Name: generation of human alloantigen-specific t cells from peripheral blood
Uploaded: 2014-11-21T15:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O&#8217;Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).

NOTE: This protocol requires use of peripheral blood samples from human volunteers. All research with human subjects should be reviewed and approved by a Human Studies Institutional Review Board to ensure compliance with the Declaration of Helsinki (2013) and the Health Insurance Portability and Accountability Act of 1996.
1. Isolation of T cells from Whole Blood
<ol>
	<li>Prior to starting, warm the density gradient medium to room temperature. Aliquot 4 ml of density gradient medium into 2-4 sterile 15 ml conical centrifuge tubes (1 tube will be used for each 10 ml total volume of diluted blood).</li>
	<li>Obtain 10-20 ml of blood in 1-2 sodium heparin spray coated (green-top) venous blood collection tubes. Collect human specimens under the supervision of trained individuals and according to an Institutional Review Board approved protocol.</li>
	<li>Wipe the outside of the tubes with 70% ethanol. Carefully remove the tops of the filled collection tubes and decant the blood into a sterile 50 ml centrifuge tube.</li>
	<li>Add 10 ml sterile phosphate buffered saline (PBS) to each blood collection tube. Collect the PBS, add to the decanted whole blood, and mix gently.</li>
	<li>Add 25 &#181;l Human T Cell Enrichment Cocktail / 2 ml total volume. Incubate at room temperature for 20 min.</li>
	<li>Using a 10 ml pipette, layer up to 10 ml of the 1:1 diluted blood gently on top of the density gradient medium. Be careful to not disrupt the surface of the density gradient medium.</li>
	<li>Centrifuge layered blood and density gradient medium at 1,200 x g for 20 min&#160;at 20 oC.</li>
	<li>Remove the tubes from the centrifuge, taking care to not disrupt the interface between the density gradient medium and the layer of leukocytes at the interface between the density gradient medium and the diluted plasma. Using a 5 ml pipette, carefully collect the leukocyte layer and transfer to a new sterile 50 ml conical tube.</li>
	<li>Add PBS to bring the volume of the collected PBL to 50 ml and mix gently.</li>
	<li>Centrifuge at 600 x g for 5 min&#160;at 20 oC. Decant.</li>
	<li>Resuspend pelleted cells in 10 ml sterile flow cytometry sorting buffer (sterile-filtered PBS containing 1% BSA). Sample 10 &#181;l of cell suspension, dilute 1:10 (add to 90 &#956;l) trypan blue to count cells using a hemocytometer (expected yield 1 &#8211; 4 x 106 cells / tube of whole blood).
	<ol>
		<li>Remove 1 ml aliquot, transfer to 5 ml tube, and keep on ice for analysis of T cells not labeled by tetramer. Keep cell suspension on ice.</li>
	</ol>
	</li>
</ol>
2. Magnetic Enrichment of Alloantigen-specific T cells
<ol>
	<li>Dilute alloantigen pMHC tetramer to 1:100 in sterile sort buffer.</li>
	<li>Centrifuge cell suspension (9 ml from step 1.11) at 600 x g for 5 min&#160;at 20 oC. Decant.</li>
	<li>Add 50 &#181;l of diluted alloantigen pMHC tetramer to pelleted cells (up to 107 cells). Mix by gentle pipetting. Transfer to a sterile 5 ml tube. Incubate for 30 min&#160;at room temperature.</li>
	<li>Add 2 ml sort buffer. Centrifuge at 600 x g for 5 min&#160;at 20 oC. Decant.</li>
	<li>Resuspend cells in 100 &#181;l sort buffer. Add 10 &#181;l Biotin Selection Cocktail and incubate for 15 min&#160;at room temperature.</li>
	<li>Add 5 &#181;l Magnetic Nanoparticles and incubate for 10 min&#160;at room temperature.</li>
	<li>Add 2.5 ml sort buffer and mix gently by pipetting. Remove 100 &#181;l aliquot, transfer to 5 ml tube, and keep on ice for analysis of pre-enrichment cells.</li>
	<li>Place the 5 ml tube containing the cell suspension in the cell separation magnet. The cap should be loosely atop the tube. Incubate for 5 min&#160;at room temperature.</li>
