Human macrophages and innate lymphoid cells: Tissue-resident innate immunity in humanized mice

Journal Pre-proofs Review Human Macrophages and Innate Lymphoid Cells: Tissue-resident Innate Immunity in Humanized Mice Arlisa Alisjahbana, Imran Mohammad, Yu Gao, Elza Evren, Emma Ringqvist, Tim Willinger PII: DOI: Reference:

S0006-2952(19)30371-5 https://doi.org/10.1016/j.bcp.2019.113672 BCP 113672

To appear in:

Biochemical Pharmacology

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19 August 2019 15 October 2019

Please cite this article as: A. Alisjahbana, I. Mohammad, Y. Gao, E. Evren, E. Ringqvist, T. Willinger, Human Macrophages and Innate Lymphoid Cells: Tissue-resident Innate Immunity in Humanized Mice, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.113672

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Human Macrophages and Innate Lymphoid Cells: Tissue-resident Innate Immunity in Humanized Mice Arlisa Alisjahbanaa,1, Imran Mohammada,1, Yu Gaoa,1, Elza Evrena, Emma Ringqvista, Tim Willingera,* aCenter

for Infectious Medicine, Karolinska Institutet, Alfred Nobels allé 8, 141 52 Stockholm,

Sweden *Corresponding author E-mail address: [email protected] (T. Willinger). 1Equal

contribution.

Keywords: Macrophages, Innate Lymphoid Cells, Humanized Mice, Tissue-resident Immunity, Single-cell RNA-sequencing, CRISPR/Cas9 genome editing

Abstract Macrophages and innate lymphoid cells (ILCs) are tissue-resident cells that play important roles in organ homeostasis and tissue immunity. Their intricate relationship with the organs they reside in allows them to quickly respond to perturbations of organ homeostasis and environmental challenges, such as infection and tissue injury. Macrophages and ILCs have been extensively studied in mice, yet important species-specific differences exist regarding innate immunity between humans and mice. Complementary to ex-vivo studies with human cells, humanized mice (i.e. mice with a human immune system) offer the opportunity to study human macrophages and ILCs in vivo within their surrounding tissue microenvironments. In this review, we will discuss how humanized mice have helped gain new knowledge about the basic biology of these cells, as well as their function in infectious and malignant conditions. Furthermore, we will highlight active areas of investigation related to human macrophages and ILCs, such as their cellular heterogeneity, ontogeny, tissue residency, and plasticity. In the near 1

future, we expect more fundamental discoveries in these areas through the combined use of improved humanized mouse models together with state-of-the-art technologies, such as singlecell RNA-sequencing and CRISPR/Cas9 genome editing.

1. Introduction Macrophages and innate lymphoid cells (ILCs) are currently two of the most topical cell types in the field of immunology. Together, they orchestrate local immune responses in organs throughout the body and control tissue homeostasis by performing “non-immunological” functions that are distinct from classical immunity based on host defense. Furthermore, macrophages and ILCs are linked by important concepts related to their origin, tissue residency, and plasticity. Given their tissue-resident “lifestyle”, macrophages and ILCs are difficult to study in human samples and human studies are restricted to end-point measurements in biopsies or explants. Recent improvements of humanized mouse models provide the opportunity to greatly increase our understanding of human macrophage and ILC function in vivo (Fig. 1). To date, humanized mice have revealed important roles for human macrophages and ILCs in viral and bacterial infections and in cancer immunity, which will be discussed in this review.

2. Humanized mouse models to study macrophages and ILCs 2.1. Generation of humanized mice In a broad sense, humanized mice are immunodeficient mice transplanted with human cells/tissues with the aim of studying human hematopoiesis, immune function, pathogens with human tropism, and other immune-mediated diseases. The idea behind their creation is to have an in vivo-experimental system with the advantages of a small animal model and the ability to produce findings that are relevant to human physiology and pathology. The various humanized models and their historical development have been extensively reviewed elsewhere [1-6]. Mice

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with a human immune system are generated by the transplantation of immunodeficient mice with human hematopoietic stem and progenitor cells (HSPCs; usually purified based on the surface marker CD34) that develop into different lineages of immune cells. Different models can be distinguished based on the type of immunodeficient mice, the source of transplanted HSPCs (fetal, neonatal, adult), whether recipient mice are pre-conditioned before transplantation (usually by irradiation), and whether any human tissue is co-transplanted with HSPCs (Table 1). The most common immunodeficient mice strains lack endogenous T, B, and natural killer (NK) cells and are the following: (i) BALB/c Rag2-/-Il2rg-/- (BRG); (ii) NOD/SCID Il2rg-/- (NSG); and (iii) NOD/Shi-SCID Il2rg-/- (NOG). These models can be combined with the co-transplantation of human fetal liver and thymus fragments in addition to engraftment with human HSPCs, generating so-called “bone marrow, liver, thymus” (BLT) mice [7]. A limitation of BLT models is the development of human T cell-mediated xenograft responses, resembling graft-versus-host disease [8, 9]. 2.2. Improvement of humanized mouse models Humanized mouse models have been improved substantially over the last decade by enhancing human hematopoiesis in the mouse host and by preventing the elimination of human cells (Table 1). The latter was achieved by making residual mouse phagocytes more tolerant of human hematopoietic cells, especially T and NK cells. To establish mouse-to-human phagocytic tolerance, the signal-regulatory protein alpha (SIRP)-CD47 receptor system was modified to establish a “Don’t Eat Me Signal” [10]. Several approaches have been used for this purpose, such as mice expressing human SIRP as a transgene [11, 12] or knock-in allele [13], BRG mice having the SirpaNOD allele (BRGS) [14], or transduction of human HSPCs with mouse CD47 [15]. Human hematopoiesis in the mouse host was improved by the provision of critical human factors, mainly cytokines [16], thereby overcoming the lack of cross-reactivity between mouse factors and human receptors. Provision of human factors can be achieved in 3

