Name: Generation of Human Chimeric Antigen Receptor Regulatory T Cells (Video) | JoVE Journal
Uploaded: 2025-01-03T13:40:00.000+00:00
Duration: 10 min 29 s
Description: Learn to generate human chimeric antigen receptor regulatory T cells using lentiviral transduction and electroporation techniques. This protocol establishes standardized methods for developing engineered immune cell therapies.

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LMRF is funded by Human Islet Research Network (HIRN) Emerging Leader in Type 1 Diabetes grant U24DK104162-07, American Cancer Society (ACS) Institutional Research Grant IRG-19-137-20, South Carolina Clinical and Translational Research (SCTR) Pilot Project Discovery Grant 1TL1TR001451-01, Diabetes Research Connection (DRC) Grant IPF 22-1224, and Swim Across America Grant 23-1579. RWC is supported by the Cellular, Biochemical and Molecular Sciences training grant T32GM132055 and Hollings Cancer Center Lowvelo Graduate Fellowship. This study was supported in part by the Flow Cytometry and Cell Sorting Shared Resource, Hollings Cancer Center, Medical University of South Carolina (P30 CA138313). Special thanks to Dr. Qizhi Tang at the University of California, San Francisco (UCSF) for kindly gifting the CAR mutant plasmids.

Engineering and Expansion of Human CAR Tregs for Immune Regulation Therapies

<ol><li>Zhang, X., Zhu, L., Zhang, H., Chen, S., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CAR-T+cell+therapy+in+hematological+malignancies%3A+current+opportunities+and+challenges%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">CAR-T cell therapy in hematological malignancies: current opportunities and challenges.</a> Front Immunol. 13, 927153(2022).</li>
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<li>Spanier, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tregs+with+an+MHC+class+II+peptide-specific+chimeric+antigen+receptor+prevent+autoimmune+diabetes+in+mice%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">Tregs with an MHC class II peptide-specific chimeric antigen receptor prevent autoimmune diabetes in mice.</a> J Clin Invest. 133 (18), e168601(2023).</li>
<li>Muller, Y. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precision+engineering+of+an+anti-HLA-A2+chimeric+antigen+receptor+in+regulatory+T+cells+for+transplant+immune+tolerance%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance.</a> Front Immunol. 12, 686439(2021).</li>
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<li>Boardman, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flagellin-specific+human+CAR+Tregs+for+immune+regulation+in+IBD%5BTitle%5D)+AND+%22J+Autoimmun%22%5BJournal%5D)">Flagellin-specific human CAR Tregs for immune regulation in IBD.</a> J Autoimmun. 134, 102961(2023).</li>
<li>Schreeb, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+design%3A+human+leukocyte+antigen+Cclass+I+molecule+A%28%2A%2902-chimeric+antigen+receptor+regulatory+T+cells+in+renal+transplantation%5BTitle%5D)+AND+%22Kidney+Int+Rep%22%5BJournal%5D)">Study design: human leukocyte antigen Cclass I molecule A(*)02-chimeric antigen receptor regulatory T cells in renal transplantation.</a> Kidney Int Rep. 7 (6), 1258-1267 (2022).</li>
<li>Zimmerman, C. M., Robino, R. A., Cochrane, R. W., Dominguez, M. D., Ferreira, L. M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Redirecting+human+conventional+and+regulatory+T+cells+using+chimeric+antigen+receptors%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Redirecting human conventional and regulatory T cells using chimeric antigen receptors.</a> Methods Mol Biol. 2748, 201-241 (2024).</li>
<li>Tang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+decrease+of+donor-reactive+T%28regs%29+after+liver+transplantation+limits+T%28reg%29+therapy+for+promoting+allograft+tolerance+in+humans%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Selective decrease of donor-reactive T(regs) after liver transplantation limits T(reg) therapy for promoting allograft tolerance in humans.</a> Sci Transl Med. 14 (669), eabo2628(2022).</li>
<li>Bender, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+phase+2+randomized+trial+with+autologous+polyclonal+expanded+regulatory+T+cells+in+children+with+new-onset+type+1+diabetes%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">A phase 2 randomized trial with autologous polyclonal expanded regulatory T cells in children with new-onset type 1 diabetes.</a> Sci Transl Med. 16 (746), eadn2404(2024).</li>
<li>Ferreira, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conference+report%3A+Advanced+Therapies+Week+2023%5BTitle%5D)+AND+%22Regen+Med%22%5BJournal%5D)">Conference report: Advanced Therapies Week 2023.</a> Regen Med. 18 (4), 297-299 (2023).</li>
<li>Cochrane, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+test+human+CAR+T+cells+in+solid+tumors%2C+the+next+frontier+of+CAR+T+cell+therapy%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">How to test human CAR T cells in solid tumors, the next frontier of CAR T cell therapy.</a> Methods Mol Biol. 2748, 243-265 (2024).</li>
<li>Roy, A., Krzykwa, E., Lemieux, R., Neron, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+efficiency+of+gamma-irradiated+versus+mitomycin+C-treated+feeder+cells+for+the+expansion+of+normal+human+cells+in+long-term+cultures%5BTitle%5D)+AND+%22J+Hematother+Stem+Cell+Res%22%5BJournal%5D)">Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures.</a> J Hematother Stem Cell Res. 10 (6), 873-880 (2001).</li>
