This work was supported by NIH grant R01 CA148673 (NH); the Ontario Institute for Cancer Research Clinical Investigator Award IA-039 (NH); BioCanRX Catalyst Grant (NH); The Princess Margaret Cancer Foundation (MOB, NH); Canadian Institutes of Health Research Canada Graduate Scholarship (TG); Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship (TG); Province of Ontario (TG, MA); and Guglietti Fellowship Award (TO). HLA and CD1d monomers were kindly provided by the NIH tetramer core facility.

1. Preparing Retroviral Construct Encoding TCR Hemichain of Interest
<ol>
	<li>Linearize pMX vector to allow the insertion of a TCR gene in subsequent steps. Digest the plasmid DNA with EcoRI and NotI restriction enzymes at 37 &#176;C for 3 hr (Table 1)8.</li>
	<li>Carry out electrophoresis of the digested plasmid on 1.2% agarose gel. Excise band of approximately 4,500 base pairs (bps), and elute in 30 &#956;l sterile water using commercially available gel extraction kits9.</li>
	<li>Design 5&#39; and 3&#39; primers for the TCR gene of interest that also encode 15-20 bps overlapping the EcoRI and NotI digestion site of pMX vector, respectively8.</li>
	<li>Amplify TCR gene with primers. Carry out electrophoresis of the PCR product and elute band of approximately 1,000 bps as described in step 1.2.</li>
	<li>Clone TCR gene into digested vector by combining each linear fragment with commercially available plasmid assembly master mix reagent and incubating at 50 &#176;C for 1 hr. 
	NOTE: Refer to manufacturer&#39;s protocol for relative volumes of each component. This assembly method is based on the technique originally described by Gibson et al.10.</li>
	<li>(Optional) Tag TCR gene to mark transduced cells with the truncated form of human nerve growth factor receptor (&#916;NGFR, amino acids 1-277)11 separated by furin cleavage site, serine-glycine linker, and 2A sequences12,13. Design primers encoding sequences overlapping the fragment ends and assemble plasmid as described in steps 1.3-1.5. 
	NOTE: These sequences can be found in references11-13.</li>
	<li>Transform chemically competent E. coli with assembled plasmid following manufacturer&#39;s protocol14. Seed transformed bacteria on agar plates (20 mg/ml lysogeny broth (LB), 15 mg/ml agar, and 1 &#956;g/ml ampicillin) and incubate at 37 &#176;C for 18 hr.</li>
	<li>Culture a single bacterium colony in 50 ml of LB medium (20 mg/ml LB and 1 &#956;g/ml ampicillin) for 16 hr in a shaking incubator at 37 &#176;C and 200 rounds per min.</li>
	<li>Purify plasmids using commercially available midiprep kits following manufacturer&#39;s protocol. Dilute plasmid to concentration of around 1 &#956;g/&#956;l. 
	NOTE: The plasmid purification protocol is based on the alkaline extraction method15.</li>
</ol>
2. Generating Stable Packaging Cell Line
NOTE: Both 293GPG and PG13 cells are adherent. Culture cells in Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 50 &#956;g/ml gentamicin. Culture 293GPG cells with 1 &#956;g/ml tetracycline before transfection. Incubate cells at 37 &#176;C with 5% CO2 between all steps.
<ol>
	<li>Transfect 293GPG packaging cells16 cultured in T75 flask with the hemichain-encoding vector obtained in step 1.9, using commercially available transfection reagent following manufacturer&#39;s protocol17. NOTE: Transfect 293GPG cells at 50-60% confluency.</li>
	<li>Aspirate medium for 293GPG cells and add 10 ml of fresh DMEM medium 1 day post transfection.</li>
	<li>Harvest transiently produced virus from transfected 293GPG cells 2 days after step 2.2 by transferring culture medium to syringe and passing through 0.45 &#956;m filter. 
	NOTE: Virus can be used immediately or stored at -80 &#176;C for future use.</li>
	<li>Culture 1 x 105 PG13 cells in T25 flask. Count cells using a hemocytometer. After one day, aspirate culture medium and add 1.5 ml of 293GPG-derived virus from step 2.3 and 1.5 ml DMEM medium, along with 8 &#956;g/ml of polybrene.</li>
	<li>Change the medium as described in step 2.4 once per day for 4 days, to establish stable PG13 packaging cell line producing retrovirus encoding the TCR hemichain of interest.</li>
	<li>Aspirate medium and replace with fresh DMEM medium for PG13 cell lines 24 hr after last infection for further culture. 
	NOTE: Freeze or sub-culture cells by detaching with 0.05% trypsin-EDTA solution.</li>
	<li>To produce virus from transduced PG13 cell lines, culture 2 x 106 cells in T75 flask with 10 ml DMEM medium, and harvest virus as described in step 2.3 three days after seeding the cells. 
	NOTE: Virus is best used immediately but can be stored at -80 &#176;C for up to two months.</li>
</ol>
3. Activation and Transduction of Human T cells
NOTE: Human samples are obtained and used in accordance with the institutional ethics committee approved protocols. Culture primary human cells in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% human serum instead of FCS and 50 &#956;g/ml gentamicin. Incubate cells at 37 &#176;C with 5% CO2 between all steps.
<ol>
	<li>Isolate human peripheral blood mononuclear cells (PBMC) by density gradient separation following manufacturer&#39;s protocol18.</li>
	<li>Activate T cells to induce proliferation required for retroviral infection. Culture 2 x 107 fresh or thawed PBMC per well in 6-well plate, in 5 ml of RPMI medium with 100 IU/ml of recombinant human interleukin-2 (rhIL-2) and 50 ng/ml of anti-CD3 monoclonal antibody (clone OKT3).</li>
