This study was supported by JDRF grants 1-INO-2015-74-S-B, 2-SRA-2016-238-S-B, and SRA-2-S-2018-648-S-B, a Diabetes Education and Action Award, and the Caroline Wiess Law Fund for Research in Molecular Medicine at Baylor College of Medicine. Cell-sorting was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the NIH (S10RR024574 and P30CA125123). All the peptide-MHC tetramers were obtained from the NIH Tetramer Core Facility.

Mice were maintained under specific pathogen-free conditions at a Transgenic Mouse Facility, and all animal experiments were performed in accordance with protocols approved by the Baylor College of Medicine animal care and use committee.
NOTE: The experiment requires preparing the virus and the T cells in parallel. Table 1 summarizes the protocol. The key reagents and buffers are listed in the Table of Materials. We focus on the generation and expansion of CAR...

<ol>
	<li>Atkinson, M. A., Eisenbarth, G. S., Michels, A. W. <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.">Type 1 Diabetes.</a> Lancet. 383, 69-82 (2014).</li><li>Bankovich, A. J., Girvin, A. T., Moesta, A. K., Garcia, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+register+shifting+within+the+MHC+groove:+theory+becomes+reality.">Peptide register shifting within the MHC groove: theory becomes reality.</a> Molecular Immunology. 40 (14-15), 1033-1039 (2004).</li><li>Nakayama, M., 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=Prime+role+for+an+insulin+epitope+in+the+development+of+type+1+diabetes+in+NOD+mice.">Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice.</a> Nature. 435, 220-223 (2005).</li><li>Crawford, F., 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=Specificity+and+detection+of+insulin-reactive+CD4++T+cells+in+type+1+diabetes+in+the+nonobese+diabetic+(NOD)+mouse.">Specificity and detection of insulin-reactive CD4+ T cells in type 1 diabetes in the nonobese diabetic (NOD) mouse.</a> Proceedings of the National Academy of Sciences of the United States of America. , 16729-16734 (2011).</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=Diabetogenic+T+cells+recognize+insulin+bound+to+IAg7+in+an+unexpected,+weak+binding+register.">Diabetogenic T cells recognize insulin bound to IAg7 in an unexpected, weak binding register.</a> Proceedings of the National Academy of Sciences of the United States of America. , 10978-10983 (2010).</li><li>Zhang, L., 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=Monoclonal+antibody+blocking+the+recognition+of+an+insulin+peptide-MHC+complex+modulates+type+1+diabetes.">Monoclonal antibody blocking the recognition of an insulin peptide-MHC complex modulates type 1 diabetes.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 2656-2661 (2014).</li><li>Zhang, L., 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=Chimeric+antigen+receptor+(CAR)+T+cells+targeting+a+pathogenic+MHC+class+II:peptide+complex+modulate+the+progression+of+autoimmune+diabetes.">Chimeric antigen receptor (CAR) T cells targeting a pathogenic MHC class II:peptide complex modulate the progression of autoimmune diabetes.</a> Journal of Autoimmunity. 96, 50-58 (2019).</li><li>Purbhoo, M. A., Irvine, D. J., Huppa, J. B., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+killing+does+not+require+the+formation+of+a+stable+mature+immunological+synapse.">T cell killing does not require the formation of a stable mature immunological synapse.</a> Nature Immunology. 5, 524-530 (2004).</li><li>Huppa, J. B., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell-antigen+recognition+and+the+immunological+synapse.+Nature+Reviews.">T-cell-antigen recognition and the immunological synapse. Nature Reviews.</a> Immunology. 3, 973-983 (2003).</li><li>Irvine, D. J., Purbhoo, M. A., Krogsgaard, M., Davis, M. 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MAb287 and its derivatives are protected by a US patent issued in 2014.

This protocol describes an efficient method for producing antigen-specific CD8 CAR-T cells by retroviral transduction. The transduction efficiency of our protocol is typically high, and robust expression of the CAR is generally observed. The expanded CAR T cells retain the essential features of the parent-activated T cells, and antibody specificity, and are suitable for both in vitro and in vivo use. We have applied Ab-CAR CD8 T cells in reprograming Type 1 Diabetes in NOD mice7.
<p class="jov...

