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 (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 &#947;&#948; T cells in PBMC by flow cytometry (section 3) (Fig. 2).</li>
</ol>
2. Expansion of &#947;&#948; 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 &#956;M, respectively. ALyS203 (Cell Science &#38; Technology Institute) or OpTmizer (Invitrogen) media support good expansion of &#947;&#948; T cells (more information in references 6 and 9). Zometa is provided in liquid form (4 mg/5-ml vial). To prepare a 5 &#956;M solution, add 50 &#956;l 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 &#956;l for each well of a 24-well plate). Place the plates in a humidified 37&#176;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 &#947;&#948; T cells by flow cytometry (see below).</li>
</ol>
3. Phenotypic analysis by flow cytometry
<ol>
	<li>Transfer 200 &#956;l 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 &#956;l FACS buffer (PBS + 1% FCS + 0.1% sodium azide). Add 5 &#956;l 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&#176;C. Carefully decant the supernatant.</li>
	<li>Resuspend the cells in 300 &#956;l FACS buffer and vortex. Analyze samples on a flow cytometer (Fig. 2).</li>
</ol>
4. IFN-&#947; 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 &#956;M) (hereafter designated Z-Daudi).</li>
	<li>Collect Z-Daudi and resuspend in RPMI-10 at 2x106 cells/ml. Add 100 &#956;l of Z-Daudi (2x105) to each well of a round-bottom 96-well plate.</li>
	<li>Prepare &#947;&#948; T cells at 2x106 cells/ml in RPMI-10 containing Brefeldin A at 20 &#956;g/ml. Transfer 100 &#956;l of &#947;&#948; T cell suspension (2x105) to each well containing Z-Daudi cells or to control wells (100 &#956;l of RPMI-10 only, or RPMI-10 with 20 ng/ml of phorbol 12-myristate 13-acetate [PMA] plus 2 &#956;g/ml of ionomycin).</li>
	<li>Mix by pipetting up and down several times. Incubate for 4 hr in a 37&#176;C, 5% CO2 incubator.</li>
	<li>Centrifuge the plate for 5 min at 400 x g at 4&#176;C and resuspend the pellets in 200 &#956;l 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&#176;C.</li>
	<li>Resuspend the pellets in 50 &#956;l of FACS buffer with FITC-conjugated anti-TCRV&#947;9 (5 &#956;l) and PE/Cy5-conjugated anti-CD3 mAb (2.5 &#956;l). Incubate protected from light for 15 min at room temperature.</li>
	<li>Add 100 &#956;l 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 &#956;l of IntraPrep reagent 2. Incubate for 5 min at room temperature without shaking.</li>
	<li>Add 5 &#956;l of PE-conjugated anti-IFN-&#947; 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+ TCRV&#947;9+ cells and examine the expression of IFN-&#947; (Fig. 4).</li>
</ol>
5. Representative Results:
It is important to determine the percentage of &#947;&#948; T cells in PBMC at the initiation of culture. As shown in Fig. 2 A, the percentage of CD3+TCRV&#947;9+ &#947;&#948; T cells in PBMC was 1.6% on day 0. The dominant populations were CD27+CD45RA+ naive or CD27+CD45RA- central memory phenotypes. When &#947;&#948; 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+ &#945;&#946; T cells or NK cells could dominate the growth of &#947;&#948; T cells (Fig. 3 C and D). After 14 days of culture, the frequency of &#947;&#948; T cells increased to more than 93.8% of the cultured cells in successful &#947;&#948; T cell cultures (Fig. 2 E). The cultured &#947;&#948; 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 &#947;&#948; T cells were evaluated with regard to cytokine production and cytotoxicity. The intracellular IFN-&#947; staining demonstrated that &#947;&#948; T cells produced IFN-&#947; 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 &#947;&#948; 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>TCR&#945;&#946;</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 V&#947;9</td>
			<td>TCR&#945;&#946;</td>
			<td>CD45</td>
			<td>CD3</td>
		</tr>
		<tr>
			<td>5</td>
			<td>TCR V&#947;9</td>
			<td>NKG2D</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6</td>
			<td>TCR V&#947;9</td>
			<td>CD69</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7</td>
			<td>TCR V&#947;9</td>
			<td>mouse IgG1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8</td>
			<td>TCR V&#947;9</td>
			<td></td>
			<td>CD45RA</td>
			<td>CD27</td>
		</tr>
		<tr>
			<td>9</td>
			<td>TCR V&#947;9</td>
			<td></td>
			<td>mouse IgG1</td>
			<td>mouse IgG1</td>
		</tr>
	</tbody>
</table>
Table 1. Monoclonal antibodies used in multicolor staining of &#947;&#948; T cells. An example of the phenotypic analysis of &#947;&#948; T cells performed in our laboratory is shown in Fig. 2.
<img alt="Figure 1" src="/files/ftp_upload/3182/3182fig1.jpg" /> 
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="Figure 2" src="/files/ftp_upload/3182/3182fig2.jpg" /> 
Figure 2. Typical surface phenotype of &#947;&#948; T cells. PBMC were stimulated with zoledronate and IL-2 for 14 days. Cells were stained with FITC-labeled anti-TCR V&#947;9 and PE/Cy5-labeled anti-CD3 to monitor the expansion of &#947;&#948; T cells (A and E). &#947;&#948; T cells were identified by their expression of TCRV&#947;9, and their expression of CD27 and CD45RA (B and F), NKG2D (C and G), or CD69 (D and H) was examined.
<img alt="Figure 3" src="/files/ftp_upload/3182/3182fig3.jpg" /> 
Figure 3. Representative &#947;&#948; T cell cultures. PBMC were stimulated with IL-2 (1000 IU/ml) and zoledronate (5 &#956;M). Representative fields are shown (IX71 inverted microscope [Olympus] x 200). Clusters and aggregates of &#947;&#948; T cells can be observed on day 3 (A) and day 5 (B), when &#947;&#948; T cells were successfully expanded. In contrast, no clusters or aggregates were observed when &#947;&#948; T cell growth was not adequate (C and D).
<img alt="Figure 4" src="/files/ftp_upload/3182/3182fig4.jpg" /> 
Figure 4. IFN-&#947; production. &#947;&#948; T cells were incubated with RPMI-10 only (A) or PMA/ionomycin (B) or Z-Daudi (C) for 4 hr. First, surface expression of TCRV&#947;9 was stained and then IFN-&#947; production was examined by intracellular IFN-&#947; staining.
<img alt="Figure 5" src="/files/ftp_upload/3182/3182fig5.jpg" /> 
Figure 5. Kinetics of &#947;&#948; T cell culture. (A) Absolute number of cultured cells, (B) percentage of &#947;&#948; T cells, and (C) absolute number of &#947;&#948; T cells at the indicated time points.

<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. 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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 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  &#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

Research

JoVE Journal

Immunology and Infection

26.5K Views.  University of Tokyo Hospital. A method to expand γδ T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived γδ T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of γδ T cells can be applied to autologous cellular immunotherapy of cancer.

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

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 ...

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 ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

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.