation3. Whether neutrophil and macrophage egress occurs in all inflammatory contexts, however, is unknown. Evidence for T lymphocyte egress from steady state4,5,6,7, infected8, and inflamed4,9,10,11,12 peripheral, non-lymphoid tissues indicates that T cells actively recirculate, though the tissue-based signals that drive this exit remain poorly understood. Several studies have identified signals necessary for directional migration towards draining lymphatic capillaries and subsequent egress including chemokine (C-C motif) ligand 21 (CCL21) and its receptor CCR74,11,13, chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor CXCR42,14, and sphingosine-1-phosphate (S1P)10,15,16. These mechanisms are not active in all contexts, however, and whether they determine egress of all cell types remains an open question. Importantly, further insight into the mechanisms that govern egress and its functional relevance in disease requires quantitative in vivo methods of analysis.

Several methods have been used to quantify egress in multiple animal models in vivo including direct cannulation of lymphatic vessels, adoptive transfer of ex vivo labeled leukocytes, transdermal application of fluorescent tracers, injection of labeled particles, and in vivo photoconversion17,18. Direct cannulation of afferent mouse lymphatic vessels is difficult and limited in small animals by the volumes of fluid that can be collected. Thus, cannulation has largely been performed in large animals (e.g., sheep) where such surgical manipulations are practical. These studies provide direct evidence for the presence of both lymphoid and myeloid cells in lymph10,19,20. Furthermore, ovine models reveal that acute and chronic inflammation increased lymphocyte presence in lymph by nearly 100-fold10,21.

Adoptive transfer of labeled and genetically manipulated lymphocytes has importantly revealed that CCR7 is required for the egress of CD4+ T cells from acutely inflamed skin5,11, while the pretreatment of lymphocytes with the small molecule S1P receptor agonist, FTY720, only partially inhibits their egress10. Interestingly, the egress of transferred lymphocytes from chronically inflamed skin is CCR7-independent10, but may partially require CXCR49. Adoptive transfer experiments, however, deliver non-physiological numbers of ex vivo activated and labeled lymphocytes into tissue through injection, which alters the biomechanical environment of tissues and elevated interstitial fluid pressures that open initial lymphatic capillaries and alter their transport properties22. As an alternative, transdermal application of fluorescein isothiocyanate (FITC) in the presence or absence of dermal irritants (e.g., dibutyl phthalate, DBP) or infection23,24 allows for the tracking of phagocytic cells that accumulate tracer and migrate to dLNs. Similarly, fluorescently labeled tumors provide a means to track phagocytic cells that have engulfed tumor material25. These methods have provided important insight into the mechanisms that govern DC egress13,14,17,26,27 but are unable to track non-phagocytic lymphocytes and, interpretation can be complicated by free lymphatic drainage of soluble FITC thus labeling non-migratory, LN resident DCs.

Alternatively, intravital microscopy is a powerful tool that allows for in vivo tracking of physiologically relevant leukocyte populations in real time28,29. Used in combination with the reporter mice and antibody-based in vivo immunofluorescent labeling, intravital microscopy has revealed the complex spatial and temporal dynamics of immune cell trafficking, including interstitial migration30, transmigration across the lymphatic endothelium, passage within the lymphatic lumen, and migration upon LN entry28,31. Broad adoption of intravital imaging techniques is limited by expense, necessary expertise for set up, and limited throughput for quantifying multiple cell types. Still, coupling quantitative methods that analyze population dynamics tissues with intravital imaging will provide additional and important mechanistic insight with respect to the mechanisms of motility and migration toward and within lymphatic capillaries18,31,32.

Consequently, in vivo photoconversion has emerged as a method that allows for in situ labeling, independent of phagocytic activity, and for the quantification of physiological leukocyte egress (when coupled with flow cytometry) in the absence or presence of challenge. Kaede-Tg mice constitutively express a protein isolated from stony coral that exhibits green fluorescence (Kaede green) until exposed to violet light, after which it irreversibly converts to red fluorescence (Kaede red)33. Photoconverted cells can be tracked as they egress from peripheral tissue sites and accumulate in dLNs. This and other similar photoconvertible mouse models34,35 have revealed important biology including constitutive egress of regulatory T cells from skin36, CXCR4-dependent B cell egress from Peyer's patches37, mobilization of resident memory T cells upon peptide re-challenge38, and broad leukocyte egress from tumor microenvironments39. Herein, we perform a head-to-head comparison of photoconversion with transdermal FITC application in the context of cutaneous inflammation and infection to allow for direct comparison of existing data with the photoconvertible method. Furthermore, we demonstrate photoconversion in implanted tumors and describe the conversion efficiency and selective egress from tumor microenvironments. As such, we argue that further application of these methods is needed to elucidate the critical biology of leukocyte egress from tumors, which will have significant implications for interpreting intratumoral leukocyte complexity, anti-tumor immunity, and response to therapy.

