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>

<ol>
	<li>Smith-Garvin, J. E., Koretzky, G. A., Jordan, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+activation.">T cell activation.</a> Annu. Rev. Immunol. 27 (1), 591-619 (2009).</li><li>Morris, G. P., Allen, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+TCR+balances+sensitivity+and+specificity+for+the+recognition+of+self+and+pathogens.">How the TCR balances sensitivity and specificity for the recognition of self and pathogens.</a> Nat. Immunol. 13 (2), 121-128 (2012).</li><li>Arstilla, T. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+estimation+of+the+human+&#945;&#946;+T+cell+receptor+diversity.">A direct estimation of the human &#945;&#946; T cell receptor diversity.</a> Science. 286 (5441), 958-961 (1999).</li><li>Robbins, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+assessment+of+T-cell+receptor+&#946;-chain+diversity+in+&#945;&#946;+T+cells.">Comprehensive assessment of T-cell receptor &#946;-chain diversity in &#945;&#946; T cells.</a> Blood. 114 (19), 4099-4107 (2009).</li><li>Alanio, C., Lemaitre, F., Law, H. K. W., Hasan, M., Albert, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+T-cell+alloreactivity.">Specificity of T-cell alloreactivity.</a> Nat. Rev. Immunol. 7 (12), 942-953 (2007).</li><li>Morris, G. P., Ni, P. P., Allen, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alloreactivity+is+limited+by+the+endogenous+peptide+repertoire.">Alloreactivity is limited by the endogenous peptide repertoire.</a> Proc. Natl. Acad. Sci. USA. 108 (9), 3695-3700 (2011).</li><li>Suri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+APCs,+the+autologous+peptides+selected+by+the+diabetogenic+I-Ag7+molecule+are+unique+and+determined+by+the+amino+acid+changes+in+the+P9+pocket.">In APCs, the autologous peptides selected by the diabetogenic I-Ag7 molecule are unique and determined by the amino acid changes in the P9 pocket.</a> J. Immunol. 168 (3), 1235-1243 (2002).</li><li>Yssl, H., Spits, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+maintenance+of+cloned+human+T+cell+lines.">Generation and maintenance of cloned human T cell lines.</a> Curr. Protoc. Immunol. 7, (2002).</li><li>Altman, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+analysis+of+antigen-specific+T+lymphocytes.">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274 (5284), 94-96 (1996).</li><li>Moon, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+CD4++T+cell+frequency+varies+for+different+epitopes+and+predicts+repertoire+diversity+and+response+magnitude.">Naive CD4+ T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude.</a> Immunity. 27 (2), 203-213 (2007).</li><li>Chicz, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+and+promiscuity+among+naturally+processed+peptides+bound+to+HLA-DR+alleles.">Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles.</a> J. Exp. Med. 178 (1), 27-47 (1993).</li><li>Ni, P. P., Allen, P. M., Morris, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ability+to+rearrange+dual+TCRs+enhances+positive+selection,+leading+to+increased+allo-+and+autoreactive+T+cell+repertoires.">The ability to rearrange dual TCRs enhances positive selection, leading to increased allo- and autoreactive T cell repertoires.</a> J. Immunol. In press, (2014).</li><li>Morris, G. P., Uy, G. L., Donermeyer, D., DiPersio, J. F., Allen, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+receptor+T+cells+mediate+pathologic+alloreactivity+in+patients+with+acute+graft-versus-host+disease.">Dual receptor T cells mediate pathologic alloreactivity in patients with acute graft-versus-host disease.</a> Sci. Transl. Med. 5 (188), (2013).</li><li>Altman, J. D., Reay, P. A., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+functional+peptide+complexes+of+class+II+major+histocompatibility+complex+proteins+from+subunits+produced+in+Escherichia+coli.">Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli.</a> Proc. Natl. Acad. Sci. USA. 90 (21), 10330-10334 (1993).</li><li>Sabatino, J. J., Huang, J., Zhu, C., Evavold, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+prevalence+of+low+affinity+peptide-MHC+II+tetramer-negative+effectors+during+polyclonal+CD4++T+cell+responses.">High prevalence of low affinity peptide-MHC II tetramer-negative effectors during polyclonal CD4+ T cell responses.</a> J. Exp. Med. 208 (1), 81-90 (2011).</li></ol>

