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 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.
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.
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
Days 3 and 5
STIMULATION 2
Day 7
Days 10 and 12
Day 14
STIMULATION 3
Days 17 and 19
Day 21
3. NK Cell Cytotoxicity Assay
4. NK Cell Purification by RosetteSep
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 7×105 NK cells resuspended in 700 uls of NKEM and 4×105 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 6×105 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 4×106 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 1×105 cells/ mL and proceed with the next step in the protocol.
6. REPRESENTATIVE RESULTS
Figure 2. When the expansion is performed as per the scheme described above, using 5×106 PBMCs as starting material, typical NK cell yields range from 1×109 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).
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).
Figure 4. PBMCs recovery from Buffy coat is donor dependent and can range from 300×106 to 800×106. 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.
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).
The authors have nothing to disclose.
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.
NK Cell Expansion and Activation Media (NKEM)
PBMC and NK Cell Isolation
NK Cell Cytotoxicity Assay
Antibodies
The list of antibodies used for NK cell phenotyping are listed in table below;
Antibody | Volume per Test | Company | Catalog number | |
Tube 1 |
Isotype FITC Isotype FITC Isotype FITC Isotype FITC FACS Buffer Total Volume |
5 5 5 5 80 100 |
BD Pharmingen BD Pharmingen BD Pharmingen BD Pharmingen |
555748 555749 557224 340442 |
Tube 2 | CD56 FITC NKp30 PE NKp44 PE NKp46 PE CD3 PE-Cy5 CD16 Alexa 647 FACS Buffer Total Volume |
5 5 5 5 5 5 70 100 |
BD Pharmingen BD Pharmingen BD Pharmingen BD Pharmingen BD Pharmingen BD Pharmingen |
340410 558407 558563 557991 555341 557710 |
Tube 3 | CD56 FITC KIR2DL1 PE KIR2DL2/3 PE KIR3DL1 PE CD3 PE-Cy5 NKG2D APC FACS Buffer Total Volume |
5 5 5 5 5 5 70 100 |
BD Pharmingen R&D Systems Miltenyi Biotec R&D Systems BD Pharmingen BD Pharmingen |
340410 FAB1844P 130-092-618 FAB12251P 555341 558071 |
Tube 4 | CD56 FITC CD11b PE CD3 PE-Cy5 CD27 APC FACS Buffer Total Volume |
5 5 5 5 80 100 |
BD Pharmingen BD Pharmingen BD Pharmingen BD Pharmingen |
340410 555388 555341 558664 |
Tube 5 | CD56 FITC CD266 (DNAM-1) PE CD3 PE-Cy5 CD160 Alexa647 FACS Buffer Total Volume |
5 5 5 5 80 100 |
BD Pharmingen BD Pharmingen BD Pharmingen eBiosciences |
340410 559789 555341 51-1609-42 |
Tube 6 | CD56 FITC CD244 (2B4) PE CD3 PE-Cy5 CD197 (CCR7) APC FACS Buffer Total Volume |
5 5 5 5 80 100 |
BD Pharmingen BD Pharmingen BD Pharmingen eBiosciences |
340410 550816 555341 17-1979-42 |