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Protocol

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All animal protocols have been approved by the Institutional Animal Care and Use Committee at the Oregon Health & Science University.

1. Induction of Inflammation and FITC Painting of Mouse Pinna

  1. In a laminar flow hood, anesthetize a C57Bl/6 mouse using vaporized isofluorane (induce at 3-5% isofluorane and maintain at 1-3% isofluorane; oxygen flow rate at 0.5-1.0 L/min). Ensure proper anesthetization by monitoring the loss of pedal reflex, involuntary movements and reduced respiratory rate.
  2. Lay an ear flat with the ventral side of the ear facing upwards. Pipet 20 µL of 5% FITC solution dissolved in 1:1 acetone:dibutyl phthalate (DBP) to the ventral side of ear pinna. Allow the ear to dry for a few seconds.
  3. 24 h after FITC application, euthanize the mice via carbon dioxide exposure followed by cervical dislocation. Collect the ear pinnae into PBS by separating the ears from the head using scissors. Collect the cervical dLNs and inguinal non-draining LNs into PBS using tweezers to separate the LNs from surrounding tissues. Inguinal non-draining LNs will serve as negative controls for the presence of FITC+/Kaede red+ leukocytes. Dispose of carcasses per institutional protocol.
    Note: The optimal time post photoconversion for analysis will depend on the cell type and known migratory behaviors. Dendritic cells, for example, can be detected in dLNs as early as 6 h post DBP application.

2. Induction of Inflammation and Photoconversion of Mouse Pinna

  1. In a laminar flow hood, anesthetize a Kaede-Tg mouse (background C57Bl/6) by intraperitoneal injection of 80 mg/kg ketamine and 10 mg/kg xylazine dissolved in saline. Ensure proper anesthetization as described in Step 1.1.
  2. Lay an ear flat with the ventral side of the ear facing upwards. Pipet 20 µL of a 1:1 acetone:DBP to the ventral side of the ear pinna. Allow the ear to dry for a few seconds.
  3. After DBP application, cut a slit in a piece of aluminum foil and pull the ear through the slit to expose the ear to the violet light source. Lay the ear flat with the dorsal side facing upward using double-sided tape to secure the ear to the foil.
  4. Position the ear directly under the light source, and photoconvert for 3 min using a 405 nm light source at 100 mW power.
  5. 24 h after photoconversion, euthanize the Kaede-Tg mice and harvest the ear pinnae, cervical LNs, and inguinal LNs as described in Step 1.3.
    Note: In addition to considerations cited in Step 1.3, the rates of proliferation will determine the timing of analysis as the loss of Kaede red will occur in rapidly dividing cells.

3. Vaccinia Infection of Mouse Pinna and FITC Application

  1. Anesthetize a C57Bl/6 mouse with vaporized isofluorane as described in Step 1.1.
  2. Lay the ventral side of the ear flat. Pipet 5 x 106 plaque-forming units (PFU) of vaccinia virus (VacV) diluted in 10 µL of PBS onto the ear pinna. Using a 29-gauge needle, poke the pinna 25 times40.
  3. 24 h post-infection, anesthetize VacV-infected mice with isofluorane as described in Step 1.1 and pipet 20 µL 5% FITC dissolved in acetone onto the ventral side of the ear pinna. Allow the ear to dry for a few seconds.
  4. 24 h after FITC application, euthanize the mice and collect the ear pinnae, cervical LNs and inguinal LNs as described in Step 1.3.
    Note: FITC may be applied at any time point post infection to determine DC trafficking at various 24 h intervals. We previously reported that DC migration to dLNs is maintained at similar levels from day 1 to 3 post infection41.

