De mekanismer som styr den interstitiella motilitet av CD4 T-celler vid inflammationsställen är relativt okänd. Vi presenterar en icke-invasiv metod för att visualisera och manipulera in vitro -primed CD4 T-celler i de inflammerade öron dermis, vilket möjliggör studier av dynamiska beteendet hos dessa celler in situ.
Förmågan hos CD4-T-celler för att utföra effektorfunktioner är beroende av en snabb och effektiv migrering av dessa celler i inflammerade perifera vävnader genom en än så länge odefinierad mekanism. Tillämpningen av multifotonmikroskop till studiet av immunsystemet ger ett verktyg för att mäta dynamiken i immunresponser inom intakta vävnader. Här presenterar vi ett protokoll för icke-invasiv intravital multifoton avbildning av CD4 T-celler i de inflammerade mus öron dermis. Användning av en anpassad avbildning plattform och en venkateter möjliggör visualisering av CD4 T-cells dynamik i huden interstitium, med möjlighet att förhöra dessa celler i realtid via tillsats av blockerande antikroppar till viktiga molekylära komponenter som motilitet. Detta system ger fördelar gentemot både in vitro modeller och kirurgiska invasiva avbildningsförfaranden. Förstå banorna används av CD4 T-celler för rörlighet i slutändan kan ge insikt i de basic funktionen hos CD4-T-celler såväl som vid patogenesen av både autoimmuna sjukdomar och patologi från kroniska infektioner.
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.
Betydelse
Här presenterar vi en komplett protokoll för 4D visualisering av överförda, antigenspecifika effektor Th1 celler i intakta mus öron dermis. Denna metod ger fördelar över några aktuella avbildningsmetoder av flera skäl. Genom avbildning av ventrala örat dermis, har vi möjlighet att avstå hårborttagning som krävs för avbildningsprotokoll som involverar andra hudytor. Även hårborttagningsmedel är vanligtvis milda, har de visat sig orsaka störningar i hudbar…
The authors have nothing to disclose.
Författarna tackar University of Rochester multifotonmikroskop Kärn anläggning för hjälp med levande avbildning. Med stöd av NIH AI072690 och AI02851 till DJF; AI114036 till AG och AI089079 till 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 |