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 diversity in the T cell response impairs the ability to examine the biochemistry of pMHC ligand recognition driving alloreactive T cell responses. The standard solution to this challenge has been to generate clonal T cell lines through repeated stimulation with a given allogeneic cell line and limiting dilution analysis. While this approach is capable of generating clonal T cell lines with a single specificity, it may not reflect the original composition of the oligoclonal response of naive T cells. Additionally, this approach does not directly identify the specific pMHC alloantigen recognized (even narrowing the response to a single HLA molecule still presents the challenge of finding the stimulatory peptide from among the estimated 2x104 endogenous peptides presented under steady-state conditions), complicating biochemical characterization of T cell ligand recognition in alloreactivity.
The protocol presented here utilizes fluorescently-labeled pMHC multimers, magnetic bead enrichment, and flow cytometry cell sorting to identify and isolate human T cells specific for individual allogeneic pMHC ligands. This technique has become a powerful tool for examining antigen-specific T cell responses12. Our increasing understanding of the highly peptide-specific nature of T cell recognition of allogeneic pMHC complexes7,8 has enabled us to adapt this approach to the study of alloreactive T cells in both mice14 and humans15. This approach uniquely enables examination of the composition of the naive T cell populations that comprise the initial oligoclonal alloreactive response. Furthermore, combination of the pMHC multimer labeling approach with in vitro tissue culture facilitates generation of clonal T cell cultures that can be used to dissect the biochemistry of allogeneic pMHC ligand recognition, a question that cannot adequately be addressed via studying polyclonal T cell responses and is significantly impaired by traditional T cell cloning methods. 
Using this approach, it can be expected that T cells specific for a given alloantigen pMHC should be identifiable for isolation and culture. However, the efficacy of this approach is dependent on multiple variables. First, the frequency of T cells from an immunologically naive donor that recognize a single alloantigen pMHC is relatively low (on the order of 1/103&#8211; 1/106) and varies between individuals. The ability to isolate pMHC-specific T cells depends on their abundance, as not all isolated cells will successfully expand to generate clonal cultures. The efficiency of the protocol can be influenced by several factors including the nature of the peptide used and the viability of the cells during preparation and isolation. The success of this technique also requires identification of allogeneic pMHC ligands capable of being re-folded into fluorescently-labeled multimers from recombinantly expressed HLA molecules and synthesized peptides. While there are multiple descriptions in the literature of identified peptides presented by specific HLA alleles, not all peptide re-fold in solution equally well. The efficacy of pMHC re-folding depends on several factors, including peptide length, solubility, and affinity for the MHC peptide binding pocket16. The ability of pMHC multimers to label antigen-specific T cells is also dependent on the peptide used. The affinity of the peptide for the MHC as well as the affinity of the TCR for the pMHC complex influences the binding of the fluorophore-labeled pMHC multimer. Weak binding can result in low shifts in fluorescence intensity, which can lead to difficulty in identifying antigen-specific cells by flow cytometry. These are significant technical hurdles that can impair the efficient isolation of alloantigen-specific T cells. In addition to these technical limitations, a significant biological caveat to this approach should be noted. T cell binding of pMHC tetramers is limited to TCRs with relatively high ligand affinity. This feature results in excluding T cells with low affinity for a ligand from identification and analysis. This is significant, as these low-affinity T cells are capable of substantial contribution to primary T cell responses17. However, even with these limitations, the approach described here is an efficient tool for the generation of clonal human T cells with defined antigen specificity. While the focus presented here is identification and isolation of human T cells specific for allogeneic pMHC ligands, the T cell identification, isolation, and culture techniques can be easily adapted to other pMHC specificities.