4. Vaccinia Infection of Mouse Pinna and Photoconversion.

  1. Anesthetize a Kaede-Tg mouse with vaporized isofluorane as described in Step 1.1.
  2. Infect the ear pinna with VacV as described in Step 3.2.
  3. 24 h post-infection, anesthetize VacV-infected Kaede mice as described in Step 2.1 and perform photoconversion as described in Steps 2.3-2.4.
  4. 24 h after photoconversion, euthanize the mice and harvest the ear pinnae, cervical LNs, and inguinal LNs as described in Step 1.3.
    Note: As mentioned above in Step 3.4, photoconversion can be administered at any time point post infection.

5. Ear and Lymph Node Processing for Flow Cytometry

  1. Create single cell suspensions of ear pinnae: peel apart the ventral and dorsal sides of the ear pinna using two pairs of tweezers and place with the inside of the ear facing down into the wells of a 24-well plate containing 1 mg/mL collagenase D and 80 U/mL DNase diluted in Hank’s Buffered Saline Solution (HBSS) (containing Ca2+ and Mg2+). Incubate at 37 °C for 30 min. Press the digested tissue through a 70 µm nylon cell strainer.
  2. Create single cell suspensions from LNs: place LNs in wells of a 24-well plate containing 1 mg/mL collagenase D and 80 U/mL DNase diluted in HBSS. Tease open the lymph node capsule using two 29-gauge needles, and then incubate the lymph nodes at 37 °C for 30 min. Press the digested tissue through a 70 µm nylon cell strainer.

6. Intradermal Melanoma Tumor Implantation and Photoconversion.

  1. Shave the fur from the center of the back of a Kaede-Tg mouse using an electric razor.
  2. Position a 29-gauge needle in the center of the back between the left and right upper scapulae, and intradermally inject 5 x 105 tumor cells (diluted in 50 µL of saline) into the skin of Kaede-Tg mice. Tumors must be carefully positioned to ensure lymphatic drainage to specified lymph nodes (i.e., left and right brachial LNs for tumors placed in the middle of the upper back). Avoid placing the tumor above dLN as this can result in direct photoconversion of the LNs through the skin/tumor.
  3. Allow the tumors to grow to desired size (100-650 mm3).
  4. One day prior to tissue collection, anesthetize a Kaede-Tg mouse as described in 2.1 and shave any newly regrown fur around the tumor.
  5. Cut a circular hole in aluminum foil and pull the tumor through to expose the tumor to the light source. Cut the hole slightly smaller than the tumor to prevent the tumor from falling back through the hole and minimize the conversion of adjacent, non-tumor skin.
  6. Position the tumor directly below the light source and photoconvert for 5 min using a 405 nm light source at 200 mW power.
  7. 24 h after photoconversion, euthanize the mice as described in Step 1.3. Collect the tumors, brachial dLNs, and inguinal non-draining LNs into PBS. Cut the tumors away from the surrounding skin using scissors and remove LNs as described in Step 1.3.

7. Tumor and Lymph Node Processing for Flow Cytometry

  1. Create single cell suspensions from the tumors: mince the tumors with scissors into wells of a 24-well plate containing 1 mg/mL collagenase D and 80 U/mL DNase diluted in HBSS. Incubate at 37 °C for 1 h. Press the digested tissue through a 70 µm nylon strainer.
  2. Create single cell suspension of lymph nodes as described in Step 5.2.

8. Antibody Staining for Flow Cytometry

  1. After digesting the tissues into single cell suspensions, perform standard flow cytometry staining techniques to label the cells with markers of interest. Briefly, pipet 2 x 106 cells into a 96-well plate. Incubate the samples with Fc block (1 µg/mL) for 20 min on ice; wash twice with FACs buffer (1% bovine serum albumin in PBS). Add live/dead stain (diluted in PBS) to the samples and incubate for 15 min on ice; wash twice with FACs buffer. Incubate with primary antibodies (Table of Materials) diluted in FACs buffer for 30 min on ice; wash twice with FACs buffer.
    Note: Kaede green and Kaede red fluorescence overlaps with FITC and PE fluorophores; thus, FITC and PE-conjugated antibodies cannot be used in combination with Kaede proteins.
  2. After staining, run the samples on a flow cytometer. Alternatively, fix the cells with 2% PFA.

