1. Isolation of PBMC
<ol>
	<li>Draw blood (7.5-8.0 ml) into a BD Vacutainer CPT Cell Preparation Tube with Sodium Heparin. The tube contains a sodium heparin anticoagulant and a Ficoll-Hypaque density fluid, plus a polyester gel barrier, which separates the two liquids. Centrifuge tube/blood sample at room temperature (18&#176;C to 25&#176;C) in a horizontal rotor (swing-out head) for 20 min at 1800 x g. Switch centrifuge brakes off.</li>
	<li>After centrifugation, the sequence of layers occurs as follows (see...

<ol>
	<li>Bonneville, M., O'Brien, R. L., Born, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#947;+T+cell+effector+functions:+a+blend+of+innate+programming+and+acquired+plasticity.">&#947; T cell effector functions: a blend of innate programming and acquired plasticity.</a> Nat Rev Immunol. 10, 467-478 (2010).</li><li>Hintz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+(E)-4-hydroxy-3-methyl-but-2-enyl+pyrophosphate+as+a+major+activator+for+human+&#947;&#948;+T+cells+in+Escherichia+coli.">Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human &#947;&#948; T cells in Escherichia coli.</a> FEBS Lett. 509, 317-322 (2001).</li><li>Gober, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T+cell+receptor+&#947;&#948;+cells+recognize+endogenous+mevalonate+metabolites+in+tumor+cells.">Human T cell receptor &#947;&#948; cells recognize endogenous mevalonate metabolites in tumor cells.</a> J Exp Med. 197, 163-168 (2003).</li><li>Kabelitz, D., Wesch, D., He, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+of+gammadelta+T+cells+in+tumor+immunology.">Perspectives of gammadelta T cells in tumor immunology.</a> Cancer Res. 67, 5-8 (2007).</li><li>Roelofs, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+monocytes+are+responsible+for+gammadelta+T+cell+activation+induced+by+zoledronic+acid+through+accumulation+of+IPP/DMAPP.">Peripheral blood monocytes are responsible for gammadelta T cell activation induced by zoledronic acid through accumulation of IPP/DMAPP.</a> Br J Haematol. 144, 245-250 (2009).</li><li>Dieli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+&#947;&#948;+T-lymphocyte+effector+functions+by+bisphosphonate+zoledronic+acid+in+cancer+patients+in+vivo.">Induction of &#947;&#948; T-lymphocyte effector functions by bisphosphonate zoledronic acid in cancer patients in vivo.</a> Blood. 102, 2310-2311 (2003).</li><li>Kondo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zoledronate+facilitates+large-scale+ex+vivo+expansion+of+functional+&#947;&#948;+T+cells+from+cancer+patients+for+use+in+adoptive+immunotherapy.">Zoledronate facilitates large-scale ex vivo expansion of functional &#947;&#948; T cells from cancer patients for use in adoptive immunotherapy.</a> Cytotherapy. 10, 842-856 (2008).</li><li>Espinosa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+and+biological+activity+of+bromohydrin+pyrophosphate,+a+potent+stimulator+of+human+&#947;&#948;+T+cells.">Chemical synthesis and biological activity of bromohydrin pyrophosphate, a potent stimulator of human &#947;&#948; T cells.</a> J Biol Chem. 276, 18337-18344 (2001).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+profile+and+anti-tumor+effects+of+adoptive+immunotherapy+using+&#947;&#948;+T+cells+against+advanced+renal+cell+carcinoma:+a+pilot+study.">Safety profile and anti-tumor effects of adoptive immunotherapy using &#947;&#948; T cells against advanced renal cell carcinoma: a pilot study.</a> Cancer Immunol Immunother. 56, 469-476 (2007).</li><li>Murali-Krishna, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+antigen-specific+CD8+T+cells:+a+reevaluation+of+bystander+activation+during+viral+infection.">Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection.</a> Immunity. 8, 177-187 (1998).</li><li>Sato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+culture+medium+on+the+expansion+of+T+cells+for+immunotherapy.">Impact of culture medium on the expansion of T cells for immunotherapy.</a> Cytotherapy. 11, 936-946 (2009).</li><li>Abe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+and+immunological+evaluation+of+zoledronate-activated+V&#947;9+&#947;&#948;T-cell-based+immunotherapy+for+patients+with+multiple+myeloma.">Clinical and immunological evaluation of zoledronate-activated V&#947;9 &#947;&#948;T-cell-based immunotherapy for patients with multiple myeloma.</a> Exp Hematol. 37, 956-968 (2009).</li><li>Nakajima, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+study+of+adoptive+immunotherapy+for+recurrent+non-small-cell+lung+cancer+patients+with+autologous+&#947;&#948;+T+cells.">A phase I study of adoptive immunotherapy for recurrent non-small-cell lung cancer patients with autologous &#947;&#948; T cells.</a> Eur J Cardiothorac Surg. 37, 1191-1197 (2010).</li></ol>

