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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.
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.
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....
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
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.......
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.......
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.
....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 |
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