	<li>Three days post stimulation, collect T cells by pipetting and centrifuge at 350 x g for 4 min. Discard supernatant and seed 0.5-1 x 106 T cells per well in 24-well plate resuspended in 1 ml of PG13 virus from step 2.7, and 1 ml of RPMI medium supplemented with 200 IU/ml of rhIL-2. Centrifuge plate at 1,000 x g and 32 &#176;C for 1 hr.</li>
	<li>After 24 hr, collect T cells by pipetting and centrifuge at 350 x g for 4 min. Discard supernatant and resuspend cells in 1 ml of PG13 virus from step 2.7, and 1 ml of RPMI medium supplemented with 200 IU/ml of rhIL-2. Repeat this step for a total of 6 infections. 
	NOTE: Number of infections should be optimized depending on titer of virus produced by packaging cell line. If necessary, passage T cells by removing 20-30% of the cells each day to prevent overgrowth.</li>
	<li>24 hr after the last infection, collect T cells by pipetting and centrifuge at 350 x g for 4 min. Discard supernatant and resuspend cells in RPMI medium with 100 IU/ml of rhIL-2 for further culture.</li>
	<li>2-3 days after step 3.5, stain T cells with HLA multimer at 4 &#176;C for 30 min, then anti-human CD3 and co-receptor monoclonal antibodies (mAbs) at 4 &#176;C for 15 min. Analyze by flow cytometry. Use irrelevant multimer and/or untransduced cells as negative controls (Figure 1-3)19.</li>
	<li>If the introduced centric hemichain is a TCR&#945; chain, analyze V&#946; usage of the de novo multimer positive cells using commercially available V&#946;-specific antibody panel by flow cytometry20.</li>
</ol>
4. Cloning De Novo TCR Counter-hemichains
<ol>
	<li>Sort the de novo multimer positive population in step 3.6 by flow-assisted cell sorting.</li>
	<li>Isolate RNA from sorted T cells using the acid guanidinium thiocyanate-phenol-chloroform extraction method21,22. 
	NOTE: RNA can be stored at -80 &#176;C, but should be used to generate cDNA as soon as possible.</li>
	<li>Synthesize cDNA library from extracted RNA using commercially available reverse transcriptase-PCR kits following manufacturer&#39;s protocol23,24.</li>
	<li>If cloning TCR&#946; counter-chains, design V&#946; gene and TCR&#946; constant region specific primers, as determined in step 3.7, and clone full-length TCR&#946; genes as described in steps 1.3-1.9. See step 4.5 if counter-chain is TCR&#945;, otherwise continue to step 5.1.</li>
	<li>Commercially available V&#945;-specific antibodies are limited, thus V&#945; gene usage cannot be determined by flow cytometry. Clone TCR&#945; counter-chains via 5&#39;-rapid amplification of cDNA ends (RACE)25, using commercially available 5&#39; RACE kits6.
	<ol>
		<li>Synthesize 5&#39; RACE compatible cDNA from RNA extracted in step 4.2 following manufacturer&#39;s protocol.</li>
		<li>Perform 1st round PCR as described in Table 2.</li>
		<li>Carry out electrophoresis of the PCR product and elute band of approximately 1,100 bps, as described in step 1.2.</li>
		<li>Perform 2nd round PCR as described in Table 3, using 1st round PCR product as template. 
		NOTE: Primer sequences shown under Table 3 were designed for cloning into EcoRI and NotI digested pMX vector.</li>
		<li>Carry out electrophoresis of the PCR product and elute band of approximately 1,000 bps, as described in step 1.2.</li>
		<li>Clone TCR&#945; genes into EcoRI and NotI digested pMX vector and purify plasmids as described in steps 1.5-1.9.</li>
	</ol>
	</li>
</ol>
5. Reconstituting Novel Antigen-specific TCR Clones
NOTE: Culture Jurkat 76 cells and subsequent cell lines in RPMI medium supplemented with 10% FCS and 50 &#956;g/ml gentamicin. Incubate cells at 37 &#176;C with 5% CO2 between all steps.
<ol>
	<li>Transduce Jurkat 76 cells26 (or equivalent TCR-/- human T cell line) with centric TCR hemichain using 293GPG virus produced in step 2.3. For Jurkat 76, seed 5 x 104 cells per well in 24-well plate with 1 ml of virus and 1 ml of RPMI medium. Centrifuge plate at 1,000 x g and 32 &#176;C for 1 hr.</li>
	<li>24 hr after infection, collect hemichain transduced Jurkat 76 cells by pipetting and centrifuge at 350 x g for 4 min. Discard supernatant and resuspend in fresh RPMI medium for further culture.</li>
	<li>Purify transduced cells if the hemichain is molecularly tagged 2-3 days after step 5.2, by staining with fluorophore conjugated anti-NGFR mAb followed by flow-assisted or magnetic-assisted cell sorting (optional)27.</li>
	<li>Following steps 2.1 to 2.3, produce 293GPG virus encoding a TCR counter-chain cloned from steps 4.4 or 4.5.</li>
	<li>To fully reconstitute the TCR, transduce T cell line stably expressing the centric TCR hemichain generated in steps 5.1-5.3 using virus from step 5.4. Perform transduction as described in steps 5.1-5.2.</li>
	<li>Purify CD3+ transfectants using anti-CD3 magnetic-assisted cell sorting following manufacturer&#39;s protocol, 2-3 days after step 5.527.</li>
	<li>To validate antigen specificity, stain the transfectants expressing clonotypic TCRs composed of the centric TCR hemichain paired with various counter-chains, with anti-CD3 and/or co-receptor mAbs, and HLA multimer, 3-5 days post CD3 purification. Analyze cells by flow cytometry (Figure 4-5)19.</li>
</ol>

