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>

<ol>
	<li>Makino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+of+a+non-obese,+diabetic+strain+of+mice.">Breeding of a non-obese, diabetic strain of mice.</a> Jikken Dobutsu. 29, 1-13 (1980).</li><li>van Belle, T. L., Coppieters, K. T., von Herrath, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+1+diabetes:+etiology,+immunology,+and+therapeutic+strategies.">Type 1 diabetes: etiology, immunology, and therapeutic strategies.</a> Physiol. Rev. 91, 79-118 (2011).</li><li>Delovitch, T. L., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nonobese+diabetic+mouse+as+a+model+of+autoimmune+diabetes:+immune+dysregulation+gets+the+NOD.">The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD.</a> Immunity. 7, 727-738 (1997).</li><li>Wicker, L. S., Miller, B. J., Mullen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+autoimmune+diabetes+mellitus+with+splenocytes+from+nonobese+diabetic+(NOD)+mice.">Transfer of autoimmune diabetes mellitus with splenocytes from nonobese diabetic (NOD) mice.</a> Diabetes. 35, 855-860 (1986).</li><li>Bendelac, A., Carnaud, C., Boitard, C., Bach, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syngeneic+transfer+of+autoimmune+diabetes+from+diabetic+NOD+mice+to+healthy+neonates.+Requirement+for+both+L3T4++and+Lyt-2++T+cells.">Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates. Requirement for both L3T4+ and Lyt-2+ T cells.</a> J. Exp. Med. 166, 823-833 (1987).</li><li>Haskins, K., McDuffie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+diabetes+in+young+NOD+mice+with+a+CD4++islet-specific+T+cell+clone.">Acceleration of diabetes in young NOD mice with a CD4+ islet-specific T cell clone.</a> Science. 249, 1433-1436 (1990).</li><li>Christianson, S. W., Shultz, L. D., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+control+of+autoimmune+diabetes+in+the+NOD+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+class+I-mediated+antigen+presentation+and+induction+of+CD8++cytotoxic+T-cell+responses+in+autoimmune+diabetes-prone+NOD+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation,+maintenance,+and+adoptive+transfer+of+diabetogenic+T-cell+lines/clones+from+the+nonobese+diabetic+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta+cell+apoptosis+in+T+cell-mediated+autoimmune+diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perforin-independent+beta-cell+destruction+by+diabetogenic+CD8(+)+T+lymphocytes+in+transgenic+nonobese+diabetic+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-lymphocyte+clone+specific+for+pancreatic+islet+antigen.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+a+diabetogenic+T+cell+from+genesis+through+pathogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromogranin+A+is+an+autoantigen+in+type+1+diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pinpointing+when+T+cell+costimulatory+receptor+CTLA-4+must+be+engaged+to+dampen+diabetogenic+T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-gamma-dependent+regulatory+circuits+in+immune+inflammation+highlighted+in+diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+defects+in+innate+and+adaptive+immunologic+function+in+NOD/LtSz-scid+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+autoimmune+diabetes+locus+Idd9+regulates+development+of+type+1+diabetes+by+affecting+the+homing+of+islet-specific+T+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+autoimmune+diabetes+in+monoclonal+T+cell+nonobese+diabetic+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+early+age-related+increase+in+the+frequency+of+CD4++Foxp3++cells+in+BDC2.5NOD+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nonobese+diabetic+scid+mouse:+model+for+spontaneous+thymomagenesis+associated+with+immunodeficiency.">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

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...

Watch this Scientific Journal Video about Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells at JoVE.com

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

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 ...

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16.6K Views.  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.

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

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Biology

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.
Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.
In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

We describe hepatic neo-islet formation in STZ (streptozotocin)-induced diabetic mice by gene transfer of Neurogenin3 (Ngn3) and Betacellulin (Btc) using helper-dependent adenoviral vector (HDAd) and the reversal of hyperglycemia. Our method takes advantages of helper-dependent adenoviral vectors with their highly efficient in vivo transduction and the long lasting gene expression.

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the ...

Medicine

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Bioengineering

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation

Source: Buchele, V., et al. <a href="https://app.jove.com/v/58871">Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice.</a> J. Vis. Exp. (2019).
This video demonstrates the induction of intestinal graft-versus-host disease, GvHD, in a lethally-irradiated recipient mouse using allogeneic hematopoietic stem cells and GvHD-inducing splenic alloreactive T lymphocytes. This protocol helps establish a severe acute intestinal GvHD model to understand the mechanism of the disease and develop innovative therapeutic options.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. GvHD ...

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

Summary

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Chapters in this video

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4⁺CD45RB^high T Cells into Immunodeficient Mice

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4⁺ T Cells to Immunodeficient Mice

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Induction of Graft Versus Host Disease in Mouse Models via Allogeneic Bone Marrow and Splenic Cell Transplantation