Name: Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells (Video) | JoVE Journal
Uploaded: 2013-05-06T13:00:00.000+00:00
Duration: 6 min 27 s
Description: Learn to rapidly induce type 1 diabetes in mice by adoptive transfer of diabetogenic CD4+ T cells from BDC 2.5 donors. This reproducible protocol enables diabetes development within two weeks for studying pathogenesis and therapeutic interventions.

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5 minutes

We thank Drs. Robert Bonneau and Neil Christensen for helpful comments.	
This work was supported by Pennsylvania State University College of Medicine funds.

1. Isolation of T Cells from Spleen and Lymph Nodes of BDC2.5 Mice
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Collect the spleens and lymph nodes in 10 ml Dulbecco's-Modified Eagle's Medium (DMEM) in a 15 ml conical tube on ice.</li>
	<li>Prepare a single cell suspension by using the end of a sterile 10 ml syringe plunger to gently press the lymphoid organs through 70 &mu;m cell strainers (one spleen per strainer and 4-6 lymph nodes per strainer) into the same 50 ml conical tube.</li>
</ol>
During the process, rinse each strainer with 1 ml DMEM several times to maximize the recovery of cells from the strainer.
<ol start="5">
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Fluorescence-activated Cell Sorting of Diabetogenic CD4+ T Cells from BDC2.5 Mice
<ol>
	<li>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.</li>
	<li>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 V&beta;4 (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 &deg;C in the dark.</li>
	<li>Wash the sample and single stain controls with FACS buffer at &gt;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 &mu;l for single stain controls) and store on ice until the next step.</li>
	<li>Pass the cell sample through a 35 &mu;m cell strainer cap tube to remove cell clumps. Sort the samples for CD4+TCR V&beta;4+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.</li>
	<li>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.</li>
</ol>
3. Adoptive Transfer of Diabetogenic CD4+ T Cells from BDC2.5 Mice
<ol>
	<li>Using a 1 ml syringe and an 18-1&frac12; gauge needle, gently re-suspend the FACS-purified CD4+ T cells and load the 1 ml syringe. In preparation for injection, replace the 18-1&frac12; gauge needle with a 27&frac12; gauge needle.</li>
	<li>Expose 6-8 week-old female NOD.SCID recipient mice to a heating lamp until they exhibit grooming behavior.</li>
	<li>Restrain recipient mice in a restrainer and wipe their tail with 70% (v/v) ethanol to disinfect the injection site.</li>
	<li>Inject 1-2 x 106 FACS sorted cells/mouse (in up to 500 &mu;l PBS) into either of the lateral tail veins.</li>
</ol>
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
<ol>
	<li>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 &gt;250 mg/dl are considered diabetic.</li>
</ol>