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 border="0" cellpadding="0" cellspacing="0" width="907">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name of Reagent/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:292px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Sodium heparin venous blood collection tube 16 x 100 mm</td>
			<td style="width:223px;">Becton, Dickenson and Company</td>
			<td style="width:119px;">366480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Lymphoprep</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">7801</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:273px;">Rosette Sep Human T Cell Enrichment Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">15061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dulbecco&#39;s PBS, 1x without Ca or Mg</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bovine serum albumin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E6635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Fluorophore-labeled pMHC tetramer</td>
			<td style="width:223px;">NIH Tetramer Facility</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Biotin Selection Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Selection magnet</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:273px;">TruStain FcX Human Fc blocking solution</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">422301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD5 PE-Cy7 (clone UCHT2)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">300621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD14 FITC (clone HCD14)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">325603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD19 FITC (clone HIB19)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">302205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Iscove&#39;s DMEM, without b-ME or L-glutamine</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">15-016-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin sulfate (50 mg/ml)</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">GT-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human AB serum, male donor</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">HS-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2</td>
			<td style="width:223px;">Peprotech</td>
			<td style="width:119px;">AF 200-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Dynabeads Human T-Activator CD3/CD28</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">11131D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Media</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Cell sorting buffer</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PBS, pH 7.4</td>
			<td style="width:223px;">1 L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BSA</td>
			<td style="width:223px;">10g</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA (0.5 M)</td>
			<td style="width:223px;">2 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human T Cell Culture Medium</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Iscove&#39;s DMEM</td>
			<td style="width:223px;">351.6 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Heat-inactivated human AB serum</td>
			<td style="width:223px;">40 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES (1 M)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax (100 x)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin (50 mg/ml)</td>
			<td style="width:223px;">0.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-mercaptoethanol (14.3 M)</td>
			<td style="width:223px;">1.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2 (1 mg/ml)</td>
			<td style="width:223px;">1 ml</td>
			<td style="width:119px;"></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-specific human T cell clones from peripheral blood. The process of single T cell isolation and setting up the cell culture is expected to take approximately 4.5 hr. Expansion of the T cell clones requires multiple rounds of non-specific T cell stimulation and culture, each taking 14 days. Clonal cultures can be tested for alloantigen specificity 28 days after isolation, and cultures can be further expanded for additional testing by repeated rounds of stimulation.
The expected yield of alloantigen-specific T cells will depend on the donor, the alloantigen used, and the corresponding frequency of reactive T cells in the donor. An average healthy donor will have 4.5 - 10.0 x 106 leukocytes/ml of blood, with approximately 20% T lymphocytes. Figure 2 illustrates the results of measuring the binding of T cells from a HLA-DR4 (HLA-DRB1*04:01)-negative donor to a specific alloantigen, amino acid residues 30-48 of the HLA-A2 protein (DTQFVRFDSDAASQRMEPR, A230-48) presented by the class II molecule HLA-DR413.
<img alt="Figure 2" src="/files/ftp_upload/52257/52257fig2highres.jpg" /> 
Figure 2: Flow cytometry sorting of alloantigen tetramer-labeled cells for generating single cell cultures. A. Flow cytometric identification and isolation of A230-48/HLA-DR4 tetramer-labeled T cells from a HLA-DR4-negative donor. T cells enriched for tetramer-labeled cells by magnetic bead selection were sorted by flow cytometry, gating on singlet CD5+CD14-CD19-tetramer+ lymphocytes as illustrated. Tetramer-unlabeled cells were used as a fluorescence-minus one control to establish gating for tetramer+ cells. B. Tetramer+ T cells were sorted into a 96-well round-bottom tissue culture plate containing 100 &#181;l human T cell culture medium. The plate set-up included positive and negative control wells as indicated.
From a starting number of 2.0 x 107 leukocytes we found a frequency of alloantigen-specific cells of 1/4776 T cells:
1.1x106 tetramer-enriched cells x 0.992 (singlets) x 0.744 (lymphocytes) x 0.477 (CD5+CD14-CD19-) x 0.0034 (tetramer+ T cells) / 2.0 x 107 peripheral blood leukocytes x 0.314 (pre-enrichment CD3+)
From the starting 2.0x107 leukocytes we were able to isolate 88 individual tetramer-labeled T cells for culture. After 2 rounds of anti-CD3/CD28 microbead stimulation and culture as described we identified 2 growth-positive cultures by microscopic examination (representative example of growth 10 days after single cell isolation and culture shown in Figure 3.A). Clonal cultures were expanded to 1 ml as described, and alloantigen specificity was assessed by examining binding of the A230-48/HLA-DR4 tetramer (Figure 3.B).
<img alt="Figure 3" src="/files/ftp_upload/52257/52257fig3highres.jpg" /> 
Figure 3. Evaluation of T cell cultures. A. Microscopic examination of 96-well plate T cell cultures. Representative example of T cell culture 10 days after isolation of individual T cells by flow cytometry sorting. Cells were stimulated with 0.5 &#181;l anti-CD3/CD28 microbeads/well (examples indicated by arrowhead) in a volume of 200 &#181;l human T cell culture medium. B. T cell alloantigen specificity of clonal T cell cultures evaluated by flow cytometry analysis of pMHC tetramer labeling. Clonal T cell cultures of flow cytometry isolated CD5+CD14-CD19- A230-48/HLA-DR4 tetramer+ lymphocytes were examined for A230-48/HLA-DR4 tetramer binding after 4 weeks of in vitro culture. Tetramer-unlabeled cells were used as a fluorescence-minus one control to establish gating for tetramer+ cells.

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

Nanyang Technological University

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

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

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 ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &#947;&#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

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 ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

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 ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

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 ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.