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°C to 25°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 (seen from top to bottom): a) plasma - b) peripheral blood mononuclear cells (PBMC) and platelets - c) density solution - d) polyester gel - e) granulocytes - f) red blood cells (Fig. 1).</li>
	<li>Collect a fraction of the plasma layer, leaving 5 to 10 mm of plasma above the interphase without disturbing the cell layer. The plasma can be used for the culture (section 2.5).</li>
	<li>Harvest the enriched fraction (PBMC) at the interphase with a pipette and transfer to a 15 ml conical tube.</li>
	<li>Wash the PBMC with 10 ml of phosphate-buffered saline (PBS), by inverting the tube 5 times, and then centrifuge for 5 min at 400 x g.</li>
	<li>Repeat washing steps twice, and then resuspend the cell pellet in 5 ml of PBS. Determine the cell number. Usually 1.3 x106 cells are recovered from 1 ml of whole blood.</li>
	<li>Determine the frequency and phenotype of γδ T cells in PBMC by flow cytometry (section 3) (Fig. 2).</li>
</ol>
2. Expansion of γδ T cells
<ol>
	<li>Centrifuge cell suspensions in 15 ml conical tubes for 5 min at 400 x g at room temperature, and discard the supernatants.</li>
	<li>Prepare culture medium (CM) by adding human IL-2 (IL-2) and zoledronate (Zometa) to final concentrations of 1000 IU/ml and 5 μM, respectively. ALyS203 (Cell Science &amp; Technology Institute) or OpTmizer (Invitrogen) media support good expansion of γδ T cells (more information in references 6 and 9). Zometa is provided in liquid form (4 mg/5-ml vial). To prepare a 5 μM solution, add 50 μl of Zometa to 30 ml of culture medium.</li>
	<li>Resuspend cell pellet in culture medium and adjust to 1x106 cells/ml.</li>
	<li>Pipet 1 ml of CM containing 1x106 cells into each well of a 24-well plate. For large-scale cultures, cells can be seeded at 0.5 x 106 cells/cm2 according to the surface areas of plate wells, dish, or flask.</li>
	<li>Add autologous plasma (section 1.3), pooled human AB sera, or FCS so that it is approximately 10% of the volume of the culture (100 μl for each well of a 24-well plate). Place the plates in a humidified 37°C, 5% CO2 incubator for 24-48 hr.</li>
	<li>Maintain the culture at a cell density of 0.5-2 x 106 cells/ml. Add fresh medium containing human IL-2 (1000 IU/ml) only (without Zometa) every 2-3 days, and transfer cultured cells into new wells or flasks as necessary, according to the degree of cell proliferation (Fig. 3). Supply plasma or serum to the medium so that the serum concentration can be maintained at least 1%.</li>
	<li>Harvest cells on day 12-14 and determine the frequency, phenotype, and functions of γδ T cells by flow cytometry (see below).</li>
</ol>
3. Phenotypic analysis by flow cytometry
<ol>
	<li>Transfer 200 μl samples containing 2 x 105 cells to fluorescence-activated cell sorting (FACS) tubes.</li>
	<li>Add 2 ml of cold PBS and centrifuge for 5 min at 400 x g. Then, resuspend the pellets in 50 μl FACS buffer (PBS + 1% FCS + 0.1% sodium azide). Add 5 μl of each antibodies to the samples (the monoclonal antibodies are listed in Table 1).</li>
	<li>Incubate on ice in the dark for 20 min.</li>
	<li>Add 2 ml FACS buffer to each sample, and then vortex. Centrifuge samples for 5 min at 400 x g at 4°C. Carefully decant the supernatant.</li>
	<li>Resuspend the cells in 300 μl FACS buffer and vortex. Analyze samples on a flow cytometer (Fig. 2).</li>
</ol>
4. IFN-γ production assay10
<ol>
	<li>The day before assay, prepare stimulator cells by culturing 3-5 x105 Daudi cells/ml in RPMI 1640 medium plus 10% FCS (RPMI-10) overnight with Zometa (5 μM) (hereafter designated Z-Daudi).</li>
	<li>Collect Z-Daudi and resuspend in RPMI-10 at 2x106 cells/ml. Add 100 μl of Z-Daudi (2x105) to each well of a round-bottom 96-well plate.</li>
	<li>Prepare γδ T cells at 2x106 cells/ml in RPMI-10 containing Brefeldin A at 20 μg/ml. Transfer 100 μl of γδ T cell suspension (2x105) to each well containing Z-Daudi cells or to control wells (100 μl of RPMI-10 only, or RPMI-10 with 20 ng/ml of phorbol 12-myristate 13-acetate [PMA] plus 2 μg/ml of ionomycin).</li>
	<li>Mix by pipetting up and down several times. Incubate for 4 hr in a 37°C, 5% CO2 incubator.</li>
	<li>Centrifuge the plate for 5 min at 400 x g at 4°C and resuspend the pellets in 200 μl of cold PBS.</li>
	<li>Transfer the samples to FACS tubes. Add 4 ml of cold PBS and centrifuge the tubes for 5 min at 400 x g at 4°C.</li>
	<li>Resuspend the pellets in 50 μl of FACS buffer with FITC-conjugated anti-TCRVγ9 (5 μl) and PE/Cy5-conjugated anti-CD3 mAb (2.5 μl). Incubate protected from light for 15 min at room temperature.</li>
	<li>Add 100 μl of IntraPrep reagent 1 and incubate for 15 min at room temperature. Add 4 ml of PBS to each tube and centrifuge for 5 min at 400 x g at room temperature.</li>
	<li>Remove the supernatant by aspiration and add 100 μl of IntraPrep reagent 2. Incubate for 5 min at room temperature without shaking.</li>
	<li>Add 5 μl of PE-conjugated anti-IFN-γ mAb to the test tube. Incubate protected from light for 15 min at room temperature.</li>
