The authors would like to thank Laurence Cooper, Harjeet Singh, and Lenka Hurton for their work in creating the initial K562 aAPC and mIL21 fusion vectors.
Funding for this work was provided by the UT MD Anderson Physician Scientist Program, the St. Baldrick's Foundation, and the Legends of Friendswood.

1. Isolation of PBMCs from Buffy Coat
Peripheral blood mononuclear cells (PBMC) are obtained by buoyant density centrifugation on Ficoll-Paque from healthy donor buffy coat samples derived by leukapheresis.
<ol>
<li>The Ficoll-Paque centrifugation is done as per manufacturer's protocol with minor modifications.</li>
<li>Add PBS to a normal-donor blood-bank buffy coat to a final volume of 140 mL (typical buffy coat volume is 40-70 mL).</li>
<li>Layer 35 mL of buffy coat sample on 15 mL of Ficoll-Paque (4 tubes).</li>
<li>Centrifuge at 400g for 20 minutes without brake.</li>
<li>Recover the PBMCs from the Ficoll-Paque: plasma interface, do not discard the RBCs at the bottom of the Ficoll-Paque.</li>
<li>Wash PBMCs three times with PBS, centrifuging each time at 400g for 10 minutes.</li>
<li>PBMCs can be used directly for NK cell expansion at this stage or NK cells can be isolated by RosetteSep (Section 4)</li>
<li>Remaining PBMCs can be frozen in FBS containing 10% DMSO in liquid nitrogen.</li>
<li>Aspirate the Ficoll and collect the RBCs from step 5 in to two 50 mL centrifuge tube, wash three times with PBS (added to 50 mL mark), each time aspirate the supernatant skimming the top of the RBC layer to remove granulocytes.</li>
<li>The RBCs can be used immediately for RosetteSep purification of NK cells (refer to Section 4) or stored in equal volume of Alsever's solution at 4&deg;C for later use (The RBCs can be store for a maximum of 4 weeks). </li>
</ol>
2. NK Cell Expansion
The NK cell expansion can be initiated using PBMCs or purified NK cells. The amount of PBMCs used for expansion can be varied based on the amount of NK cells desired at the end of a three week expansion, refer to representative results section for details. (See Note 1)

