Human Primary NK Cells from Humanized BLT-IL15 Mice Show Superior Expansion and Cytotoxicity in Comparison with Humanized BLT-mice Using Membrane Bound IL21-modified 721.221 Feeder Cell Expansion System

Human Primary NK Cells from Humanized BLT-IL15 Mice Show Superior Expansion and Cytotoxicity in Comparison with Humanized BLT-mice Using Membrane Bound IL21-modified 721.221 Feeder Cell Expansion System | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 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Data may be preliminary. 9 June 2024 V1 Latest version Share on Human Primary NK Cells from Humanized BLT-IL15 Mice Show Superior Expansion and Cytotoxicity in Comparison with Humanized BLT-mice Using Membrane Bound IL21-modified 721.221 Feeder Cell Expansion System Authors : Yan Yang , Jianshui Zhang , Minh Ma , Yilun Cheng , Saroj Chandra Lohani [email protected] , Qingsheng Li , and Dongfang Liu 0000-0002-7295-8088 Authors Info & Affiliations https://doi.org/10.22541/au.171797475.54380127/v1 596 views 225 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: Natural Killer (NK) cells play a critical role in host defense. Studying human NK immunobiology is mainly focused on using in vitro assays with limited NK cells from peripheral blood. It is challenging to study human NK cell biology in vivo due to potential ethical issues in human study and the lack of suitable animal models. Developing a suitable animal model to study human NK cell biology in vivo is critical to support NK-based clinical immunotherapy. Results: Here, we develop a novel method to study human NK cells in vivo by using hu-BLT (humanized bone marrow-liver-thymus) mice that constitutively express human IL-15 (henceforth, hu-BLT-IL15). We also compare human NK cells between hu-BLT-IL15 and hu-BLT mice without IL-15 expression by a newly developed approach for the rapid propagating of primary human NK cells from various sources (including peripheral blood, spleen, and bone marrow). NK cells from hu-BLT-IL15 show superior number, purity, and cytotoxicity (including natural cytotoxicity and antibody-dependent cellular cytotoxicity [ADCC]), compared with NK cells from hu-BLT. Unexpectedly, we also identify a significantly increased percentage of NK-like T cells (CD3+ CD16+ CD56+) from hu-BLT-IL15, indicating that IL-15 signaling enhances both NK and NKT cell development. Conclusions: A better understanding of the immunobiology of the NK-like T cells in the hu-BLT-IL15 mouse model may provide critical information for determining the clinical value of these cells in predicting disease progression. Thus, we propose that the hu-BLT-IL15 mouse model in combination with the 721.221-mIL21 feeder cell expansion system can serve as a superior model to study human NK and NK-like T cells in comparison with hu-BLT. INTRODUCTION NK cells play a critical role in cancer, infection, inflammation, and autoimmune diseases 1 . Further, immunotherapy based on NK cells, such as CAR-NK cells 2 and NK-based engagers 3 , is in clinical trials 4, 5 . NK cells distinguish “self” and “non-self” through inhibitory and activating receptor-mediated signaling pathways without MHC restriction, which makes it possible to be one of the ‘off-the-shelf’ products in the cell therapy markets. Clinically, human peripheral blood NK cells are defined by CD3 - and CD56 + surface staining 1 . Usually, the CD3 - CD56 + NK cells can be further classified into CD56 bright and CD56 dim with the expression profile of the CD16 surface molecule 6 . There are three main functions exerted by NK cells, which include cytokine production, direct cytotoxicity against tumors and virus-infected cells, and regulations of adaptive immune via interacting with dendritic cells 7 . Human peripheral blood is the easiest and most convenient way to obtain enough NK cells for research and clinical application. To study human NK cells, peripheral blood NK cells are usually interrogated with several in vitro assays, such as cytokine production and cytotoxic killing, as well as non-conventional immunological approaches such as several imaging technologies. However, it is imperative