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The University Health Network has filed a patent related to this methodology on which N.H., M.N., and T.O. are named as inventors. The other authors have no financial conflicts of interest.

The first requirement for successful application of this method is achieving sufficient transduction efficiency of primary T cells with the hemichain of interest. In our experience, the combination of using PG13 as packaging cell line and pMX as retroviral vector results in stable and efficient expression of the introduced gene in human primary T cells. PG13 packaging cells can be single-cell cloned to select for high-titer packaging cells to improve transduction efficiency. Furthermore, proliferation of T cells is also ...

T cell receptors (TCRs) are heterodimeric adaptive immune receptors expressed by T lymphocytes, composed of a TCR&#945; and TCR&#946; chain. They are generated via somatic rearrangement of V(D)J gene segments, which produces a highly diverse repertoire capable of recognizing virtually unlimited configurations of HLA/peptide complexes. Clinically, T cells engineered to express clonotypic TCRs specific for tumor-associated antigens have demonstrated efficacy in a variety of cancers1. However, many TCRs cloned for this purpose lack sufficient affinity for the antigen of interest, which limit their therapeutic application.
Here, we describe a method to overcome this limitation for existing TCRs by exploiting chain-centricity. It has been reported that one TCR hemichain could play a more dominant role in recognition of the target antigen2, here termed centricity. Crystal structural analyses have shown that one centric hemichain of a TCR could account for the majority of the footprint on the MHC/peptide complex3,4. Using this concept, we have previously demonstrated that the SIG35&#945; TCR&#945; can pair with a diverse repertoire of TCR&#946; chains and maintain reactivity against the MART127-35 peptide presented by HLA-A25. Similar results were obtained with the TAK1 TCR, where the centric TCR&#946; hemichain paired with various TCR&#945; chains and maintained reactivity for the WT1235-243 peptide presented by HLA-A246. Both MART1 and WT1 are tumor-associated antigens. Chain-centricity was also applied to study antigen recognition of CD1d-restricted invariant natural killer (iNKT) TCRs, by pairing the invariant V&#945;24-J&#945;18 (V&#945;24i) TCR&#945; chain of human iNKT TCRs with different V&#946;11 TCR&#946; chains7.
In all cases, we were able to generate a de novo repertoire of TCRs by transducing the centric TCR hemichain to peripheral blood T cells, where the introduced hemichain paired with the endogenous TCR&#945; or TCR&#946; counter-chains. In essence, the centric hemichain serves as a bait that can be used to identify the appropriate counter-chains, which when paired together form TCRs that maintain the antigen specificity of interest, yet varying in affinity. From these novel repertoires, we were able to isolate clonotypic TCRs with improved interaction strength against the target antigen compared to pre-existing TCRs. Therefore, we believe this method will accelerate the pipeline of identifying optimal TCRs for clinical application.