	<li>Gently remove the cap from the tube. Holding the tube and magnet together, decant the tube contents into a fresh 5 ml tube. Do not tap or shake the tube contents to remove the last drops of liquid.</li>
	<li>Remove the tube from the magnet. Add 2 ml cold sort buffer and mix gently by pipetting. Sample 10 &#181;l of cell suspension, dilute 1:2 (add to 10 &#181;l) trypan blue to count cells using a hemocytometer.</li>
</ol>
3. Preparation of T cells for Single-cell Flow Cytometry Cell Sorting
<ol>
	<li>Add cold sort buffer to all tubes of cells (tetramer unlabeled, tetramer-labeled un-enriched, and tetramer-labeled enriched) to bring volume to 3 ml.</li>
	<li>Centrifuge at 600 x g for 5 min&#160;at 4 oC and decant buffer to recover cells.</li>
	<li>Resuspend pelleted cells in 25 &#181;l cold sort buffer. Add 5 &#181;l human Fc block. Incubate on ice for 20 min.</li>
	<li>Prepare antibodies for flow cytometry sorting. Mix 15 &#181;l sort buffer, 15 &#181;l anti-CD5, 15 &#181;l anti-CD14, and 15 &#181;l anti-CD19.</li>
	<li>Add 20 &#181;l of antibody mixture to each tube of cells. Incubate on ice for 20 min.</li>
	<li>Add 2 ml of cold sort buffer to each tube.</li>
	<li>Centrifuge at 600 x g for 5 min&#160;at 4&#160;oC and decant buffer to recover cells.</li>
	<li>Resuspend cells at a concentration of 1 -2 x 106 cells/ml in sort buffer.</li>
	<li>Aliquot 100 &#181;l of human T cell culture medium (Iscove&#8217;s DMEM supplemented with 2 mM Glutamax, 10 mM HEPES, 50 &#181;g/ml gentamycin, 50 &#181;M 2-mercaptoethanol, 10% heat-inactivated human AB serum, and 2.5 ng/ml recombinant human IL-2) to each well of a 96-well round bottom cell culture plate. Keep on ice.</li>
</ol>
4. Isolation of Tetramer-labeled T cells by Single-cell Flow Cytometry Sorting
<ol>
	<li>Establish flow cytometry gating parameters to identify alloantigen-pMHC tetramer labeled T cells (Figure 2.A). Use the pre-enrichment tetramer labeled fraction to establish gating strategies to eliminate doublets, gate on lymphocytes, exclude CD19 expressing B cells and CD14 expressing monocytes, and identify T cells by CD5 expression. Use the sample not labeled with tetramer as a fluorescence minus one control to establish the gating for identification of tetramer-binding T cells (with 0% of CD5+CD14-CD19- cells from the sample not labeled by tetramer should fall within this gate). 
	NOTE: Gating strategies that are not adequately stringent will result in isolation of non-T cells or T cells that are not antigen specific, while overly restrictive gating strategies will reduce the number of T cells isolated.</li>
	<li>Program the plate parameters for the single cell sort. Direct the sorter to place 1 CD5+CD14-CD19-tetramer+ cell in each well. Direct the plate set up to contain 1 negative control well (no cells sorted into the well) and 1 positive control well (100 CD5+CD14-CD19-tetramer- cells) in each row (Figure 2.B).</li>
	<li>Using the established flow cytometry gating strategy and plate setup, isolate individual tetramer-binding T cells directly into the 96-well plate using a flow cytometric cell sorter.</li>
</ol>
5. Culture and Expansion of Alloantigen-specific T cell Clones
<ol>
	<li>After completion of cell sorting, centrifuge plate at 600 x g for 5 min&#160;at 20 oC. Place culture plate at 37 oC in a 6% CO2 incubator.</li>
	<li>Vortex stimulatory anti-CD3/anti-CD28 microbeads for 30 sec&#160;to resuspend beads.</li>
	<li>Calculate volume of activator beads required for stimulation of collected T cells (0.5 &#181;l of activator microbeads / well). Transfer calculated volume of stimulator beads to a sterile 5 ml tube. Add 1 ml human T cell culture medium and vortex.</li>
	<li>Place tube with microbead suspension in magnet. The cap should be loosely atop the tube. Incubate for 2 min&#160;at room temperature.</li>
