Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Summary

We describe a method to visualize GFP-labeled γδ IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions.  

Abstract

Intraepithelial lymphocytes expressing γδ T cell receptor (γδ IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the γδ T cell receptor, our understanding of the regulation of γδ IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating γδ IEL function and the responsiveness of these cells to the local microenvironment. Although γδ IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP γδ T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

Introduction

Intraepithelial lymphocytes (IEL) are located within the intestinal epithelium, and are found both along the basement membrane and between adjacent epithelial cells in the lateral intercellular space¹. There is approximately one IEL for every 5-10 epithelial cells; these IELs serve as sentinels to provide immune surveillance of the large expanse of the intestinal epithelial barrier². IELs expressing the γδ T cell receptor (TCR) comprise up to 60% of the total IEL population in the murine small intestine. Studies in γδ T-cell-deficient mice demonstrate a largely protective role of these cells in respon....

Protocol

All studies were conducted in an Association of the Assessment and Accreditation of Laboratory Animal Care (AALAC)-accredited facility according to protocols approved by Rutgers New Jersey Medical School Comparative Medicine Resources.

1. Mouse Preparation

NOTE: The following procedure, including animal preparation and surgery, will take 30–40 min. Prior to the surgery, turn on the microscope and warm up the enclosed incubator on the microscope to 37 °C.

1. Perform experiments on 8–10 weeks old TcrdEGFP mice on a C57BL/6 background, which were obtained from Bernard Malis....

Results

Using intravital imaging of TcrdEGFP reporter mice, we have previously shown that γδ IELs exhibit a dynamic surveillance behavior, in which they patrol the epithelium by migrating along the basement membrane and into the lateral intercellular space (LIS) at steady state (Figure 2, Movie 1).

This approach can also be used to evaluate how the inhibition of specific cell signaling pathways and/or receptors influences γδ IEL migratory behavior. For ex.......

Discussion

The development of intravital microscopy techniques has provided an unprecedented opportunity to observe the reorganization of subcellular structures^8,9,22, cell-cell interactions^12,25 and cell migratory behavior^13,14,15,16,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm dish, No. 1.5 Coverslip	MatTek	P35G-1.5-14-C
Alexa Fluor 633 Hydrazide	Invitrogen	A30634
BD PrecisionGlide Hypodermic needles - 27 G	Thermo Fisher Scientific	14-826-48
BD Slip Tip Sterile Syringe - 1 mL	Thermo Fisher Scientific	14-823-434
BD Tuberculin Syringe	Thermo Fisher Scientific	14-829-9
Dissecting scissors	Thermo Fisher Scientific	08-940
Electrocautery	Thermo Fisher Scientific	50822501
Enclosed incubation chamber	OKOLAB		Microscope
Eye Needles, Size #3; 1/2 Circle, Taper Point, 12 mm Chord Length	Roboz	RS-7983-3
Hank's Balanced Salt Solution	Sigma-Aldrich	55037C
Hoechst 33342	Invitrogen	H3570
Imaris (v. 9.2.1) with Start, Track, XT modules	Bitplane		Software
Inverted DMi8	Leica		Microscope
IQ3 (v. 3.6.3)	Andor		Software
Ketamine	Putney		Anesthesia
Kimwipes	VWR	21905-026
McPherson-Vannas scissors 3” (7.5 cm) Long 5 x 0.15 mm Straight Sharp	Roboz	RS-5600
Non-absorbable surgical suture, Silk Spool, Black Braided	Fisher Scientific	NC0798934
Nugent Forceps 4.25” (11 cm) Long Angled Smooth 1.2 mm Tip	Roboz	RS-5228
Puralube Vet Ointment	Dechra		Lubricating Eye Ointment
Spinning disk Yokogawa CSU-W1 with a 63x 1.3 N.A. HC PLAN APO glycerol immersion objective, iXon Life 888 EMCCD camera, 405 nm diode laser, 488 nm DPSS laser, 640 nm diode laser	Andor		Confocal system
Xylazine	Akorn		Anesthesia