Summary

成像的CD4 T细胞间质迁移发炎真皮

Published: March 25, 2016
doi:

Summary

支配CD4效应T细胞的间质动力在炎症部位的机制比较陌生。我们提出了一种非侵入性的方式来可视化和体外 -primed CD4T细胞操纵在发炎耳朵真皮,允许这些细胞在原位的动态行为的研究。

Abstract

CD4 + T细胞进行的效应子功能的能力取决于通过一个尚未未定义机制这些细胞在发炎外周组织的迅速和有效的迁移。多光子显微镜对免疫系统的研究中的应用提供了测量的完整组织内的免疫应答的动力学的工具。这里,我们提出在发炎的小鼠耳真皮的CD4 T细胞的非侵入性活体多光子成像的协议。使用定制成像平台和静脉导管允许的CD4 T细胞动力学在真皮间质的可视化,通过加入阻断抗体中涉及能动性关键分子组分来询问这些细胞中的实时的能力。该系统提供了在体外模型和微创手术成像程序既有优势。了解的CD4 T细胞的活力所使用的途径,最终可能提供洞察到基CD4 + T细胞的C函数以及自身免疫性疾病的慢性感染的发病机制和病理。

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

所有涉及小鼠的程序是由罗切斯特大学的机构动物护理和使用委员会批准,并严格按照动物福利法和人文关怀和实验动物的使用公共卫生服务政策由国家机构管理的实施卫生,实验室动物福利办公室。 1.效应CD4 + T细胞的制备注:特异性识别从鸡蛋卵清蛋白(:ISQAVHAAHAEINEAGR POVA)肽BALB / c小鼠TCR转基因小鼠DO11.10。其他TCR转基因系统可以被取代,使用适当的?…

Representative Results

研究原位免疫反应,而不改变免疫环境的能力是研究与发炎组织T效应细胞的实时交互必需的。由这个协议中,在图1A和B中概述的完好耳真皮的成像,允许转移荧光标记的T效应细胞在皮肤间质的可视化。这允许两个高分辨率( 图1C)和延时( 图1D, 电影1)在发炎的真皮效应T细胞动力学的图片。 <p cl…

Discussion

意义

这里,我们提出一个完整的协议为传输,抗原特异性效应Th1细胞在完整小鼠耳真皮4D可视化。此方法提供了以下几个原因一些当前成像模式的优点。通过成像腹耳真皮,我们能够放弃所需的涉及其他部位皮肤成像方案脱毛。虽然脱毛剂通常是温和的,它们已被证明导致破坏皮肤屏障42,这个过程可以刺激免疫应答43,44。还通过避免侵入外科手术以暴…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

作者感谢罗切斯特多光子显微镜核心设施的大学与实时成像帮助。支持NIH AI072690和AI02851到DJF; AI114036以股份公司和AI089079到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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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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