	<li>Gently remove the cap from the tube. Holding the tube and magnet together, decant tube contents into a fresh 5 ml tube. Do not tap or shake the tube contents.</li>
	<li>Remove the tube from the magnet. Add human T cell culture medium (100 &#181;l medium / 0.5 &#181;l of microbeads initially added).</li>
	<li>Aliquot 100 &#181;l of activator bead suspension to each well of the 96-well plate. Incubate culture plate at 37 oC in a 6% CO2 incubator.</li>
	<li>Monitor cell growth daily by microscopic examination. Small clusters of proliferating cells may be observed microscopically after 5 &#8211; 7 days of culture (Figure 3.A). 
	NOTE: Visualization of T cell cultures at this point can be difficult, and the absence of observable cell clusters at this stage may not be indicative of a lack of growing T cells.</li>
	<li>At 14 days after cell isolation, feed cultures by carefully removing 100 &#181;l of media off of the top of the culture and replace with 100 &#181;l fresh human T cell culture medium. Continue incubating culture plate at 37 oC in a 6% CO2 incubator.</li>
	<li>Monitor cell growth daily by microscopic examination and evaluation of the color of the culture medium. Large cell clusters should be visible microscopically 2 &#8211; 3 days after the medium change. Macroscopically, cell pellets should become visible during the 14 days following the medium change.</li>
	<li>At 28 days following cell isolation, identify growing clones by microscopic examination. Transfer the 200 &#181;l volume of the growth-positive 96-well plate cultures to individual wells of a 48-well tissue culture plate containing 200 &#181;l human T cell culture medium.</li>
	<li>Add 100 &#181;l containing 12.5 &#181;l of stimulatory anti-CD3/anti-CD28 microbeads (prepared as described in sections 5.2 &#8211; 5.6). Incubate culture plate at 37 oC in a 6% CO2 incubator.</li>
	<li>Monitor culture growth daily by microscopic examination and evaluation of the color of the culture medium. When T cell culture reaches &#62;2x106/ml (typically 3-5 days after re-stimulation, can be estimated by the day that the media begins turning straw-yellow), transfer culture to a well of a 24-well tissue culture plate and add 500 &#181;l human T cell medium.</li>
	<li>At 10-14 days follow re-stimulation, collect 200 &#181;l of T cell culture to assess alloantigen specificity by flow cytometry analysis of pMHC tetramer binding (using the labeling and gating strategy described in sections 3 and 4.1).</li>
</ol>
6. Long-term Re-stimulation and Culture of T cell Clones
<ol>
	<li>Continually re-stimulate and expand clonal T cell cultures with the desired specificity can be every 14 days. Collect T cell cultures into a sterile 5 ml tube at day 14 following the last anti-CD3/CD28 microbead stimulation. Combine multiple wells of the same T cell clone in a single tube, up to a volume of 2.5 ml.</li>
	<li>Add medium to bring the final volume to 2.5 ml and mix gently by pipetting.</li>
	<li>Place the 5 ml tube containing the T cell culture into the cell separation magnet to remove old microbeads from the culture. The cap should be loosely atop the tube. Incubate for 5 min&#160;at room temperature.</li>
	<li>Gently remove the cap from the tube. Holding the tube and magnet together, decant the cell suspension into a fresh 5 ml tube. Do not tap or shake the tube contents to remove the last drops of liquid.</li>
	<li>Add medium to bring the final volume to 4 ml. Sample 10 &#181;l to count cells using a hemocytometer.</li>
	<li>Recover T cells by centrifugation at 600 x g for 5 min&#160;at room temperature.</li>
	<li>Decant supernatant. Resuspend T cells at 106 cells/ml human T cell culture medium. Transfer 1 ml aliquots to each well of a 24-well tissue culture plate.</li>
	<li>Re-stimulate T cells by adding 100 &#181;l media containing 12.5 &#181;l of stimulatory anti-CD3/anti-CD28 microbeads (as described in sections 5.2 &#8211; 5.6). Incubate at 37 oC in a 6% CO2 incubator.</li>
</ol>

