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.

<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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+ligand+recognition+by+T+cells.">Direct observation of ligand recognition by T cells.</a> Nature. 419, 845-849 (2002).</li><li>Barrett, D. M., Singh, N., Porter, D. L., Grupp, S. A., June, C. H. <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+therapy+for+cancer.">Chimeric antigen receptor therapy for cancer.</a> Annual Review of Medicine. 65, 333-347 (2014).</li><li>Kochenderfer, J. N., Yu, Z., Frasheri, D., Restifo, N. P., Rosenberg, S. A. <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+syngeneic+T+cells+transduced+with+a+chimeric+antigen+receptor+that+recognizes+murine+CD19+can+eradicate+lymphoma+and+normal+B+cells.">Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells.</a> Blood. 116, 3875-3886 (2010).</li><li>Kochenderfer, J. N., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treating+B-cell+cancer+with+T+cells+expressing+anti-CD19+chimeric+antigen+receptors.">Treating B-cell cancer with T cells expressing anti-CD19 chimeric antigen receptors.</a> Nature Reviews Clinical Oncology. 10, 267-276 (2013).</li><li>Fishman, 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=Adoptive+Transfer+of+mRNA-Transfected+T+Cells+Redirected+against+Diabetogenic+CD8+T+Cells+Can+Prevent+Diabetes.">Adoptive Transfer of mRNA-Transfected T Cells Redirected against Diabetogenic CD8 T Cells Can Prevent Diabetes.</a> Molecular Therapy. 25 (2), 456-464 (2017).</li><li>Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (1982).</li><li>Holst, 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=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nature Protocols. 1 (1), 406-417 (2006).</li><li>Johnstone, A., Thorpe, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunochemistry in practice. , (1996).</li><li>Rowland-Jones, S. L., McMichael, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lymphocytes: a practical approach. , (2000).</li><li>Pear, W. S., Nolan, G. P., Scott, M. L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+high-titer+helper-free+retroviruses+by+transient+transfection.">Production of high-titer helper-free retroviruses by transient transfection.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (18), 8392-8396 (1993).</li><li>Sena-Esteves, M., Saeki, Y., Camp, S. M., Chiocca, E. A., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+conversion+of+cells+to+retrovirus+vector+producers+with+herpes+simplex+virus-Epstein-Barr+virus+hybrid+amplicons.">Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons.</a> Journal of Virology. 73 (12), 10426-10439 (1999).</li><li>Carrero, J. A., Calderon, B., Towfic, F., Artyomov, M. N., Unanue, E. 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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=T-cell+activation+by+transitory+neo-antigens+derived+from+distinct+microbial+pathways.">T-cell activation by transitory neo-antigens derived from distinct microbial pathways.</a> Nature. 509 (7500), 361-365 (2014).</li><li>Griffith, I. 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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.
Our protocol incorporates several critical modifications to previously described methods. First, we use an optimized T cell culturing medium that allows an extended activation time. The complete medium described contains optimal levels of several key supplements, and significantly improves both T cell viability and the extent of proliferation following activation. It should be noted that mouse IL-2 can be substituted for the human protein with equivalent results, although at present, human IL-2 is more affordable. Of note, a significantly higher transduction efficiency is obtained using T cells activated for 40-48 h than if a 24 h activation step is used.
Second, we use an improved transduction procedure that eliminates polybrene B (which is toxic to the T cells) and uses fibronectin instead. This further improves cell viability. It should be noted that to guarantee good transduction efficiency it is critical to maintain the T cells in an optimized medium at an appropriate cell density and to use fresh high-titer viral supernatants rather than previously frozen virus. Using our modified procedure, a third transduction step is unnecessary and indeed is undesirable as viability typically drops if a third spin infection step is included. It must also be emphasized that it is critical to never let the cells overgrow during the expansion phase. Once cells are overgrown, they tend to rapidly lose their phenotype and die.
In addition to the parameters described above, two other potential causes of low transduction efficiency/viability must be avoided. First, as antibiotics should not be present during the transfection steps it is important to make sure that the plasmids are prepared using an endotoxin-free kit, and dissolved in sterile water, and that good sterile technique is used at all times. Second, the presence of high levels of dead or dying T cells must be avoided. If the activated parental CD8 T cell suspension contains high levels of dead cells or cell debris this should be removed prior to transduction using commercial kits.
We have deliberately not included a CAR-T cell freezing step in this protocol, as in our experience a significant proportion of the transduced cells die during cryopreservation and thawing. Similarly, although the expanded CAR-T cells can be re-stimulated in vitro, they have an increased tendency to lose expression of the transgene. Accordingly, given the high degree of proliferation we observe using freshly sorted CAR-T cells, we highly recommend that only freshly generated CAR-T cells are used for functional assays and adoptive transfer.
In summary, the significance of this protocol is that it describes a procedure that provides high transduction efficiency and generates large numbers of healthy antigen specific mouse CD8 T cells for use in vitro and in vivo. Our protocol thus provides a useful tool for researchers undertaking CAR-T cell studies in mouse models of disease.