Given the likely reduced risk of unwanted off-target effects, antigen-specific immune therapies (ASI) are promising treatments for autoimmune diseases such as T1D. Accumulating evidence suggests that immune responses to (prepro)insulin may be particularly important in T1D1. In the past decade, studies from multiple groups, including our own, strongly suggest that presentation of an epitope containing insulin B chain amino acids 9 to 23 by specific MHC class II molecules (B:9-23/MHCII), plays an important role in the development of T1D in mice and humans2,3,4,5. To selectively target the B:9-23/MHCII complex, we generated a monoclonal antibody, named mAb287, that has no cross reactivity to the hormone insulin or complexes containing other peptides6. MAb287 blocks antigen presentation in vitro, and weekly administration of mAb287 to pre-diabetic NOD mice delayed the development of T1D in 35% of the treated mice6. To block antigen presentation in vivo, frequent injections are typically required in order to maintain a high circulating concentration. We hypothesized that we could overcome this difficulty by taking advantage of the high specificity of Ab287 to reprogram T cells, thereby providing an improved antigen-specific T cell therapy for T1D7.
Cytotoxic T cells are reported to be able to kill their target if even a single copy of their cognate ligand is expressed8,9,10. Thus, B:9-23/MHCII specific CD8 T cells are expected to have higher efficiency in eliminating the unwanted antigen presentation than the parent antibody, which will likely need to bind to multiple complexes on the same APC to exert its effect. CAR T cells have been used for treating multiple human cancers11,12,13, and may also be efficacious in autoimmunity14. However, CAR-T cells with specificity for pathogenic peptide-MHC complexes have not so far been used to modify the progression of T1D. By using the optimized CD8 T cell transduction technique described below, we recently demonstrated proof of principle that this indeed represents a viable approach7.
In this protocol, we outline an efficient and streamlined transduction and expansion method. Our protocol is applicable to other studies requiring the generation of mouse CD8 CAR T cells with high efficiency.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol (50mM)</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td>50 uM</td>
		</tr>
		<tr>
			<td>5&#8217; RACE PCR</td>
			<td>Clontech</td>
			<td>634859</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD28 antibodies</td>
			<td>eBioscience</td>
			<td>14-0281-86</td>
			<td>final concentration at 1&#181;g/ml</td>
		</tr>
		<tr>
			<td>anti-mouse CD3e antibody</td>
			<td>eBioscience</td>
			<td>145-2C11</td>
			<td>final concentration at 1&#181;g/ml</td>
		</tr>
		<tr>
			<td>Biotin Rat Anti-Mouse IFN-&#947;</td>
			<td>BD Biosciences</td>
			<td>554410</td>
			<td>Working concentration at 0.5 &#181;g/ml</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>Endo-free Maxi-Prep kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td>Final 50 &#181;g/ml.</td>
		</tr>
		<tr>
			<td>Heat inactivated FCS</td>
			<td>Hyclone</td>
			<td>SH30087.03</td>
			<td>Final 10% FCS</td>
		</tr>
		<tr>
			<td>HEPES (100X)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>1X</td>
		</tr>
		<tr>
			<td>IAg7-CLIP tetramer-BV421</td>
			<td>NIH tetramer Facility at Emory</td>
			<td>per approval</td>
			<td>Working concentration at 6 &#181;g/ml</td>
		</tr>
		<tr>
			<td>IAg7-insulin P8E tetramer-BV421</td>
			<td>NIH tetramer Facility at Emory</td>
			<td>per approval</td>
			<td>Working concentration at 6 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-Ethanolamine (ITS 100x)</td>
			<td>ThermoFisher</td>
			<td>51500056</td>
			<td>Final concentraion is 1x</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Separation Buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse CD8a+ T Cell Isolation Kit</td>
			<td>Miletenyi Biotec Inc</td>
			<td>130-104-075</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse CD8a+ T Cell Isolation Kit</td>
			<td>Miltenyi Biotec</td>
			<td>130-104-075</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM medium</td>
			<td>ThermoFisher</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (5000U/ml)</td>
			<td>ThermoFisher</td>
			<td>15070063</td>
			<td>50 U/ml</td>
		</tr>
		<tr>
			<td>Phoenix-ECO cells</td>
			<td>ATCC</td>
			<td>CRL-3214</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>pMIG II</td>
			<td>Addgene</td>
			<td>52107</td>
			<td></td>
		</tr>
		<tr>
			<td>pMSCV-IRES-GFP II</td>
			<td>Addgene</td>
			<td>52107</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse IFN-&#947;</td>
			<td>BD Biosciences</td>
			<td>551216</td>
			<td>Working concentration at 3 &#181;g/ml</td>
		</tr>
		<tr>
			<td>Red cell lysis buffer</td>
			<td>Sigma</td>
			<td>R7767</td>
			<td></td>
		</tr>
		<tr>
			<td>RetroNectin</td>
			<td>Takara</td>
			<td>T100A</td>
			<td>Working concentration at 50 &#181;g/ml in PBS</td>
		</tr>
		<tr>
			<td>rhIL-2 (stock concentration 105 IU/ul)</td>
			<td>Peprotech</td>
			<td>200-02</td>
			<td>Final concentration at 200 IU/ml</td>
		</tr>
		<tr>
			<td>rmIL-7 ( stock concentration 50ng/ul)</td>
			<td>R&#38;D</td>
			<td>407-ML-005</td>
			<td>Final concentration at 0.5ng/ml</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell Strainers</td>
			<td>Fisher Scientific</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryple</td>
			<td>Gibco</td>
			<td>12605-028</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Typically, the transduction efficiency using this protocol is ~60-90%. In the experiment shown in Figure 3, prior to sorting approximately, 70% of the CD8 T cells co-expressed GFP. They also co-expressed CD28 and CD3 (Figure 3C). Importantly, all of the &#8220;test&#8221; GFP+ cells also co-stained with IAg7-B:R3 tetramers, but not with the control tetramer (Figure 4). Similarly...