STIMULATION 1
Day 0
<ol>
<li>For each 5x106 PBMCs to be expanded, count and irradiate 10x106 K562 Cl9 mIL21 using a gamma irradiator at 100 Gy.</li>
<li>Post irradiation, wash the cells with PBS and resuspend in NK cell expansion media (NKEM).</li>
<li>Seed 5x106 PBMCs with 10x106 irradiated K562 Cl9 mIL21 (1:2 ratio) in 40 mL of NKEM in a T75 flask and place it upright in an incubator at 37&deg;C and 5% CO2.</li>
</ol>
Days 3 and 5
<ol start="4">
<li>Recover cells by centrifugation at 400g for 5 min and replace half of the media with fresh NKEM (adding fresh IL2 for the entire media volume) and continue culture.</li>
</ol>
STIMULATION 2
Day 7
<ol>
<li>Count the number of cells in culture at the end of one week.</li>
<li>Set aside 5x105 cells for phenotyping by flow cytometry (See Note 2)</li>
<li>For each 5x106 cells to be restimulated, count and irradiate 5x106 K562 Cl9 mIL21 using a gamma irradiator at 100 Gy.</li>
<li>Add an equal number of irradiated K562 Cl9 mIL21 (1:1 ratio) and resuspend in NKEM at 2.5x105 total cells/mL (See Note 3).</li>
<li>Seed cells in T75 flasks (maximum 50 mL per flask).</li>
</ol>
Days 10 and 12
<ol start="6">
<li>Count the number of cells.</li>
<li>Change entire media with fresh NKEM based on the cell numbers (See Note 3).</li>
</ol>
Day 14
<ol start="8">
<li>At the end of two weeks of expansion count the number of cells in culture.</li>
<li>Set aside 5x105 cells for phenotyping by flow cytometry (See Note 2)</li>
<li>If expansion was started from PBMCs the NK cells can be purified at this stage of expansion using the RosetteSep purification protocol (refer to Section IV). If expansion was started from purified NK cells proceed to stimulation 3.</li>
<li>After purification set aside 5x105 cells for phenotyping by flow cytometry to verify purity of the NK cells (as in Step 8).</li>
<li>Proceed with Stimulation 3 using all of the purified NK cells (See Note 4).</li>
</ol>
STIMULATION 3
<ol>
<li>Resuspend NK cells with irradiated K562 Cl9 mIL21 (1:1 ratio) in NKEM based on cell numbers (See Note 3).</li>
</ol>
Days 17 and 19
<ol start="2">
<li>Count the number of cells.</li>
<li>Change media with fresh NKEM based on the cell numbers (See Note 3).</li>
</ol>
Day 21
<ol start="4">
<li>At the end of three weeks of expansion count the number of cells in culture.</li>
<li>Recover 1x106 cells for flow cytometry analysis for full NK cell phenotyping antibody panel (See Table 1).</li>
<li>Freeze cells in FBS containing 10% DMSO at a maximum density of 5x107 cells per vial for future use.</li>
</ol>
3. NK Cell Cytotoxicity Assay
<ol>
<li>Thaw a vial of NK cells and seed in NKEM, one day prior to performing cytotoxicity assay to allow recovery.</li>
<li>For each NK cell cytotoxicity assay using a single target cell line, 6x105 NK cells and 3x105 target cells are required (See Note 5).</li>
<li>Prepare CAM-Media by diluting Calcein-AM (stock 1 mg/mL in DMSO) in NKEM (See Note 6).</li>
<li>Resuspend 106 target cells in 1 mL of CAM-media (See Note 7).</li>
<li>Incubate for 1 h at 37 &deg;C, with occasional shaking.</li>
<li>Resuspend NK cells at 1x106 cells/ mL and add 200uL of NK cell suspension to each of the 3 wells of a U-bottom 96-well plate corresponding to10:1 E:T ratio shown in Figure 1. (See Note 8)</li>
<li>Add 100uL of complete media to all remaining wells except for "Maximum". </li>
<li>Add 100uL of 2% Triton X-100 to "Maximum".</li>
<li>Perform serial dilutions of the NK cells for the 5 subsequent E:T ratios by transferring 100uL of cells each time, mix well. Discard 100uL from the last wells (E:T ratio of 0.3125:1).</li>
<li>After 1 hour of calcein loading, wash target cells in NKEM twice, centrifuging for 5 min at 1200 rpm. (See Note 9)</li>
<li>Re-count the target cells and resuspend at 1x105 cells/ mL.</li>
<li>Add 100 &mu;L of target cells to each well (1x104/well). Centrifuge for 1 min at 100g to initiate cell contact.</li>
<li>Incubate at 37 &deg;C and 5% CO2 for 4 hours.</li>
<li>Mix the culture gently by pipetting with a 100 &mu;L pipetter in order to uniformly suspend the released calcein, spin down plate at 100g for 5 minutes to pellet the cells and transfer 100 &mu;L of the supernatant to a new plate taking care to avoid bubbles. Pop any bubbles that may form using fine needle.</li>
<li>Read the plate using a fluorescent plate reader (excitation filter 485 nm, emission filter 530 nm). Bottom read is recommended.</li>
<li>Calculate Percent Specific Lysis according to the formula [(test release-spontaneous release)/(maximum release - spontaneous release)] x 100.</li>
</ol>
4. NK Cell Purification by RosetteSep
<ol>
<li>Take 100-fold excess of RBCs to that of PBMCs or expanded cells, into a 50 mL tube (100:1 RBC:PBMC).</li>