various ways, e.g. by injection of recombinant proteins, by hydrodynamic injection of plasmids, through viral delivery systems, and in transgenic or knock-in mice. The knock-in approach has the advantage of achieving physiological human cytokine expression in terms of amount, space, and time. Furthermore, in homozygous knock-in mice, only the human factor is expressed, which gives a competitive advantage to human hematopoietic cells in the mouse host [16]. 2.2.1. Improved humanized mouse models for macrophages Macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage colonystimulating factor (GM-CSF) are two critical cytokines for the development and homeostasis of macrophages. Accordingly, various humanized mouse strains expressing human M-CSF and/or GM-CSF have been created, which show enhanced human macrophage development (Fig. 1 and Table 1). For example, human interleukin-3 (IL-3)/GM-CSF knock-in BRG mice support the development of human lung macrophages after engraftment with human CD34+ cells [17], while knock-in of human M-CSF improves human monocyte and macrophage reconstitution [18]. Likewise, hydrodynamic injection of M-CSF into NSG mice promoted the development of mature monocytes and macrophages from human CD34+ cells [19]. Human IL3/GM-CSF/stem cell factor (SCF) triple transgenic NSG mice [20-22] and human IL-3/GMCSF transgenic NOG mice [23] have also been generated. One problem with human IL-3/GMCSF/SCF triple transgenic mice is the development of fatal macrophage activation syndrome after transplantation with human CD34+ cells [24]. IL-34 is required for specific macrophage populations (Fig. 1), and it has been reported that NOG mice expressing transgenic human IL34 harbor human microglia-like cells [25]. 2.2.2. Improved humanized mouse models for ILCs As for T lymphocytes, IL-2 family cytokines are important for ILCs and NK cells (Fig. 1). For example, IL-7 is essential for the development and homeostasis of (non-NK cell) ILCs. Several humanized mouse models expressing human IL-7 through various means have been created,

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with modest effects on human T lymphocytes [26-28], but human (non-NK cell) ILCs have not been examined in these studies. In any case, human ILCs are present in humanized mouse models that do not have a non-hematopoietic source of human IL-7 [29-35], and own unpublished observation), suggesting that mouse IL-7 may be sufficient to support human ILCs. In contrast, mouse IL-15 is not sufficient to fully support human NK cells [36, 37] and provision of exogenous human IL-15 is required for the development of fully-differentiated human NK cells in BRG mice [36]. The requirement for IL-15 from hematopoietic versus nonhematopoietic sources varies between IL-15-dependent cell types [38]. Hematopoietic-derived human IL-15 can be provided for instance by human monocytes that develop in the humanized MISTRG model described below, which supports the development of functional human NK cells in various tissues [12]. To provide a non-hematopoietic source, human IL-15 knock-in mice were generated on a human SIRP knock-in BRG background. After engraftment with human CD34+ cells, these mice harbored higher numbers of human NK cells and ILC1s, including intraepithelial NK cells in the intestine, than mice without human IL-15 [39]. Finally, IL-2 is known to promote NK cell activation and their cytotoxic activity. Consequently, human IL-2 transgenic NOG mice have increased numbers of activated human NK cells due to supraphysiological amounts of IL-2 [40]. Similarly, adenoviral vector-mediated expression of human IL-2 increases human NK cell numbers in BLT-NSG mice [41]. Apart from cytokines, human MHC class I is also important for the proper education and licensing of human NK cells in the mouse host [42-44]. Human ILC homeostasis is also indirectly improved in humanized mice by boosting human myeloid cells, such as monocytes (see MISTRG mice below) and dendritic cells, which then likely provides a more supportive cytokine milieu for ILCs. For example, injection of recombinant human Fms-related tyrosine kinase 3 (FLT3) ligand expands human dendritic cells in Flk2-/- BRGS mice, thereby promoting human ILC homeostasis [31]. 2.2.3. MISTRG humanized mouse model

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A model of particular interest in terms of studying human macrophages and ILCs is the “MISTRG” humanized mouse strain [12]. MISTRG mice express the human proteins M-CSF, IL-3/GM-CSF, SIRP, and thrombopoietin (TPO) in the BRG background. This "genetic preconditioning" creates a highly permissive environment for human hematopoiesis in the mouse host, with superior myelopoiesis compared to other models and allowing efficient human engraftment even in the absence of irradiation [12, 45]. Specifically, MISTRG mice engrafted with human CD34+ cells support the development of human macrophages in lung, liver, and intestine [12]. Moreover, all three subsets of human blood monocytes (see below) are present in MISTRG mice, including CD16+ monocytes that are not found in other models [12, 45]. This is likely mediated by the knock-in of human M-CSF since non-classical CD14loCD16+ monocytes are absent in humans with mutations in CSFR1, encoding the M-CSF receptor [46]. As mentioned above, functional human NK cells also develop in CD34+-engrafted MISTRG mice, without the need for providing exogenous human IL-15 [12]. This is likely due to the trans-presentation of human IL-15 by human monocytes present in MISTRG mice [12]. Apart from NK cells, all other types of human ILCs are present in the MISTRG model (own unpublished observation). One limitation of the MISTRG humanized mouse model is the development of anemia over time due to destruction of mouse red blood cells by human macrophages [12].

3. Macrophages and ILCs: Two major players in tissue-resident innate immunity 3.1. Macrophages Macrophages are tissue-resident myeloid cells that belong to the mononuclear phagocyte system, which also includes monocytes and dendritic cells [47-51]. Macrophages are dependent on colony stimulating factor receptor 1 (CSFR1; CD115), the receptor for M-CSF and IL-34. A distinctive hallmark of macrophages is phagocytosis, enabling them to clear microbes, dead

6

cells, and other material. This scavenger function is critical for their role in host defense and maintenance of tissue homeostasis. For example, the lack of alveolar macrophages in the lung results in pulmonary alveolar proteinosis (PAP), an inflammatory syndrome caused by the defective clearance of lung surfactant [52]. Furthermore, macrophages play a crucial role in tissue repair and as orchestrators of the immune response through the production of cytokines, chemokines, and growth factors; not only in the lung, but also in other tissues, e.g. brain, liver, and skin [51, 53]. In contrast, monocytes are circulating cells that develop from HSPCs in the bone marrow [54]. Monocytes can enter tissues from the blood, especially in states of altered homeostasis, such as infection, and differentiate into macrophages in order to support host defense. In human blood, three monocyte subsets have been described based on the surface markers CD14 and CD16: classical (CD14+CD16lo), intermediate (CD14+CD16+), and non-classical (CD14loCD16+) monocytes [54-57]. Recent single-cell RNA-sequencing (RNA-seq) studies suggest that the intermediate CD14+CD16+ population does not separate into a distinct cluster of monocytes, but rather exists as a diverse intermediate [58]. Classical monocytes either extravasate into tissues or differentiate via CD14+CD16+ intermediate monocytes into patrolling CD14loCD16+ monocytes [59, 60]. In humans, subsets of blood monocytes, e.g. CD14+CD16+ intermediate monocytes, are expanded in states of chronic inflammation [46, 57, 61-64]. However, the functional significance of the different monocyte subsets in human disease settings remains unknown and warrants further investigation with the use of humanized mice as invaluable invivo experimental systems. In contrast, studies of monocytes and other blood cells in human samples only gives a snapshot without any direct information on migration, lineage relationships, and function. 3.2 ILCs

