Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

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 T1D^1,2. 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 splenocytes^4,5, 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 on the left side of the mouse. Make a 1-inch incision in the peritoneum using the small scissors and gently grasp the spleen in the center with a pair....

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.......

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(TcrαBDC 2.5, TcrβBDC 2.5)	JAX	004460
NOD.SCID mice (NOD.CB17-Prkdcscid/J)	JAX	001303
Dulbecco's Modified Eagle's Medium (DMEM)	Themo Scientific	SH30022.01
Bayer Diastix	Fisher Scientific	AM2803
15 ml conical tubes	Falcon	352095
50 ml conical tubes	Falcon	352070
Sterile surgical tweezers
Sterile small pair scissors
Sterile large pair scissors
70 μm cell strainers	Fisher Scientific	22363548
35 μm cell strainer cap tubes	BD Biosciences	352235
Ammonium-Chloride-Potassium (ACK) buffer			0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2 in dH2O
BD FACSFlowTM sheath fluid	BD Biosciences	342003
FACS staining buffer			PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized
Phase contrast microscope
Trypan blue
Hemocytometer
Anti-CD4 (APC) mAb	Biolegend	1005616	clone RM4-5
Anti-TCR Vβ4 (FITC) mAb	BD Biosciences	553365	clone KT4
Anti-CD62L (PE) mAb	BD Biosciences	553151	clone MEL-14
Cell sorter	BD Biosciences		e.g. BD FACSAria III
Heat lamp
Mouse restrainer
1 ml syringes	Becton Dickinson	309602
18-1½ gauge needles (sterile)	Becton Dickinson	305196
27½ gauge needles (sterile)	Becton Dickinson	305109