Human NK cell development in hIL-7 and hIL-15 knockin NOD/SCID/IL2rgKO mice

A new humanized mouse expressing human IL-7 and IL-15 facilitates development and maturation of human NK cells and can be useful as a preclinical in vivo model for testing new treatment modalities.


Introduction
Cytokine receptor signaling is indispensable for reconstitution of the human immune system following hematopoietic stem cell (HSC) therapy. Among multiple cytokines, IL-7 promotes differentiation and maturation of T cells, B cells (Mackall et al, 2011), and innate lymphoid cells (Moro et al, 2010). In addition to the development of mature lymphoid cells, IL-7 signaling plays a pivotal role at the level of progenitor cells. Studies of IL-7-or IL-7R-deficient mice revealed multiple defects in T-and B-cell development (Peschon et al, 1994;von Freeden-Jeffry et al, 1995). Defective IL-7R expression in humans results in T − B + NK + SCID (Puel et al, 1998).
IL-15 supports innate lymphoid cell development (Ali et al, 2015). Studies using IL-15 transgenic mice (Fehniger et al, 2001) and IL-15 knockout (IL-15KO) mice (Kennedy et al, 2000) have shown IL-15 to be essential in the development of NK cells, natural killer T (NKT) cells, and memory CD8 + T cells. Knocking out the genes encoding IL-15 or IL-15Rα results in complete loss of NK cells in the thymus, BM, and spleen. NKT cells and CD44 high memory phenotype CD8 + T cells were also reduced in IL-15KO and IL-15Rα knockout mice (Lodolce et al, 1998;Kennedy et al, 2000). A recent report demonstrated a role of IL-15 in anticancer immunity in that the frequencies of breast cancer metastasis were more frequent in IL-15KO mice than those in IL-15 transgenic mice or in C57BL/6 control mice (Gillgrass et al, 2014).
We developed NOD/SCID/IL2rgKO (NSG) mice to investigate the in vivo dynamics of the human immune system (Ishikawa et al, 2005;Shultz et al, 2005). In studies of humanized mice engrafted with human HSC, we and others reported development of human T and B cells. However, the frequencies of human NK cells did not reach physiological levels in NSG humanized mice (Andre et al, 2010). The decreased NK cell development could be due to the species barrier between human lymphoid or NK cell progenitors and recipient microenvironment (Mestas & Hughes, 2004).
To investigate the in vivo function of human IL-7 and IL-15 in the development of the human immune system, we created new strains of NSG mice expressing either hIL-7 alone (hIL-7TG NSG mice and hIL-7 KI NSG mice) and mice expressing hIL-7 and hIL-15 (hIL-7xhIL-15 KI NSG mice). Analyses of these mice engrafted with human HSCs showed that hIL-15 is required for NK cell development. In addition, we found multiple subsets of human T cells in NSG recipient mice expressing human IL-7 and IL-15, demonstrating the roles of these cytokines in human T-cell development. These new humanized mouse models may support studies of human monoclonal antibody therapy in vivo and for studies of human acquired and innate tumor immunity.
In addition to human NK cell development, we examined whether the cytokines facilitate the development of human T cells or induce skewing of specific human T-cell subsets. To this end, we first analyzed development of human T-cell subsets in the thymus and spleen. Within the thymic hCD45 + CD56 − CD3 + fraction, we found higher frequency of CD4 − CD8 + single positive T cells in hIL-7xhIL-15 KI NSG humanized mice (NSG, n = 18: CD4 − CD8 + cells 15.6 ± 3.1%; hIL7xhIL-15 KI NSG, n = 12: CD4 − CD8 + cells 59.5 ± 5.9%. P < 0.001 by two-tailed t test; Fig 2A and B, absolute numbers of cells are shown in Table S3).