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ILCs are an emerging subset of innate immune cells with diverse roles in barrier immunity, tissue repair, lymphoid tissue formation, and metabolism [65-71]. ILCs belong to the lymphoid lineage, but, in contrast to T and B lymphocytes, do not exhibit any antigen specificity due to the lack of re-arranged antigen-specific receptors. Besides cytotoxic NK cells, ILCs are classified into non-cytotoxic ILC1s, ILC2s, and ILC3s based on signature transcription factors and effector cytokines, similar to CD4+ T helper cells: (i) ILC1s depend on the transcription factor T-BET and produce interferon gamma (IFN); (ii) ILC2s depend on the transcription factor GATA3 and produce the cytokines IL-5 and IL-13; (iii) ILC3s depend on the transcription factor RORt and produce the cytokines IL-17 and/or IL-22. Furthermore, ILC3s consist of two major subsets: (i) fetal lymphoid tissue inducer (LTi) cells and adult LTi-like ILC3s; and (ii) ILC3s expressing natural cytotoxicity receptors, such as NKp46. ILCs lack cell lineage markers for other immune cells and, with the exception of NK cells, are defined by their expression of IL-7 receptor alpha (IL-7Rα; CD127). Accordingly, ILCs are mainly dependent on the cytokine IL-7, whereas NK cells require IL-15 for their development and homeostasis. In both mice and humans [29, 72-74], ILCs are present in many organs, but are most abundant in tissues that are exposed to the environment, such as intestine and lung. Furthermore, parabiosis studies in mice suggest that, in contrast to NK cells, ILC2s and ILC3s are mainly tissue-resident cells, at least in steady-state [75]. 3.3. Species-specific macrophage and ILC differences Due to divergent evolution, the immune systems of mice and humans show important differences in terms of development, cellular composition, surface markers, migratory receptors, tissue localization, and effector function [76-79]. For example, recent single-cell RNA-seq studies indicate that the human lung macrophage compartment is more diverse than its mouse counterpart [80]. In contrast, the main monocyte populations are relatively wellconserved between the two species [80, 81], with some differences in gene expression [81]. 8

However, in humans classical monocytes predominate in blood, whereas classical Ly6Chi and non-classical Ly6Clo monocytes are equally represented in the mouse [54]. Similarly, based on single-cell RNA-seq data, NK cells in spleen seem to be more heterogeneous in humans than in mice [82]. Moreover, it has been suggested that the tissue compartmentalization of human ILCs is less strict than in mice [74]. The recent discovery of circulating ILC precursors (ILCPs) in humans [33] has raised the possibility that human and mouse ILCs may also differ in terms of how strictly tissue-resident they are. Finally, there are differences in immune receptor expression between humans and mice, e.g. related to MHC class II, CD11b, and NK cell receptors [69]. For example, human monocytes are HLA-DR+, whereas their mouse counterparts show heterogeneous expression of MHC class II [54].

4. Important common features of macrophages and ILCs Macrophages and ILCs have several interesting common characteristics (Fig. 2) that have mainly been investigated in mouse studies. Accordingly, there is a need to explore these concepts further in human macrophages and ILCs in vivo and, in this context, humanized mice are a valuable experimental system. 4.1. Ontogeny of macrophages and ILCs Since blood monocytes can enter tissues and differentiate into macrophages, macrophages were thought to constitute a “tissue-resident form of monocytes”. However, mouse studies over the last decade have led to a paradigm shift, demonstrating that in many tissues macrophages originate from embryonic precursors and self-renew locally [83-86]. Specifically, lineage tracing in mice has defined three developmental waves of macrophages from early embryonic to adult life [85]. The first wave of macrophage precursors derives from yolk sac, e.g. forming microglia in the fetal brain. The second wave consists of fetal liver monocytes that give rise to macrophages in multiple organs, such as lung and liver. The third and final wave is derived

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from adult hematopoiesis in the bone marrow, i.e. the development of circulatory monocytes into monocyte-derived macrophages, such as in the steady-state intestine. The contribution of each wave to the macrophage compartment differs between organs and even between distinct macrophage populations within each tissue. Therefore, macrophages can now be classified according to their origin as: (i) Adult/HSPC-derived (blood monocyte-dependent) macrophages; (ii) Embryonic-derived (blood monocyte-independent) macrophages. The origin, timing, and tissue specific cues for these three developmental waves have been thoroughly investigated in mice (Fig. 2). In contrast, much less known about the origin and ontogeny of human macrophages. The macrophage concept of “layered ontogeny” also seems to apply to ILCs [71]. Similar to macrophages, ILCs seed tissues early during development and temporal fate mapping recently demonstrated that mouse ILC2s derive from fetal, neonatal, and adult waves [87]. A topical area relates to the question whether cellular origin determines macrophage function or whether imprinting by the local tissue environment is the predominant factor (“nature versus nurture”). This question is particularly relevant in the context of tissue injury and inflammation, where depletion of the resident macrophage compartment (embryonic origin) allows blood monocytes to enter the tissue niche and differentiate into macrophages (adult monocyte origin). This results in a macrophage compartment of dual origin with potential implications for subsequent inflammatory responses, e.g. in the lung [88, 89]. It has been shown that yolk sac macrophages, fetal liver monocytes, and bone marrow monocytes can all reconstitute an empty alveolar niche and become transcriptionally and functionally similar to resident alveolar macrophages [90], suggesting that tissue imprinting determines macrophage function. However, further studies are needed to clarify this issue, especially in an inflammatory context. 4.2. Tissue residency and homeostasis of macrophages and ILCs

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Macrophages and ILCs occupy distinct anatomical niches within the same organ to carry out specialized functions. For example, in the lung there are interstitial and alveolar macrophages [52, 91, 92]. Interestingly, macrophages residing in distinct niches within an organ can have different cellular origins, e.g. in the lung [92-94]. Similarly, in the intestine there are lymphoidtissue resident LTi-like ILC3s as well as NKp46+ ILC3s that localize to the lamina propria. The chemotactic signals directing the occupation of different tissue niches by ILCs in the intestine are being identified [95]. Thus, CXCL16 ensures localization of NKp46+CXCR6+ ILC3s to the small intestinal villi [96], whereas the cholesterol metabolite 7,25-dihydroxycholesterol positions GPR183+ LTi-like ILC3s in intestinal cryptopatches and isolated lymphoid follicles [97, 98]. Once positioned within their tissue niche, macrophages and ILCs are thought to self-renew locally. In this context, it has been shown that macrophages have a stem cell-like capacity for self-renewal in steady-state, independent of circulating monocytes [83, 84, 99]. Tissue injury and associated inflammation may impair this self-renewal ability, leading to depletion of resident macrophages, thereby inducing recruited blood monocytes to replace resident macrophages. It is currently controversial to what extent ILCs are maintained through local self-renewal or, alternatively, through the recruitment of circulating or locally-resident ILCPs. In steady-state, the majority of mouse ILC2s and ILC3s are tissue-resident, i.e. independent of circulating cells [75]. In contrast, during helminth infection, inter-organ trafficking of ILC2s occurs from the intestine to the lung, supporting the local ILC2 pool to provide host defense [100]. In humans, ILCPs are present in blood and within tissue, giving rise to the concept of local, on-demand, “ILC-poiesis” [33]. This is conceptually similar to how blood monocytes can contribute to the macrophage compartment in various tissues. 4.3. Tissue specialization, cellular heterogeneity, and plasticity of macrophages and ILCs

