Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK cells.
Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, particularly CAR-NK cell, is one of the most promising ‘off-the-shelf’ development without severe life-threatening toxicity. However, the bottleneck for developing a successful CAR-NK therapy is achieving sufficient numbers of non-exhaustive, long-lived, ‘off-the-shelf’ CAR-NK cells from a third party. Here, we developed a new CAR-NK expansion method using an Epstein-Barr virus- (EBV) transformed B cell line expressing a genetically modified membrane form of interleukin-21 (IL-21). In this protocol, step-by-step procedures are provided to expand NK and CAR-NK cells from cord blood and peripheral blood, as well as solid organ tissues. This work will significantly enhance the clinical development of CAR-NK immunotherapy.
Natural killer (NK) cells are an important subset of lymphocytes that express CD56 and lack expression of the T cell marker, CD31,2. Conventional NK cells are classified as innate immune cells responsible for immunosurveillance of virally infected cells and cancerous cells. Unlike T cells, NK cells recognize infected or malignant cells using CD16 or other activating receptors and do not require the formation of a complex between antigen peptides and major histocompatibility complex (MHC) class I molecules3,4. Recent clinical investigations using chimeric antigen receptor (CAR)-NK cells to treat relapsed or refractory CD19-positive cancers (non-Hodgkin's lymphoma or chronic lymphocytic leukemia [CLL])showed the outstanding safety advantages of CAR-NK cells5. For instance, CAR-NK cell infusion is associated with minimal or negligible graft versus host disease (GVHD), neurotoxicity, cardiotoxicity, and cytokine release syndrome (CRS)6,7,8,9,10. However, conventional methods to expand human NK cells showed exhaustive phenotypes with strong fratricidal killing and telomere shortage, which presents a major challenge in obtaining an adequate number of functional NK cells for adoptive immunotherapy11.
To overcome these challenges, a method was developed to expand primary NK cells directly from unfractionated peripheral blood mononuclear cells (PBMCs) or cord blood (CB) using an irradiated and genetically engineered 721.221 (hereinafter, 221) cell line, a human B-lymphoblastoid cell line with low expression of MHC class I molecules3. Previous studies showed the importance of IL-21 in NK cell expansion; therefore, a genetically engineered membrane-bound IL-21 expressing a version of the 721.221 cell line (starting now, 221-mIL-21) was developed11,12,13,14,15. The results showed that 221-mIL-21 feeder-cell-expanded primary NK cells were expanded to an average of >40,000-fold with persistent high NK cell purity for approximately 2-3 weeks. Additional information regarding the application of this protocol can be found in Yang et al.16.
This protocol aims to demonstrate the step-by-step procedure of the novel expansion of PBNK, CBNK, tissue-derived NK, and CAR-NK cells ex vivo.
Human tissues and blood-related work in this protocol follows the guidelines of the Rutgers University Institutional Review Board (IRB)
1. NK cell expansion from liver tissues (Day 0), as shown in Figure 1.
NOTE: Initial cell number and viability are strongly correlated with the time since organ removal and the initial tissue sample amount. However, if tissues are placed in 30 mL of Hank's Balanced Salt Solution (HBSS) and kept on ice or in the fridge at 4 °C overnight, NK cells can still be expanded at high purity and viability up to 24 h later.
2. Primary NK cell expansion from PBMCs (or CB or organ tissues) (Day 0), as shown in Figure 2.
3. Attachment of 293T cells (Day 2), as shown in Figure 2.
4. Retrovirus transfection (Day 3)
5. Retronectin plate-coating (Day 3)
6. Transduction (Day 4)
7. CAR-NK cells collection (Day 6 or 7), as shown in Figure 2.
A schematic workflow of tissue-infiltrating NK cell isolation and PBNK cell expansion using the 221-mIL-21 feeder cell methodology is shown in Figure 1 and Figure 2. Expanded PBNK cells were collected every 3 or 4 days for flow cytometry to determine the NK cell purity by staining cells with anti-human CD56 and anti-human CD3. The experiment was repeated using two different donors to show the reproducibility of the expansion system (Figure 3). PBNK cells expanded by 221-mIL-21 were shown to expand nearly 5 × 104 folds (Figure 3A). Furthermore, the NK cell purity was highly maintained, around 85% throughout the 21-day expansion (Figure 3B). Using the 221-mIL-21 feeder cell expansion system, the NK cell purity consistently ranged between 85%-95%, independent of the donors (data not shown). To demonstrate the robustness of the 221-mIL-21 expansion system, PBMCs were stained for anti-CD56 and anti-CD3 prior to the expansion, which showed a cell purity of 7.09% for NK cells and a high percentage of T cells (Figure 4A). PBMCs were cocultured with 221-mIL-21 to expand NK cells; the NK purity was checked prior to CAR-NK transduction on Day 4 (Figure 4A). CAR-NK cells were collected and stained for anti-CD56, anti-CD3, and anti-hIgG(H+L) F(ab')2, which showed a high NK cell population (86.9% on Day 7) and a high CAR transduction efficiency of approximately 70% (Figure 4). Higher transduction efficiencies (up to 95%) were also observed using the retrovirus packaging system. Altogether, these data show that the 221-mIL-21 feeder cells could successfully expand NK cells and preserve the NK cell purity ex vivo.