to develop an animal model to study NK cells in vivo because tissue microenvironmental factors are important in shaping NK cell functions. IL-15 plays an important role in NK cell differentiation and maturation 8, 9 . Recent studies show that the hu-BLT (humanized Bone marrow, Liver, and Thymus) mouse can be an excellent model to study virus infections, such as Viral Hemorrhagic Fevers research 10 , HIV 11, 12 , and Kaposi’s sarcoma-associated herpesvirus infection 13 . Hu-BLT mice can develop several lineages of human immune cells, such as T, B, monocyte/macrophage, and dendritic cells 14 . However, few NK cells can be developed in hu-BLT mice, which makes the NK cell study challenge in hu-BLT mice. In this study, we compare human NK cells between hu-BLT-IL15 and hu-BLT mice by a newly developed approach for the rapid propagating of primary NK cells from various sources (including peripheral, spleen, and bone marrow). NK cells from hu-BLT-IL15 show superior number, purity, and functions (including natural cytotoxicity and ADCC), compared with NK cells from hu-BLT. Unexpectedly, we also identify a significantly increased percentage of NK-like T cells (CD3 + CD16 + CD56 + , a chronic lymphocytic leukemia predictive maker) from hu-BLT-IL15. Understanding the immunobiology of the NK-like T cells may provide critical information for determining the clinical value of these cells in disease progression. Thus, we propose that the hu-BLT-IL15 mouse model in combination with the newly-created 721.221-mIL21 feeder cell expansion system can serve as a superior model to study human NK and NK-like T cells in comparison with hu-BLT. RESULTS Superior propagation of NK cells by 221-mIL21 cells in hu-BLT-IL15 mice Previous studies have shown that IL-21 plays a critical role in NK cell proliferation. 15, 16 We developed an artificial antigen-presenting cell line using the 721.221 cell line expressing a membrane form of IL-21 17 . To set up a human primary NK cell expansion system, IL-21 was cloned into an SFG vector that contains a human IgG1 CH2-CH3 domain, CD28-transmembrane (TM) domain, CD28 intracellular domain, 4-1BB-Ligand, and CD3zeta. After the establishment of the 221-mIL21 cell line as feeder cells, we determine whether the 221-mIL21 feeder cell line can optimally expand human primary NK cells from hu-BLT-IL15 mice. To expand human primary NK cells from peripheral blood, spleen, and bone marrow isolated from hu-BLT-IL15 mice, we co-cultured lymphocytes from these tissues with feeder cells, 200 U/ml of IL-2, and 5 ng/mL of IL-15. To compare their ability to stimulate NK cell expansion, cells from both hu-BLT-IL15 and hu-BLT were evaluated directly. The initial number of PBMCs and the proportion of NK cells were 5 million and 10-20%, respectively. A representative NK expansion profile between hu-BLT-IL15 and hu-BLT mice at Day 10, gated on CD3 and CD56 by flow cytometry is shown ( Figure 1A ). Importantly, the increased percentage of NK cells gated on CD3 - and CD56 + with 221-mIL21 feeder cells and the relative purity of expanded NK cells was higher than that of K562-mIL21 feeder cells (data not shown). Additionally, the percentage of CD16 + CD56 + NK cells is increased in the presence of mIL21-expressing feeder cells ( Figure 1B ). We also analyzed the dynamics of CD3-CD56+ NK cell expansion at different time points (day 0, day 6, day 13, and day 21). A clear increase in the percentage of NK cells was observed in hu-BLT-IL15 mice, indicating the importance of IL-15 signaling in human NK cell proliferation in vivo . Thus, 221-mIL21 cells can be used to successfully expand human primary NK cells from hu-BLT-IL15 and hu-BLT mice. The percentage of NK cells from hu-BLT-IL15 is superior to that of those from hu-BLT mice. Increased percentage of CD3 + CD16 + CD56 + NK-like T cells from hu-BLT-IL15 mice Several studies show that the percentage of CD3 + CD16 + CD56 + cells correlates with the outcome of in vitro fertilization and leukemia disease progression 18-20 , indicating the important clinical value of this population. Thus, we compared the percentage of CD3 + CD16 + CD56 + cells stimulated