<li>Eyquem, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+a+CAR+to+the+TRAC+locus+with+CRISPR%2FCas9+enhances+tumour+rejection%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.</a> Nature. 543 (7643), 113-117 (2017).</li>
<li>Negrini, M., Wang, G., Heuer, A., Bjorklund, T., Davidsson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AAV+Production+everywhere%3A+a+simple%2C+fast%2C+and+reliable+protocol+for+in-house+AAV+vector+production+based+on+chloroform+extraction%5BTitle%5D)+AND+%22Curr+Protoc+Neurosci%22%5BJournal%5D)">AAV Production everywhere: a simple, fast, and reliable protocol for in-house AAV vector production based on chloroform extraction.</a> Curr Protoc Neurosci. 93 (1), e103(2020).</li>
<li>Velasco Cardenas, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Harnessing+CD3+diversity+to+optimize+CAR+T+cells%5BTitle%5D)+AND+%22Nat+Immunol%22%5BJournal%5D)">Harnessing CD3 diversity to optimize CAR T cells.</a> Nat Immunol. 24 (12), 2135-2149 (2023).</li>
<li>Boomer, J. S., Green, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+enigmatic+tail+of+CD28+signaling%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Biol%22%5BJournal%5D)">An enigmatic tail of CD28 signaling.</a> Cold Spring Harb Perspect Biol. 2 (8), a002436(2010).</li>
<li>Suhoski, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+artificial+antigen-presenting+cells+to+express+a+diverse+array+of+co-stimulatory+molecules%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Engineering artificial antigen-presenting cells to express a diverse array of co-stimulatory molecules.</a> Mol Ther. 15 (5), 981-988 (2007).</li>
<li>Fung, V. C. W., Rosado-Sanchez, I., Levings, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transduction+of+human+T+cell+subsets+with+lentivirus%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Transduction of human T cell subsets with lentivirus.</a> Methods Mol Biol. 2285, 227-254 (2021).</li>
<li>Bailey-Bucktrout, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-antigen-driven+activation+induces+instability+of+regulatory+T+cells+during+an+inflammatory+autoimmune+response%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Self-antigen-driven activation induces instability of regulatory T cells during an inflammatory autoimmune response.</a> Immunity. 39 (5), 949-962 (2013).</li>
<li>Nakagawa, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Instability+of+Helios-deficient+Tregs+is+associated+with+conversion+to+a+T-effector+phenotype+and+enhanced+antitumor+immunity%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Instability of Helios-deficient Tregs is associated with conversion to a T-effector phenotype and enhanced antitumor immunity.</a> Proc Natl Acad Sci U S A. 113 (22), 6248-6253 (2016).</li>
<li>Dawson, N. A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+effects+of+chimeric+antigen+receptor+co-receptor+signaling+domains+in+human+regulatory+T+cells%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Functional effects of chimeric antigen receptor co-receptor signaling domains in human regulatory T cells.</a> Sci Transl Med. 12 (557), eaaz3866(2020).</li>
<li>Rana, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CAR-+and+TRuC-redirected+regulatory+T+cells+differ+in+capacity+to+control+adaptive+immunity+to+FVIII%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">CAR- and TRuC-redirected regulatory T cells differ in capacity to control adaptive immunity to FVIII.</a> Mol Ther. 29 (9), 2660-2676 (2021).</li>
<li>Battaglia, M., Stabilini, A., Tresoldi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanding+human+T+regulatory+cells+with+the+mTOR-inhibitor+rapamycin%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Expanding human T regulatory cells with the mTOR-inhibitor rapamycin.</a> Methods Mol Biol. 821, 279-293 (2012).</li>
<li>Brady, B. L., Steinel, N. C., Bassing, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigen+receptor+allelic+exclusion%3A+an+update+and+reappraisal%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Antigen receptor allelic exclusion: an update and reappraisal.</a> J Immunol. 185 (7), 3801-3808 (2010).</li>
<li>Samarasinghe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+characterization+of+alloreactive+T+cells+identifies+CD25+and+CD71+as+optimal+targets+for+a+clinically+applicable+allodepletion+strategy%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Functional characterization of alloreactive T cells identifies CD25 and CD71 as optimal targets for a clinically applicable allodepletion strategy.</a> Blood. 115 (2), 396-407 (2010).</li>
<li>Voss, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FOXP3+protects+conventional+human+T+cells+from+premature+restimulation-induced+cell+death%5BTitle%5D)+AND+%22Cell+Mol+Immunol%22%5BJournal%5D)">FOXP3 protects conventional human T cells from premature restimulation-induced cell death.</a> Cell Mol Immunol. 18 (1), 194-205 (2021).</li>
<li>Collison, L. W., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+Treg+suppression+assays%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">In vitro Treg suppression assays.</a> Methods Mol Biol. 707, 21-37 (2011).</li>
</ol>