<li>If using fresh RBCs proceed directly to next step or if the RBCs were stored in Alsever s solution, count the number of RBCs and wash appropriate (100 fold excess) amount of RBCs with PBS supplemented with 2% FBS three times, centrifuging at 1200 rpm for 10 minutes each time.</li>
<li>Combine RBCs with PBMCs from step 1.7 or expanded cells from step 2.10 in PBS + 2% FBS to a final volume of 1 mL per 5x107 of PBMCs or expanded cells.</li>
<li>Add 1&mu;L of RosetteSep Human NK Cell Enrichment Cocktail per 1x106 of PBMCs or expanded cells.</li>
<li>Mix well and incubate at room temperature for 20 minutes with gentle mixing every 5 minutes.</li>
<li>Add equal volume of PBS + 2% FBS mix gently and layer on top of Ficoll-Paque.</li>
<li>Repeat the steps of Ficoll-Paque centrifugation described for PBMC isolation (Section I).</li>
<li>Count the NK cells recovered after purification and set aside 5x105 cells for phenotpying by flow cytometry for NK cell purity (Step 8).</li>
</ol>
5. Notes
NOTE 1. NK cells can be expanded directly from PBMCs, or from RosetteSep purified NK cells. We have noted similar expansion efficiency, but some donors may have very low NK cell numbers resulting in difficulty purifying by RosetteSep prior to expansion.
NOTE 2. We routinely use CD56-FITC, CD16-PE, and CD3-PE-Cy5 for phenotyping during the expansion, enumerating NK cells as those which are CD3-negative and CD16- or CD56-positive.
NOTE 3. At each media change or stimulation, resuspend cells at 2.5 x 105/mL to keep PBMC/NK cell numbers at or under 2 million per mL at peak stages of expansion. This will prevent depletion of nutrients and help achieve maximal expansion and survival.
NOTE 4. The NK expansion rate is donor dependent and at the end of stimulations 1 or 2 part of the cells can be frozen and part expanded further depending on the experimental need. We have had good success in using the frozen cells for expansions at a later time.
NOTE 5. In order to allow room for error we recommend using a minimum of 7x105 NK cells resuspended in 700 uls of NKEM and 4x105 Calcein-AM stained target cells resuspended in 4 mL of NKEM for setting up the cytotoxicity assay. If using multichannel pipette for seeding target cells higher volumes of cells (up to 6x105 in 6 mL) may be required based on the size of media basin being used. Also the recommended NK cell numbers are specifically for the E:T ratios show in the protocol, for using higher E:T ratios increase the NK cell numbers per mL accordingly (eg. For a 40:1 E:T ratio use 4x106 cells/ mL)
NOTE 6. We recommend performing a preliminary Calcein-AM loading titration for the target cell line of choice, using the following dilutions of 1:500, 1:400. 1:300, 1:200 and 1:100 to achieve optimal difference between maximum and spontaneous release.
NOTE 7. When using an adherent cell line as target, first prepare single cell suspension using non-enzymatic cell dissociation buffer. If performing ADCC, prepare a duplicate tube of target cells in CAM-media.
NOTE 8. If performing ADCC, add same NK cells to 3 wells corresponding to 10:1 E:T for ADCC. Repeat for additional donors. Repeat for additional target cells.
NOTE 9. If performing an ADCC experiment, after 45 minutes of calcein loading, add 10ug of antibody specific for inducing ADCC against the target cells. After 15 more minutes, wash target cells in complete medium twice, centrifuging for 5 min at 1200 rpm. Resuspend cells at 1x105 cells/ mL and proceed with the next step in the protocol.
6. REPRESENTATIVE RESULTS
<img src="/files/ftp_upload/2540/2540fig2.jpg" alt="Figure 2" /> 
Figure 2. When the expansion is performed as per the scheme described above, using 5x106 PBMCs as starting material, typical NK cell yields range from 1x109 to 1010 cells (donor dependent variability). The figure shows NK cell fold expansion (n=19) compared to NK cells present in the original product (median +/- quartile).
<img src="/files/ftp_upload/2540/2540fig3.jpg" alt="Figure 3" /> 
Figure 3. The expanded NK cells express various NK cell receptors that are comparable to the unexpanded primary NK cells with a few exceptions (CD11b, CD160 and CD244).
<img src="/files/ftp_upload/2540/2540fig4.jpg" alt="Figure 4" /> 
Figure 4. PBMCs recovery from Buffy coat is donor dependent and can range from 300x106 to 800x106. NK cells may comprise 2% - 18% of the PBMCs. For RosetteSep purification of expanded cells, recovery of pure NK cells on day 14 ranges from 40-70%. By following the recommended protocol of expansion and purification, NK cell purity of 99% can be expected. 
<img src="/files/ftp_upload/2540/2540fig5.jpg" alt="Figure 5" /> 
Figure 5. Expanded NK cells have demonstrated cytotoxicity against a range of tumor cell lines including neuroblastoma, AML, osteosarcoma and melanoma (representative AML killing shown as percent specific lysis).