by 221-mIL21 between hu-BLT-IL15 and hu-BLT without IL-15 expression mice ( Figure 3A ). Unexpectedly, CD3 + CD16 + CD56 + populations increased in hu-BLT-IL15 mice, compared with hu-BLT mice without IL-15 expression ( Figure 3B ). In conclusion, the percentage of CD3 + CD16 + CD56 + NK-like T cells from hu-BLT-IL15 mice increase, indicating a critical role of IL-15 in the development and proliferation of CD3 + CD16 + CD56 + NK-like T cells in hu-BLT-IL15 mice Characteristics of expanded NK cells in hu-BLT-IL15 mice To determine the immunophenotyping of 221-mIL21 expanded NK cells, we used flow cytometry to characterize several important activating and inhibitory receptors. The activating receptors include CD16, NKG2D, NKp46, 2B4, DNAM-1, CD94, CD8a, NKG2C ( Figure 4A and 4B ). The inhibitory receptors include NKG2A, CTLA-4, KLRG1, PD-1, LIR1, TIM-3, TIGIT, LAG-3, total KIR, KIR2DL1, KIR2DL2/L3, KIR3DL1, and KIR3DL2 ( Figure 4C-4E ). The expression of these activating and inhibitory receptors on expanded NK cells is comparable. Interestingly, CD69, an activation marker of NK cells, as well as CD8alpha, were increased in the 221-mIL21 expanded NK cells from hu-BLT-IL15 mice, compared with that of NK cells in hu-BLT mice. In summary, expanded 221-mIL21 NK cells from hu-BLT-IL15 mice show different surface receptors with NK cells from hu-BLT mice. Superior cytotoxicity of NK from hu-BLT-IL15 mice NK cells kill tumor cells and virus-infected cells via two distinct mechanisms: natural cytotoxicity (NC) through activating receptors, such as NKG2D 21 , and antibody-dependent cellular cytotoxicity (ADCC) through CD16 7,22 . To test NC and ADCC of human primary NK cells from hu-BLT-IL15 mice, K562 and Raji cell lines were used as target cells for evaluating NC. To evaluate the ADCC of NK cells, we pre-treated Raji target cells with Rituximab because the Raji cell line is CD20 positive. To compare the cytotoxicity of human NK cells, we examined the NC and ADCC ability of NK cells using a chromium-51 ( 51 Cr) release assay. For NC, NK cells were co-incubated for 4 hours with 51 Cr-loaded K562 cells, whereupon the amount of 51 Cr in the medium was determined. The results showed that NK cells displayed superior cytotoxicity against K562 ( Fig. 5A ), as well as ADCC ( Fig. 5B and 5C). Thus, we conclude that human primary NK cells from hu-BLT-IL15 mice show superior cytotoxicity to that of NK cells from hu-BLT without IL-15 expression. DISCUSSION We developed and characterized a new mouse model that makes human primary NK cell in vivo study possible. We demonstrated that human primary NK cells in hu-BLT-IL15 mice excelled in superior expansion and cytotoxicity, which provides a new mouse model to study human NK cells in vivo for human tumor and infection studies. Meanwhile, we identified an increased percentage of CD56 + CD16 + CD3 + NK-like T cells in hu-BLT-IL15 mice. Few studies show human NK cell development and proliferation in hu-BLT-IL15 mice due to low levels of CD56 + NK cell development in hu-BLT mice (Refs). In this study, we demonstrated that a higher level of CD56 + NK can be developed in the hu-BLT-IL15 mice. Meanwhile, we proposed a novel method to expand these NK cells derived from hu-BLT-IL15. Phenotyping and functional data suggest that the NK cells from hu-BLT-IL15 can be functional. Adoptive transfer of human hematopoietic progenitor cells into NOD-scid IL2Rγ null mice represents a novel approach to investigating human NK cells in vivo . However, the reconstituted NK cells have low levels of IFN-γ and degranulation due to the terminally differentiated CD56 dim CD16 + NK populations 23 . The significant contribution of this study includes: 1) we proposed that hu-BLT-IL15 mice can serve as a novel in vivo platform to study human primary NK cells and NK cell-based immunotherapy. This platform provides a physiological environment beyond in vitro ‘live incubator’ for human NK cells because of another immune cell, immune tissue, and immune organ development in this platform, 2) NK cells from hu-BLT-IL15 mice display superior functions, including ADCC and NC, compared with NK cells