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The authors declare no competing financial interests.

T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This ...

T lymphocytes are critical components of the adaptive immune system. T cells are responsible for not only directly mediating protective immune responses to pathogens through a variety of effector mechanisms, but also actively maintaining immunological self-tolerance and directing the responses of other cells in the immune system. These functions are directed through a number of integrated signals, including T cell receptor (TCR) ligation, cytokines and chemokines, and metabolites1. Of these signals, the TCR is of particular importance, as it provides the characteristic specificity that defines the T cell&#8217;s role in adaptive immunity. A TCR interacts with linear peptide antigens presented by MHC (human ortholog HLA) molecules (pMHC complexes) in a highly specific and sensitive manner to provide the signals that initiate T cell effector function. The biochemical parameters of TCR interactions with pMHC ligands provide not only the specificity for T cell activation, but also have a qualitative impact on subsequent T cell function2. Thus, studying T cell function often requires examining the responses of clonal T cells with defined antigenic specificity.
The human T cell compartment, containing approximately 1012&#945;&#946; T cells, contains an estimated 107&#8211; 108 distinct &#945;&#946;TCRs3-4. This diverse repertoire provides opportunity for recognition of the vast array of peptides from potential pathogens that would necessitate a T cell response for protective immunity. It is estimated that the frequency of T cells responding to a given foreign antigen presented by self-MHC is on the order of 10-4&#8211; 10-7 in the absence of prior immune response to that antigen5. The naive T cell repertoire is shaped by thymic selection to ensure the ability to recognize self-MHC presenting peptide antigens and limit reactivity against self-peptide antigens, maximizing the potential utility for mediating protective immunity2. However, in violation of this designed reactivity, a relatively large frequency, 10-3&#8211; 10-4, of T cells from immunologically naive individuals respond to stimulation with allogeneic cells, recognizing both the foreign MHC molecules as well as the endogenous peptides they present6. The recognition of allogeneic pMHC ligands is structurally similar to the recognition of foreign antigens presented by self-MHC; the TCR makes critical biochemical interactions with both the allogeneic MHC molecule as well as the presented peptide7. The robust nature of the response of T cells to allogeneic stimulation results from the diversity of pMHC complexes present on the surface of allogeneic cells8. It is estimated that each MHC presents approximately 2 x 104 different endogenous peptide antigens9. This breadth of response to allogeneic stimulation is a significant aspect of the clinically-relevant pathology, such as allograft rejection or graft versus host disease (GVHD), resulting from T cell alloreactivity.
Study of human T cell alloreactive responses has traditionally relied upon examining polyclonal responses of naive T cells following stimulation with allogeneic cells. Repeated stimulation with the same allogeneic cell line combined with limiting dilution analyses is capable of generating clonal T cells with defined recognition of allogeneic HLA10. However, this approach is problematic for examining responses to individual allogeneic pMHC ligands, as the large and diverse repertoire of endogenous pMHC complexes present for a given allogeneic HLA stimulates a broad repertoire of T cells. This bulk population stimulation and limiting dilution approach would require screening of large numbers of clones to isolate T cells with the desired reactivity against a single pMHC ligand. Additionally, the frequency of T cells responding to an individual allogeneic pMHC ligand is relatively low among naive T cell populations, which presents a barrier to efficient generation of human T cell clones responsive to a given antigen.
Identification and isolation of antigen-specific T cells from polyclonal populations have been enabled by the development of fluorophore-labeled pMHC multimers11. This approach utilizes specific peptide antigens loaded into recombinant soluble biotinylated MHC molecules, which are labeled by binding to a streptavidin-labeled fluorophore. Multimerization of pMHC increases the avidity, compensating for the intrinsically low (&#181;M) affinity of TCR for soluble pMHC ligands. Labeled cells can be identified and isolated by flow cytometry. However, this approach is still limited by the low frequency of antigen-specific T cells among naive T cell populations, which are typically orders of magnitude less than the limit of accurate identification and quantification on most flow cytometers. To address this limitation, a method of pMHC tetramer labeling and subsequent magnetic bead enrichment for tetramer-labeled cells has been developed12. This method has demonstrated reliable detection, enumeration, and isolation of low-frequency antigen-specific T cells.
This protocol describes an effective protocol for the generation of human T cell clones that specifically respond to individual allogeneic pMHC ligands. The protocol applies pMHC (HLA) multimer labeling and enrichment for the isolation of alloantigen-specific human T cells with flow cytometry cell sorting and a robust method for in vitro culture of human T cells to enable production of T cell clones from single sorted cells (overview in Figure 1).