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Macrophages functionally adapt to each tissue they reside in (Fig. 2). This tissue adaptation is enforced by the expression of tissue-specific transcription factors, which are induced by tissuespecific cues, such as locally produced cytokines [50, 101, 102]. For example, GM-CSF produced by alveolar epithelial type II cells induces expression of the master transcription factor peroxisome proliferator-activated receptor gamma (PPAR), a key regulator of lipid and surfactant metabolism, in alveolar macrophages [52]. This concept of tissue-specific transcription factors does not seem to apply to ILCs. In this case, tissue-specific functions may be achieved through the differential distribution of ILC subsets in different organs. For example, ILC2s are enriched in adipose tissue, where they regulate lipid metabolism [69]. Heterogeneous populations of macrophages and ILCs can be generated through distinct developmental paths (cellular origins) and through cellular plasticity in response to local cues from the tissue microenvironment (Fig. 2). In this scenario, cellular origin, anatomical location, and local tissue signals dictate the level of immune specification and the potential to respond to environmental challenges. This process tailors the local immune response to the type of tissue insult, thereby enabling an optimal outcome for the host.

5. Discoveries in human macrophage and ILC biology using humanized mice Humanized mice have provided important insights into the biology of human macrophages and ILCs (Table 2) by allowing in-vivo experimentation that is not possible in humans. 5.1. Development and function of human monocytes and macrophages Humanized mice have helped to dissect the lineage relationship and function of human blood monocytes. For example, transfer into immunodeficient mice revealed that human non-classical monocytes patrol blood vessels [103]. Subsequent studies with human subjects, using in vivo deuterium labeling, determined the lifespan of the three blood monocyte subsets [59, 60]. Classical CD14+CD16lo monocytes circulate in blood for up to 1 day, whereas intermediate

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CD14+CD16+ monocytes have a lifespan of ~4 days. Non-classical, patrolling, CD14loCD16+ monocytes have the longest life span of ~7 days. These data suggested that classical monocytes convert into CD16+ monocytes. To confirm this hypothesis, purified human CD14+CD16lo monocytes were intravenously injected into MISTRG mice [59]. Transferred CD14+CD16lo monocytes gave first rise to CD14+CD16+ and then to CD14loCD16+ monocytes, consistent with a linear differentiation model [59]. Earlier, the MISTRG model was employed to examine how human NK cells are maintained in vivo. MISTRG mice engrafted with human CD34+ cells had increased amounts of human IL15 and IL-15R, which allowed the development of human NK cells [12]. Moreover, human monocytes expressed both IL-15 and IL-15R; and the depletion of blood monocytes reduced human NK cells in MISTRG mice [12]. These data support the notion that IL-15 transpresentation by monocytes is critical for human NK cell homeostasis in vivo. Humanized mice have also given insight into the origin of human macrophages. Using human IL-3/GM-CSF knock-in mice as well as MISTRG mice, Willinger et al. first demonstrated that human alveolar macrophages can develop from human fetal liver CD34+ cells [12, 17]. This finding supports the concept that human GM-CSF supports human macrophage reconstitution of an empty alveolar niche. Subsequent work showed that not only fetal liver CD34+ cells, but also CD34+ cells from umbilical cord blood ([45] and own unpublished observation) and granulocyte colony-stimulating factor (G-CSF)-mobilized CD34+ cells from peripheral blood [104] are capable of giving rise to human alveolar macrophages. Overall, these results demonstrate that human alveolar macrophages can develop from HSPCs of different developmental age (fetal, neonatal, adult) and can therefore have a monocytic origin. Importantly, human alveolar macrophages of monocytic origin are functional since they are able to prevent PAP [17]. HSPC-derived human macrophages are also found in other tissues, such as liver and intestine, in MISTRG mice engrafted with human CD34+ cells [12]. 13

Following up on the finding that functional human macrophages can develop from human CD34+ cells, it was tested whether in vitro-generated human macrophages can be harnessed to treat lung disease. In this approach, human CD34+ cells from cord blood were differentiated into macrophage-like cells in vitro and then administered intra-tracheally into human IL-3/GMCSF knock-in mice [105]. The transplanted macrophage-like cells could reconstitute the alveolar niche of human IL-3/GM-CSF knock-in mice and rescue PAP [105]. Subsequently, the same group reported that induced pluripotent stem (iPSC)-derived macrophages are also able to ameliorate PAP [106]. Therefore, pulmonary transplantation of macrophage progenitors or in vitro-generated macrophages may be a viable treatment strategy for human lung disease. Difficult-to-study brain tissue and human microglia development has been another useful area of humanized mice. For example, one study showed that human CD34+ HSPCs can give rise to human microglia-like cells after transplantation into NSG mice [107]. Furthermore, Bennett et al. used human M-CSF knock-in BRG mice [18] crossed with Csfr1-/- mice to demonstrate engraftment of human blood cells (giving rise to HSPC-derived microglia-like cells), fetal brain macrophages, and post-natal microglia after transplantation into the mouse brain [108]. Furthermore, the authors identified MS4A7 as a conserved ontogeny marker across mice and humans, since it was specific for HSPC-derived microglia-like cells. In addition, MITRG mice support the engraftment of iPSC-derived human microglia [109, 110]. The MITRG model also revealed that human iPSC-derived hematopoietic progenitors differentiate into functional human microglia (and other brain macrophages) in a human M-CSF-dependent manner, but independently of human GM-CSF or TPO [111]. Finally, the iPSC-derived human microglia in MITRG mice had human-specific transcriptional responses to systemic inflammation and betaamyloid plaques [111]. Macrophages also play an important role in skin homeostasis [53] and humanized mouse models have been used to investigate the role of macrophages in tolerance to skin