from hu-BLT mice, 3) higher percentage of CD56 + CD16 + CD3 + cell population from Hu-BLT-IL15 mice. The percentage of CD3 + CD16 + CD56 + cells correlates with disease progression and outcomes such as infectious diseases and cancers. The hu-BLT-IL15 platform can be used to study the development of CD3 + CD16 + CD56 + cells. Investigation of underlying molecule mechanisms of this subset NK-like T cells will support the clinical application of CD3 + CD16 + CD56 + , and 4) Different immunophenotyping of human primary NK cells from hu-BLT-IL15 mice. We showed that human primary NK cells from hu-BLT-IL15 mice expressed higher levels of NKG2D and CD69, compared with NK cells from hu-BLT and human peripheral blood. Expectedly, higher surface expression of TIM-3 and KIR, but not PD-1, on NK cells from hu-BLT-IL15 mice, compared with NK cells from hu-BLT and human peripheral blood. It will be interesting to investigate the molecular basis using this hu-BLT-IL15 platform. In summary, we have characterized NK cells in hu-BLT-hIL15 mice and propose that the hu-BLT-IL15 mouse model can serve as a superior model to study human NK and NK-like T cells in comparison with hu-BLT without IL-15 expression. This model can be useful for studying human NK cells in human tumors and infections 24 . Online content Any methods, additional references, source data, and statements of code and data availability are available online. METHODS AND MATERIALS Antibodies and Reagents PE and APC anti-human CD3 antibody (clone OKT3, BioLegend), FITC, BV605, PE/Cy7, and BV 510 anti-human CD56 antibody (clone HCD56, BioLegend), PE anti-human CD69 antibody (clone FN50, BioLegend), PE/Cy7 anti-human CD8a antibody (clone HIT8a, BioLegend), APC/Fire 750 anti-human CD226 antibody (DNAM-1) (clone 11A8, BioLegend), APC/Fire 750 anti-human KLRG1 (MAFA) antibody (clone SA231A2, BioLegend), BV 421 anti-human CD335 (NKp46) antibody (clone 9E2, BioLegend), PE/Cy7 anti-human CD158b (KIR2DL2/L3, BioLegend) antibody (clone DX27, BioLegend), PE/Cy7 anti-human CD244 (2B4) antibody (clone C1.7, BioLegend), PE anti-human CD152 (CTLA-4) antibody (clone BNI3), APC anti-human CD366 (Tim-3) antibody (clone F38-2E2), PerCP/Cy5.5 anti-human TIGIT (VSTM3) antibody (clone A15153G), FITC anti-human CD223 (LAG-3) antibody (clone 11C3C65, BioLegend), and PerCP/Cy5.5 anti-human CD94 (clone DX22, BioLegend) were purchased from BioLegend (San Diego, CA, USA). APC anti-human CD16 antibody (clone B73.1, BD Biosciences), FITC anti-human CD3 antibody (clone UCHT1, BD Biosciences), BV480 anti-human CD85j antibody (LIR-1) antibody (clone GHI/75, BD Biosciences), and BV711 anti-human CD314 (NKG2D) antibody (clone 1D11, BD Biosciences). FITC anti-human KIR/CD158 antibody (clone 180704, R&D Systems), PE anti-human KIR2DL1/KIR2DS5 antibody (clone 143211, R&D Systems), APC anti-human KIR3DL1 antibody (clone DX9, R&D Systems), AF405 anti-human KIR3DL2/CD158k antibody (clone 539304, R&D Systems), APC anti-human NKG2A/CD159a antibody (clone 131411, R&D Systems), and PE anti-human NKG2C/CD159c antibody (clone 134591, R&D Systems) were purchased from R&D Systems. Cell lines 721.221 cell line was received from Dr. Eric O. Long (National Health of Allergy and Infectious Diseases in National Health Institutes). K562 and Raji cell lines were purchased from the American Type Culture Collection (ATCC). 721.221 cells were engineered to express membrane bound IL-21 as previously described 17 and cultured in R-10 media at 37 ℃ under 5% (v/v) CO 2 . Preparation of Humanized BLT-IL-15 and humanized mice The generation of hu-BLT-IL15 and hu-BLT mice was conducted by following our previous publications 13, 28 . Female NSG-Tg (Hu-IL15) mice of 6-8 weeks old, expressing the human IL15 gene on the NSG background, from The Jackson Laboratory (Strain #:030890) and female NSG mice of 6-8 weeks old without hIL-15 Tg from the Jackson Laboratory (Strain #:005557) were used. The mice were irradiated at the dose of 12 cGy/gram of mouse body weight with RS200 X-ray irradiator (RAD.Source Technologies, Inc., GA) and surgically implanted with human thymic and hepatic tissue into the left renal