Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Summary

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Abstract

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

Introduction

The thymus is an organ in which critical events in T cell development occur¹. Resident thymocytes, upon rearrangement of the T cell receptor (TCR) α and β genes, undergo a series of interactions with lymphostromal and antigen presenting cells (APC) in the cortical and medullary regions of the thymus². Thymic positive selection is mediated by cortical thymic epithelial cells (TEC) to produce thymocytes that recognize antigenic peptides in the context of host major histocompatibility complex (MHC) class I and II molecules^2-3. Subsequent thymic negative selection entails purging of autoreactive thymocytes, driven by an interacti....

Protocol

The murine studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina Chapel Hill and all animal care was in accordance with the IACUC guidelines.

1. Preparation of Newborn and Adult Thymi

1. Prepare all reagents and equipment prior to euthanizing donor mice.
2. Sterilize surgical instruments by autoclaving or other appropriate methods. All surgical procedures must be performed under a laminar flow hood to maintain sterile conditions and avoid contamination. Assemble the tools needed for extraction of thymi from donor mice.
3. Fill a 60 mm dish with sterile 1x PBS (pH 7.4) and plac....

Results

The success of this procedure is dependent on minimal surgical trauma as well as the accurate positioning of the graft underneath the kidney capsule. The thymic graft should be cut to ensure appropriately sized thymic sections for subsequent transplantation as shown in Figure 1. Following the schematic in Figure 1, thymi from newborn or adult mice can be used for successful subcapsular transplantation with consistent and reproducible T cell engraftment results. As mentioned, appropriate .......

Discussion

Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge synd.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol	Decon Laboratories	2705HC
PBS	GIBCO	14190-144	pH 7.4
Betadine Solution	Purdue Products	67618-150-17
18 gauge needle	BD	305195
Sutures (1.0 metric)	Ethicon	J493G	18" (45cm)
AUTOCLIP Wound Clips (9mm)	Clay Adams® Brand	427631
Transfer pipette	Fisher Scientific	13-711-20	Sterile, disposable
Mouse anti-CD3 Ab	eBioscience	11-0031-85	Clone: 1452C11
Mouse anti-CD4 Ab	eBioscience	48-0042-82	Clone: RM4-5
Mouse anti-CD8 Ab	eBioscience	25-0081-82	Clone: 53-6.7
Lancet	Medipoint	Goldenrod 4mm
Pacific Orange Succinimidyl Ester, Triethylammonium Salt	Invitrogen	P30253
96-well round botton polypropylene plates	Corning	3365
1.2 ml polypropylene cluster tubes	Corning	4401
5 ml polypropylene round-bottom tubes	BD	352002
40uM Nylon Cell Strainer	Falcon	352340
16% Paraformaldehyde Solution, EM Grade	Electron Microscopy Services	15710	Hazardous
Puralube Optical Ointment	Fisher Scientific	NC0138063
Lympholyte	Cedarlane	CL5030
60 mm cell culture dish	Corning	430196	60 mm x 15 mm, Sterile