	<li>Add 4 ml of PBS to each tube and centrifuge for 5 min at 400 x g at room temperature. Remove the supernatant by aspiration and resuspend the cell pellet in 0.5 ml of FACS buffer.</li>
	<li>Analyze the cells by flow cytometer. Gate on CD3+ TCRVγ9+ cells and examine the expression of IFN-γ (Fig. 4).</li>
</ol>
5. Representative Results:
It is important to determine the percentage of γδ T cells in PBMC at the initiation of culture. As shown in Fig. 2 A, the percentage of CD3+TCRVγ9+ γδ T cells in PBMC was 1.6% on day 0. The dominant populations were CD27+CD45RA+ naive or CD27+CD45RA- central memory phenotypes. When γδ T cells were efficiently stimulated, they formed clusters on days 3-5 (Fig. 3 A and B). When cluster formation was delayed, the growth of other cell types, such as CD4+ or CD8+ αβ T cells or NK cells could dominate the growth of γδ T cells (Fig. 3 C and D). After 14 days of culture, the frequency of γδ T cells increased to more than 93.8% of the cultured cells in successful γδ T cell cultures (Fig. 2 E). The cultured γδ T cells upregulated NKG2D and CD69 expression (Fig. 2 G and H). They displayed CD27-CD45RA- effector memory phenotype (Fig. 2 F). The functions of γδ T cells were evaluated with regard to cytokine production and cytotoxicity. The intracellular IFN-γ staining demonstrated that γδ T cells produced IFN-γ in response to PMA/ionomycin treatment or Z-Daudi cells that accumulated IPP after zoledronate treatment (Fig. 4). These results indicate that zoledronate can efficiently stimulate and expand functional γδ T cells.
<table border="1">
	<tbody>
		<tr>
			<td>tube</td>
			<td>FITC</td>
			<td>PE</td>
			<td>ECD</td>
			<td>PE/Cy5</td>
		</tr>
		<tr>
			<td>1</td>
			<td>CD3</td>
			<td>CD19</td>
			<td>CD45</td>
			<td>CD14</td>
		</tr>
		<tr>
			<td>2</td>
			<td>CD3</td>
			<td>TCRαβ</td>
			<td>CD4</td>
			<td>CD8</td>
		</tr>
		<tr>
			<td>3</td>
			<td>CD3</td>
			<td></td>
			<td></td>
			<td>CD56</td>
		</tr>
		<tr>
			<td>4</td>
			<td>TCR Vγ9</td>
			<td>TCRαβ</td>
			<td>CD45</td>
			<td>CD3</td>
		</tr>
		<tr>
			<td>5</td>
			<td>TCR Vγ9</td>
			<td>NKG2D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6</td>
			<td>TCR Vγ9</td>
			<td>CD69</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7</td>
			<td>TCR Vγ9</td>
			<td>mouse IgG1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8</td>
			<td>TCR Vγ9</td>
			<td></td>
			<td>CD45RA</td>
			<td>CD27</td>
		</tr>
		<tr>
			<td>9</td>
			<td>TCR Vγ9</td>
			<td></td>
			<td>mouse IgG1</td>
			<td>mouse IgG1</td>
		</tr>
	</tbody>
</table>
Table 1. Monoclonal antibodies used in multicolor staining of γδ T cells. An example of the phenotypic analysis of γδ T cells performed in our laboratory is shown in Fig. 2.
<img alt="Blood separation layers in centrifuge tube; plasma, PBMC, density, gel, granulocytes, RBCs." src="/files/ftp_upload/3182/3182fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Separation of PBMC. Blood (7.5-8.0 ml) is drawn into a BD Vacutainer CPT Cell Preparation Tube with Sodium Heparin and directly centrifuged for 20 min at 1800 x g. After centrifugation, the resulting layers as seen from top to bottom: a) Plasma - b) PBMC and platelets - c) Density solution - d) Polyester gel - e) Granulocytes - f) Red blood cells.
<img alt="Flow cytometry graphs showing immune cell markers TCR Vγ9, CD3, CD45RA, CD27, NKG2D, CD69 on days 0, 14." src="/files/ftp_upload/3182/3182fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Typical surface phenotype of γδ T cells. PBMC were stimulated with zoledronate and IL-2 for 14 days. Cells were stained with FITC-labeled anti-TCR Vγ9 and PE/Cy5-labeled anti-CD3 to monitor the expansion of γδ T cells (A and E). γδ T cells were identified by their expression of TCRVγ9, and their expression of CD27 and CD45RA (B and F), NKG2D (C and G), or CD69 (D and H) was examined.
<img alt="Cell culture differentiation process; microscopy images; stages A-D; in vitro study; stem cells." src="/files/ftp_upload/3182/3182fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Representative γδ T cell cultures. PBMC were stimulated with IL-2 (1000 IU/ml) and zoledronate (5 μM). Representative fields are shown (IX71 inverted microscope [Olympus] x 200). Clusters and aggregates of γδ T cells can be observed on day 3 (A) and day 5 (B), when γδ T cells were successfully expanded. In contrast, no clusters or aggregates were observed when γδ T cell growth was not adequate (C and D).
<img alt="Flow cytometry result; TCR Vγ9 vs. IFN-γ levels in different treatments; scatter plots." src="/files/ftp_upload/3182/3182fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. IFN-γ production. γδ T cells were incubated with RPMI-10 only (A) or PMA/ionomycin (B) or Z-Daudi (C) for 4 hr. First, surface expression of TCRVγ9 was stained and then IFN-γ production was examined by intracellular IFN-γ staining.
<img alt="Cell growth analysis chart; cultured cells vs. % γδT cells over 14 days; research data visualizations." src="/files/ftp_upload/3182/3182fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Kinetics of γδ T cell culture. (A) Absolute number of cultured cells, (B) percentage of γδ T cells, and (C) absolute number of γδ T cells at the indicated time points.