9. Flow Cytometry Analysis

  1. Set the FITC (Kaede green) and PE (Kaede red) channel PMT voltages to 70-80% of the total range (center population at approximately 104) using ex vivo Kaede green or Kaede red single-color splenocytes or blood.
    Note: Do not compensate for the Kaede green (FITC) and Kaede red (PE) channels as this will result in greater signal spread.
    1. To create single-color Kaede controls, collect a spleen or blood from a Kaede-Tg mouse. Lyse the red blood cells with ACK buffer, then divide the cells into two groups: unconverted Kaede green and converted Kaede red.
    2. For single color Kaede red controls, suspend the cells in 1 mL of PBS and photoconvert in a 24-well plate for 5 min using a 405 nm light sources at a power of 100 mW. Use these cells to set the Kaede green (FITC) and Kaede red PMT voltages on the flow cytometer (Step 9.1).
  2. Once the voltages for the Kaede proteins has been set, compensate for all other primary antibody stains using single-fluorophore labeled compensation beads per manufacturer’s instructions.
  3. Run the samples on a flow cytometer to collect data.
    Note: The Kaede protein is continually being produced by the cells. This means that 24 h after photoconversion, converted cells will be double positive for Kaede green and Kaede red as newly synthesized Kaede (green) protein has accumulated in the cell.
  4. Analyze the data using FlowJo software or a similar software.

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Results

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We first sought to replicate photoconversion results published in the literature to evaluate the efficiency and determine the associated inflammation in the mouse skin. The ear pinna was exposed to 100 mW violet light (405 nm) for 3 min as previously described33. Single cell suspensions generated from the ear skin or cervical dLNs immediately following the exposure revealed a 78% conversion efficiency of all CD45+ leukocytes in the skin with no converted...

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Discussion

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Although the leukocyte egress from peripheral, non-lymphoid tissues is critical for the initiation and resolution of immune responses, the molecular mechanisms that govern egress are poorly understood. This gap in knowledge is largely due to ready availability of tools for the quantification in vivo. Here, we describe the use of photoconvertible mice (Kaede-Tg) to quantify endogenous leukocyte egress from the skin and tumors and provide a direct head-to-head comparison with FITC paint in inflammatory and infecti...

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Disclosures

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The authors have no conflicts to disclose.

Acknowledgements

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The authors would like to thank Dr. Marcus Bosenberg for providing YUMM 1.1 and YUMM 1.7 murine melanoma lines and Dr. Deborah J. Fowell for providing B6.Cg-Tg(CAG-tdKaede)15Utr mice in agreement with RIKEN BRC through the National Bio-Resource of the MEXT, Japan.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Collagenase DRoche11088866001
DNaseRoche4536282001
Silver-LED-405B light source with optical fiber and collimtorPrizmatix LtdV8144
Fluorescein isothiocyanate isomer ISigma-AldrichF4274
dibutyl phthalateSigma-Aldrich524980
acetoneMacron Fine Chemicals2440-02
29-guage syringesExel International26029
Evans BlueSigma-AldrichE2129
70 um cell strainersVWR732-2758
paraformaldehydeSigma-AldrichP6148
HBSSCaissonHBL06
LIVE/DEAD Fixable Aqua Dead Cell Stain KitInvitrogenL34966
Purified Anti-mouse CD16/CD32Tonbo Biosciences70-0161-M001
BV605 CD11c (clone N418)Biolegend117334
PerCP-Cy5.5 MHCII (clone M5/114.15.2)BD Pharmingen562363
BV421 CD3e (clone 145-2C11)Biolegend100341
APC CD8a (clone 53-6.7)TonBo Biosciences20-0081-u100
APC-Cy7 CD45 (clone 30-F11)Biolegend103116
BV650 CD19 (clone 6D5)Biolegend115541
PercCP-Cy5.5 Ly6C (clone HK1.4)Biolegend128011
Alexa Fluor 647 F4/80 (clone BM8)Biolegend123121
APC-Cy7 Ly6G (clone 1A8)Biolegend127623
BV711 CD11b (clone M1/70)Biolegend101241
BV605 CD45 (clone 30-F11)Biolegend103155
BV711 CD4 (clone RM4-5)BD Biosciences563726
Bovine serum albumin (Fraction V)Fisher ScientificBP1600-100
Anit-Rat and Anti-Hamster Igk / Negative Control Compensation Particle SetBD Biosciences552845
Fortessa Flow CytometerBD Biosciences
FlowJo v10 SoftwareFlowJo

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Photoconversion TechniqueFlow Cytometry AnalysisKaede Transgenic MouseMurine Skin TumorsCollagenase D Digestion405 Nanometer LightSingle Cell SuspensionsDraining Lymph Nodes

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