Figure 1: Diagram of NK cell expansion from solid human organ samples. Briefly, the obtained human liver samples are minced into small cubes for mechanical digestion. Dissociated cells are then isolated using PVP-coated silica and Lymphocyte Separation Media. Further, the NK cells are expanded using the expansion protocol described in Figure 2. Please click here to view a larger version of this figure.
Figure 2: Schematic workflow of CAR-NK cell generation from PBMCs. Briefly, 221-mIL21 feeder cells were irradiated at 100 Gy prior to coculturing with PBMCs supplemented with IL-2 and IL-15 on Day 0. In parallel, 293T cells were transfected with the retrovirus packaging system to produce CAR retrovirus that was then transduced into the expanded PBNK cells in the presence of IL-2 and IL-15. Primary CAR-NK cells were harvested on Day 7 and continued expansion for 21 days. This figure has been modified from Yang et al.16. Please click here to view a larger version of this figure.
Figure 3: Dynamic time-lapsed expansion of PBNK cells. (A) Fold expansion of PBNK during a 22- day time course. Cells were stained with anti-CD56 and anti-CD3 at indicated days for flow cytometry. The total number of NK cells was determined by multiplying NK cell purity to the total number of cells. Expansion rate was generated as follows: (Number of NK cells)Tn/(Number of NK cells)T0, where Number of NK cells = (percentage of NK cell purity) × (total number of cells), T0 is the number of NK cells at time day 0, and Tn is the number of NK cells at time day n. (B) NK cell purity during a 22-day time course. The NK cell expansion was repeated two times with two different donors. Error bars represent ± SEM. Please click here to view a larger version of this figure.
Figure 4: Representative flow cytometric analysis of CAR-NK cells. (A) Representative dot plots showing the dynamic time-lapse of NK cell purity of the CAR-NK cells during an 18-day course. Flow cytometry analysis was assessed by staining the cells with anti-CD56 and anti-CD3 at indicated timepoints. Day 0 indicates pre-expansion of PBNK. Day 4 indicates post-expansion of PBNK and pre-transduction of CAR-NK cells. Day 7 indicates the post-transduction of CAR-NK cells. (B) Representative dot plots showing the transduction efficiency of CAR-NK cells using the retrovirus packaging system. Cells were stained with anti-CD56, anti-CD3, and anti-hIgG(H+L) F(ab')2 for flow cytometry. (C) Representative dot plots showing the CAR expression in various subsets, including CD56+CD3–, CD56–CD3+, CD56+CD3+, and CD56–CD3– on Day 18. Cells were stained with anti-CD56, anti-CD3, and anti-hIgG(H+L) F(ab')2 (indicating CAR expression) for flow cytometry. Please click here to view a larger version of this figure.
Most of the current CAR-NK products in clinical trials utilize NK cell lines17, such as NK-92, a cell line isolated from a non-Hodgkin's lymphoma patient18, NK-92MI, IL-2 independent NK-92 cell line19, and NKL, isolated from a large granular lymphocyte patient20, as these cell lines are easily proliferative for 'off-the-shelf' products. However, these cell lines, e.g., NK-92 cells, have marginal clinical efficacies and in vivo expansion, as they require irradiation prior to infusion, thus limiting their proliferation and cytotoxicity in vivo21. Given these reasons, various strategies are currently being explored to expand primary NK cells from several sources, including peripheral blood, CB, bone marrow (BM), human embryonic stem cells (HSCs), induced pluripotent stem cells (iPSCs), and tumor tissues21,22,23. For instance, NK cells can be expanded ex vivo using interleukins including IL-15, IL-18, and IL-21. Lymphoblastoid cell lines such as K562 cells or Epstein-Barr Virus-transformed lymphoblastoid cell lines such as 721.221 cells, are also used for NK cell expansion16. However, the aforementioned strategies often generate insufficient number of NK cells for an adoptive transfer of CAR-NK immunotherapy22,24. To help solve the problem, the study here shows a protocol for an ex vivo NK cell expansion using a genetically modified EBV-transformed cell line, 221-mIL-21 feeder cells.
The expansion methodology using 221-mIL-21 feeder cells shown in this protocol is optimized to expand NK cells with an expansion rate of at least 10 to 100-fold higher than other leukemia cell lines, including HL-60 and OCl-AML3 expressing membrane IL-21, K562, and K652-mIL21 expressing OX40 ligand22,24,25. The CAR expression is also evaluated for approximately 2 weeks ex vivo. More significantly, the 221-mIL-21 feeder cell expansion strategy can be applied to expand NK cells from various sources, including PBMCs, CB, and solid organs such as the liver, without an initial NK enrichment step. Although the 221-mIL-21 feeder system is not as donor-dependent as the aforementioned feeder cell lines, it is not entirely independent of donors. On average, the 221-mIL-21 expansion system can achieve 90% of NK cell purity with a high NK cell number, with approximately <5% of T cell contamination on day 14 post-expansion. Therefore, to eliminate the possibilities of T cell contamination, it is necessary to isolate NK cells from obtained samples prior to the ex vivo expansion or use a CD3+ selection system to eliminate T cells after an ex vivo expansion.