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	<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>

No conflicts of interest declared.

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><tbody><tr><td>ZOMETA</td><td>Novartis AG</td><td></td><td>zoledronate</td></tr><tr><td>PROLEUKIN</td><td>Novartis AG</td><td></td><td>human recombinant IL-2</td></tr><tr><td>BD Vacutainer CPT Cell Preparation Tube with Sodium Heparin</td><td>BD Biosciences</td><td>362753</td><td></td></tr><tr><td>RPMI1640</td><td>Invitrogen</td><td>21870-076</td><td></td></tr><tr><td>ALyS203- medium</td><td>Cell Science &amp;amp; Technology Institute</td><td>0301-7</td><td></td></tr><tr><td>OpTmizer</td><td>Invitrogen</td><td>0080022SA</td><td></td></tr><tr><td>brefeldin A</td><td>Sigma-Aldrich</td><td>B5936-200UL</td><td></td></tr><tr><td>phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma-Aldrich</td><td>P1585-1MG</td><td></td></tr><tr><td>ionomycin</td><td>Sigma-Aldrich</td><td>13909-1ML</td><td></td></tr><tr><td>IntraPrep</td><td>Beckman Coulter Inc.</td><td>A07803</td><td></td></tr><tr><td>anti-human CD3-FITC or PE/Cy5</td><td>Beckman Coulter Inc.</td><td>A07746 FITC A07749 PE/Cy5</td><td></td></tr><tr><td>anti-human CD4-ECD</td><td>Beckman Coulter Inc.</td><td>6604727</td><td></td></tr><tr><td>anti-human CD8-PE/Cy5</td><td>Beckman Coulter Inc.</td><td>6607011</td><td></td></tr><tr><td>anti-human CD14-PE/Cy5</td><td>Beckman Coulter Inc.</td><td>A07765</td><td></td></tr><tr><td>anti-human CD19-PE</td><td>Beckman Coulter Inc.</td><td>A07769</td><td></td></tr><tr><td>anti-human CD45-ECD</td><td>Beckman Coulter Inc.</td><td>A07784</td><td></td></tr><tr><td>anti-human CD56-PE/Cy5</td><td>Beckman Coulter Inc.</td><td>A07789</td><td></td></tr><tr><td>anti-human TCR&amp;alpha;&amp;#946;-PE</td><td>Beckman Coulter Inc.</td><td>A39499</td><td></td></tr><tr><td>anti-human TCR V&amp;#947;9-FITC</td><td>Beckman Coulter Inc.</td><td>IM1463</td><td></td></tr><tr><td>anti-human CD27-PE/Cy5</td><td>Beckman Coulter Inc.</td><td>6607107</td><td></td></tr><tr><td>anti-human CD45RA-ECD</td><td>Beckman Coulter Inc.</td><td>IM2711</td><td></td></tr><tr><td>anti-human CD69-PE</td><td>BD Biosciences</td><td>555531</td><td></td></tr><tr><td>anti-human NKG2D-PE</td><td>Beckman Coulter Inc.</td><td>A08934</td><td></td></tr><tr><td>Anti-humal IFN&amp;#947;-PE</td><td>Beckman Coulter Inc.</td><td>IM2717U</td><td></td></tr><tr><td>Mouse IgG1 isotype control-PE</td><td>Beckman Coulter Inc.</td><td>A07796</td><td></td></tr><tr><td>Mouse IgG1 isotype control-ECD or PE/Cy5</td><td>Beckman Coulter Inc.</td><td>A07797A07798</td><td></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  &#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.

