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 border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>BDC 2.5 TCR transgenic NOD mice (NOD.Cg-Tg(Tcr&alpha;BDC 2.5, Tcr&beta;BDC 2.5)</td>
			<td>JAX</td>
			<td>004460</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NOD.SCID mice (NOD.CB17-Prkdcscid/J)</td>
			<td>JAX</td>
			<td>001303</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle's Medium (DMEM)</td>
			<td>Themo Scientific</td>
			<td>SH30022.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bayer Diastix</td>
			<td>Fisher Scientific</td>
			<td>AM2803</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352095</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile surgical tweezers</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile small pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile large pair scissors</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>70 &mu;m cell strainers</td>
			<td>Fisher Scientific</td>
			<td>22363548</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 &mu;m cell strainer cap tubes</td>
			<td>BD Biosciences</td>
			<td>352235</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ammonium-Chloride-Potassium (ACK) buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2 in dH2O</td>
		</tr>
		<tr>
			<td>BD FACSFlowTM sheath fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS staining buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>PBS, 0.2 mM EDTA, 0.5% BSA/FCS, filter sterilized</td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</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&beta;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>&nbsp;</td>
			<td>e.g. BD FACSAria III</td>
		</tr>
		<tr>
			<td>Heat lamp</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse restrainer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 ml syringes</td>
			<td>Becton Dickinson</td>
			<td>309602</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>18-1&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>27&frac12; gauge needles (sterile)</td>
			<td>Becton Dickinson</td>
			<td>305109</td>
			<td>&nbsp;</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...

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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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JoVE Journal

Immunology and Infection

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4+ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during na&#239;ve CD4+ T cell activation. Also, real-time PCR experiments using &#60;40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human na&#239;ve CD4+ T cells. Also described are real-time PCR experiments using &#60;40,000 cells that do not require additional low input RNA kits.

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into na&#239;ve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

A High-Throughput Multiplexed Screening for Type 1 Diabetes, Celiac Diseases, and COVID-19

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and celiac disease in the United States. With the coronavirus disease 2019 (COVID-19) pandemic, the epidemiology of COVID-19 in the general population and knowledge about the association between COVID-19 infection and T1D development are urgently needed. The currently standard screening method of the radio-binding assay (RBA) has met two great challenges: low efficiency with a single assay format and low disease specificity with a large proportion of low-affinity antibodies generated in screening. With the platform of the multiplex electrochemiluminescence (ECL) assay we established previously, a novel 6-Plex ECL assay was developed that combines, in a single well, all four islet autoantibodies (IAbs) to insulin, glutamic acid decarboxylase (GAD65), insulinoma antigen 2 (IA-2), and Zinc transporter 8 (ZnT8) for T1D, transglutaminase autoantibodies (TGA) for celiac disease, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) antibodies for COVID-19. The assay was validated in blind using 880 samples from the ASK study, including 325 positive samples and 555 all antibody-negative samples, and compared with the standard RBAs and a single ECL assay. With the advantages of high efficiency, low cost, and low serum volume, this assay has been accepted as the primary screening tool for the ASK study.

In line with the urgent need for screening for type 1 diabetes, celiac disease, and coronavirus disease 2019, we developed a high throughput 6-Plex electrochemiluminescence assay to simultaneously detect all four islet autoantibodies, tissue transglutaminase autoantibodies, and antibodies to the receptor binding domain of severe acute respiratory syndrome coronavirus 2.

An ongoing clinical trial, Autoimmunity Screening for Kids (ASK), is the first screening study in the general population for type 1 diabetes (T1D) and ...