Summary

Imaging CD4 T cellule interstiziali migrazione nel derma infiammate

Published: March 25, 2016
doi:

Summary

I meccanismi che regolano la motilità interstiziale di cellule CD4 T effettrici in siti di infiammazione sono relativamente sconosciuti. Presentiamo un approccio non invasivo per visualizzare e manipolare cellule CD4 T vitro -primed nel derma dell'orecchio infiammate, permettendo studio del comportamento dinamico di queste cellule in situ.

Abstract

La capacità delle cellule T CD4 per svolgere funzioni effettrici dipende dalla migrazione rapida ed efficiente di queste cellule in tessuti periferici infiammati attraverso un meccanismo non ancora definito. L'applicazione della microscopia multiphoton allo studio del sistema immunitario fornisce uno strumento per misurare la dinamica delle risposte immunitarie all'interno tessuti intatti. Qui vi presentiamo un protocollo per l'imaging non invasivo intravitale multifotonica delle cellule T CD4 nel derma per le orecchie del mouse infiammate. Uso di una piattaforma di imaging personalizzato e un catetere venoso permette la visualizzazione delle dinamiche di cellule CD4 T nell'interstizio dermico, con la possibilità di interrogare queste cellule in tempo reale mediante l'aggiunta di anticorpi bloccanti di componenti molecolari coinvolti nella motilità. Questo sistema fornisce vantaggi rispetto ai due modelli in vitro e procedure di imaging invasivi di tipo chirurgico. La comprensione dei percorsi utilizzati dalle cellule T CD4 per la motilità può infine fornire una conoscenza delle basifunzione c di cellule CD4 T e patogenesi di entrambe le malattie autoimmuni e patologia da infezioni croniche.

Introduction

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.

Protocol

Tutte le procedure che coinvolgono i topi sono stati approvati dal Comitato Cura e uso istituzionale degli animali dell'Università di Rochester, e condotte in stretta conformità con la legge sulla protezione degli animali e la politica Public Health Service su Humane cura e l'uso di animali da laboratorio amministrato dal National Institutes della Salute, Ufficio di laboratorio Animal Welfare. 1. Preparazione di cellule effettrici T CD4 NOTA: BALB / c top…

Representative Results

La possibilità di studiare le risposte immunitarie in loco senza alterare l'ambiente immunitario è essenziale per lo studio delle interazioni in tempo reale delle cellule T effettrici con un tessuto infiammato. Imaging del derma orecchie intatte di questo protocollo, descritto nella Figura 1A e B, permette la visualizzazione di trasferire cellule T effettrici fluorescente nell'interstizio cutanea. Questo permette sia ad alta risoluzion…

Discussion

Importanza

Qui vi presentiamo un protocollo completo per la visualizzazione 4D di, antigene-specifica effettrici cellule Th1 trasferiti nel derma per le orecchie del mouse intatti. Questo metodo fornisce vantaggi rispetto alcune modalità di imaging correnti per diversi motivi. Con l'imaging del derma orecchie ventrali, siamo in grado di rinunciare depilazione che è necessario per i protocolli di imaging che coinvolgono altri siti cutanei. Anche se per la depilazione sono gener…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Gli autori ringraziano l'Università di Rochester struttura Multiphoton Microscopio Nucleo per un aiuto con immagini dal vivo. Supportato da NIH AI072690 e AI02851 a DJF; AI114036 di AG e AI089079 di MGO.

Materials

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

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Cite This Article
Gaylo, A., Overstreet, M. G., Fowell, D. J. Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis. J. Vis. Exp. (109), e53585, doi:10.3791/53585 (2016).

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