Los mecanismos que regulan la motilidad intersticial de las células T CD4 efectoras en sitios de inflamación son relativamente desconocidos. Se presenta un enfoque no invasivo para visualizar y manipular en las células T CD4 in vitro -primed en la dermis para los oídos inflamados, lo que permite el estudio del comportamiento dinámico de estas células in situ.
La capacidad de las células CD4 T para llevar a cabo funciones efectoras depende de la migración rápida y eficiente de estas células en los tejidos periféricos inflamados a través de un mecanismo que aún no definido. La aplicación de la microscopía multifotónica para el estudio del sistema inmune proporciona una herramienta para medir la dinámica de la respuesta inmune dentro de tejidos intactos. Aquí se presenta un protocolo para no invasiva de imágenes multifotónica intravital de las células T CD4 + en la dermis del oído del ratón inflamadas. El uso de una plataforma de formación de imágenes a medida y un catéter venoso permite la visualización de la dinámica de células T CD4 en el intersticio dérmica, con la capacidad de interrogar a estas células, en tiempo real a través de la adición de anticuerpos de bloqueo a los componentes moleculares clave que participan en la motilidad. Este sistema ofrece ventajas sobre ambos modelos in vitro y procedimientos de imagen invasivos de tipo quirúrgico. Comprensión de las vías utilizadas por las células T CD4 para la motilidad última instancia, puede dar una idea de las Basic función de las células CD4 T, así como la patogénesis de ambas enfermedades autoinmunes y patología de las infecciones crónicas.
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.
Significado
Aquí se presenta un protocolo completo para la visualización de 4D, efectoras específicas de antígenos células Th1 transferidos en la dermis del oído del ratón intactas. Este método proporciona ventajas sobre algunas modalidades de imagen actuales para varias razones. Formando imágenes de la dermis del oído ventrales, somos capaces de renunciar a la eliminación del vello que se requiere para los protocolos de imágenes que incluye otros sitios de la piel. Aunq…
The authors have nothing to disclose.
Los autores agradecen a la Universidad de Rochester instalación Multifotónica Microscopio Núcleo para obtener ayuda con imágenes en directo. Apoyado por el NIH AI02851 y AI072690 a DJF; AI114036 a AG y AI089079 de MGO.
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 |