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transplantation. Using immunodeficient SCID/beige mice transplanted with human CD34+ HSPCs, the authors showed that human CD68+ macrophages of monocytic origin contribute to the rejection of human skin (and artery) allografts [112]. Conversely, another study suggested that the depletion of human macrophages in BRG mice injected with human peripheral blood mononuclear cells may cause more severe graft-versus-host disease in the skin and other organs [113]. 5.2. Development and function of human ILCs The development of ILCs has been well studied in the mouse and reviewed extensively [68, 114]. In contrast, the study of ILC and NK cell development in humans has been more limited. Nevertheless several human precursors have been defined and a more complete picture of human ILC development has been emerging. Here, humanized mouse models have been invaluable because they allow studying the development of human ILCs in vivo. First, studies in humanized mice have provided insights into the human equivalent of mouse common lymphoid progenitors (CLPs) that give rise to T, B, NK cells and ILCs. Previously, human lymphoid-primed multipotent progenitors were thought to be the equivalent of mouse CLPs, but they are not lymphoid-restricted [115]. Hussen et al. used humanized NGS mice to define human bone marrow CLPs and early lymphoid progenitors (ELPs) as CD34+CD45RA+, which can be further divided into two subsets based on CD127 expression [116], where CD127+ ELPs are biased towards developing into early B cell precursors and CD127- ELPs into T cell precursors. NK cells and ILCs were shown to be able to develop from both CD127+ and CD127ELPs, suggesting that there are multiple paths for human ILCs and NK cell development. The

first

human

NK

cell

committed

precursors

(NKPs)

were

defined

as

CD34+CD45RA+CD10+CD7+ cells, lacking expression of CD127 and mature NK cell markers [117]. The NK cell-restricted differentiation potential of NKPs was confirmed in vivo by the adoptive transfer into NSG mice [117]. Another important discovery was that human common

15

innate lymphoid progenitors (CILPs) can give rise to human NK cells and all ILC subsets, but not T cells, B cells or dendritic cells [118]. The authors showed that human CD34+CD117+CD45RA+IL1R+ CILPs expressing RORγt were able to differentiate into all mature ILC subsets including NK cells in vitro. However, the in-vivo transfer of CILPs into NSG mice generated mature NK cells, but not ILCs [118]. This study revealed an important species-specific difference because RORγt expression in human ILCs is more generalized than in mice and is important for both ILC and NK cell development [118]. More recently, circulating human CD7+CD127+CD117+CD45RA+IL1R1+ ILCPs have been identified [33]. Like ILC3s, they express CD117, but do not produce cytokines, although mature ILC genes are in an epigenetically-poised state. In-vitro cloning experiments demonstrated that this population contains multi- and uni-potent cells that can differentiate into mature NK cells, ILC1s, ILC2s and/or ILC3s. In-vivo adoptive transfer experiments using newborn BRGS mice revealed that human ILCPs derive from CD34+ HSPCs and confirmed the precursor-product relationship between ILCPs and mature ILC subsets [33]. These ILCPs were not only present in the circulation, but also in various organs, showing that circulating ILCPs can enter tissues to develop into mature ILC subsets depending on tissue-specific signals. Lim et al. termed this concept of local, on-demand ILC differentiation within tissue “ILC-poiesis” [119]. However, even with these discoveries the picture of human ILC development is far from complete and several unanswered questions remain. For example, human LTi cells have been described [120, 121], but their progenitor is currently unknown. Similarly, despite one study questioning their existence [73], human ILC1s have been described in several reports [29, 35, 74, 122-124], but a specific precursor for ILC1s has not been described, unlike for human ILC2s and ILC3s [125, 126]. Humanized mice could potentially be used to identify further ILC precursors, along with answering other questions in the field of human ILC development. 5.3. Human ILC plasticity

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Analogous to T lymphocytes, ILCs have been found to be highly plastic immune cells, i.e. being able to convert from one subset into another in response to cytokines. ILC plasticity was first demonstrated in vitro and humanized mice have been important in confirming that this plasticity can also occur in vivo. For example, the Spits laboratory demonstrated that IL-12 drives the conversion of human ILC3s into IFN-producing ILC1s and that these ILC1s accumulate in the inflamed colon of NSG mice engrafted with human CD34+ cells [29]. Furthermore, using adoptive transfer into NSG mice, the same authors showed that human ILC1s are able to revert to ILC3s in the absence of inflammation [127]. Similarly, it was found that, upon adoptive transfer into NSG mice, human ILC2s converted into ILC1s expressing the signature transcription factor T-BET [30]. Finally, a recent study demonstrated that MISTRG mice (additionally expressing human IL-6 and IL-15) supported the conversion of human ILC3s into ILC1s [35]. Interestingly, the degree of ILC3-ILC1 plasticity varied between spleen and liver, suggesting that cues from the local tissue microenvironment control ILC plasticity in vivo [35]. In addition, ILC2-ILC3 plasticity has recently been demonstrated in vitro for ILCs isolated from human skin [128]. However, this skin ILC plasticity has not been confirmed in vivo (i.e. using humanized mice), yet. In conclusion, humanized mice have demonstrated ILC plasticity in human inflammatory diseases, specifically ILC3-ILC1 plasticity in the context of inflammatory bowel disease [29, 127] and ILC2-ILC1 plasticity in the context of chronic obstructive pulmonary disease [30]. These findings support the notion that human ILC1s may have proinflammatory function, thereby contributing to disease pathology. 5.4. Human NK cell education Education of NK cells and their function is regulated by interactions between inhibitory NK cell receptors, such as killer-cell immunoglobulin-like receptors (KIRs), with cognate MHC class I ligands. Humanized mice have been instrumental in elucidating this process in human NK cells [42, 43]. For this purpose, one study used NSG mice with transgenic expression of

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human HLA-B*27:05 (a ligand for KIR3DL1), either engrafted with human CD34+ cells or adoptively transferred with human NK cells [42]. This approach demonstrated that inhibitory KIR-cognate MHC class I interactions in cis and as well as in trans are required for educating developing human NK cells [42]. 5.5. Human innate memory Although they are part of the innate immune system, there is now sufficient evidence that macrophages have also features previously exclusively associated with adaptive immunity. Thus, “trained immunity” in macrophages has been shown to result in stronger responses to secondary stimuli, therefore representing a form of innate memory [129]. Similarly, mouse studies demonstrated that adaptive immunity, such as delayed-type hypersensitivity and antiviral responses, can be carried out by hepatic NK cells [130, 131]. A recent study extended this concept of innate memory to human NK cells. Using NSG-BLT mice transplanted with human fetal liver CD34+ cells, the authors showed that human liver-resident (but not splenic) CXCR6hi NK cells are able to mount an antigen-specific memory response after vaccination with human immunodeficiency virus (HIV)-encoded envelope protein [132].