<table><tbody><tr><td>Sodium heparin venous blood collection tube 16 x 100 mm</td><td>Becton, Dickenson and Company</td><td>366480</td><td></td></tr><tr><td>Lymphoprep</td><td>Stemcell Technologies</td><td>7801</td><td></td></tr><tr><td>Rosette Sep Human T Cell Enrichment Kit</td><td>Stemcell Technologies</td><td>15061</td><td></td></tr><tr><td>Dulbecco&#039;s PBS, 1x without Ca or Mg</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6635</td><td></td></tr><tr><td>Fluorophore-labeled pMHC tetramer</td><td>NIH Tetramer Facility</td><td>NA</td><td></td></tr><tr><td>EasySep Biotin Selection Kit</td><td>Stemcell Technologies</td><td>18553</td><td></td></tr><tr><td>EasySep Selection magnet</td><td>Stemcell Technologies</td><td>18000</td><td></td></tr><tr><td>TruStain FcX Human Fc blocking solution</td><td>Biolegend</td><td>422301</td><td></td></tr><tr><td>Anti-CD5 PE-Cy7 (clone UCHT2)</td><td>Biolegend</td><td>300621</td><td></td></tr><tr><td>Anti-CD14 FITC (clone HCD14)</td><td>Biolegend</td><td>325603</td><td></td></tr><tr><td>Anti-CD19 FITC (clone HIB19)</td><td>Biolegend</td><td>302205</td><td></td></tr><tr><td>Iscove&#039;s DMEM, without b-ME or L-glutamine</td><td>Corning</td><td>15-016-CV</td><td></td></tr><tr><td>HEPES</td><td>Corning</td><td>25-060-CI</td><td></td></tr><tr><td>b-Mercaptoethanol&amp;nbsp;</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Gentamicin sulfate (50 mg/ml)</td><td>Omega Scientific</td><td>GT-50</td><td></td></tr><tr><td>Human AB serum, male donor</td><td>Omega Scientific</td><td>HS-30</td><td></td></tr><tr><td>Recombinant human IL-2</td><td>Peprotech</td><td>AF 200-02</td><td></td></tr><tr><td>Dynabeads Human T-Activator CD3/CD28</td><td>Life Technologies</td><td>11131D</td><td></td></tr><tr><td>Media</td><td></td><td></td><td></td></tr><tr><td>Cell sorting buffer</td><td></td><td></td><td></td></tr><tr><td>PBS, pH 7.4</td><td>1 L</td><td></td><td></td></tr><tr><td>BSA</td><td>10 g</td><td></td><td></td></tr><tr><td>EDTA (0.5 M)</td><td>2 ml</td><td></td><td></td></tr><tr><td>Human T Cell Culture Medium</td><td></td><td></td><td></td></tr><tr><td>Iscove&#039;s DMEM</td><td>351.6 ml</td><td></td><td></td></tr><tr><td>Heat-inactivated human AB serum</td><td>40 ml</td><td></td><td></td></tr><tr><td>HEPES (1 M)</td><td>4 ml</td><td></td><td></td></tr><tr><td>Glutamax (100x)</td><td>4 ml</td><td></td><td></td></tr><tr><td>Gentamicin (50 mg/ml)</td><td>0.4 ml</td><td></td><td></td></tr><tr><td>b-mercaptoethanol (14.3 M)</td><td>1.4 ml</td><td></td><td></td></tr><tr><td>Recombinant human IL-2 (1 mg/ml)</td><td>1 ml</td><td></td><td></td></tr></tbody></table>

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 protocol describes the generation of clonal human T cell cultures with defined alloantigen specificity via a magnetic bead enrichment and single-cell flow cytometry sorting strategy. Figure 1 provides an outline of the process.
<img alt="Figure 1" src="/files/ftp_upload/52257/52257fig1highres.jpg" /> 
Figure 1: Protocol overview. The protocol described here provides a reliable method for generation of alloantigen...

Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

generation-of-human-alloantigen-specific-t-cells-from-peripheral-blood

Research

JoVE Journal

Immunology and Infection

12.9K Views.  University of California, San Diego. 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.

Video: Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

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 ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Developmental Biology

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Cancer Research

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Biology

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

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.

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.

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

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 ...

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 ...

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 ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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Rozdziały w tym wideo

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

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

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

Generation of Human CD40-activated B cells

Isolation and Ex Vivo Culture of Vδ1⁺CD4⁺γδ T Cells, an Extrathymic αβT-cell Progenitor

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

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

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

Generation of Human Regulatory T Cell Clones