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<li>van Belle, T. L., Coppieters, K. T., von Herrath, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type+1+diabetes%3A+etiology%2C+immunology%2C+and+therapeutic+strategies%5BTitle%5D)+AND+%22Physiol.+Rev%22%5BJournal%5D)">Type 1 diabetes: etiology, immunology, and therapeutic strategies.</a> Physiol. Rev. 91, 79-118 (2011).</li>
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<li>Christianson, S. W., Shultz, L. D., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adoptive+transfer+of+diabetes+into+immunodeficient+NOD-scid%2Fscid+mice.+Relative+contributions+of+CD4%2B+and+CD8%2B+T-cells+from+diabetic+versus+prediabetic+NOD.NON-Thy-1a+donors%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Adoptive transfer of diabetes into immunodeficient NOD-scid/scid mice. Relative contributions of CD4+ and CD8+ T-cells from diabetic versus prediabetic NOD.NON-Thy-1a donors.</a> Diabetes. 42, 44-55 (1993).</li>
<li>Wicker, L. S., Todd, J. A., Peterson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+control+of+autoimmune+diabetes+in+the+NOD+mouse%5BTitle%5D)+AND+%22Annual+Reviews+in+Immunology%22%5BJournal%5D)">Genetic control of autoimmune diabetes in the NOD mouse.</a> Annual Reviews in Immunology. 13, 179-200 (1995).</li>
<li>Serreze, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MHC+class+I-mediated+antigen+presentation+and+induction+of+CD8%2B+cytotoxic+T-cell+responses+in+autoimmune+diabetes-prone+NOD+mice%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">MHC class I-mediated antigen presentation and induction of CD8+ cytotoxic T-cell responses in autoimmune diabetes-prone NOD mice.</a> Diabetes. 45, 902-908 (1996).</li>
<li>Milton, M. J., Poulin, M., Mathews, C., Piganelli, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation%2C+maintenance%2C+and+adoptive+transfer+of+diabetogenic+T-cell+lines%2Fclones+from+the+nonobese+diabetic+mouse%5BTitle%5D)+AND+%22Methods+Mol.+Med%22%5BJournal%5D)">Generation, maintenance, and adoptive transfer of diabetogenic T-cell lines/clones from the nonobese diabetic mouse.</a> Methods Mol. Med. 102, 213-225 (2004).</li>
<li>Kurrer, M. O., Pakala, S. V., Hanson, H. L., Katz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beta+cell+apoptosis+in+T+cell-mediated+autoimmune+diabetes%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Beta cell apoptosis in T cell-mediated autoimmune diabetes.</a> Proc. Natl. Acad. Sci. U.S.A. 94, 213-218 (1997).</li>
<li>Amrani, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perforin-independent+beta-cell+destruction+by+diabetogenic+CD8%28%2B%29+T+lymphocytes+in+transgenic+nonobese+diabetic+mice%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Perforin-independent beta-cell destruction by diabetogenic CD8(+) T lymphocytes in transgenic nonobese diabetic mice.</a> J. Clin. Invest. 103, 1201-1209 (1999).</li>
<li>Dobbs, C., Haskins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+a+T+cell+clone+and+of+T+cells+from+a+TCR+transgenic+mouse%3A+TCR+transgenic+T+cells+specific+for+self-antigen+are+atypical%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Comparison of a T cell clone and of T cells from a TCR transgenic mouse: TCR transgenic T cells specific for self-antigen are atypical.</a> J. Immunol. 166, 2495-2504 (2001).</li>
<li>Haskins, K., Portas, M., Bradley, B., Wegmann, D., Lafferty, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T-lymphocyte+clone+specific+for+pancreatic+islet+antigen%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">T-lymphocyte clone specific for pancreatic islet antigen.</a> Diabetes. 37, 1444-1448 (1988).</li>
<li>Katz, J. D., Wang, B., Haskins, K., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Following+a+diabetogenic+T+cell+from+genesis+through+pathogenesis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Following a diabetogenic T cell from genesis through pathogenesis.</a> Cell. 74, 1089-1100 (1993).</li>
<li>Stadinski, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromogranin+A+is+an+autoantigen+in+type+1+diabetes%5BTitle%5D)+AND+%22Nat.+Immunol%22%5BJournal%5D)">Chromogranin A is an autoantigen in type 1 diabetes.</a> Nat. Immunol. 11, 225-231 (2010).</li>
<li>Luhder, F., Chambers, C., Allison, J. P., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pinpointing+when+T+cell+costimulatory+receptor+CTLA-4+must+be+engaged+to+dampen+diabetogenic+T+cells%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">Pinpointing when T cell costimulatory receptor CTLA-4 must be engaged to dampen diabetogenic T cells.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 12204-12209 (2000).</li>
<li>Tang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.</a> J. Exp. Med. 199, 1455-1465 (2004).</li>
<li>Calderon, B., Suri, A., Pan, X. O., Mills, J. C., Unanue, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IFN-gamma-dependent+regulatory+circuits+in+immune+inflammation+highlighted+in+diabetes%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">IFN-gamma-dependent regulatory circuits in immune inflammation highlighted in diabetes.</a> J. Immunol. 181, 6964-6974 (2008).</li>
<li>Shultz, L. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiple+defects+in+innate+and+adaptive+immunologic+function+in+NOD%2FLtSz-scid+mice%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice.</a> J. Immunol. 154, 180-191 (1995).</li>
<li>Waldner, H., Sobel, R. A., Price, N., Kuchroo, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+autoimmune+diabetes+locus+Idd9+regulates+development+of+type+1+diabetes+by+affecting+the+homing+of+islet-specific+T+cells%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">The autoimmune diabetes locus Idd9 regulates development of type 1 diabetes by affecting the homing of islet-specific T cells.</a> J. Immunol. 176, 5455-5462 (2006).</li>
<li>Verdaguer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+autoimmune+diabetes+in+monoclonal+T+cell+nonobese+diabetic+mice%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.</a> J. Exp. Med. 186, 1663-1676 (1997).</li>
<li>Thomas, D. C., Mellanby, R. J., Phillips, J. M., Cooke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+early+age-related+increase+in+the+frequency+of+CD4%2B+Foxp3%2B+cells+in+BDC2.5NOD+mice%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">An early age-related increase in the frequency of CD4+ Foxp3+ cells in BDC2.5NOD mice.</a> Immunology. 121, 565-576 (2007).</li>
<li>Prochazka, M., Gaskins, H. R., Shultz, L. D., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+nonobese+diabetic+scid+mouse%3A+model+for+spontaneous+thymomagenesis+associated+with+immunodeficiency%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.S.A%22%5BJournal%5D)">The nonobese diabetic scid mouse: model for spontaneous thymomagenesis associated with immunodeficiency.</a> Proc. Natl. Acad. Sci. U.S.A. 89, 3290-3294 (1992).</li>
</ol>

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.