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 &amp;mu;M</td></tr><tr><td>5&amp;rsquo; 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&amp;micro;g/ml</td></tr><tr><td>anti-mouse CD3e antibody</td><td>eBioscience</td><td>145-2C11</td><td>final concentration at 1&amp;micro;g/ml</td></tr><tr><td>Biotin Rat Anti-Mouse IFN-&amp;gamma;</td><td>BD Biosciences</td><td>554410</td><td>Working concentration at 0.5 &amp;micro;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 &amp;micro;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 &amp;micro;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 &amp;micro;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-&amp;gamma;</td><td>BD Biosciences</td><td>551216</td><td>Working concentration at 3 &amp;micro;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 &amp;micro;g/ml in PBS</td></tr><tr><td>rhIL-2 (stock concentration 10&lt;sup&gt;5&lt;/sup&gt; 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&amp;amp;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, the sorted test and control CAR-T cells each secreted high levels of IFN-&#947; only after co-culture with targets cells expressing their cognate ligands (Figure 5). This confirms that the transduced cells have a CD8 effector T cell phenotype directed towards the target of the parent antibodies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59985/59985fig1.jpg" /> 
Figure 1: Schematic of the CAR retroviral construct. The CAR comprises a targeting domain derived from the Fab fragment of a suitable mouse monoclonal antibody, and a spacer/ membrane anchor/ signaling domain from mouse CD28, CD137 and CD247. The synthetic cDNA is inserted into the pMIG-II retroviral expression vector. Restriction endonuclease sites used for generating the mAb287-CAR are shown. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59985/59985fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59985/59985fig2.jpg" /> 
Figure 2: Effect of different plating methods on cell distribution. (Left) Cells pipetted using a swirling motion show an even distribution. (Right) Cells were pipetted directly into the center of the well. Images were captured after spinning at 350 x g for 5 min. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59985/59985fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59985/59985fig3.jpg" /> 
Figure 3: Flow cytometric analysis of transduced T cells. Cells were co-stained with PE-Cy7 conjugated anti-CD8, AF647 conjugated anti-CD3, and BV421 conjugated anti-CD28, as described in step 6.4. Profiles gated on single viable cells are shown. (A) Un-stained parental CD8 T cells. (B) PE-Cy7/GFP profile of transduced cells. The CAR expressing cells are identified by the GFP reporter. (C) Stained transduced cells were gated on PE-Cy7/GFP double positivity. The AF647/BV421 profile is shown. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59985/59985fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59985/59985fig4.jpg" /> 
Figure 4: Tetramer staining of un-sorted CAR-T cells. Cells were stained with BV421 conjugated tetramers as described in step 8.1. Profiles gated on single viable cells are shown. (A) Test IAg7-insulin tetramer. (B) Control I-Ag7-HEL tetramer. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59985/59985fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59985/59985fig5.jpg" /> 
Figure 5: Antigen-specific cytokine secretion by CAR T cells. Sorted CD8 T cells expressing the test mAb287 or control mAb24.1 CAR were co-cultured with M12C3 cells expressing IAg7-B:R3, &#8220;empty&#8221; IAg7, or TFR-MBP-DTRL (the ligand for mAb24.1) as described in step 8.2. After 24 h, secreted IFN-&#947; ELISA was quantified by ELISA. Specific stimulation of both T cell lines was observed. Data represent mean &#177; SD of 3 repeated experiments. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59985/59985fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time</td>
			<td>Virus preparation</td>
			<td>T cell preparation</td>
		</tr>
		<tr>
			<td>DAY -4 (Fri)</td>
			<td>Thaw Phoenix cells (PM)</td>
			<td></td>
		</tr>
		<tr>
			<td>Day -3 (Sat)</td>
			<td>Re-plate Phoenix</td>
			<td></td>
		</tr>
		<tr>
			<td>Day -2 (Sun)</td>
			<td>Holiday</td>
			<td></td>
		</tr>
		<tr>
			<td>Day-1 (Mon)</td>
			<td>Irradiate Phoenix cells (PM)</td>
			<td>Coat non-treated plate with CD3/CD28 Ab</td>
		</tr>
		<tr>
			<td rowspan="2">Day 0 (Tue)</td>
			<td rowspan="2">Transfecting Phoenix cells (AM)</td>
			<td>Prepare splenocytes and isolate CD8 T cells.</td>
		</tr>
		<tr>
			<td>T cell activation.</td>
		</tr>
		<tr>
			<td>Day 1 (Wed)</td>
			<td>Change Phoenix cells medium to 4 ml (AM).</td>
			<td>Coat RetroNectin</td>
		</tr>
		<tr>
			<td>Day 2 (Thu)</td>
			<td>Collect virus and refill.</td>
			<td>Transduction, CD8 T cells</td>
		</tr>
		<tr>
			<td>Day 3 (Fri)</td>
			<td>Collect virus.</td>
			<td>Transduction, CD8 T cells</td>
		</tr>
		<tr>
			<td>Day 4 (Sat)</td>
			<td></td>
			<td>Wash cells and expand cells.</td>
		</tr>
		<tr>
			<td>Day 5 (Sun)</td>
			<td></td>
			<td>Cell expansion if needed.</td>
		</tr>
		<tr>
			<td>Day 6 (Mon)</td>
			<td></td>
			<td>CAR-T cell sorting.</td>
		</tr>
		<tr>
			<td>Day 7 -10 ( Tue to Fri)</td>
			<td></td>
			<td>Expansion of T CAR- cells. Experiments.</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of the CAR-T generation protocol.