Maturation and location of human NK cells in hIL-7xhIL-15 KI NSG humanized mice
After the development of human NK cells in the primary immune organs (BM and thymus), they undergo maturation in peripheral organs such as spleen (Freud et al, 2014;Bjorkstrom et al, 2016 Table S3). We next assessed functional capacity of human NK cells developing in conventional NSG humanized mice and hIL-7xhIL-15 NSG humanized mice using in vitro cytotoxicity assay. hIL-7xhIL-15 NSG spleen NK cells exhibited cytotoxicity at a similar level compared with human PB NK cells (NSG, n = 3: 5.7% ± 2.2%; hIL-7xhIL-15 KI NSG, n = 8: 12.4 ± 1.8%; human PB, n = 2: 8.1 ± 1.8%; Fig 3E). When we analyzed PFA-fixed, paraffin-embedded thin sections of the recipient spleens, we found cells that were specifically stained with anti-hCD56 and NKp46 antibody. Whereas hCD3 + T cell and hCD19 + B cells appeared to form lymphoid clusters, human NK cells were distributed outside lymphoid clusters, consistent with physiological distribution of human NK cells in human spleen (Witte et al, 1990) (Figs 4 and S4). These findings indicate that hIL-7 and hIL-15 support multiorgan development and maturation of human NK cells in humanized mice.

Maintenance of human NKT cells in hIL-7xhIL-15 KI NSG mice
Although TCRVα24 + Vβ11 + CD3 + NKT cells were not detected in the BM and spleen of hIL-7xhIL-15 KI NSG humanized mice, we examined whether human IL-7 and human IL-15 maintain human NKT cells for longer term in the BM and lungs. We intravenously injected 2 × 10 6 cells, human NKT cells, or iPS-NKT cells into NSG mice or hIL-7xhIL-15 KI NSG mice. In hIL-7xhIL-15 KI NSG mice, we detected NKT cells or iPS-NKT cells in the BM and lungs at higher frequencies at 14 d postinjection (NSG, n = 4: BM NKT cells 0.000%, lung NKT cells 0.001 ± 0.002%; hIL-7xhIL-15 n = 11 BM NKT cells 0.084 ± 0.025%, lung NKT cells 0.079 ± 0.014%, P = 0.07 in BM and P < 0.01 in the lungs by twotailed t test; Fig S5). This finding indicates that hIL-7 and hIL-15 play an important role for the survival of human NKT cells.

