Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Abstract

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

Introduction

The NOD mouse develops autoimmune diabetes spontaneously and has been widely used as an animal model for human T1D1,2. Pathogenesis of T1D in NOD mice is characterized by infiltration, beginning at 3-4 weeks of age, of the pancreatic islets of Langerhans by dendritic cells and macrophages, followed by T and B cells. This phase of non-destructive peri-insulitis leads to a slow, progressive destruction of insulin-producing pancreatic β cells, resulting in overt diabetes by 4-6 months of age3. Transfer of splenocytes4,5, CD4+6,7 or CD8+8,9 T cells from diabetic NOD mice have been shown to mediate diabetes in imm....

Protocol

1. Isolation of T Cells from Spleen and Lymph Nodes of BDC2.5 Mice

  1. Use 6-week-old pre-diabetic female BDC2.5 mice as donors of diabetogenic CD4+ T cells. Mice should be diabetes-free as determined by urine glucose measurement (see below).
  2. Euthanize each mouse using CO2 asphyxiation and remove the spleen, axillary and brachial lymph nodes under sterile conditions. To remove the spleen, soak the fur with 70% ethanol, then cut and retract skin. The spleen will be visible as a dark red organ .......

Representative Results

Our results show the isolation of transgenic BDC2.5 cells expressing CD62L, which is critical for T cells to home to secondary lymphoid organs such as pancreatic lymph nodes. Our findings further demonstrate the potent ability of this monospecific T cell population to transfer rapidly and efficiently T1D to NOD.SCID recipient mice.

Isolation of diabetogenic CD4+ T cells from BDC2.5 mice is shown in Figure 2. Approximately 5 x 107 cells from pooled spleen and lymph n.......

Discussion

T1D can be induced in recipient mice at varying efficiencies by adoptive transfer of whole spleen cells or T cell subsets from diabetic NOD mice or mice transgenic for TCRs derived from diabetogenic T cell clones. We report herein a reproducible method to induce T1D in recipient mice within two weeks at 100% incidence by transferring FACS-purified CD62L+ BDC2.5 transgenic CD4+ T cells into NOD.SCID mice.

Specific advantages of the BDC2.5 T cell transfer model described here include the very s.......

Disclosures

All mice were housed at the Penn State College of Medicine specific pathogen-free (SPF) facility in accordance with the guidelines of the Penn State Institutional Animal Care and Use Committee.

The authors declare that they have no competing financial interests.

Acknowledgements

We thank Drs. Robert Bonneau and Neil Christensen for helpful comments.

This work was supported by Pennsylvania State University College of Medicine funds.

....

Materials

NameCompanyCatalog NumberComments
Name of Reagent/MaterialCompanyCatalog NumberComments
BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(TcrαBDC 2.5, TcrβBDC 2.5)JAX004460 
NOD.SCID mice (NOD.CB17-Prkdcscid/J)JAX001303 
Dulbecco's Modified Eagle's Medium (DMEM)Themo ScientificSH30022.01 
Bayer DiastixFisher ScientificAM2803 
15 ml conical tubesFalcon352095 
50 ml conical tubesFalcon352070 
Sterile surgical tweezers   
Sterile small pair scissors   
Sterile large pair scissors   
70 μm cell strainersFisher Scientific22363548 
35 μm cell strainer cap tubesBD Biosciences352235 
Ammonium-Chloride-Potassium (ACK) buffer  0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2 in dH2O
BD FACSFlowTM sheath fluidBD Biosciences342003 
FACS staining buffer  PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized
Phase contrast microscope   
Trypan blue   
Hemocytometer   
Anti-CD4 (APC) mAbBiolegend1005616clone RM4-5
Anti-TCR Vβ4 (FITC) mAbBD Biosciences553365clone KT4
Anti-CD62L (PE) mAbBD Biosciences553151clone MEL-14
Cell sorterBD Biosciences e.g. BD FACSAria III
Heat lamp   
Mouse restrainer   
1 ml syringesBecton Dickinson309602 
18-1½ gauge needles (sterile)Becton Dickinson305196 
27½ gauge needles (sterile)Becton Dickinson305109 

References

  1. Makino, S., et al. Breeding of a non-obese, diabetic strain of mice. Jikken Dobutsu. 29, 1-13 (1980).
  2. van Belle, T. L., Coppieters, K. T., von Herrath, M. G. Type 1 diabetes: etiology, immunology, and therapeutic strategies.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Accelerated Type 1 DiabetesAdoptive TransferDiabetogenic CD4 T CellsNOD MouseAutoimmune DiabetesT Cell Receptor BDC2 5NOD SCID RecipientT1D PathogenesisTherapeutic Interventions

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved