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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR-T+cell+therapy+in+hematological+malignancies:+current+opportunities+and+challenges.">CAR-T cell therapy in hematological malignancies: current opportunities and challenges.</a> Front Immunol. 13, 927153 (2022).</li><li>Cappell, K. M., Kochenderfer, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+outcomes+following+CAR+T+cell+therapy:+what+we+know+so+far.">Long-term outcomes following CAR T cell therapy: what we know so far.</a> Nat Rev Clin Oncol. 20 (6), 359-371 (2023).</li><li>Choi, 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=Intraventricular+CARv3-TEAM-E+T+cells+in+recurrent+glioblastoma.">Intraventricular CARv3-TEAM-E T cells in recurrent glioblastoma.</a> N Engl J Med. 390 (14), 1290-1298 (2024).</li><li>Brown, C. E., 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=Locoregional+delivery+of+IL-13Ralpha2-targeting+CAR-T+cells+in+recurrent+high-grade+glioma:+a+phase+1+trial.">Locoregional delivery of IL-13Ralpha2-targeting CAR-T cells in recurrent high-grade glioma: a phase 1 trial.</a> Nat Med. 30 (4), 1001-1012 (2024).</li><li>Bagley, S. 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=Intrathecal+bivalent+CAR+T+cells+targeting+EGFR+and+IL13Ralpha2+in+recurrent+glioblastoma:+phase+1+trial+interim+results.">Intrathecal bivalent CAR T cells targeting EGFR and IL13Ralpha2 in recurrent glioblastoma: phase 1 trial interim results.</a> Nat Med. 30 (5), 1320-1329 (2024).</li><li>Ghobadinezhad, 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=The+emerging+role+of+regulatory+cell-based+therapy+in+autoimmune+disease.">The emerging role of regulatory cell-based therapy in autoimmune disease.</a> Front Immunol. 13, 1075813 (2022).</li><li>Sakaguchi, S., Yamaguchi, T., Nomura, T., Ono, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+T+cells+and+immune+tolerance.">Regulatory T cells and immune tolerance.</a> Cell. 133 (5), 775-787 (2008).</li><li>Sakaguchi, 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=Regulatory+T+cells+and+human+disease.">Regulatory T cells and human disease.</a> Annu Rev Immunol. 38, 541-566 (2020).</li><li>Ferreira, L. M. R., Muller, Y. D., Bluestone, J. A., Tang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+regulatory+T+cell+therapy.">Next-generation regulatory T cell therapy.</a> Nat Rev Drug Discov. 18 (10), 749-769 (2019).</li><li>Rosenblum, M. D., Gratz, I. K., Paw, J. S., Abbas, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treating+human+autoimmunity:+current+practice+and+future+prospects.">Treating human autoimmunity: current practice and future prospects.</a> Sci Transl Med. 4 (125), 125sr121 (2012).</li><li>Sawitzki, B., 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=Regulatory+cell+therapy+in+kidney+transplantation+(The+ONE+Study):+a+harmonised+design+and+analysis+of+seven+non-randomised,+single-arm,+phase+1/2A+trials.">Regulatory cell therapy in kidney transplantation (The ONE Study): a harmonised design and analysis of seven non-randomised, single-arm, phase 1/2A trials.</a> Lancet. 395 (10237), 1627-1639 (2020).</li><li>Spanier, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tregs+with+an+MHC+class+II+peptide-specific+chimeric+antigen+receptor+prevent+autoimmune+diabetes+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+engineering+of+an+anti-HLA-A2+chimeric+antigen+receptor+in+regulatory+T+cells+for+transplant+immune+tolerance.">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><li>MacDonald, K. G., 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=Alloantigen-specific+regulatory+T+cells+generated+with+a+chimeric+antigen+receptor.">Alloantigen-specific regulatory T cells generated with a chimeric antigen receptor.</a> J Clin Invest. 126 (4), 1413-1424 (2016).</li><li>Boardman, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flagellin-specific+human+CAR+Tregs+for+immune+regulation+in+IBD.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+design:+human+leukocyte+antigen+Cclass+I+molecule+A(*)02-chimeric+antigen+receptor+regulatory+T+cells+in+renal+transplantation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redirecting+human+conventional+and+regulatory+T+cells+using+chimeric+antigen+receptors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+decrease+of+donor-reactive+T(regs)+after+liver+transplantation+limits+T(reg)+therapy+for+promoting+allograft+tolerance+in+humans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+2+randomized+trial+with+autologous+polyclonal+expanded+regulatory+T+cells+in+children+with+new-onset+type+1+diabetes.">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. 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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=Self-antigen-driven+activation+induces+instability+of+regulatory+T+cells+during+an+inflammatory+autoimmune+response.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instability+of+Helios-deficient+Tregs+is+associated+with+conversion+to+a+T-effector+phenotype+and+enhanced+antitumor+immunity.">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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adeno-associated virus&#160;(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&#160;</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&#160;</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&#160;</td>
			<td>302610</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD4 FITC (clone SK3)</td>
			<td>Biolegend&#160;</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&#160;</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>Ca2+ and Mg2+ free Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)&#160;</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&#160;</td>
			<td>STEMCELL Technologies</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human CD4+ T cell Enrichment Kit</td>
			<td>STEMCELL Technologies</td>
			<td>19052</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Human CD8+ T cell Enrichment Kit</td>
			<td>STEMCELL Technologies</td>
			<td>19053</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep magnet&#160;</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&#956;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 &#956;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&#160;</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&#160;</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&#160;</td>
			<td>Sigma&#160;</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

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...

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

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

1.0K Views.  Medical University of South Carolina. This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Video: Generation of Human Chimeric Antigen Receptor Regulatory T Cells

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

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve 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.

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

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.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG-/- recipients) are reconstituted with naive CD4+ T cells from healthy wild type hosts.
This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4+ T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG-/- hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

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

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

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

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

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

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

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

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

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

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

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

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Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells