Beeldvorming CD4 T Cell interstitiële Migratie in de ontstoken Dermis
Summary
De mechanismen die de interstitiële beweeglijkheid van CD4-effector-T-cellen regelen op plaatsen van ontsteking zijn relatief onbekend. Wij presenteren een niet-invasieve benadering visualiseren en manipuleren in vitro geprimed CD4 T-cellen in het ontstoken oor dermis, waardoor studie van het dynamisch gedrag van deze cellen in situ.
Abstract
Het vermogen van CD4 T-cellen om effectorfuncties uitvoeren is afhankelijk van de snelle en efficiënte migratie van deze cellen in ontstoken perifere weefsels door middel van een vooralsnog ongedefinieerde mechanisme. De toepassing van multifoton microscopie voor de studie van het immuunsysteem een werktuig om de dynamiek van immuunresponsen in intacte weefsels te meten. Hier presenteren we een protocol voor niet-invasieve beeldvorming van intravitale multifoton CD4 T-cellen in de ontstoken muisoor dermis. Gebruik van een aangepaste imaging platform en een veneuze katheter maakt de visualisatie van CD4 T-cel dynamica in de huid interstitium, met de mogelijkheid om deze cellen in real-time uitlezen via de toevoeging van blokkerende antilichamen tegen belangrijke moleculaire componenten betrokken bij motiliteit. Dit biedt voordelen ten opzichte van in vitro modellen en chirurgisch invasieve beeldvorming procedures. Het begrijpen van de paden die worden gebruikt door CD4 T-cellen voor de beweeglijkheid kan uiteindelijk geven inzicht in de basic functie van CD4 T-cellen en de pathogenese van zowel auto-immuunziekten en pathologie van chronische infecties.
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
Representative Results
Discussion
Betekenis
Hier presenteren wij u een protocol voor 4D visualisatie overgedragen, antigeen-specifieke effector Th1 cellen in het intacte muizenoor dermis. Deze werkwijze biedt voordelen ten opzichte van sommige van de huidige beeldvormingsmodaliteiten om verschillende redenen. Door de beeldvorming van de ventrale oor dermis, zijn we in staat om ontharing die nodig is voor de beeldvorming protocollen waarbij andere huid websites af te zien. Hoewel ontharingsmiddelen algemeen mild, ble…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
De auteurs danken de Universiteit van Rochester Multiphoton Microscope Core faciliteit voor hulp bij het live-imaging. Ondersteund door NIH AI072690 en AI02851 naar DJF; AI114036 naar AG en AI089079 te 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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