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. 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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, and allergies9. Utilizing CAR Tregs enables antigen-specific therapy, holding the potential to avoid the side effects associated with broad-spectrum immunosuppressants11. Here, we describe in vitro protocols to validate Treg lineage stability and function upon engineering with a CAR. While not discussed in this article, immunodeficient NSG mice provide the opportunity to test human CAR Treg function in vivo in models of graft-versus-host disease and organ transplant13,14, an important step before translating into humans.
A critical step in this protocol involves isolating and purifying Tregs from human peripheral blood, using sequential magnetic separation and fluorescence-activated cell sorting (FACS) to achieve high purity of CD4+CD25highCD127- Tregs. The quality of Treg isolation is crucial, as it directly influences the therapeutic efficacy of CAR Tregs. Contamination by conventional T (Tconv) cells must be strictly avoided, as they can proliferate faster than Tregs, especially under high IL-2 concentrations (1,000 IU/mL). Other published methodologies include the addition of rapamycin in their Treg expansion protocol to selectively limit Tconv expansion while minimally impacting Treg expansion33. Magnetic negative selection of CD4+ T cells is employed to shorten the duration of FACS, which is less gentle on the cells than magnetic isolation of untouched cells. Moreover, negative selection preserves the CD4 epitope for antibody staining. These steps are pivotal due to the low frequency of Tregs in peripheral blood (1% of PBMC, 3-8% of CD4+ T cells).
Two methods for genetic modification of human Tregs are described here: lentiviral transduction and CRISPR/Cas9-mediated gene knock-in. Lentiviral transduction ensures efficient gene delivery and lentivirus is easier to manufacture. However, it involves random genomic integration and variable gene copy numbers per cell. In contrast, CRISPR/Cas9 and HDR allow for genomic editing at a precise location (the TRAC locus in our case) and, if targeting the TCR genomic region, a single gene copy per cell (only one TCR allele can be targeted per T cell due to TCR allelic exclusion34). Yet, electroporation, used to deliver the CRISPR/Cas9 RNP, is more disruptive to the cells than the addition of lentivirus to the medium and centrifugation, requiring more Tregs at the start (4 &#215; 106 Tregs for electroporation with a 100 &#181;L electroporation tip versus as low as 2.5 &#215; 105 Tregs for lentiviral transduction in a 24-well) and resulting in lower CAR Treg expansion (typically 20-70 fold between the day of lentivirus transduction and the day of functional assessment).
Three benchmarks to assess human CAR Treg engineering are presented here: activation, stability, and suppression. We gauge CAR Treg activation in the presence of CAR antigen-expressing target cells by measuring the upregulation of CD71, the transferrin receptor. Although CD69 and CD25 are also activation markers, CD71 was chosen due to its stable post activation expression for various days compared to the transient expression of CD6935. CD25 has low dynamic range in Tregs due to their high basal levels of this molecule. Stability testing is crucial as Tregs can convert to an effector phenotype under prolonged ex vivo expansion, multiple rounds of activation, or certain inflammatory conditions, which could counteract the desired immunosuppressive effects29,30. This protocol assesses CAR Treg phenotypic stability 9 days post CAR-mediated activation as that is the typical duration of a human T cell activation cycle. However, it can be important to look at further time points as for some donors, Tregs are not fully rested and continue to expand until up to 12 days post stimulation. Additionally, it can be important to evaluate the impact of multiple activation cycles on CAR Treg stability. We utilize FOXP3 and HELIOS simultaneous expression as a hallmark of Treg stability. Inclusion of HELIOS is key, as, in humans, Tconv cells upregulate FOXP3 upon activation36.
The suppression assay evaluates the immunosuppressive function of CAR Tregs, measuring the division of CD4+ and CD8+ T cells through CellTrace Violet (CTV) fluorescence tracking. This assay&#39;s sensitivity and donor dependency require careful setup and troubleshooting. Parameters that can be established prior to the utilization of CAR Tregs, whose number can be a limiting factor, include determining how fast labeled T cells proliferate for a specific donor and the optimal ratio of anti-CD3/CD28 beads to T cells for a given donor and batch of beads17,28,37.
A challenge in human CAR Treg engineering lies in the interdonor variability in Treg abundance, genetic manipulation efficiency, expansion, and immunosuppressive function. This variability can&#8239;be attributed&#8239;to donor-specific genetic and environmental differences and to the increased sensitivity of Tregs during in vitro handling as compared to Tconv cells, which may affect the reproducibility and scalability of the human CAR Treg generation and testing pipeline. By detailing this protocol and its nuances here, we aim to enable other researchers to replicate and build upon our findings, advancing the field of CAR Treg therapy. Future research should focus on developing CARs for new targets, tailoring the CAR signaling to Tregs&#39; specific needs, exploring non-viral delivery systems, refining our understanding of CAR Treg mechanisms of suppression, and expanding the application of this technology to various immune-mediated diseases.