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 ...

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7.8K Views.  University of Colorado Denver. 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.

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

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

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.

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 ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

Biology

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

Medicine

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-&#947;- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified ...

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 ...

A Technique for the Generation of Antigen-Specific CAR T Cells

Source: Davidson, H. W., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59985">High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model.</a> J. Vis. Exp. (2019).
This video demonstrates a technique for generating antigen-specific chimeric antigen receptor (CAR) T cells through retroviral transduction. Activated murine CD8+ T cells are added to a microwell plate coated with recombinant fibronectin fragments. Retroviral vectors containing transgenic RNA encoding an antigen-specific chimeric antigen receptor (CAR) are then introduced, and the plate is centrifuged to facilitate transduction. After incubation, the transduced cells express antigen-specific CARs on their surface.

1. Generation and validation of single chain Fab antibody (scFab)-CARs.
NOTE: CARs typically contain 3 critical domains&#8212;an antigen-targeting domain, ...

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Cellular Immunotherapy

Imaging Transgenic Beta-Cells in an Immunodeficient Mouse Challenged with Diabetic Splenocytes

Source: Stewart, T. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/64836">Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates an imaging-based method to visualize transplanted luciferase-expressing beta cells into a non-obese diabetic immunodeficient mouse. The introduction of splenocytes from a diabetic mouse leads to progressive beta cell destruction, visually confirmed by declining luminescence after substrate introduction.

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

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

Summary

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Chapters in this video

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

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

Antigenic Liposomes for Generation of Disease-specific Antibodies

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8⁺ T Cell Responses

Extraction of Tissue Antigens for Functional Assays

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

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

A Technique for the Generation of Antigen-Specific CAR T Cells

Imaging Transgenic Beta-Cells in an Immunodeficient Mouse Challenged with Diabetic Splenocytes