Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

Summary

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

Abstract

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.

As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

Introduction

At the heart of the adaptive immune system lies the ability of a T cell to recognize and respond to a specific antigen. When and where a T cell responds to its cognate antigen determines the balance of infection and autoimmunity, homeostasis and cancer, health and disease¹. It follows that the study of T cells in a specific context of immunity should focus on the cells with specificity for a relevant antigen of interest. Among the technological advancements that have greatly enhanced the ability to characterize antigen-specific T cell populations are fluorescently labeled soluble multimers (usually tetramers) of major histocompatibility co....

Protocol

The procedures described in this protocol are approved and developed in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) of Massachusetts General Hospital, an American Association for the Accreditation of Laboratory Animal Care (AAALAC)-accredited animal management program. Experiments were performed on 8-12-week-old male and female mice on a C57BL/6 genetic background bred and maintained in the MGH animal facility under specific pathogen-free conditions.

Discussion

Prior characterizations of antigen-specific T cells from the lungs have benefitted from the robust numbers of antigen-specific T cells that expand following an acute priming event such as intranasal immunization or infection^20,21,22. However, rarer T cell populations in the lungs, such as self antigen-specific T cells or tissue-resident memory T cells, are difficult to detect without some form of sample enrichment for the T cell.......

Materials

Name	Company	Catalog Number	Comments
100 mm cell strainer	Fisher Scientific	22-363-549
10x PBS without Ca++ or Mg++	Corning	46-013-CM
1x PBS without Ca++ or Mg++	Corning	21-031-CV
AccuCheck Counting Beads	Invitrogen	PCB100
Aminoguanidine Hemisulfate Salt	Sigma-Aldrich	A7009
CD90.2 microbeads, mouse	Miltenyi	130-121-278
Cell separation magnet (MidiMACS Separator)	Miltenyi	130-042-302	Holds single LS column
Cell separation magnet (QuadroMACS Separator)	Miltenyi	130-090-976	Holds 4 LS columns
Dasatinib	Sigma-Aldrich	CDS023389
DNase I	Roche	10104159001
Eagle’s Ham’s Amino Acids medium	Sigma-Aldrich	C5572
gentleMACS	Miltenyi	130-093-235	Automated tissue dissociator
gentleMACS C Tubes	Miltenyi	130-093-237	Automated tissue dissociator tubes
Hank's Balanced Salt Solution with Ca++ or Mg++	Corning	21-020-CM
HEPES	Gibco	15630080
Ketamine	Vedco	NDC 50989-996-06
Liberase TM	Roche	5401119001
Pacific Blue anti-mouse CD45 antibody (Clone: 30-F11)	Biolegend	103126
Paramagnetic cell separation columns (LS Columns)	Miltenyi	130-042-401	Comes with plunger
Purified anti mouse CD16/32 antibody (Clone: 93)	Biolegend	101302
RPMI 1640 medium without L-glutamine	Corning	15-040-CM
Sodium Chloride 0.9% (Normal Saline)	Cytiva	Z1376
Xylazine	Pivetal	NDC 466066-750-02