6. Humanized mice and the role of macrophages and ILCs in human disease Humanized mice have been employed as models for human diseases, where the immune system is involved, such as infection and cancer [6, 133, 134]. Here we highlight studies relevant to human macrophages and ILCs. 6.1. Infection 6.1.1. Viral infections Humanized mouse models have been used extensively for studies of pathogens with human tropism [6, 133-137]. Historically, the first humanized mice were generated to have an in-vivo

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model for HIV infection. Accordingly, many studies showed that humanized mouse models are capable of sustaining HIV infection, mostly focusing on human CD4+ T cells [138-141]. Besides CD4+ T cells, monocytes and macrophages are targets of HIV infection. Deng et al. used an improved version of MISTRG mice, where human SIRP is expressed as a knock-in allele instead of as a transgene [13]. Similar to the original MISTRG model [12], these mice supported the development of human monocytes/macrophages and functional human T cells [13]. The authors then created patient-derived humanized mice by engrafting MISTRG mice with bone marrow CD34+ cells obtained from a HIV patient. Upon infection with primary HIV1 isolates grown from resting CD4+ T cells from the same patient, HIV replication was demonstrated not only in T cells but also in human CD14+ monocytes/macrophages from spleen and lung [13]. Another study, using the NSG-BLT model, found that human CD169+ macrophages lining splenic sinuses capture blood-borne cell-free HIV, thereby potentially contributing to viral trans-infection of T lymphocytes [142]. Honeycutt et al. transplanted NOD/SCID mice with human CD34+ cells, which reconstitutes the human myeloid, but not the human T lymphocyte compartment [143, 144]. Using these humanized “myeloid-only” mice, the authors demonstrated HIV persistence in human macrophages in vivo [144]. A recent report, using NSG mice engrafted with human CD34+ cells, examined the early response of monocytes/macrophages to HIV infection [145]. Furthermore, Mathews et al. used NOG mice expressing a human IL-34 transgene to show that microglia can act as a main reservoir for HIV in the brain [25]. An earlier study had reported HIV replication in bone marrow-derived human microglia-like cells in NSG mice engrafted with human CD34+ cells [146]. In contrast, another humanized mouse study concluded that T cells maintain HIV infection in the brain in the absence of human myeloid cells [143]. Finally, BLT mice were used to reveal that human osteoclasts can also serve as a target of HIV infection [147].

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As with CD4+ T cells, depletion of ILCs in peripheral blood, lymphoid tissues, and the gastrointestinal tract has been observed in HIV infection [148-150]. These studies suggested that the loss of ILCs may contribute to intestinal pathology in HIV and that ILC function is not fully restored after anti-HIV treatment. Further investigations using humanized NRG mice identified CD4+ ILC1s as one of the ILC subsets affected by HIV infection in vivo and demonstrated that type I IFN signaling contributes to the depletion of ILC1s [151]. Interestingly, unlike previous reports in humans, combined anti-retroviral therapy was not sufficient to restore ILC1 function in humanized mice. Finally, humanized mice have also been used to investigate intestinal barrier dysfunction in HIV infection. Hofer et al. combined HIV infection in the BRG model with chemically-induced disruption of the intestinal epithelial barrier, thereby causing bacterial translocation [152]. Interestingly, HIV-infected macrophages failed to eliminate translocated bacterial products, such as lipopolysaccharide (LPS), which triggered T cell activation, thereby promoting viral replication and T cell depletion [152]. Other medically-relevant viruses with human tropism are hepatitis virus B and C (HBV and HCV). Modeling of HBV/HCV infection requires the co-engraftment of human hepatocytes to create mice with a combined human liver-human hematopoietic system [153-156]. Studies with these human liver-immune system mice demonstrated infiltration of the virus-infected liver with activated human NK cells as well as with human CD68+ cells resembling Kupffer cells [157]. Other studies, using HLA-A2 transgenic NSG mice, also found liver infiltration by human M2-like macrophages, which could contribute to virus-induced liver fibrosis [158, 159]. Similar observations were made for HCV-infected humanized mice [160]. Epstein-Barr virus (EBV), a very common herpes virus in humans, is another human-tropic virus that has been investigated using humanized mice [161, 162]. One study in NSG mice transplanted with human CD34+ cells showed that human NK cells control lytic (but not latent)

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EBV infection, CD8 T cell expansion, and EBV-associated tumorigenesis [163]. Similarly, human NK cell responses against intravaginal herpes simplex virus infection have been demonstrated in humanized mice [164]. Another study used NOG mice transplanted with human CD34+ cells to examine the role of human macrophages in EBV-induced lymphoproliferative disease (LPD) [165]. The authors found that more aggressive LPD induced by different EBV strains was associated with greater infiltration of the lymphoma by CD68+ and CD163+ macrophages. Moreover, depletion of human CD163+ macrophages abolished lymphoma formation. The authors concluded that exosomes containing EBV-coding micro RNAs act in trans on non-infected macrophages to promote EBV-associated tumorigenesis [165]. Another common herpes virus in humans is cytomegalovirus (CMV). Studies in NSG mice transplanted with human CD34+ cells showed that G-CSF reactivates latent CMV in human monocytes/macrophages [166]. Finally, humanized BLT mice were used to demonstrate that Kaposi’s sarcoma-associated herpes virus replicates in human macrophages [167]. Besides investigating viruses with human tropism, humanized mice have been used to study other important viruses. Willinger et al. demonstrated that human IL-3/GM-CSF knock-in [17] and MISTRG mice (own unpublished observation) engrafted with human CD34+ cells are able to mount an immune response to influenza virus. Specifically, influenza infection induced human type I IFN production in the lung, with human lung macrophages representing a likely source [17]. A subsequent study employing NSG mice, where exogenous M-CSF was provided by hydrodynamic injection, reported similar findings [19]. Finally, Zika virus has been shown to infect human macrophages in BLT-BRG mice [168]. 6.1.2. Bacterial infections Humanized mice can also be used to study bacteria that preferentially infect human hosts. One example represents the human pathogen Salmonella Typhi that causes typhoid fever, but does not normally replicate in mice. Several groups demonstrated that NSG or BRG mice