Watch this Scientific Journal Video about High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model at JoVE.com

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

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 ...

This protocol allows you to generate a large number of functional antigen-specific T cells which are a desired safe approach in treating autoimmune diseases. The main advantage of this technique is that it provides you with a highly efficient transduction process and a large number of functional T cells. This technique can be extended to treat type 1 diabetes and other autoimmune diseases in which antigens are known.Furthermore, CAR T cells may be a promising treatment for certain types of cancer. This method can be used to generate immune cells expressing other CARs. It is important to have good accepted technique, read the entire protocol and plan the whole experiment in advance.This two week long protocol includes mini-steps and success is dependent on proper performance of each step. Visual demonstrations of this method is critical for learners to follow correctly. Prepare Phoenix cells for transfection as described in the manuscript.It is good practice to mark the side wall where medium will be added and removed to minimize blowing off cells. On day zero aspirate the supernatant from the cells and wash them with five milliliters of PBS. Carefully add seven milliliters of reduced serum medium dropwise to the side wall of the plate and transfer the cells back to the incubator.Add 1.5 milliliters of reduced serum medium to two 14 millimeter round bottom polypropylene tubes. Add 40 micrometers of transfection reagent to one of the tubes and 15 micrograms of antibody redirected CAR plasmid along with five micrograms of packaging plasmid to the other. Incubate the tubes at room temperature for five minutes and then add transfection reagent to the plasmid tube.Mix by gently pipetting the solution up and down three times and incubate it at room temperature for at least 20 minutes. Add three milliliters of the mixture to the Phoenix cells and place them in a tissue culture incubator. After four to five hours add one milliliter of FCS to the cells and culture them overnight at 37 degrees Celsius.On the next day, remove the supernatant from the cells and add four milliliters of fresh prewarmed culture medium. Harvest the virus on day two by collecting the virus containing medium from the Phoenix cells and filtering it to remove residual cell debris. Add recombinant human IL-2 stock to the virus for a final concentration of 200 international units per milliliter and use it immediately for transduction.Add four milliliters of fresh medium to the Phoenix cells and place them in the incubator overnight. Repeat virus collection on the next day for a second transduction and discard the cells. One day prior to seeding murine CD8 T cells, add one milliliter of a mixture of anti-mouse CD3 and CD28 antibodies to each well of a 24 well plate and incubate the plate overnight at four degree Celsius.On day zero, harvest mouse spleens and put them on a cell strainer soaking in 10 milliliters of PBS in a cell culture dish on ice. In a cell culture hood, cut each spleen into three to five pieces and use a sterile plunger of a syringe to press the tissue through the wire mesh. Gently remove red blood cells by resuspending splenocytes in one to four diluted red cell lysis buffer and incubating them at room temperature for five minutes then dilute 10 microliters of the cell suspension with Trypan Blue for cell counting.And pellet the rest of the cells by centrifuging at 350 times g for seven minutes. Use a mouse CD8 T cell isolation kit to enrich CD8 T cells and suspend the cell pellets in 400 microliters of buffer with 100 microliters of biotin antibody cocktail per one times 10 to the eighth cells. Mix the cell suspension well and incubate it for five minutes at four degrees Celsius to allow for antibody binding.Then add 300 microliters of labeling buffer and 200 microliters of Anti-Biotin MicroBeads per one times 10 to the eighth cells. Mix well and incubate for another 10 minutes at four degrees Celsius. Meanwhile, set up a separation column on the separator and wash it with three milliliters of labeling buffer.Put a 40 micrometer cell strainer on the top of a column and pass