<ol>
	<li>McKenna, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+manufacturing+practices+production+of+natural+killer+cells+for+immunotherapy:+a+six-year+single-institution+experience.">Good manufacturing practices production of natural killer cells for immunotherapy: a six-year single-institution experience.</a> Transfusion. 47, 520-520 (2007).</li><li>Koehl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ex+vivo+expansion+of+highly+purified+NK+cells+for+immunotherapy+after+haploidentical+stem+cell+transplantation+in+children.">ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children.</a> Klinische Padiatrie. 217, 345-345 (2005).</li><li>Klingemann, H. G., Martinson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ex+vivo+expansion+of+natural+killer+cells+for+clinical+applications.">ex vivo expansion of natural killer cells for clinical applications.</a> Cytotherapy. 6, 15-15 (2004).</li><li>Ayello, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cord+blood+natural+killer+and+lymphokine+activated+killer+lymphocytes+following+ex+vivo+cellular+engineering.">Characterization of cord blood natural killer and lymphokine activated killer lymphocytes following ex vivo cellular engineering.</a> Biol Blood Marrow Transplant. 12, 608-608 (2006).</li><li>Carlens, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+in+vitro+expansion+of+cytotoxic+human+CD3-CD56++natural+killer+cells.">A new method for in vitro expansion of cytotoxic human CD3-CD56+ natural killer cells.</a> Hum Immunol. 62, 1092-1092 (2001).</li><li>Boissel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Umbilical+cord+mesenchymal+stem+cells+increase+expansion+of+cord+blood+natural+killer+cells.">Umbilical cord mesenchymal stem cells increase expansion of cord blood natural killer cells.</a> Biol Blood Marrow Transplant. 14, 1031-1031 (2008).</li><li>North, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-primed+human+natural+killer+cells+lyse+NK-resistant+tumor+targets:+evidence+of+a+two-stage+process+in+resting+NK+cell+activation.">Tumor-primed human natural killer cells lyse NK-resistant tumor targets: evidence of a two-stage process in resting NK cell activation.</a> J Immunol. 178, 85-85 (2007).</li><li>Berg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical-grade+ex+vivo-expanded+human+natural+killer+cells+up-regulate+activating+receptors+and+death+receptor+ligands+and+have+enhanced+cytolytic+activity+against+tumor+cells.">Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells.</a> Cytotherapy. 11, 341-341 (2009).</li><li>Fujisaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+highly+cytotoxic+human+natural+killer+cells+for+cancer+cell+therapy.">Expansion of highly cytotoxic human natural killer cells for cancer cell therapy.</a> Cancer Res. 69, 4010-4010 (2009).</li><li>Fujisaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+potential+of+human+natural+killer+cells.">Replicative potential of human natural killer cells.</a> Br J Haematol. 145, 606-606 (2009).</li><li>Gong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+of+natural+killer+cells+with+high+cytotoxicity+by+K562+cells+modified+to+co-express+major+histocompatibility+complex+class+I+chain-related+protein+A,+4-1BB+ligand,+and+interleukin-15.">Ex vivo expansion of natural killer cells with high cytotoxicity by K562 cells modified to co-express major histocompatibility complex class I chain-related protein A, 4-1BB ligand, and interleukin-15.</a> Tissue Antigens. 76, 467-467 (2010).</li></ol>