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transplanted with human CD34+ cells support Salmonella Typhi infection in vivo after intravenous or intraperitoneal administration [169-171]. In one study, Salmonella Typhi infection in humanized mice was accompanied by mononuclear cell infiltration of liver and spleen as well as clinical symptoms and mortality [170]. Although it was not directly investigated, human macrophages are a likely cellular site of Salmonella Typhi replication. Furthermore, Salmonella infection in humanized mice was associated with a type 1 immune response characterized by the production of human cytokines, such as tumor necrosis factor alpha (TNFα) and IL-12 that are produced by macrophages [169]. The report by Song et al. also demonstrated the potential of humanized mice to study human-specific bacterial virulence factors [169]. Other bacteria do not have human-restricted host specificity, but cause characteristic inflammatory responses and immunopathology specific to humans [16]. A prominent example is infection with Mycobacterium tuberculosis (M. tuberculosis), leading to the formation of characteristic granulomas consisting of CD4+ T cells and macrophages. M. tuberculosis infection of mice fails to reproduce the organized granulomas typically found in humans. In contrast, the formation of granuloma-like structures containing human macrophages, T cells, and mycobacteria has been reported in NSG mice engrafted with human CD34+ cells after intravenous Mycobacterium bovis bacillus Calmette-Guérin (BCG) or M. tuberculosis administration [172]. Similar human-like granulomas were reported when using NSG-BLT mice [173]. In contrast, another study, employing HLA-A2 transgenic NSG-BLT mice failed to observe organized human granulomas in response to BCG [174], possibly due to differences in BCG doses used. In the studies by Heuts et al. and Lee et al., humanized mice had increased bacterial loads compared to control mice [172, 174], suggesting a failure to control infection and that human macrophages and/or T cells may instead promote mycobacterial dissemination.

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Finally, Li et al. reported better control of BCG replication when humanized NSG mice received human M-CSF through hydrodynamic injection [19]. Another clinically-relevant pathogen is Staphylococcus aureus (S. aureus), which has been studied in humanized mice. Compared to non-humanized mice, NSG mice engrafted with human CD34+ cells showed enhanced development of S. aureus-induced skin lesions [175]. Humanized NSG mice also had greater S. aureus replication after intraperitoneal administration [176]. Moreover, bacterial burden in the lung was higher in NSG mice engrafted with human CD34+ cells compared to control mice not harboring human cells after S. aureus infection, which was further increased in NSG mice with transgenic expression of human IL-3, GM-CSF, and SCF [177]. The authors also used this model to study the effect of Staphylococcus aureus toxins on human macrophages and on the severity of pneumonia [177]. Increased S. aureus replication in humanized mice has been suggested to result from preferential targeting of the bacteria to human macrophages [6]. Finally, humanized mice have been used to investigate cytokine storm induced by septicemia. In contrast to non-transplanted mice, NSG mice transplanted with human CD34+ cells mounted an inflammatory response in a model of bacterial sepsis [178, 179] and were more susceptible to sepsis [180]. This was characterized by the production of human pro-inflammatory cytokines as well as inflammatory mediators, such as high-mobility group protein 1 (HMGB1). Humanized mice quickly succumbed to this cytokine storm [179]. Furthermore, the authors demonstrated that RNA interference-mediated targeting of HMGB1 in human macrophages protects from cytokine storm [179]. 6.2. Cancer The tumor microenvironment, including immune cells, has an essential role in cancer outcome [181] and immunotherapy represents the most promising new cancer treatment [182]. In this respect, human immune system mice transplanted with tumor cell lines or patient-derived tumor

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xenografts, ideally as an autologous cancer-immune system model, have served as useful preclinical models [183-185]. 6.2.1. Human macrophages in cancer immunity Besides T lymphocytes, monocytes and macrophages are an abundant cellular component of the immune environment in cancer and have emerged as new targets for cancer immunotherapy [186, 187]. Recent single-cell RNA-seq studies revealed the large heterogeneity of human macrophages in the tumor microenvironment, e.g. in lung cancer [80, 188, 189]. In general, tumor-associated macrophages (TAMs) are associated with a poor prognosis in cancer due to their tumor-promoting features, such as suppression of anti-tumor T cell responses, stimulation of angiogenesis, and facilitation of cancer metastasis [186]. In addition, monocytic myeloidderived suppressor cells (MDSCs) are often present in the tumor microenvironment [54] and MDSCs have been described in humanized mouse models [190]. The tumor-promoting function of human TAMs has been demonstrated in humanized mouse models. For example, tumorinfiltrating CD206+CD163+HLA-DRlow TAMs were shown to enhance growth of human melanoma in MISTRG mice engrafted with human CD34+ cells through the production of vascular endothelial growth factor [12]. Studies in humanized mice have revealed other mechanisms by which TAMs inhibit anti-tumor responses. Using NSG mice transplanted with human CD34+ cells, Liu et al. found that TAMs enriched in hypoxic tumor areas inhibit NKT cell activity against neuroblastoma, which could be overcome by the provision of human IL-15 [191]. Finally, Hanazawa et al. reported an increased number of CD163+ human TAMs in tumor-bearing NOG mice transgenic for human IL-6 [192]. In the future, humanized mice could be used to better understand the functional diversity of human TAMs and to examine the impact of human TAM origin on cancer progression. On the other hand, macrophages can also contribute to anti-tumor immunity through the phagocytosis of tumor cells. As mentioned above, a major receptor system controlling

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macrophage phagocytosis consists of SIRP, expressed on macrophages, interacting with CD47 on target cells. SIRP engagement by CD47 leads to phagocytic tolerance, which is exploited by tumors since they often upregulate CD47 expression, thereby evading phagocytosis by macrophages. Therefore, efforts have been made to re-sensitize macrophage phagocytosis in order to clear cancer cells, e.g. through the use of antibodies blocking SIRP on macrophages. However, the effects of human anti SIRP antibodies are difficult to evaluate in mice as these antibodies do not bind to mouse SIRP. To overcome this limitation, mice expressing human SIRP have been used. Using this approach, it has been shown that antibodymediated SIRP blockade stimulates potent anti-tumor activity of human macrophages against lymphoma xenografts in human SIRP knock-in mice [193]. 6.2.2. Human ILCs in cancer immunity Due to their cytolytic activity, tumor-reactive NK cells represent a target for next-generation cancer immunotherapy [184, 194]. Humanized mice have demonstrated the potent anti-tumor activity of NK cells, either after transplantation with human CD34+ cells that develop into human NK cells or by the adoptive transfer of human NK cells. However, often NK cells are underrepresented in the tumor microenvironment, highly cytotoxic CD56dim NK cells in particular [188, 194, 195], suggesting impaired recruitment to the tumor. Nevertheless, studies in humanized mice have shown that NK cells derived from transplanted human CD34+ cells are able to exert anti-tumor activity against different types of tumor xenografts [196-198]. In contrast, studies in mice suggest a more complex role of ILCs other than NK cells in cancer, with both pro- and anti-tumor activities having been reported [194]. This might be due to the pleiotropic roles of ILC1s, ILC2s, and ILC3s in tissue remodeling, tertiary lymphoid tissue formation, and inflammation, as well as due to diverse tumor microenvironments. Although present in the tumor microenvironment, human ILC function in cancer has not yet been examined in humanized mouse models, despite the observation that ILC subsets are often 25

