Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

Summary

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

Abstract

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

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 sites^1-4 and extravasation^5-7 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 matrices^8-10 or microfluidic devices^11,12....

Protocol

All procedures involving mice were approved by the Institutional Animal Care and Use Committee of the University of Rochester, and carried out in strict accordance with the Animal Welfare Act and the Public Health Service Policy on Humane Care and Use of Laboratory Animals administered by the National Institutes of Health, Office of Laboratory Animal Welfare.

1. Preparation of Effector CD4 T Cells

NOTE: BALB/c TCR-transgenic DO11.10 mice that specifically recognize a peptide from chicken egg ovalbumin (pOVA: ISQAVHAAHAEINEAGR). Other TCR-transgenic systems can be substituted, using the appropriate cognate peptide i....

Results

The ability to study immune responses in situ without altering the immune environment is essential in studying real-time interactions of T effector cells with an inflamed tissue. Imaging of the intact ear dermis by this protocol, outlined in Figure 1A and B, allows for the visualization of transferred fluorescently labeled T effector cells in the dermal interstitium. This permits both high-resolution (Figure 1C) and time-lapse (<.......

Discussion

Significance

Here we present a complete protocol for the 4D visualization of transferred, antigen-specific effector Th1 cells in the intact mouse ear dermis. This method provides advantages over some current imaging modalities for several reasons. By imaging the ventral ear dermis, we are able to forego hair removal that is required for imaging protocols involving other skin sites. Although depilatories are generally mild, they have been shown to cause disruption to the skin barrier <.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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