LMRF is an inventor and has received royalties from patents on engineered immune cells and consults for Guidepoint Global and McKesson. The remaining authors declare no competing interests.

This protocol provides a streamlined and comprehensive methodology for generating and evaluating human chimeric antigen receptor regulatory T cells (CAR Tregs). The success of CAR technology in treating hematological cancers has inspired its application to the immunosuppressive subset of T cells, Tregs. Unlike conventional T cells, Tregs inhibit immune responses, offering potential treatments for conditions resulting from unwanted immunity, such as autoimmune disease, organ transplant rejection, graft-versus-host disease...

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of hematological malignancies, achieving remarkably high remission rates in previously untreatable cancers1,2. Encouraging early results using CAR T cells to treat glioblastoma3,4,5 highlight CAR technology&#39;s versatility and future potential to target a wide range of malignancies. As the field explores further applications of CARs, regulatory T cells (Tregs) have emerged as a promising cell type. Tregs play a crucial role in maintaining immune homeostasis, and regulating immune responses through several mechanisms, including sequestering IL-2, secreting immunosuppressive cytokines, and modulating antigen-presenting cells6,7.
With CAR technology, Tregs could be leveraged for the treatment of organ transplant rejection, autoimmune disease, and inflammatory disorders like allergies and asthma6,8,9. CAR Tregs could lead to significant improvements in patient outcomes and quality of life by reducing the use of immunosuppressive drugs, which inhibit the immune system as a whole and are associated with noxious side effects10,11. Preclinical models have shown promising results in translating CAR technology to Tregs, with successful applications in diseases such as type 1 diabetes, multiple sclerosis, graft-versus-host disease, and inflammatory bowel disease9,12,13,14,15. In the clinic, CAR Tregs are currently being explored to prevent solid organ transplant rejection16.
This article presents a detailed methodology for generating human chimeric antigen receptor regulatory T cells (CAR Tregs). This protocol involves isolating Tregs from human peripheral blood and genetically modifying them using techniques such as lentiviral transduction and precise gene knock-in using CRISPR/Cas9 gene editing and adeno-associated virus (AAV) vectors. We also describe the assessment of these engineered Tregs&#39; phenotypic stability and suppressive function, which are crucial steps to validate their therapeutic potential17,18,19. This approach streamlines the design and early testing of CAR Treg therapies, which hold the potential to extend the transformative impact of CAR T-cell therapy to regulate the immune system. By sharing our methodology, we hope to inspire further research and innovation in the burgeoning CAR Treg therapy space9,20.