Discussion
NK cells were first reported in 1975 (Herberman et al, 1975;Kiessling et al, 1975;Sendo et al, 1975). NK cells exert potent cytotoxic function   cells, development, maturation, and function of NK cells have been extensively studied using genetically engineered mouse models (Kennedy et al, 2000;Fehniger et al, 2001). Although mouse and human NK cell development share some aspects in their biological function and transcriptome, there are differences between human and mouse NK cells such as the use of Ly49 receptors by mouse NK cells versus the use of KIR by human NK cells for the recognition of MHC (Colucci et al, 2002). In the present study, we aimed to develop an in vivo model supporting human NK cell maturation and to assess roles of cytokine receptor signaling in human NK cell development. In particular, we aimed to address the following two questions: (1) How do human NK cells develop and distribute in primary and secondary lymphoid organs? (2) How do human NK cells become functionally mature in the microenvironment? To answer these questions, we created NSG mice expressing hIL-7 alone and those expressing both hIL-7 and hIL-15. Unlike NSG mice expressing hIL-7 alone, concurrent expression of hIL-15 resulted in enhanced in vivo human NK development in multiple organs, but not in T cells or NKT cells. Histologically, whereas human T cells and human B cells existed mainly near white pulp-like structures, most human NK cells were located outside the white pulp-like area, consistent with human splenic architecture (Witte et al, 1990). The (E) In vitro cytotoxicity of human NK cells isolated from spleen of conventional NSG, hIL-7xhIL-15 KI NSG humanized mouse spleen (circles) and BM (triangles), and from normal human PB samples (squares) against K562 are shown (NSG n = 3; IL7xIL15 n = 7; normal human PB n = 2). Error bars represent mean ± SEM. *P < 0.05, **P < 0.001, by two-tailed t test.
BM is considered as a primary organ for NK cell development and maturation (Huntington et al, 2007). Although emerging evidence has suggested that thymus is also important for NK cell development, it has yet to be understood whether thymus provides essential environmental factors for mouse and human NK cells (Freud et al, 2014;Bjorkstrom et al, 2016). In our xenograft, we found CD56 + NK cells in both BM and thymus. Thymic NK cells were reported to express IL-7R in mice and human (Vosshenrich et al, 2006). Consistent with the report, we found IL-7R + CD56 + NK cells in the thymus of hIL-7xhIL-15 KI NSG humanized mice (representative flow cytometry plots were shown in Fig S7). This result may suggest that hIL-7 signaling is important for human NK cell development particularly in thymus.
To date, the frequency of CD56 + CD16 + mature human NK cells in xenotransplantation models have not fully reflected physiological levels. Our report is consistent with previous publications using in vivo administration of hIL-15 or hIL-15-hIL-15Rα complex to humanized mice (Huntington et al, 2009;Strowig et al, 2010) and more recently, using hIL-15 expressing BALBc/Rag2KO/Il2rgKO mice assessing differentiation and function of human NK cells in vivo (Herndler-Brandstetter et al, 2017). The new NSG mouse model described in the present work further supports critical roles of IL-15 in human NK cell development. In our new strain, we found CXCR6 + CD56 + tissue-resident NK cells in the BM and liver. Because tissue-resident NK cells and circulating NK cells are different in cytotoxicity or cytokine production (Melsen et al, 2018), hIL-7xhIL-15 KI NSG humanized mice could be useful for studying the two distinct subsets of human NK cells. In addition to flow cytometric analysis of engrafted human NK cells, we performed transcriptome analysis by RNA sequencing. We found that the gene expression signature of human NK cells in hIL-7xhIL-15 KI NSG humanized mice was more similar to those in human PB as compared with human NK cells in conventional NSG humanized mice. It would be notable that maturation or cytotoxic markers such as KIRs, chemokine ligands (CCL4L1 and CCL4), cytotoxicityrelated genes (GZMA and GZMB), as well as PRDM1 were upregulated in the NK cells in hIL-7xhIL-15 KI NSG humanized mice because all these genes were known as NK-specific functional molecules (Fehniger et al, 1999;Parham, 2005;Yawata et al, 2006;Smith et al, 2010), and that the new humanized mice could be a better in vivo model for assessing interaction between NK cells and diseased cells.
We also assessed potential development and survival of human NKT cells in the NSG mice expressing hIL-7 and hIL-15. In the mice engrafted with human CB HSCs, we did not see differentiation of human NKT cells in organs such as the BM, spleen, and lungs, suggesting that these two cytokines are not sufficient to support human NKT cell development. On the other hand, with in vivo transfer of human NKT cells into hIL-7xhIL-15 KI NSG mice, the injected human NKT cells were detected at higher frequencies in multiple organs as compared with NSG mice. Because NKT cells are known to activate NK cells in vivo, the model would be useful to analyze activation of human NK cells with intravenous injection of human NKT cells (Carnaud et al, 1999;Fujii et al, 2010;Yamada et al, 2016).
Together, expression of hIL-7 and hIL-15 in the NSG humanized mice resulted in efficient development of human NK cells in multiple organs in the presence of multiple human immune subsets, and the human NK cells undergo physiological maturation process and exhibit cytotoxicity. The new humanized mouse model with hIL-7 and hIL-15 expression could be used as a valuable tool for examining in vivo biology of human NK cells and for in vivo testing of NK cell-mediated cytotoxicity against cancer.

Human samples
Human CB cells were obtained from Tokai Cord Blood Bank and Chubu Cord Blood Bank under written informed consent. All experiments were authorized by the institutional review boards at RIKEN.