capsule. Within 6 hours of surgery, the mouse was injected via the tail vein of the human hepatic CD34+ hematopoietic stem and progenitor cells (1.04 x 10 5 ). Six mice from the hu-BLT-hIL15 and Hu-BLT group each were euthanized at 12 weeks post-surgery to collect single cells from peripheral blood, spleen, and bone marrow to evaluate the NK cells. This study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Nebraska-Lincoln (UNL). Primary NK cell expansion from PBMCs and bone marrow of Hu-BLT or Hu-BLT-IL15 mice PBMCs were isolated from buffy coats (New York Blood Center) using Lymphocyte Separation Medium (Corning). Human primary NK cells were expanded as described previously 17 . For NK cell expansion from the bone marrow of hu-BLT or hu-BLT-IL15, 5 × 10 6 cells from the bone marrow were cocultured with 10 × 10 6 100 Gy irradiated 721.221-mIL21 cells in a 6-well G-Rex plate (Wilson Wolf) in R-10 media (RPMI 1640 (Corning) with glutamine containing 10% fetal bovine serum (FBS, Corning) and 100 U/mL Penicillin streptomycin (Corning)) in the presence of 200 U/mL of IL-2 (Peprotech) and 5 ng/mL of IL-15 (Peprotech) at 37 ℃ under 5% (v/v) CO 2 . Media is changed every 3-4 days until the end of expansion on day 21. Flow Cytometry Analysis PBMCs and expanded NK cells were stained with fluorescence-conjugated antibodies in FACS staining buffer (PBS with 1% FBS) on ice for 30 minutes, washed with PBS, and analyzed on a FACS LSRII or an LSR Fortessa flow cytometer (BD). PMT voltages were adjusted, and compensation values were calculated before data collection. Data were acquired using FACS Diva software (BD) and analyzed using FlowJo software (Tree Star). Chromium 51 release assay NK cell cytotoxicity toward K562 or Raji target cells was examined using a 51 Cr-release assay, as described 27 . Briefly, the target cells were labeled with 51 Cr at 37°C for 1 hour. After washing 3 times with fresh R10 medium, cells were re-suspended in R10 medium at a density of 1 × 10 5 cells/ml. An aliquot of the target cells (0.1 ml for 1×10 4 cells) was mixed with a serial dilution of NK cells, and the mixture was incubated at 37 o C for 4 hours. For antibody-dependent cellular cytotoxicity (ADCC) assays, Raji cells were incubated with 20 µg/ml Rituximab on ice for 30 minutes before the addition of NK cells and the Rituximab was also present during the 4-hour incubation at 37 o C. At the end of the incubation, the medium was collected, and radioactivity was measured using a Microbeta2 Microplate Counter (Perkin Elmer, USA). The mean percentage of specific lysis of triplicate wells was calculated as follows: [(test counts-spontaneous counts)/(maximum counts-spontaneous counts)] ×100%. Statistical Analysis Data were represented as means ± SEM. The statistical significance was determined using two-tailed unpaired Student’s t-tests, two-tailed paired Student’s t-tests, and a two-way ANOVA, where indicated. P < 0.05 was considered statistically significant. Figure legends: Figure 1. Comparison of human NK cell expansion from humanized BLT mice in the presence and absence of IL-15 . ( A) Representative flow cytometry plots of the purity of NK cells expanded from the bone marrow of humanized Hu-BLT and Hu-BLT-IL15 mice using irradiated 221-mIL21 feeder cells at indicated days (day 7 and day 10). ( B) Representative flow cytometry plots of the expression of CD16 on expanded NK cells at day 10 of expansion. The cells are gated at CD3 negative populations. Figure 2. Dynamics of Expansion of Human NK cells from humanized Hu-BLT-IL15 mice . Representative flow cytometry plots of the purity of NK cells expanded from the bone marrow of two Hu-BLT-IL15 mice using irradiated 221-mIL21 feeder cells on days 0, 6 13, and 21). Figure 3. Higher frequency of CD56 + CD16 + CD3 + cell population from hu-BLT-IL15 mice in comparison with hu-BLT mice . ( A) Representative flow cytometry plots of the frequency of CD56 positive cells in the subsets expanded from the bone marrow of hu-BLT mice and hu-BLT-IL15 mice using irradiated 221-mIL21 feeder cells on day 10. ( B) Representative flow cytometry plots of the population of CD3 + CD16 + CD56 + cells. Figure 4. Comparison of Phenotypes of NK cells Expanded from hu-BLT mice, hu-BLT-IL15 mice, and human PBMCs. ( A ) Representative histograms of CD16, NKG2D, NKp46, 2B4, and CD226 (DNAM-1) expressions on NK cells expanded from the bone marrow of hu-BLT mice, hu-BLT-IL15 mice and human PBMCs, respectively, using 221-mIL21 as feeder cells. ( B ) Representative histograms of the expression of CD69, CD94, CD8a, and NKG2C on the expanded NK cells. ( C ) Representative histograms of the expression of NKG2A, CTLA-4, KLRG1, and PD-1 on the expanded NK cells. ( D ) Representative histograms of the expression of LIR1, TIM-3, TIGIT, and LAG-3 on the expanded NK cells. ( E ) Representative histograms of the expression of KIR, KIR2DL1, KIR2DL2/L3, KIR3DL1, and KIR3DL2 on the expanded NK cells. The MFI is noted in the respective histograms. Fluorescence Minus One (FMO) controls were used to identify the fluorochrome signal, and the same FMO was used when the same fluorochrome was used. Figure 5. Superior Cytotoxicity of NK cells expanded from hu-BLT-IL15 mice . (A) Natural Cytotoxicity of NK cells expanded from the bone marrow of Hu-BLT mice and Hu-BLT-IL15 mice against K562 and (B) Raji using a 4-hour 51 Cr killing assay. (C) ADCC of NK cells expanded from the bone marrow of hu-BLT mice and hu-BLT-IL15 mice against Raji in the presence of Rituximab. Ethics approval and consent to participate All animal experiments have been approved by the Rutgers and University of Nebraska – Lincoln Institutional Animal Care and Use Committee (IACUC). NSG-Tg(Hu-IL15) mice (Strain: #030890) were purchased from The Jackson Laboratory (Bar Harbor, ME) for humanization experiments. Consent for publication Not applicable Availability of data and materials The raw data supporting the conclusions will be available by the authors without undue reservation. Competing interests The authors declare no competing interests. Funding This work was supported in part by HL125018 (Liu), AI124769 (Liu), AI129594 (Liu), CA267368 (Liu), AI130197 (Liu), U.S. National Science Foundation (NSF) grant, the NIH/NCI CCSG (P30CA072720) and Rutgers Cancer Institute of New Jersey FY23 Seeding funding, New Jersey Commission on Cancer Research (NJCCR)/New Jersey Department of Health, and Rutgers University-New Jersey Medical School Liu Laboratory Startup funding. This work was also supported by the R01AI136756 grant (Li Y and Li Q) from the NIAID, NIH. Authors’ contributions Q.L. and D.L. designed the study and wrote the manuscript, and Y.Y. and J.Z. assisted with experiments. D.L. and Q.L. conceived and supervised the study. Acknowledgments We would like to thank the members of the Liu laboratory for their comments on the manuscripts. We also would like to thank Dr. Eric Long (NIAID/NIH) for the 721.221 cell line and Dr. Gianpietro Dotti (UNC) for the SFG vectors. 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Keywords animals cells human natural killer cells Authors Affiliations Yan Yang Rutgers New Jersey Medical School View all articles by this author Jianshui Zhang University of Nebraska-Lincoln View all articles by this author Minh Ma Rutgers New Jersey Medical School View all articles by this author Yilun Cheng School of Biological Sciences and Nebraska Center for Virology View all articles by this author Saroj Chandra Lohani [email protected] University of Nebraska-Lincoln View all articles by this author Qingsheng Li University of Nebraska-Lincoln View all articles by this author Dongfang Liu 0000-0002-7295-8088 Rutgers New Jersey Medical School View all articles by this author Metrics & Citations Metrics Article Usage 596 views 225 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yan Yang, Jianshui Zhang, Minh Ma, et al. Human Primary NK Cells from Humanized BLT-IL15 Mice Show Superior Expansion and Cytotoxicity in Comparison with Humanized BLT-mice Using Membrane Bound IL21-modified 721.221 Feeder Cell Expansion System. Authorea . 09 June 2024. DOI: https://doi.org/10.22541/au.171797475.54380127/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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