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

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 transcription factors FOXP3 and HELIOS after ex vivo expansion (Figure 1B).
We present two methods to perform gene modification in human Tregs: lentivirus transduction (Figure 2) and CRISPR/Cas9-mediated gene knock-in (Figure 3), both with high efficiencies. We use a third party to synthesize lentiviral backbone constructs and produce and titrate lentivirus, which is then shipped to our laboratory and stored at -80 &#176;C until use. However, it is also possible to generate lentivirus in-house by transfecting HEK293T cells with CAR and packaging plasmids, followed by titration in Jurkat cells using flow cytometry, as described elsewhere17. With regards to gene knock-in, we target the T cell receptor alpha constant (TRAC) locus with Cas9 and a single guide RNA (sgRNA) previously shown by us and others to result in high editing efficiency13,23 and knock in the CAR transgene, delivered in the form of a non-integrating adeno-associated virus (AAV) with homology arms specific for the TRAC locus. This strategy has been shown to result in CAR expression that is more uniform and consistent across blood donors in conventional T cells23, while avoiding the potential oncogenic risk of random genomic integration with lentivirus. The AAV used here was produced and titrated by a third party and shipped to our laboratory, where it was stored at -80 &#176;C until use. Yet, it is possible to produce AAV in-house using HEK293T cells transfected with an AAV construct and AAV packaging plasmids as producer cells and commercially available kits to purify and titrate AAV, as described elsewhere13,24.
To showcase our protocol to test multiple CARs in human Tregs, we took the well-established CD19 CAR construct and generated variants with different signaling architectures, featuring either a CD3delta or CD3epsilon chain instead of the typical CD3zeta chain25, or containing mutations in the known signaling motifs of CD2826. Figure 4 shows the extent of activation, as measured by surface expression of CD71 (transferrin receptor), of different CD19 CAR Tregs upon co-incubation with irradiated CD19-expressing K562 cells. K562 cells are a common choice as target cells in T cell activation assays due to their ease of culture and lentiviral transduction, and absence of HLA, CD80, and CD86, ensuring no background from endogenous TCR or costimulatory receptor activation by unmodified cells21,27.
Alternatively, one can use magnetic beads conjugated to the CAR target protein to activate CAR Tregs28. Absence of co-stimulation signal delivered by the CAR, either due to the complete absence of a CD28 domain (Zeta construct) or to all three known CD28 signaling motifs being inactivated (CD28(xxx)zeta construct), resulted in lower CD71 expression than a CD28zeta CD19 CAR (Figure 4). A very important parameter for both efficacy and safety of CAR Treg therapies is Treg stability, as Tregs may lose suppressive function and become pro-inflammatory T cells upon prolonged ex vivo culture or exposure to inflammatory environments in vivo29,30. With our protocol, human CAR Tregs remain positive for FOXP3 and HELIOS, the gold standard of Treg identity, following isolation, gene modification, polyclonal expansion, and activation via the CAR (Figure 5). Of note, we use a spectral flow cytometer to analyze samples that underwent intracellular antibody staining for increased signal intensity and resolution. Finally, we show in Figure 6 an immune assay to gauge human CAR Treg function. Conventional T cells are labeled with a fluorescent dye to track their proliferation using flow cytometry and then activated. These activated responder T cells (Tresp) are then co-incubated with activated CAR Tregs at different Treg:Tresp ratios. The higher the number of CAR Tregs, the less the Tresp cells proliferate, a direct measure of CAR Treg suppressive function. Interestingly, swapping the CD3 chain or mutating the CD28 domain of the CAR had only a small effect on CAR Treg-mediated suppression of T cell proliferation in vitro (Figure 6), in agreement with results reported by other groups31,32.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67200/67200fig01.jpg" /> 
Figure 1: Isolation of human regulatory T cells. (A) Fluorescence-assisted cell sorting gating strategy for isolating regulatory T cells and conventional T cells from CD4+ T cells magnetically enriched from peripheral blood mononuclear cells. The plot shows CD25 and CD127 expression, with Tregs identified as CD25highCD127- cells and Tconv as CD25lowCD127+ cells. (B) Intracellular staining of isolated cell populations analyzed by flow cytometry to confirm cellular identity based on FOXP3 and HELIOS expression. Tregs typically exhibit high expression of FOXP3 and HELIOS expression, Treg lineage transcription factors. Tconv cells show minimal expression of both markers, confirming their conventional T cell phenotype. (C) Gating strategy to assess Treg stability after CAR transduction. Abbreviations: Treg = regulatory T cell; Tconv = conventional T cell; FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; CAR = chimeric antigen receptor; GFP = green fluorescent protein. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67200/67200fig01large.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/67200/67200fig02.jpg" /> 