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 ...

expansion-of-human-peripheral-blood-t-cells-using-zoledronate

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Immunology and Infection

27.5K Views.  University of Tokyo Hospital. Een methode om γδ T-cellen uit het perifere bloed mononucleaire cellen (PBMC) uit te breiden wordt beschreven. PBMC-afgeleide γδ T-cellen worden gestimuleerd en uitgebreid met behulp van zoledronaat en interleukine-2 (IL-2). Grootschalige uitbreiding van γδ T-cellen kunnen worden toegepast op autologe cellulaire immunotherapie van kanker.

Video: Expansion of Human Peripheral Blood  γδ T Cells using Zoledronate

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Cancer Research

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment challenges persist. Robust and disease-relevant animal models are vital resources for continued preclinical research and development in order to address a range of additional immune checkpoints. Here, we describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft model. BGB-A317 (Tislelizumab), an investigational humanized anti-PD-1 antibody in late-stage clinical development, is used as an example to discuss platform set-up, model characterization and drug efficacy evaluations. These humanized mice support the growth of most human tumors tested, thus allowing the assessment of I-O therapies in the context of both human immunity and human cancers. Once established, our model is comparatively time- and cost-effective, and usually yield highly reproducible results. We suggest that the protocol outlined in this article could serve as a general guideline for establishing mouse models reconstituted with human PBMC and tumors for I-O research.

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy assessment.

The discovery and development of immuno-oncology (I-O) therapy in recent years represents a milestone in the treatment of cancer. However, treatment ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major histocompatibility complex (MHC)-independent manner and develop strong cytolytic effector function that makes it an ideal candidate for cancer immunotherapy. Due to the low frequency of Gamma-Delta (&#947;&#948;) T cells in the peripheral blood, we developed an effective protocol to greatly expand a highly pure &#947;&#948; T cells drug product for first-in-human use of allogeneic &#947;&#948; T cells in patients with acute myeloid leukemia (AML). Using healthy donor apheresis as an allogenic cell source, the lymphocytes are isolated using a validated device for a counterflow centrifugation method of separating cells by size and density. 
The lymphocyte-rich fraction is utilized, and the &#947;&#948; T cells are preferentially activated with zoledronic acid (FDA-approved) and interleukin (IL)-2 for 7 days. Following the preferential expansion of &#947;&#948; T cells, a clinical-grade magnetic cell-separation device and TCR&#945;&#946; beads are used to deplete contaminating T-cell receptor (TCR)&#945;&#946; T cells. The highly enriched &#947;&#948; T cells then undergo a second expansion using engineered artificial antigen-presenting cells (aAPCs) derived from K562 cells-genetically engineered to express single-chain variable fragment (scFv) for CD3 and CD28, 41BBL (CD137L) and IL15-RA-together with zoledronic acid and IL-2. Seeding all day-7 enriched &#947;&#948; T cells in co-culture with the aAPCs facilitates the manufacture of highly pure &#947;&#948; T cells with an average fold expansion of &gt;229,000-fold from healthy donor blood.

Expansion and Enrichment of Gamma-Delta &#947;&#948; T Cells from Apheresed Human Product

Presented is a protocol for the expansion of Gamma-Delta (&#947;&#948;) T cell drug product. Lymphocytes are isolated by elutriation and &#947;&#948;-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The &#947;&#948; cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Medicine

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 ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

Bioengineering

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

Expansion of Human Peripheral Blood γδ T Cells using Zoledronate

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