<table><tbody><tr><td>Adeno-associated virus&amp;nbsp;(AAV)</td><td>Charles River Laboratories</td><td></td><td></td></tr><tr><td>CAR target-expressing K562 cells</td><td></td><td></td><td>e.g., CD19-K562</td></tr><tr><td>Cesium-137 irradiator</td><td></td><td></td><td></td></tr><tr><td>Anti-human CD8 PerCP (clone SK1)</td><td>Biolegend</td><td>344708</td><td></td></tr><tr><td>Anti-human CD4 PE/Cy7 (clone SK3)</td><td>Biolegend</td><td>344612</td><td></td></tr><tr><td>DynaMag-15 magnet</td><td>ThermoFisher</td><td>12301D</td><td></td></tr><tr><td>Ghost BV510 viability dye&amp;nbsp;</td><td>TONBO</td><td>13-0870-T100</td><td></td></tr><tr><td>K562 cells</td><td>American Type Culture Collection</td><td>CCL-243</td><td></td></tr><tr><td>0.5 M EDTA, pH 8.0</td><td>Gibco&amp;nbsp;</td><td>15575020</td><td></td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>Ammonium chloride solution</td><td>STEMCELL Technologies</td><td>7850</td><td></td></tr><tr><td>Anti-human CD127 PE (clone hIL-7R-M21)</td><td>BD Biosciences</td><td>557938</td><td></td></tr><tr><td>Anti-human CD25 APC (clone BC96)</td><td>Biolegend&amp;nbsp;</td><td>302610</td><td></td></tr><tr><td>Anti-human CD4 FITC (clone SK3)</td><td>Biolegend&amp;nbsp;</td><td>344604</td><td></td></tr><tr><td>Anti-human CD71 PE (clone SK1)</td><td>Biolegend</td><td>334106</td><td></td></tr><tr><td>Anti-human CD8 PerCP (clone SK1)</td><td>Biolegend&amp;nbsp;</td><td>344707</td><td></td></tr><tr><td>Anti-human CTLA-4 PerCP-e710</td><td>ThermoFisher</td><td>46-1529-42</td><td></td></tr><tr><td>Anti-human EGFR APC (clone AY13)</td><td>Biolegend</td><td>352905</td><td></td></tr><tr><td>Anti-human FOXP3 eFluor 450</td><td>ThermoFisher</td><td>48-4776-42</td><td></td></tr><tr><td>Anti-human HELIOS PE</td><td>Biolegend</td><td>137216</td><td></td></tr><tr><td>Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt; free Dulbecco&amp;rsquo;s Phosphate Buffered Saline (DPBS)&amp;nbsp;</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Cell counter (TC20 Automated Cell Counter)</td><td>Bio-Rad</td><td>1450102</td><td></td></tr><tr><td>Cell Counting Slides</td><td>Bio-Rad</td><td>1450016</td><td></td></tr><tr><td>CellTrace Violet Cell Proliferation Kit</td><td>ThermoFisher</td><td>C34571</td><td></td></tr><tr><td>DNA LoBind Tubes</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>Easy 50 EasySep magnet&amp;nbsp;</td><td>STEMCELL Technologies</td><td>18002</td><td></td></tr><tr><td>EasySep Human CD4&lt;sup&gt;+&lt;/sup&gt; T cell Enrichment Kit</td><td>STEMCELL Technologies</td><td>19052</td><td></td></tr><tr><td>EasySep Human CD8&lt;sup&gt;+&lt;/sup&gt; T cell Enrichment Kit</td><td>STEMCELL Technologies</td><td>19053</td><td></td></tr><tr><td>EasySep magnet&amp;nbsp;</td><td>STEMCELL Technologies</td><td>18000</td><td></td></tr><tr><td>eBioscience Foxp3 transcription factor staining buffer set</td><td>ThermoFisher</td><td>00-5523-00</td><td></td></tr><tr><td>Falcon Round-Bottom Polystyrene Test Tubes with Cell Strainer Snap Cap, 5 mL</td><td>Fisher Scientific</td><td>08-771-23</td><td>40&amp;mu;m</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>26140079</td><td></td></tr><tr><td>Flow cytometer</td><td>Beckman Coulter</td><td></td><td>CytoFLEX LX U3-V5-B3-Y5-R3-I2</td></tr><tr><td>Fluorescence-activated cell sorter</td><td>BD Biosciences</td><td></td><td>FACS Aria III Cell Sorter</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>Human CD3/28 T Cell Expansion and Activation Dynabeads</td><td>Gibco</td><td>11131D</td><td></td></tr><tr><td>Invitrogen Neon Transfection System</td><td>ThermoFisher</td><td>10431915</td><td></td></tr><tr><td>Invitrogen Neon Transfection System 100 &amp;mu;L Kit</td><td>ThermoFisher</td><td>10114334</td><td></td></tr><tr><td>Lentivirus</td><td>VectorBuilder</td><td></td><td></td></tr><tr><td>MEM Non-Essential Amino Acids Solution&amp;nbsp;</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Myc Tag antibody A647 (clone 9B11)</td><td>Cell Signaling Technologies</td><td>2233S</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>ThermoFisher</td><td>31985062</td><td></td></tr><tr><td>Penicilin-Streptomycin solution</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Recombinant human interleukin-2 (rhIL-2)</td><td>Peprotech</td><td>200-02</td><td></td></tr><tr><td>RPMI 1640 medium, no glutamine&amp;nbsp;</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360070</td><td></td></tr><tr><td>Spectral Flow Cytometer</td><td>Cytek</td><td></td><td>Northern Lights</td></tr><tr><td>TRAC gRNA</td><td>Synthego</td><td></td><td>Sequence (CAGGGTTCTGGATATCTGT)</td></tr><tr><td>TrueCut Cas9 Protein v2</td><td>ThermoFisher</td><td>A36496</td><td></td></tr><tr><td>Trypan Blue solution&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>T8154-100ML</td><td></td></tr><tr><td>1/10 Leukopak</td><td>STEMCELL Technologies</td><td>200-0092</td><td>1-2 billion PBMC</td></tr></tbody></table>

