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Immunology and Infection

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

Published: May 6th, 2013

DOI:

10.3791/50389

1Department of Microbiology & Immunology, Pennsylvania State University College of Medicine

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.

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.

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....

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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 .......

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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.......

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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.......

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

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

....

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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  

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