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 immunocompromised NOD mice, indicating that islet-reactive T cells play a central role in T1D pathogenesis. Depending on the experimental conditions, diabetes developed in recipient mice slowly, over several weeks in these studies. Similarly, various T cell clones, derived by time-consuming and costly culturing of diabetogenic T cells, have been reported to mediate diabetes several weeks after transfer into recipient mice^7,10. With the availability of transgenic mice expressing TCRs derived from CD4- or CD8-restricted diabetogenic T cell clones, several laboratories have subsequently shown that splenic T cells from such mice were able to transfer diabetes to recipients^11-13. Specifically, BDC2.5 NOD mice are transgenic for the BDC2.5 TCR, which is specific for chromogranin A, a protein in pancreatic beta cells^14-16. Transfer of in vitro-activated or un-activated whole or fractionated spleen cells from overtly diabetic or prediabetic BDC2.5 mice transferred diabetes to neonatal or immunodeficient NOD mice at varying efficiencies^11,17-19.

We describe a simple method that utilizes purified transgenic CD4+ T cells from pre-diabetic BDC2.5 mice to induce T1D in recipient mice at high efficiency and consistency. Large numbers of naive, islet antigen-specific CD4+ T cells are isolated from these mice by fluorescence-activated cell sorting (FACS) for CD4+CD62L+ T cells expressing the transgenic TCR Vβ4 chain. Purified transgenic T cells are then transferred without activation into NOD.SCID mice, which lack functional T and B cells and are insulitis- and diabetes-free²⁰. The recipient mice are monitored for elevated concentrations of urine glucose indicating T1D, which develops rapidly within two weeks after the T cell transfer.

In contrast to other methods that transfer diabetogenic T cells with heterogenous specificities, our protocol uses FACS-sorted CD4+ T cells that almost exclusively express the diabetogenic BDC2.5 TCR. Due to their homogeneity, only small numbers of transferred T cells (~1x10⁶ cells/mouse) are required for rapid T1D development within 2 weeks at 100% incidence. Another advantage of our protocol is that irradiation of recipient mice is not necessary as it is for some other methods. A potential limitation of this method is that it does not allow the investigation into the contribution of both CD4 and CD8 T cell subsets or specifically CD8 T cells in diabetes.

The described protocol will be useful for studying rapid T1D development, mediated by naïve, monospecific CD4+ T cells, as well as therapeutic strategies to intervene in homing of islet antigen-specific Th cells to the target organ.

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 of small tweezers. Carefully trim the connective tissue and attached fat as much as possible and remove the spleen.
3. Collect the spleens and lymph nodes in 10 ml Dulbecco's-Modified Eagle's Medium (DMEM) in a 15 ml conical tube on ice.
4. Prepare a single cell suspension by using the end of a sterile 10 ml syringe plunger to gently press the lymphoid organs through 70 μm cell strainers (one spleen per strainer and 4-6 lymph nodes per strainer) into the same 50 ml conical tube.

During the process, rinse each strainer with 1 ml DMEM several times to maximize the recovery of cells from the strainer.

5. Transfer the collected cells from the 50 ml conical tube into a 15 ml conical tube and centrifuge at 300-400 x g for 7 min at RT.
6. To lyse red blood cells, discard the supernatant, re-suspend the cells in 5 ml Ammonium-Chloride-Potassium (ACK) buffer (pH 7.2), and incubate the ACK-cell suspension at RT for 5 min.
7. Add 10 ml of DMEM to the ACK-cell suspension and centrifuge the tube as above. Wash the cell pellet once in 10 ml DMEM.

2. Fluorescence-activated Cell Sorting of Diabetogenic CD4+ T Cells from BDC2.5 Mice

1. Re-suspend cells in 5 ml FACS staining buffer and count the number of viable cells (using a phase contrast microscope and a hemocytometer) by trypan blue dye exclusion.
2. Using FACS buffer, adjust the cell suspension volume to 5 x 10⁷ cells/ml. Remove ~1 x 10⁶ cells per staining control (1 no stain, 3 single stains). Stain the sample for non-activated transgenic CD4+ T cells with anti-CD4 (APC), anti-TCR Vβ4 (FITC) and anti-CD62L (PE) monoclonal antibodies (mAb) in a 15 ml tube. Perform cell staining using the mAb concentrations suggested by the manufacturer for 20-30 min at 4 °C in the dark.
3. Wash the sample and single stain controls with FACS buffer at >3X the staining reaction volume. Centrifuge the tube at 300-400 x g, remove the supernatant, and re-suspend the cells in FACS buffer (1-2 x 10⁷ cells/ml for sorting sample and 300 μl for single stain controls) and store on ice until the next step.
4. Pass the cell sample through a 35 μm cell strainer cap tube to remove cell clumps. Sort the samples for CD4+TCR Vβ4+CD62L+ cells on a cell sorter with a trained operator. Collect the sorted cells into 3 ml DMEM in a 15 ml tube. Allow 2.5-3 hr, including setup, to sort 1.5 x 10⁸ cells (the approximate number of cells obtained from 3 donor mice) at a sort rate of 2 x 10⁴ total events/sec. Expect ~2.5 x 10⁶ non-activated transgenic CD4+ T cells per mouse after cell sorting.
5. Record the absolute number of sorted cells at the end of the sort and centrifuge them for 7 min at 300-400 x g. Discard the supernatant and re-suspend the cells (2 x 10⁶ cells/ml) in sterile phosphate-buffered saline (PBS) (Mg²⁺/Ca²⁺ free) for the adoptive T cell transfer.

3. Adoptive Transfer of Diabetogenic CD4+ T Cells from BDC2.5 Mice

1. Using a 1 ml syringe and an 18-1½ gauge needle, gently re-suspend the FACS-purified CD4+ T cells and load the 1 ml syringe. In preparation for injection, replace the 18-1½ gauge needle with a 27½ gauge needle.
2. Expose 6-8 week-old female NOD.SCID recipient mice to a heating lamp until they exhibit grooming behavior.
3. Restrain recipient mice in a restrainer and wipe their tail with 70% (v/v) ethanol to disinfect the injection site.
4. Inject 1-2 x 10⁶ FACS sorted cells/mouse (in up to 500 μl PBS) into either of the lateral tail veins.

Do not force the plunger. If the needle is located appropriately in the vein, the injection will take place with almost no resistance.

4. Monitoring Recipient Mice for Hyperglycemia and T1D

1. Beginning five days after the T cell transfer, NOD.SCID recipient mice are monitored daily for elevated urine glucose using reagent strips (Bayer Diastix) according to the manufacturer's instructions. Mice with two consecutive urine glucose readings >250 mg/dl are considered diabetic.

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

List of materials used in this article

Name	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