炎症部位でのCD4エフェクターT細胞の間質運動を支配するメカニズムは、比較的知られていません。我々は、in vitroで視覚化して操作するための非侵襲的なアプローチは、その場でこれらの細胞の動的挙動の研究を可能にする、炎症を起こした耳の真皮中のCD4 T細胞を-primed提示します。
エフェクター機能を実行するCD4 T細胞の能力は、未未定義の機構を介して炎症の末梢組織におけるこれらの細胞の迅速かつ効率的な移行に依存します。免疫系の研究に多光子顕微鏡の応用は、無傷の組織内の免疫応答の動態を測定するためのツールを提供しています。ここでは、炎症を起こしたマウス耳真皮におけるCD4 T細胞の非侵襲的な生体内多光子イメージングのためのプロトコルを提示します。カスタムイメージングプラットフォームの使用及び静脈カテーテルは、運動性に関与する重要な分子成分に対する抗体をブロッキング添加によってリアルタイムでこれらの細胞に問い合わせする能力を、皮膚間質におけるCD4 T細胞の動態の可視化を可能にします。このシステムは、in vitroモデルおよび外科的侵襲的な画像化手順の両方に勝る利点を提供します。運動のためのCD4 T細胞によって使用される経路を理解することは、最終的にバシへの洞察を提供することができますCのCD4 T細胞の機能だけでなく、慢性感染症からの両方の自己免疫疾患の病因と病理。
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.
意義
ここでは、無傷のマウス耳真皮における転送、抗原特異的なエフェクターTh1細胞の4D可視化のための完全なプロトコルを提示します。この方法は、いくつかの理由のために、いくつかの現在の撮像モダリティ上の利点を提供します。腹側の耳の真皮を画像化することによって、我々は他の皮膚部位を含むイメージング・プロトコルのために必要とされる脱毛?…
The authors have nothing to disclose.
著者は、ライブイメージングのヘルプのためのロチェスター大学の多光子顕微鏡中核施設に感謝します。 DJFにNIH AI072690とAI02851によってサポートされています。 MGOへAGおよびAI089079にAI114036。
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 |