One of the criticisms in using an NK cell expansion system is that the feeder cells may not have been fully eradicated after the expansion or prior to a transfusion, which may possess significant regulatory concerns; therefore, complete eradication of feeder cells before a transfusion is crucial. However, recent CAR-NK clinical trials in which K562-mIL21-4-1BBL feeder cells were used for the ex vivo CBNK cell expansion24,25 showed no concerning complications. Furthermore, our preliminary data showed a gradual decrease of the irradiated 221-mIL-21 population as the expansion progressed (data not shown). However, more extensive studies are required for this expansion method to be implemented in a clinical setting. Collectively, the 221-mIL-21 expansion system helps solve the challenge of expanding primary CAR-NK cells, and therefore will significantly contribute to the broader use of CAR-NK cell-based immunotherapy in the near future.
The authors have nothing to disclose.
We would like to thank the members of the Liu laboratory (Dr. Hsiang-chi Tseng, Dr. Xuening Wang, and Dr. Chih-Hsiung Chen) for their comments on the manuscripts. We would like to thank Dr. Gianpietro Dotti for the SFG vectors and Dr. Eric Long for the 721.221 cells. This work was supported in part from HL125018 (D. Liu), AI124769 (D. Liu), AI129594 (D. Liu), AI130197 (D. Liu), and Rutgers-Health Advance Funding (NIH REACH program), U01HL150852 (R. Panettieri, S. Libutti, and R. Pasqualini), S10OD025182 (D. Liu), and Rutgers University-New Jersey Medical School Startup funding for D. Liu Laboratory.
100 mm surface treated sterile tissue culture dishes | VWR | 10062-880 | For transfection |
293T cells | ATCC | CRL-3216 | For transfection |
6-well G-REX plate | Wilson Wolf | 80240M | For NK cell expansion |
AF647-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG (H+L) | Jackson ImmunoResearch | 109-606-088 | For flow cytometry |
CAR construct in SFG vector | Generated in Dr. Dongfang Liu's lab | For transfection | |
Collagenase IV | Sigma | C4-22-1G | For TILs isolation |
Cryopreserve media Ingredient: Fetal Bovine Serum (FBS) |
Corning | 35-010-CV | 90% |
Cryopreserve media Ingredient: Dimethyl sulfoxide (DMSO) |
Sigma | D2050 | 10% |
D-10 media Ingredient: DMEM |
VWR | 45000-304 | |
D-10 media Ingredient: Fetal Bovine Serum (FBS) |
Corning | 35-010-CV | 10% |
D-10 media Ingredient: Penicillin Streptomycin |
VWR | 45000-652 | 1% |
FastFlow & Low Binding Millipore Express PES Membrane | Millex | SLHPR33RB | For transduction |
Genejuice transfection reagent | VWR | 80611-356 | For transfection |
gentleMACS C-tubes | Miltenyi Biotec | 130-093-237 | For TILs isolation |
gentleMACS Octo | Miltenyi Biotec | Quote | For TILs isolation |
Hank's Balanced Salt Solution (HBSS – w/o calcium or magnesium) | ThermoFisher | 14170120 | For TILs isolation |
Human IL-15 | Peprotech | 200-15 | For NK cell expansion |
Human IL-2 | Peprotech | 200-02 | For NK cell expansion |
Irradiated 221-mIL21 feeder cells | Generated in Dr. Dongfang Liu's lab | Frozen in cryopreserve media | |
Lymphocyte Separation Media | Corning | 25-072-CV | For TILs isolation |
OPTI-MEM | ThermoFisher | 31895 | For transfection |
PE anti-human CD3 clone HIT3a | Biolegend | 300308 | For flow cytometry |
PE/Cy7 anti-human CD56 (NCAM) clone 5.1H11 | BioLegend | 362509 | For flow cytometry |
Pegpam3 plasmid | Generated in Dr. Dongfang Liu's lab | For transfection | |
Percoll | GE Healthcare | 17089101 | For TILs isolation |
Peripheral blood mononuclear cells (PBMCs) | New York Blood Center | Isolated from plasma of healthy donors, frozen in cryopreserve media | |
Phosphate Buffer Saline (PBS) | Corning | 21-040-CV | For transduction |
pRDF plasmid | Generated in Dr. Dongfang Liu's lab | For transfection | |
R-10 media Ingredients: RPMI 1640 |
VWR | 45000-404 | |
R-10 media Ingredient: L-glutamine |
VWR | 45000-304 | 1% |
R-10 media Ingredient: Penicillin Streptomycin |
VWR | 45000-652 | 1% |
R-10 media Ingredient: Fetal Bovine Serum (FBS) |
Corning | 35-010-CV | 10% |
Retronectin | Generated in Dr. Dongfang Liu's lab | home-made | For transduction |
Trypan Blue | Corning | 25-900-Cl | For cell counting |
Untreated 24-well plate | Fisher Scientific | 13-690-071 | For transduction |