one milliliter of the bead and cell mixture through the strainer into the separation column. Collect the flow through into a prechilled 15 milliliter tube and wash the column according to the manufacturer's directions, collecting effluent into the same tube. Count the cells and collect them by centrifugation at 350 times g for five minutes.Wash them with two milliliters of T cell medium and centrifuge again. Then resuspend them in prewarmed T cell medium at a concentration of 250, 000 to 500, 000 cells per milliliter. Wash the previously prepared CD3 and CD28 antibody coated plates with one milliliter of sterile PBS three times and add two milliliters of the cell suspension to each well.Use a swirling motion to dispense cells evenly. As a control, plate the same number of CD8 T cells into a single non-coated well of the plate and incubate the cells at 37 degrees Celsius in a 10%carbon dioxide gas tank incubator for 48 hours. On day one, prepare human fibronectin fragment coated plates by adding 0.5 milliliters of fibronectin to the wells of a 24-well plate and incubating it over night at four degrees Celsius.On the next day, remove the fibronectin solution and add one milliliter of 2%BSA and PBS per well. Incubate the plate at room temperature for 30 minutes and wash the treated wells with one milliliter of sterile PBS. The plate is ready to use after removing wash solution.Count the activated CD8 T cells using Trypan Blue. Collect them by centrifugation. And resuspend them at five million viable cells per milliliter for transduction.Add 100 microliters of the activated CD8 cell suspension to each well of the fibronectin coated plate. Then add 1.5 to two milliliters of the previously prepared virus containing medium and mix using a swirling motion to evenly dispensed the cells. Seal the plate in a Ziploc bag and centrifuge it at 2, 000 times g for 90 minutes at 37 degrees Celsius.Remove the plate from the centrifuge and take it to a biological safety cabinet. Carefully remove the plastic bag and ensure that the outside of the plate is not contaminated with medium. Transfer the plate to a 37 degrees Celsius carbon dioxide incubator.After four hours, remove one milliliter of medium from each well, replace it with one milliliter of prewarmed complete T cell medium and put the plate back in the incubator. The critical aspects of this protocol are a high titer virus, healthy activated T cells, and the appropriate transduction method. Cells were co-stained with PE size seven conjugated anti-CD8, AF647 conjugated anti-CD3, and BV 421 conjugated anti-CD28 for flow cytometric analysis.Approximately, 70%of the CD8 T cells co-expressed GFP which indicates CAR expression. They also co-expressed CD28 and CD3. Importantly, all of the test GFP positive cells also co-stained with I-Ag7 insulin BR3 tetramers which indicates expression of CAR on cell surface but not with the control tetramer.Furthermore, the sorted test and control CAR T cells each secreted high levels of interferon gamma only after coculture with target cells expressing their cognate ligands which confirms that the transduced cells have a CD8 effector T cell phenotype directed toward the target of the parent antibodies. Transduction of virus producer cells, isolation of primary CD8 T cells, and activation of these T cells are the most important steps of this experiment. Having healthy activated T cells and transduction of these T cells with appropriate reagents ensures favorable results.This method can be used to transduce other T cells but it must be customized for the specific T cell type. This protocol paves the way for the prevention of type 1 diabetes with antigen-specific chimeric antigen receptor T cell therapy.

high-efficiency-generation-antigen-specific-primary-mouse-cytotoxic-t

Research

JoVE Journal

Immunology and Infection

7.7K Görüntüleme.  University of Colorado Denver. Bu protokol, otoimmün hastalıkların tedavisinde istenilen güvenli bir yaklaşım olan fonksiyonel antijene özgü T hücrelerinin çok sayıda oluşturmanızı sağlar. Bu tekniğin en büyük avantajı, size çok verimli bir transdüksiyon süreci ve çok sayıda fonksiyonel T hücresi sağlamasıdır. Bu teknik tip 1 diyabet ve antijenlerin bilindiği diğer otoimmün hastalıkların tedavisinde genişletilebilir.Ayrıca, CAR T hücreleri kanser belirli türleri için umut verici...