<table><tbody><tr><td>0.05% Trypsin-EDTA</td><td>Wisent Bioproducts</td><td>325-043-CL</td><td></td></tr><tr><td>293GPG cells</td><td>Generated by Ory &lt;em&gt;et al.&lt;/em&gt; (ref 8)</td><td></td><td></td></tr><tr><td>Agar</td><td>Wisent Bioproducts</td><td>800-010-CG</td><td></td></tr><tr><td>Agarose</td><td>Wisent Bioproducts</td><td>800-015-CG</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Wisent Bioproducts</td><td>400-110-IG</td><td></td></tr><tr><td>Chloroform</td><td>BioShop</td><td>CCL402</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) Solution Mix</td><td>New England Biolabs</td><td>N0447L</td><td></td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life Technologies</td><td>11995065</td><td></td></tr><tr><td>EcoRI</td><td>New England Biolabs</td><td>R0101S</td><td></td></tr><tr><td>EZ-10 Spin Column DNA Gel Extraction Kit</td><td>BS353</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Life Technologies</td><td>12483020</td><td></td></tr><tr><td>Ficoll-Paque Plus</td><td>GE Healthcare</td><td>17-1440-02</td><td></td></tr><tr><td>Filter</td><td>Corning</td><td>431220</td><td>0.45 mm pore SFCA membrane</td></tr><tr><td>FITC-conjugated anti-human CD271 (NGFR) mAb</td><td>Biolegend</td><td>345104</td><td>clone ME20.4</td></tr><tr><td>FITC-conjugated anti-human CD3 mAb</td><td>Biolegend</td><td>300440</td><td>clone UCHT1</td></tr><tr><td>Gentamicin</td><td>Life Technologies</td><td>15750078</td><td></td></tr><tr><td>Gibson Assembly Master Mix</td><td>New England Biolabs</td><td>E2611L</td><td>used for multi-piece DNA assembly</td></tr><tr><td>HLA-A2 pentamer</td><td>Proimmune</td><td>depends on antigenic peptide</td><td>HLA-A2/MART1 multimer used here was purchased from Proimmune</td></tr><tr><td>HLA/CD1d monomers</td><td>NIH Tetramer Core Facility</td><td></td><td>multimerize monomers according to protocol provided by NIH tetramer core facility</td></tr><tr><td>Human AB serum</td><td>Valley Biomedical</td><td>HP1022</td><td></td></tr><tr><td>human CD3 microbeads</td><td>Miltenyi Biotec</td><td>130-050-101</td><td></td></tr><tr><td>IOTest Beta Mark TCR V beta Repertoire Kit</td><td>Beckman Coulter</td><td>IM3497</td><td></td></tr><tr><td>Jurkat 76 cells</td><td>Generated by Heemskerk &lt;em&gt;et al.&lt;/em&gt; (ref 10)</td><td></td><td></td></tr><tr><td>LB Broth</td><td>Wisent Bioproducts</td><td>800-060-LG</td><td></td></tr><tr><td>LS MACS column</td><td>Miltenyi Biotec</td><td>130-042-401</td><td></td></tr><tr><td>NEB 5-alpha Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>New England Biolabs</td><td>C2987I</td><td></td></tr><tr><td>NEBuffer 3.1</td><td>New England Biolabs</td><td>B7203S</td><td>used for EcoRI and NotI digestion</td></tr><tr><td>NotI</td><td>New England Biolabs</td><td>R0189S</td><td></td></tr><tr><td>NucleoBond Xtra Midi</td><td>Macherey-Nagel</td><td>740410</td><td>used for plasmid purification</td></tr><tr><td>PC5-conjugated anti-human CD8 mAb</td><td>Beckman Coulter</td><td>B21205</td><td>clone B9.11</td></tr><tr><td>PG13 cells</td><td>ATCC</td><td>CRL-10686</td><td></td></tr><tr><td>Phusion HF Buffer Pack</td><td>New England Biolabs</td><td>B0518S</td><td></td></tr><tr><td>Phusion&amp;nbsp;High-Fidelity DNA Polymerase</td><td>New England Biolabs</td><td>M0530L</td><td></td></tr><tr><td>pMX retroviral vector</td><td>Cell Biolabs</td><td>RTV-010</td><td></td></tr><tr><td>polybrene</td><td>Sigma-Aldrich</td><td>H-9268</td><td></td></tr><tr><td>Proleukin (recombinant human interleukin-2)</td><td>Novartis</td><td>by Rx only</td><td>equivalent product can be purchased from Sigma-Aldrich</td></tr><tr><td>Purified anti-human CD3 antibody</td><td>Biolegend</td><td>317301</td><td>clone OKT3, used for T cell stimulation</td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>11875119</td><td></td></tr><tr><td>SA-PE</td><td>Life Technologies</td><td>S866</td><td>used for multimerizing monomers from NIH tetramer core facility</td></tr><tr><td>SMARTer RACE 5&#039;/3&#039;&amp;nbsp; Kit</td><td>Clontech</td><td>634858</td><td></td></tr><tr><td>Sterile water</td><td>Wisent Bioproducts</td><td>809-115-LL</td><td></td></tr><tr><td>SuperScript III First-Strand Synthesis System</td><td>Invitrogen</td><td>18080051</td><td>for cDNA synthesis</td></tr><tr><td>Syringe</td><td>BD</td><td>301604</td><td>10 ml, slip tip</td></tr><tr><td>Tetracycline hydrochloride</td><td>Sigma-Aldrich</td><td>T7660</td><td></td></tr><tr><td>TransIT-293</td><td>Mirus Bio</td><td>MIR 2700</td><td>used to transfect 293GPG cells</td></tr><tr><td>TRIzol Reagent</td><td>Life Technologies</td><td>15596026</td><td></td></tr></tbody></table>