No conflicts of interest declared.

NK Cell Expansion and Activation Media (NKEM)
<ul>
<li>90% RPMI 1640 (Cellgro)</li>
<li>10% Fetal Bovine Serum (Gibco)</li>
<li>1x Penicillin / Streptomycin (Cellgro)</li>
<li>1x L-Glutamine (Gibco)
<ul>
<li>Filter Sterilize media before use.</li>
</ul></li>
<li>50 U/ mL IL2 (Proleukin, Novartis Vaccines and Diagnostics, Inc)
<ul>
<li>-	Diluted from a 200 IU/&mu;l stock. Add IL2 to desired amount of media just before use each time.</li>
</ul></li>
</ul>
PBMC and NK Cell Isolation
<ul>
<li>Ficoll-Paque (GE Healthcare)</li>
<li>Alsever's solution (Sigma)</li>
<li>RosetteSep Human NK Cell Enrichment Cocktail (Stemcell Technologies)</li>
</ul>
NK Cell Cytotoxicity Assay
<ul>
<li>Calcein-AM (Invitrogen)
<ul>
<li>Stock solution is at 1mg/ mL, store frozen in 50 &mu;L aliquots. Dissolve in NKEM media to achieve desired dilution for cell staining.</li></ul>
</li>
</ul>

Antibodies
The list of antibodies used for NK cell phenotyping are listed in table below;
<table border="1">
<tbody>
 <tr>
 <td>&nbsp;</td>
 <td>Antibody</td>
 <td>Volume per Test</td>
 <td>Company</td>
 <td>Catalog number</td>
 </tr>
 <tr>
 <td>Tube 1 </td>
 <td valign="top">Isotype FITC 
 Isotype FITC 
 Isotype FITC 
 Isotype FITC 
 FACS Buffer 
 Total Volume</td>
 <td valign="top">5 
 5 
 5 
 5 
 80 
 100</td>
 <td valign="top">BD Pharmingen 
 BD Pharmingen 
 BD Pharmingen 
 BD Pharmingen</td>
 <td valign="top">555748 
 555749 
 557224 
 340442</td>
 </tr>
 <tr>
 <td>Tube 2</td>
 <td valign="top">CD56 FITC 
 NKp30 PE 
 NKp44 PE 
 NKp46 PE 
 CD3 PE-Cy5 
 CD16 Alexa 647 
 FACS Buffer 
 Total Volume</td>
 <td valign="top">5 
 5 
 5 
 5 
 5 
 5 
 70 
 100</td>
 <td valign="top">BD Pharmingen 
 BD Pharmingen 
 BD Pharmingen 
 BD Pharmingen 
 BD Pharmingen 
 BD Pharmingen</td>
 <td valign="top">340410 
 558407 
 558563 
 557991 
 555341 
 557710</td>
 </tr>
 <tr>
 <td>Tube 3</td>
 <td valign="top">CD56 FITC 
 KIR2DL1 PE 
 KIR2DL2/3 PE 
 KIR3DL1 PE 
 CD3 PE-Cy5 
 NKG2D APC 
 FACS Buffer 
 Total Volume</td>
 <td valign="top">5 
 5 
 5 
 5 
 5 
 5 
 70 
 100</td>
 <td valign="top">BD Pharmingen 
 R&amp;D Systems 
 Miltenyi Biotec 
 R&amp;D Systems 
 BD Pharmingen 
 BD Pharmingen</td>
 <td valign="top">340410 
 FAB1844P 
 130-092-618 
 FAB12251P 
 555341 
 558071</td>
 </tr>
 <tr>
 <td>Tube 4</td>
 <td valign="top">CD56 FITC 
 CD11b PE 
 CD3 PE-Cy5 
 CD27 APC 
 FACS Buffer 
 Total Volume</td>
 <td valign="top">5 
 5 
 5 
 5 
 80 
 100</td>
 <td valign="top">BD Pharmingen 
 BD Pharmingen 
 BD Pharmingen 
 BD Pharmingen</td>
 <td valign="top">340410 
 555388 
 555341 
 558664</td>
 </tr>
 <tr>
 <td>Tube 5</td>
 <td valign="top">CD56 FITC 
 CD266 (DNAM-1) PE 
 CD3 PE-Cy5 
 CD160 Alexa647 
 FACS Buffer 
 Total Volume</td>
 <td valign="top">5 
 5 
 5 
 5 
 80 
 100</td>
 <td valign="top">BD Pharmingen 
 BD Pharmingen 
 BD Pharmingen 
 eBiosciences</td>
 <td valign="top">340410 
 559789 
 555341 
 51-1609-42</td>
 </tr>
 <tr>
 <td>Tube 6</td>
 <td valign="top">CD56 FITC 
 CD244 (2B4) PE 
 CD3 PE-Cy5 
 CD197 (CCR7) APC 
 FACS Buffer 
 Total Volume</td>
 <td valign="top">5 
 5 
 5 
 5 
 80 
 100</td>
 <td valign="top">BD Pharmingen 
 BD Pharmingen 
 BD Pharmingen 
 eBiosciences</td>
 <td valign="top">340410 
 550816 
 555341 
 17-1979-42</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells at JoVE.com

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

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 ...

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Immunology and Infection

47.9K Views.  MD Anderson Cancer Center - University of Texas. Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

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

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

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

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

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

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

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

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 ...

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 ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

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

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

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

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

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 ...