. Distribution of splenic T cells, B cells, and NK cells in NSG hL-7xhIL-15 humanized mice.
Thin sections of a hIL-7xhIL-15 NSG recipient spleen stained with anti-hCD56, anti-CD3 and anti-CD19 antibodies. Low and high magnification images are shown. hCD3 + T cell and hCD19 + B cells were found within lymphoid clusters, whereas CD56 + NK cells are located outside the clusters. Scale bars: low magnification, 50 μm; high magnification, 20 μm.
All mice were bred and maintained under specific pathogen free conditions at the animal facility of RIKEN Integrative Medical Sciences. All animal experiments were performed in accordance with Genotyping of NSG mice hIL-7 TG mice, hIL-7 KI mice, and hIL-15 KI mice were genotyped by PCR (Bio-Rad, Takara Bio, or BM Equipment). Primer information used for this genotyping is shown in Fig S8B. The microsatellite markers used for marker-assisted selection protocol were selected based on sequence length polymorphisms between B6 mice and NOD mice (according to Mouse Microsatellite Date Base of Japan [MMDBJ]; https://shigen.nig.ac.jp/mouse/mmdbj/top.jsp, Table S5). All primers were purchased from Life Technologies Japan.
For analysis of regulatory T cells, cells stained with mAbs for surface antigens were fixed and permeabilized using FoxP3/Transcription Factor Staining Buffer Set (eBioscience). Permeabilized cells were stained with anti-hFoxp3-APC (clone PCH101; eBioscience) and analyzed using FACSCanto II (BD Biosciences). To analyze the expression of intracellular granzyme B and perforin, cells stained with mAbs for surface antigens were fixed and permeabilized using BD Cytofix/Cytoperm Kit (BD Biosciences). Permeabilized cells were stained with anti-human granzyme B-FITC (GB11; BD Biosciences) and anti-human perforin-PE (δG9; BD Biosciences) and analyzed using FACSCanto II (BD Biosciences).

Measurement of plasma cytokine levels
To measure concentrations of cytokines in recipient plasma samples, PB samples were obtained from NSG, hIL-7 Tg NSG, hIL-7 KI NSG, and hIL-7xhIL-15 KI NSG mice using heparinized capillary tubes (Drummond Scientific Company). Plasma human IL-7 concentration was measured by ELISA (R&D Systems). Plasma human IL-15 concentration was measured by Bio-Plex Systems (Bio-Rad).

NKT cell injection
Human NKT cells and human iPS-NKT cells were prepared using a previously established method (Yamada et al, 2017). 2.0 × 10 6 NKT cells were intravenously injected into NSG mice and hIL-7xhIL-15 KI NSG mice. At 14 d postinjection, the recipients were euthanized and analyzed for the presence of human NKT cells in the BM and lungs.

RNA extraction and qPCR
BM cells of NSG and hIL7xhIL-15 KI NSG humanized mice were labeled with anti-mGra1-FITC, anti-hCD33-PE, anti-mCD45-APC-Cy7, anti-Mac1-BV421, and anti-hCD45-BV510 to purify Mac1 + Gra1 + mouse myeloid cells and hCD45 + CD33 + human myeloid cells using FACSAria III (BD Biosciences). Total RNA was extracted using Trizol reagent (Invitrogen). Total RNA was quantified using a fluorimetric Ribo-Green assay, and RNA quality was assessed using Bioanalyzer (Agilent Technologies). Extracted RNA was converted into cDNA using Superscript III reverse transcriptase (Invitrogen). Real-time PCR reactions were performed using Platinum Quantitative PCR SuperMix-UDG (Invitrogen) on a LightCycler 480 real-time PCR system (Roche) using the following primers: Relative expression levels were calculated for each gene using ACTB for normalization.

RNA sequencing analysis
Total RNA extracted from splenic CD56 + NK cells in NSG recipient, and hIL-7xhIL-15 KI NSG recipient was prepared for RNA sequencing using NEBNext Ultra RNA Library Prep kit for Illumina (catalog number E7530; New England Biolabs). Final library size distribution was validated using Bioanalyzer and quantified using quantitative PCR. The DNA libraries were hybridized to a flow cell, amplified on the Illumina cBot, and subsequently run on the Hiseq 2500 (Illumina, on a 50-base single-end read mode). Raw data are deposited at the National Bioscience Database Center (accession number: hum0171). The sequence reads were mapped to the human genome (NCBI version 19) using TopHat2 version 2.0.8 and botwie2 version 2.1.0 with default parameters, and gene annotation was provided by NCBI RefSeq. The transcript abundances were estimated using Cufflinks (version 2.1.1). Cufflinks was run with the same reference annotation with TopHat2 to generate FPKM (fragments per kilobase per million mapped reads) values for known gene models.

Quantification and statistical analysis
Quantification and statistical analysis were performed using excel. The numerical data are presented as means ± SEM. The differences were determined by two-tailed t tests, and P value < 0.05 was considered statistically significant.