generating de novo antigen-specific human t cell receptors by retroviral transduction of centric hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Without prior knowledge of which hemichain is chain-centric, the TCR&#945; and TCR&#946; chain should be separately cloned and transduced to peripheral blood T cells, which was done in the case of HLA-A24/WT1 reactive TAK1 TCR (Figure 1). Transduction of TAK1&#946; yielded a noticeably higher frequency of antigen-specific T cells. Conversely, transduction of a non-centric hemichain would not yield de novo multimer positive T cells, as seen with TAK1&#945; chain (...

Watch this Scientific Journal Video about Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain at JoVE.com

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

generating-de-novo-antigen-specific-human-t-cell-receptors-retroviral

Research

JoVE Journal

Immunology and Infection

8.4K Views.  University of Toronto.  Hierin beschrijven we een nieuwe methode om antigeenspecifieke T-celreceptoren (TCR's) door het koppelen van de TCRα of TcR van een bestaande TCR, bezit de antigeen-specificiteit van interesse, met aanvullende hemichain van de perifere T-cel receptor repertoire genereren. De novo geproduceerde TCRs behouden antigeen-specificiteit met verschillende affiniteit. 

Video: Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

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 ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

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 ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...

Engineering and Expansion of Human CAR Tregs for Immune Regulation Therapies

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.

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 ...

Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates

Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) to target a specific tumor-associated antigen. Reliance on viral vectors for the production of therapeutic T cells significantly increases the timeline, cost, and complexity of manufacturing while limiting the translation of new therapies, particularly in the academic setting. A process is presented for efficient non-viral engineering of T cells using CRISPR/Cas9 and homology-mediated end joining to achieve targeted integration of large, multicistronic DNA cargo. This approach has achieved integration frequencies comparable to those of viral vectors while yielding highly functional T cells capable of potent anti-tumor efficacy both in vitro and in vivo. Notably, this method is rapidly adaptable to current good manufacturing practices (cGMP) and clinical scale-up, providing a near-term option for the manufacturing of therapeutic T cells for use in clinical trials.

A detailed protocol is provided for using CRISPR/Cas9 technology to achieve highly efficient targeted knock-in of large, multicistronic constructs in primary human T cells via the homology-mediated end joining (HMEJ) DNA repair pathway. T cells engineered with this cGMP-adaptable protocol maintain excellent cell expansion, cytotoxicity, and cytokine production.

Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

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