The method presented here enables efficient expansion of &gamma;&delta; T cells from PBMC. &gamma;&delta; T cells activated and expanded by zoledronate and IL-2 develop complete effector functions, reflected by cytokine production and cytotoxicity. It has been reported that the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP) and 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP) also expand &gamma;&delta; T cells; however, they are not commercially available. In contrast, zoledronate is already licensed for clinica...

<table border="1">
<tbody>
<tr>
	<th>Reagent name</th>
	<th>Company</th>
	<th>Catalogue number</th>
 <th>Comments (optional)</th>
</tr>
<tr>
	<td>ZOMETA</td>
	<td>Novartis Pharma K. K</td>
	<td>&nbsp;</td>
 <td>zoledronate</td>
</tr>
<tr>
	<td>PROLEUKIN</td>
	<td>Novartis Pharmaceuticals</td>
	<td>&nbsp;</td>
 <td>human recombinant IL-2</td>
</tr>
<tr>
	<td>BD Vacutainer CPT Cell Preparation Tube with Sodium Heparin</td>
	<td>BD</td>
	<td>362753</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>RPMI1640</td>
	<td>Invitrogen</td>
	<td>21870-076</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>ALyS203- medium</td>
	<td>Cell Science &amp; Technology Institute</td>
	<td>0301-7</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>OpTmizer</td>
	<td>Invitrogen</td>
	<td>0080022SA</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>brefeldin A</td>
	<td>Sigma</td>
	<td>B5936-200UL</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>phorbol 12-myristate 13-acetate (PMA)</td>
	<td>Sigma</td>
	<td>P1585-1MG</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>ionomycin</td>
	<td>Sigma</td>
	<td>13909-1ML</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>IntraPrep</td>
	<td>BECKMAN COULTER</td>
	<td>A07803</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human CD3-FITC or PE/Cy5</td>
	<td>BECKMAN COULTER</td>
	<td>A07746 FITC 
	A07749 PE/Cy5</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human CD4-ECD</td>
	<td>BECKMAN COULTER</td>
	<td>6604727</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human CD8-PE/Cy5</td>
	<td>BECKMAN COULTER</td>
	<td>6607011</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human CD14-PE/Cy5</td>
	<td>BECKMAN COULTER</td>
	<td>A07765</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human CD19-PE</td>
	<td>BECKMAN COULTER</td>
	<td>A07769</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human CD45-ECD</td>
	<td>BECKMAN COULTER</td>
	<td>A07784</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human CD56-PE/Cy5</td>
	<td>BECKMAN COULTER</td>
	<td>A07789</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human TCR&alpha;&beta;-PE</td>
	<td>BECKMAN COULTER</td>
	<td>A39499</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human TCR V&gamma;9-FITC</td>
	<td>BECKMAN COULTER</td>
	<td>IM1463</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human CD27-PE/Cy5</td>
	<td>BECKMAN COULTER</td>
	<td>6607107</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human CD45RA-ECD</td>
	<td>BECKMAN COULTER</td>
	<td>IM2711</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human CD69-PE</td>
	<td>BD</td>
	<td>555531</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>anti-human NKG2D-PE</td>
	<td>BECKMAN COULTER</td>
	<td>A08934</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Anti-humal IFN&gamma;-PE</td>
	<td>BECKMAN COULTER</td>
	<td>IM2717U</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Mouse IgG1 isotype control-PE</td>
	<td>BECKMAN COULTER</td>
	<td>A07796</td>
 <td>&nbsp;</td>
</tr>
<tr>
	<td>Mouse IgG1 isotype control-ECD or PE/Cy5		
</td>
	<td>BECKMAN COULTER</td>
	<td>A07797
A07798</td>
 <td>&nbsp;</td>
</tr>
</tbody>
</table>

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

Watch this Scientific Journal Video about Expansion of Human Peripheral Blood  ÃŽÂ³ÃŽÂ´ T Cells using Zoledronate at JoVE.com

Expansion of Human Peripheral Blood  ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ 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.

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 ...

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