Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Summary

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

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

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 node....

Protocol

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....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Collagenase D	Roche	11088866001
DNase	Roche	4536282001
Silver-LED-405B light source with optical fiber and collimtor	Prizmatix Ltd	V8144
Fluorescein isothiocyanate isomer I	Sigma-Aldrich	F4274
dibutyl phthalate	Sigma-Aldrich	524980
acetone	Macron Fine Chemicals	2440-02
29-guage syringes	Exel International	26029
Evans Blue	Sigma-Aldrich	E2129
70 um cell strainers	VWR	732-2758
paraformaldehyde	Sigma-Aldrich	P6148
HBSS	Caisson	HBL06
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit	Invitrogen	L34966
Purified Anti-mouse CD16/CD32	Tonbo Biosciences	70-0161-M001
BV605 CD11c (clone N418)	Biolegend	117334
PerCP-Cy5.5 MHCII (clone M5/114.15.2)	BD Pharmingen	562363
BV421 CD3e (clone 145-2C11)	Biolegend	100341
APC CD8a (clone 53-6.7)	TonBo Biosciences	20-0081-u100
APC-Cy7 CD45 (clone 30-F11)	Biolegend	103116
BV650 CD19 (clone 6D5)	Biolegend	115541
PercCP-Cy5.5 Ly6C (clone HK1.4)	Biolegend	128011
Alexa Fluor 647 F4/80 (clone BM8)	Biolegend	123121
APC-Cy7 Ly6G (clone 1A8)	Biolegend	127623
BV711 CD11b (clone M1/70)	Biolegend	101241
BV605 CD45 (clone 30-F11)	Biolegend	103155
BV711 CD4 (clone RM4-5)	BD Biosciences	563726
Bovine serum albumin (Fraction V)	Fisher Scientific	BP1600-100
Anit-Rat and Anti-Hamster Igk / Negative Control Compensation Particle Set	BD Biosciences	552845
Fortessa Flow Cytometer	BD Biosciences
FlowJo v10 Software	FlowJo