Figure 2: Generation of CAR Tregs using lentiviral transduction. Flow cytometry dot plot showing CAR surface expression (Myc-tag) and GFP reporter gene expression in Tregs after transduction with Myc-tag-CD19CAR-2A-GFP lentivirus (A) before and (B)&#160;after fluorescence-assisted cell sorting. Abbreviations: Treg = regulatory T cell; CAR = chimeric antigen receptor; GFP = green fluorescent protein. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67200/67200fig02large.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/67200/67200fig03.jpg" /> 
Figure 3: Generation of CAR Tregs using CRISPR/Cas9-mediated knock-in. (A) Flow cytometry dot plot of Tregs electroporated with a CRISPR/Cas9 RNP complex targeting the TRAC locus, along with an AAV containing a Myc-tag-CD19CAR-2A-EGFRt gene as a homology-directed repair template. CAR expression, indicated by EGFRt reporter gene expression, was analyzed in conjunction with loss of CD3 expression to measure CAR knock-in in the TRAC locus. (B) Control flow cytometry dot plot of Tregs mock-electroporated without the CRISPR-RNP complex and AAV showing absence of EGFRt reporter and presence of CD3. (C) Gating strategy to assess knock-in efficiency in Tregs. Abbreviations: Treg = regulatory T cell; FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; CAR = chimeric antigen receptor; RNP = ribonucleoprotein; AAV = adeno-associated virus; EGFRt = truncated epidermal growth factor receptor. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67200/67200fig03large.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/67200/67200fig04.jpg" /> 
Figure 4: CAR Treg activation with different CAR constructs. (A) Flow cytometry histograms representing surface expression of CD71, a T cell activation marker, on CAR Tregs 48 h post activation with target, irradiated CD19-expressing K562 cells. Each color represents Tregs transduced with a different CD19 CAR construct incorporating various CD3 chain signaling domains-CD28zeta, CD28delta, CD28epsilon-or mutated CD28 co-stimulatory domains-CD28(x23)zeta, CD28(1x3)zeta, CD28(12x)zeta, CD28(xxx)zeta-with &#39;x&#39; denoting the location of a tyrosine to phenylalanine mutation along the three signaling motifs of CD28 (1. PI3K binding motif, 2. ITK binding motif, 3. LCK binding motif). UT are shown as a negative control. (B) CD71 mean fluorescence intensity in activated CAR Tregs. Values represent technical replicates of representative experiments. Bars represent mean &#177; SD with statistical significance computed by one-way ANOVA with Tukey HSD. *, p &#60; 0.05, ***, p &#60; 0.001, in comparison with CD28zeta CD19 CAR Tregs. (C) Schematic of CAR constructs containing different mutations in the CD28 co-stimulatory signaling domain. (D) Schematic of CAR constructs containing different CD3 chain signaling domains. Abbreviations: Treg = regulatory T cell; CAR = chimeric antigen receptor; UT = untransduced; MFI = mean fluorescence intensity. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67200/67200fig04large.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/67200/67200fig05.jpg" /> 
Figure 5: CAR Treg identity stability upon CAR activation. Flow cytometry dot plots of intracellular FOXP3 and HELIOS expression, key markers for Treg identity, in Tregs 9 days post activation (A) via CD19 CAR with target, irradiated CD19-expressing K562 cells or (B) via TCR and CD28 with anti-CD3/28 beads. (C) Tconv cells activated with CD3/28 beads are included as a negative staining control for FOXP3 and HELIOS. Abbreviations: Treg = regulatory T cell; Tconv = conventional T cell; CAR = chimeric antigen receptor. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67200/67200fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67200/67200fig06.jpg" /> 
Figure 6: CAR Treg immunosuppressive function with different CAR constructs. (A) Representative flow cytometry histogram of proliferation of CTV-labeled CD4+ Tresp cells co-cultured with activated CD28zeta CD19 CAR Tregs at various Treg to Tresp ratios (1:1, 1:2, 1:4, and 1:8). (B) Percentage suppression of proliferation of CD4+ Tresp cells by Tregs featuring CD19 CARs with CD28 domain mutants or alternative CD3 signaling domains. (C) Representative flow cytometry histogram of proliferation of CTV-labeled CD4+ Tresp cells co-cultured with activated CD28zeta CD19 CAR Tregs at various Treg to Tresp ratios. (D) Percentage suppression of proliferation of CD8+ Tresp cells by Tregs featuring CD19 CARs with CD28 domain mutants or alternative CD3 signaling domains. Values represent technical replicates of representative experiments. Bars represent mean &#177; SD with statistical significance computed by one-way ANOVA with Tukey HSD *, p &#60; 0.05, **, p &#60; 0.01, ***, p &#60; 0.001, ****, p &#60; 0.0001, in comparison with CD28zeta CD19 CAR Tregs. (E) Gating strategy to assess CAR Treg suppression of Tresp cells. Abbreviations: Treg = regulatory T cell; CAR = chimeric antigen receptor; CTV = Cell Trace Violet; Tresp = T responder cells.<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/67200/67200fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

1.2K 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 Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

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

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

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

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...