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...

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 &beta; 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 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 mice7,10. 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 recipients11-13. Specifically, BDC2.5 NOD mice are transgenic for the BDC2.5 TCR, which is specific for chromogranin A, a protein in pancreatic beta cells14-16. 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 efficiencies11,17-19.
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 V&beta;4 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 na&iuml;ve, monospecific CD4+ T cells, as well as therapeutic strategies to intervene in homing of islet antigen-specific Th cells to the target organ.

<table><tbody><tr><td>BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(Tcr&amp;alpha;BDC 2.5, Tcr&amp;#946;BDC 2.5)</td><td>JAX</td><td>004460</td><td></td></tr><tr><td>NOD.SCID mice (NOD.CB17-&lt;em&gt;Prkdc&lt;sup&gt;scid&lt;/sup&gt;&lt;/em&gt;/J)</td><td>JAX</td><td>001303</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Themo Scientific</td><td>SH30022.01</td><td></td></tr><tr><td>Bayer Diastix</td><td>Fisher Scientific</td><td>AM2803</td><td></td></tr><tr><td>15 ml conical tubes</td><td>Falcon</td><td>352095</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Sterile surgical tweezers</td><td></td><td></td><td></td></tr><tr><td>Sterile small pair scissors</td><td></td><td></td><td></td></tr><tr><td>Sterile large pair scissors</td><td></td><td></td><td></td></tr><tr><td>70 &amp;mu;m cell strainers</td><td>Fisher Scientific</td><td>22363548</td><td></td></tr><tr><td>35 &amp;mu;m cell strainer cap tubes</td><td>BD Biosciences</td><td>352235</td><td></td></tr><tr><td>Ammonium-Chloride-Potassium (ACK) buffer</td><td></td><td></td><td>0.15 M NH&lt;sub&gt;4&lt;/sub&gt;Cl, 1 mM KHCO&lt;sub&gt;3, &lt;/sub&gt;0.1 mM Na&lt;sub&gt;2&lt;/sub&gt;EDTA, pH 7.2 in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>BD FACSFlowTM sheath fluid</td><td>BD Biosciences</td><td>342003</td><td></td></tr><tr><td>FACS staining buffer</td><td></td><td></td><td>PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized</td></tr><tr><td>Phase contrast microscope</td><td></td><td></td><td></td></tr><tr><td>Trypan blue</td><td></td><td></td><td></td></tr><tr><td>Hemocytometer</td><td></td><td></td><td></td></tr><tr><td>Anti-CD4 (APC) mAb</td><td>Biolegend</td><td>1005616</td><td>clone RM4-5</td></tr><tr><td>Anti-TCR V&amp;#946;4 (FITC) mAb</td><td>BD Biosciences</td><td>553365</td><td>clone KT4</td></tr><tr><td>Anti-CD62L (PE) mAb</td><td>BD Biosciences</td><td>553151</td><td>clone MEL-14</td></tr><tr><td>Cell sorter</td><td>BD Biosciences</td><td></td><td>&lt;em&gt;e.g.&lt;/em&gt; BD FACSAria III</td></tr><tr><td>Heat lamp</td><td></td><td></td><td></td></tr><tr><td>Mouse restrainer</td><td></td><td></td><td></td></tr><tr><td>1 ml syringes</td><td>Becton Dickinson</td><td>309602</td><td></td></tr><tr><td>18-1&amp;frac12; gauge needles (sterile)</td><td>Becton Dickinson</td><td>305196</td><td></td></tr><tr><td>27&amp;frac12; gauge needles (sterile)</td><td>Becton Dickinson</td><td>305109</td><td></td></tr></tbody></table>

accelerated type 1 diabetes induction in mice by adoptive transfer of diabetogenic cd4+ t cells

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic 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.

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...

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 ...

