Name: High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model (Video) | JoVE Journal
Uploaded: 2019-08-16T14:00:00.000+00:00
Duration: 11 min 31 s
Description: Learn to generate functional antigen-specific CD8 T cells using an efficient transduction protocol for autoimmune disease and cancer research. This method enables large-scale expansion of therapeutic T cells.

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5 minutes

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/pubmed?term=((Type+1+Diabetes%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">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/pubmed?term=((Peptide+register+shifting+within+the+MHC+groove%3A+theory+becomes+reality%5BTitle%5D)+AND+%22Molecular+Immunology%22%5BJournal%5D)">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/pubmed?term=((Prime+role+for+an+insulin+epitope+in+the+development+of+type+1+diabetes+in+NOD+mice%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Specificity+and+detection+of+insulin-reactive+CD4%2B+T+cells+in+type+1+diabetes+in+the+nonobese+diabetic+%28NOD%29+mouse%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Diabetogenic+T+cells+recognize+insulin+bound+to+IAg7+in+an+unexpected%2C+weak+binding+register%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Monoclonal+antibody+blocking+the+recognition+of+an+insulin+peptide-MHC+complex+modulates+type+1+diabetes%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Chimeric+antigen+receptor+%28CAR%29+T+cells+targeting+a+pathogenic+MHC+class+II%3Apeptide+complex+modulate+the+progression+of+autoimmune+diabetes%5BTitle%5D)+AND+%22Journal+of+Autoimmunity%22%5BJournal%5D)">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/pubmed?term=((T+cell+killing+does+not+require+the+formation+of+a+stable+mature+immunological+synapse%5BTitle%5D)+AND+%22Nature+Immunology%22%5BJournal%5D)">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/pubmed?term=((T-cell-antigen+recognition+and+the+immunological+synapse.+Nature+Reviews%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">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/pubmed?term=((Direct+observation+of+ligand+recognition+by+T+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Chimeric+antigen+receptor+therapy+for+cancer%5BTitle%5D)+AND+%22Annual+Review+of+Medicine%22%5BJournal%5D)">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/pubmed?term=((Adoptive+transfer+of+syngeneic+T+cells+transduced+with+a+chimeric+antigen+receptor+that+recognizes+murine+CD19+can+eradicate+lymphoma+and+normal+B+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Treating+B-cell+cancer+with+T+cells+expressing+anti-CD19+chimeric+antigen+receptors%5BTitle%5D)+AND+%22Nature+Reviews+Clinical+Oncology%22%5BJournal%5D)">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/pubmed?term=((Adoptive+Transfer+of+mRNA-Transfected+T+Cells+Redirected+against+Diabetogenic+CD8+T+Cells+Can+Prevent+Diabetes%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aManiatis%2C+T+%22Molecular+cloning%3A+a+laboratory+manual%22">Molecular cloning: a laboratory manual</a>. , Cold Spring Harbor. N.Y. (1982).</li>
<li>Holst, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+T-cell+receptor+retrogenic+mice%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJohnstone%2C+A+%22Immunochemistry+in+practice%22">Immunochemistry in practice</a>. , 3rd edn, Blackwell Science. Cambridge, MA. (1996).</li>
<li>Rowland-Jones, S. L., McMichael, A. J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRowland-Jones%2C+SL+%22Lymphocytes%3A+a+practical+approach%22">Lymphocytes: a practical approach</a>. , 2nd ed, Practical approach series. New York. (2000).</li>
<li>Pear, W. S., Nolan, G. P., Scott, M. L., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+high-titer+helper-free+retroviruses+by+transient+transfection%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Single-step+conversion+of+cells+to+retrovirus+vector+producers+with+herpes+simplex+virus-Epstein-Barr+virus+hybrid+amplicons%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+transcriptional+and+cellular+landscape+of+type+1+diabetes+in+the+NOD+mouse%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Defining the transcriptional and cellular landscape of type 1 diabetes in the NOD mouse.</a> PLoS One. 8 (3), e59701(2013).</li>
<li>Jansen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunohistochemical+characterization+of+monocytes-macrophages+and+dendritic+cells+involved+in+the+initiation+of+the+insulitis+and+beta-cell+destruction+in+NOD+mice%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Immunohistochemical characterization of monocytes-macrophages and dendritic cells involved in the initiation of the insulitis and beta-cell destruction in NOD mice.</a> Diabetes. 43 (5), 667-675 (1994).</li>
<li>Corbett, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T-cell+activation+by+transitory+neo-antigens+derived+from+distinct+microbial+pathways%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">T-cell activation by transitory neo-antigens derived from distinct microbial pathways.</a> Nature. 509 (7500), 361-365 (2014).</li>
<li>Griffith, I. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+mutation+affecting+intracellular+transport+and+cell+surface+expression+of+murine+class+II+molecules%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">Structural mutation affecting intracellular transport and cell surface expression of murine class II molecules.</a> Journal of Experimental Medicine. 167 (2), 541-555 (1988).</li>
<li>Kozono, H., White, J., Clements, J., Marrack, P., Kappler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+soluble+MHC+class+II+proteins+with+covalently+bound+single+peptides%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Production of soluble MHC class II proteins with covalently bound single peptides.</a> Nature. 369 (6476), 151-154 (1994).</li>
<li>Allicotti, G., Borras, E., Pinilla, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+time-resolved+fluorescence+immunoassay+%28DELFIA%29+increases+the+sensitivity+of+antigen-driven+cytokine+detection%5BTitle%5D)+AND+%22Journal+of+Immunoassay+%26+Immunochemistry%22%5BJournal%5D)">A time-resolved fluorescence immunoassay (DELFIA) increases the sensitivity of antigen-driven cytokine detection.</a> Journal of Immunoassay & Immunochemistry. 24 (4), 345-358 (2003).</li>
</ol>

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 &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

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 “test” GFP+ cells also co-stained with IAg7-B:R3 tetramers, but not with the control tetramer (Figure 4). Similarly, the sorted...

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 ...

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

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Learn to generate functional antigen-specific CD8 T cells using an efficient transduction protocol for autoimmune disease and cancer research. This method enables large-scale expansion of therapeutic T cells.

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model (Video) | JoVE Journal

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

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