Bioengineering

Streamlined Protocol for Mouse CAR-T Cell Generation in Syngeneic Models

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

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Author Spotlight: Enhancing CAR-T Cell Function in Syngeneic Tumor Models 

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

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

Automated Processor for Clinical-Scale CAR-T Cell Manufacturing

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant diseases. CAR-T cells are genetically modified T cells that express a chimeric protein that recognizes and binds to a cell surface target, resulting in the killing of the target cell. Traditional CAR-T cell manufacturing methods are labor-intensive, expensive, and may carry the risk of contamination. The CliniMACS Prodigy, an automated cell processor, allows for manufacturing cell therapy products at a clinical scale in a closed system, minimizing the risk of contamination. Processing occurs semi-automatically under the control of a computer and thus&#160;minimizes human involvement in the process, which saves time and reduces variability and errors.
This manuscript and video describes the T cell transduction (TCT) process for manufacturing CAR-T cells using this processor. The TCT process involves CD4+/CD8+ T cell enrichment, activation, transduction with a viral vector, expansion, and harvest. Using the Activity Matrix, a functionality that allows ordering and timing of these steps, the TCT process can be customized extensively. We provide a walk-through of CAR-T cell manufacturing in compliance with current Good Manufacturing Practice&#160;(cGMP) and discuss required release testing and preclinical experiments that will support an Investigational New Drug (IND) application. We demonstrate&#160;the feasibility and discuss the advantages and disadvantages of using a&#160;semi-automatic process for clinical CAR-T cell manufacturing. Finally, we describe an ongoing investigator-initiated clinical trial that targets pediatric B-cell malignancies [NCT05480449] as an example of how this manufacturing process can be applied in a clinical setting.

Author Spotlight: Advancements in CAR-T Cell Manufacturing and Gene Therapy Production 

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 (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant ...

Medicine

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

Source: Riccione, K., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52397">Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care.</a> J. Vis. Exp. (2015).
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 genome and produce surface-bound CARs targeting the tumor antigen.

Technika ex vivo do generowania specyficznych dla nowotworu chimerycznych komórek T receptora antygenowego

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

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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.

Technika wytwarzania limfocytów CAR T specyficznych dla antygenu

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

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

Activate fresh or cryopreserved primary human T cells by mixing them with anti-CD3/CD28 magnetic beads at a ratio of 3 beads per T cell in the wells of the culture dishes. Culture the T cells in X-VIVO 15 medium supplemented with normal human AB serum, L-glutamine, HEPES, and IL-2.
Activate the T cells at a concentration of 1 million T cells per milliliter during expansion, and culture them at 37 degrees Celsius with 20% oxygen, 5% carbon dioxide, and 95% humidity. After overnight stimulation, add lentiviral supernatant to the activated T cells. Calculate the volume of supernatant necessary to achieve a multiplicity of infection between 3 and 5. Continue culturing the cells using the same conditions.
On day 3, collect a representative aliquot of the cells of cryopreservation. Prior to cryopreservation, remove the magnetic beads by gently pipetting and magnetic separation. Prepare the freezing medium which contains PBS with 0.5% DMSO and store it at 4 degrees Celsius until ready to use.
Next, centrifuge the T cells at 300 times g for 5 minutes. Discard the supernatant and add 5 milliliters of PBS. Centrifuge the cells again at 300 times g for 5 minutes and discard the PBS. Resuspend the pellet in 1 milliliter of cold cryopreservation medium. Freeze the T cells in a chilled freezing container and store at minus 80 degrees Celsius for 48 hours.
After this, transfer the frozen cells to liquid nitrogen. Wash the rest of the T cells once in 5 milliliters of PBS to eliminate any residual vector. Centrifuge at 300 times g for 5 minutes, then decant the PBS and resuspend the cell pellet in T cell culture medium at a concentration of 500,000 cells per milliliter.
Split the T cells into two cultures designed for day 5 and 9. Count the T cells by flow cytometry using counting beads and monoclonal antibodies to human CD4 and CD8 as well as a viability dye. Refeed the cultures every other day to maintain them at a concentration of 500,000 cells per milliliter.
On day 5, counter and cryopreserve the day 5 cultures as previously described. On day 7, wash 500,000 cells in PBS and resuspend them in 100 microliters of fluorescence-activated cell sorting buffer. Then detect CAR surface protein expression by immunostaining with a fluorescently-conjugated anti-CAR19 idiotype by flow cytometry. On day 9, count and cryopreserved the day 9 cultures as previously described.

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

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