The overall goal of this procedure is to rapidly induce type one diabetes in mice by transferring diabetogenic T cells from transgenic mice into mice, which lack endogenous t and b cells. Begin by harvesting the spleens and lymph nodes from the donor mice and purifying the CD four positive T cells. Next adoptively, transfer the purified donor T cells into non skid recipient mice.Then monitor the recipient mice for type one diabetes development. Ultimately, this protocol can be used to study diabetes pathogenesis and to investigate therapeutic interventions Compared to other methods. The main advantage of this technique of transferring BDC 2.5, CD four positive T-cells into non-skid recipients is the rapid and reproducible development of type one diabetes.For donors of the Diabetogenic CD four positive T cells use six week old pre-diabetic female BDC 2.5 mice that have been determined to be diabetes free by urine glucose measurement. To remove the spleen and lymph nodes, soak the fur with 70%ethanol, then cut the skin, retract the skin until the muscles of the front limbs are exposed. Near the base of the front limbs, two lymph nodes on each side should be visible Carefully Grasp each lymph node with small tweezers and remove it by trimming the connective tissue.Next, make a one inch incision in the peritoneum using the small scissors. Now gently grasp the spleen in the center. Carefully trim away the connective tissue and the attached fat.Then remove the spleen, lace, the spleens and lymph nodes in 10 milliliters of DMEM on ice. With the end of a sterile 10 milliliter syringe plunger, gently press the lymphoid organs through a 70 micrometer cell strainer. To obtain a single cell suspension.Rinse each strainer with one milliliter of DMEM several times to maximize the recovery of cells. Then transfer the collected cells into a 15 milliliter conical tube and centrifuge at 400 Gs for seven minutes. At room temperature, re suspend the cell pellet in three milliliters of a CK buffer.Now incubate every room temperature for five minutes. To lye the red blood cells now add 12 milliliters of DMEM and pellet the cells by centrifugation. Wash the cell pellet once in 10 milliliters of DMEM.Next, we suspend the cells in five milliliters of fact staining buffer, and use a phase contrast microscope and hemo cytometer to count the number of viable cells. Now remove about one times 10 of the six cells per staining control. Also stain the cells with mouse monoclonal antibodies to sort for nonactivated transgenic CD four positive T-cell.Incubate all those samples at four degrees Celsius in the dark for 30 minutes. Now wash the cells with three times the staining volume of fax buffer and resuspend the cells in fax buffer one to two times 10 to the seven cells per milliliter for sorting sample and 300 microliters for single stain controls. Next, pass the cell sample through a 35 micrometer cell strainer cap tube.To remove cell clumps process the samples for a cell sorter with a trained operator Allow about three hours, including setup time to sort about 1.5 times 10 to the eighth totals Transgenic naive CD four positive T cells. You can expect about 2.5 times 10 of the six cells per mouse and the sort rate's gonna be about two times 10 of the fourth events per second on an instrument such as the BD Fox Aria. Collect the sorted cells into three milliliters of DMEM.Record the absolute number of sorted cells and centrifuge them for seven minutes. At 400 Gs, we suspend the cell pellets in sterile phosphate buffered saline for the adoptive T-cell transfer. Using a one milliliter syringe in an 18 gauge needle, gently resuspend the fax purified CD four positive T cells.Load the one milliliter syringe then in preparation for injection, attach a 27 point a half gauge needle to the syringe. Restrain the knot skid recipient mice and wipe their tail with 70%ethanol to disinfect the injection site. Then inject one times 10th of the six purified CD four positive T-cells per mouse into either of the lateral tail veins.When doing your tail vein injections, make sure that you don't force the plunger. If the needle is actually in the vein, your injection should occur with almost no resistance Beginning five days after the T-cell transfer. Monitor the nod skid recipient mice daily for development of type one diabetes.Measure the urine glucose using reagent strips according to the manufacturers and instructions. Note that mice with two consecutive urine glucose readings of greater than 250 milligrams per deciliter are considered diabetic. In a typical experiment, each BDC 2.5 donor mouse yields about two and a half times 10 of the six naive transgenic CD four positive T cells after cell sorting after adoptive transfer of diabetogenic CD four positive T cells, the mice were monitored for urine glucose concentrations.The adoptive transfer of small numbers of cells from BDC 2.5 donor mice induced type one diabetes in all non-skid recipients by day 11. When done properly, this technique can be done in approximately eight hours from organ harvest to T-cell transfer.

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Research

JoVE Journal

Immunology and Infection

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Learn to rapidly induce type 1 diabetes in mice by adoptive transfer of diabetogenic CD4+ T cells from BDC 2.5 donors. This reproducible protocol enables diabetes development within two weeks for studying pathogenesis and therapeutic interventions.

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

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