generation of human chimeric antigen receptor regulatory t cells

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

The protocol described here provides a streamlined and standardized pipeline to assess new chimeric antigen receptor (CAR) constructs in human regulatory T cells (Tregs), with the aim of creating living therapeutics for autoimmune disease, graft-versus-host disease, organ transplant rejection, and allergy. Figure 1 depicts how we obtain highly pure human Tregs from peripheral blood using FACS (Figure 1A), as evaluated by their high levels of the Treg lineage tra...

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

generation-of-human-chimeric-antigen-receptor-regulatory-t-cells

Research

JoVE Journal

Immunology and Infection

We describe a method for generating regulatory, memory and naïve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

This video demonstrates a technique to manufacture chimeric antigen receptor (CAR) T cells ex vivo. Human T cells are incubated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, along with a medium supplemented with interleukin-2 (IL-2), leading to T cell activation and proliferation. Transgenic lentiviral vectors are introduced, allowing the transgenic RNA to integrate into the host genome and express the transgene. This results in the transduced T cells expressing chimeric...

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

This video demonstrates a technique for the ex vivo generation of tumor antigen-specific chimeric antigen receptor (CAR) T cells. Retroviral vectors carrying transgenic RNA are mixed with spleen cells in a medium supplemented with interleukin-2 to promote T-cell survival and proliferation. The mixture is then transferred to a plate coated with recombinant human fibronectin fragments and centrifuged to facilitate the fusion of the virus with the cell, allowing the RNA to integrate into the host...

An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

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...

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

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial.

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Here we present a protocol for the generation and functional verification of hypoxia-sensitive chimeric antigen receptor (CAR)-T cells. This protocol presents the lentivirus-based generation of hypoxia-sensitive CAR-T cells and their characterization, including the validation of hypoxia-dependent CAR expression and selective cytotoxicity.

Generation and Functional Verification of Hypoxia-Sensitive Chimeric Antigen Receptor-T Cells

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

Learn to generate human chimeric antigen receptor regulatory T cells using lentiviral transduction and electroporation techniques. This protocol establishes standardized methods for developing engineered immune cell therapies.

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

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