altered in cancer patients [194]. An interesting area to be studied with humanized mouse models represents human ILC plasticity in cancer. For example, Koh at al. reported recently that IL-23 produced by pulmonary squamous cell carcinoma triggers the conversion of ILC1s into IL-17secreting ILC3s, which was associated with poor cancer outcome [199]. 6.2.3. Humanized mice as pre-clinical platform for cancer immunotherapy As mentioned above, humanized mice are useful as pre-clinical models for testing new cancer treatments and to predict the patient’s response to specific immunotherapies [185]. Diverse approaches based on NK cell-based immunotherapy have been shown to have great promise [194, 200], especially for the treatment of hematological malignancies, where allo-reative NK cells exert potent graft-versus-leukemia effects [184]. Human NK cells for cancer immunotherapy can be obtained from blood or, alternatively, can be derived from embryonic stem cells (ESCs) and iPSCs. Adoptively transferred fresh or ex vivo-expanded human NK cells are able to reject transplanted tumor cell lines as well as primary human tumors in humanized mice. This applies not only to hematological malignancies, but also to solid tumors [201-204]. Moreover, even in the absence of a HLAinhibitory KIR mismatch, allo-reactive NK cells are able to exert potent anti-tumor activity through activating KIRs. This has been demonstrated for KIR2DS2+ NK cells against glioblastoma in humanized mice [205]. Importantly, humanized mice demonstrated that adoptively transferred NK cells are capable of targeting cancer stem cells in NSG mice [206]. Similar to T cells, NK cells expressing chimeric antigen receptors (CARs) against tumor antigens have been developed. For example, NSG mice xeno-grafted with non-Hodgkin’s lymphoma have been treated with anti-CD20 CAR NK cells [207]. Co-administration of IL-2 and/or IL-15 was found to enhance the anti-tumor activity of NK cells in humanized mice [196, 208]. Another approach is based on targeting activating NK cell receptors. For example, antiNKG2A antibody treatment enhanced the killing of leukemic cells by human NK cells in NSG

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mice [209]. Moreover, NK also participate in the elimination of antibody-targeted tumor cells. Accordingly, antibody-dependent NK cell cytoxicity against CCR4+ lymphoma was demonstrated in human IL-2 transgenic NOG mice after treatment with a humanized anti-CCR4 antibody [40]. To closely mimic the anti-tumor response of patients, autologous human tumorimmune systems have been developed. For this purpose, Fu and colleagues engrafted the primary tumor and CD34+ cells from the same patient into immunodeficient mice [210]. This kind of experimental system will allow personalized screening of immunotherapies in vivo. Finally, humanized mice are also useful for investigating mechanisms underlying immunotherapy-associated toxicity. For example, using the NSG model expressing SCF, GMCSF, and IL-3, it was recently shown that monocyte-produced IL-1 and IL-6 underlie cytokinerelease syndrome and neurotoxicity associated with CAR T cell immunotherapy [211].

7. New technologies to study human macrophages and ILCs in humanized mice Humanized mice can be combined with powerful new technologies, such as such as single-cell RNA-seq and gene editing using CRISPR/Cas9, to investigate human immune function (Fig. 3). Single-cell RNA-seq in particular has been already invaluable for dissecting the cellular heterogeneity of the immune system [212, 213], which has led to the discovery of new immune cell populations. Moreover, this technology has the ability to shed light on the origin of specific immune cells as well as defining developmental paths of immune cells through trajectory analysis [212, 213]. We have recently used this technology in humanized mice to dissect the developmental path of human lung macrophages from their precursors (unpublished). Furthermore, CRISPR/Cas9 has emerged as an exceptionally powerful tool for genome engineering [214, 215]. This technology has already been successfully used for gene editing in human CD34+ cells, which allows studying the function of human immune genes in vivo. Examples of successfully targeted human genes include the genes encoding IL-2R, CCR5, and

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-globin [216-218]. In addition, it is possible to track successfully CRISPR-edited cells in humanized mice by flow cytometry based on fluorescent reporter expression [219]. Another study generated human CD34+ cells lacking CD33 that were transplanted into NSG mice, which demonstrated that CD33 is dispensable for the development and function of human myeloid cells [220]. Future studies could extend this approach to genes with potential roles in human macrophages and ILCs. Other interesting future applications of CRISPR/Cas9-edited human CD34+ cells in humanized mice are performing genome-wide screens as well as introducing immune gene

polymorphisms and disease risk alleles (that likely affect

macrophages and ILCs) to determine their significance in vivo.

8. Future improvements of humanized mouse models Humanized mouse models are constantly being further refined to more closely mimic human physiology, which should open up new applications (Fig. 3). 8.1. Further optimization of human hematopoiesis in humanized mice CRISPR/Cas9 genomic engineering of the mouse host allows the rapid provision of additional human factors to improve human immunity in humanized mice. This has been achieved in NSG mice, where several factors have been introduced by CRISPR/Cas9 [221]. Therefore, further factors relevant for human macrophages and ILCs could be provided in this way. Limitations of current humanized mouse models are mostly related to adaptive immunity. This includes suboptimal thymopoiesis, insufficient proper selection of T and B lymphocytes, lack of mature B cell differentiation, and poor development of human lymphoid structures. Future improvements in these areas are necessary to be able to study the interaction of macrophages and ILCs with adaptive immune cells. Recent developments in this context are the generation of BRG SIRPNOD mice expressing human thymic stromal lymphopoietin, which supports the development of lymph nodes [222]. Since lymphoid tissues are communication centers of the 28

immune system, this new humanized mouse model will allow studies of T and B lymphocyte interaction with lymph node-resident human macrophages and ILCs. 8.2. Tissue engineering to humanize non-hematopoietic mouse tissue Tissue-resident immunity relies on interaction of immune cells with surrounding nonhematopoietic cells. In current humanized mouse models, human hematopoietic cells interact with mouse host cells such as epithelial or stromal cells. This represents a challenge because factors either produced by human immune cells (acting on non-hematopoietic cells) or produced by host non-hematopoietic cells (acting on human hematopoietic cells) may not be crossreactive between mice and humans. In its simplest form, humanization of mouse tissue can be achieved by the co-transplantation of human non-hematopoietic tissue. Examples are cotransplantation of a fetal thymus in BLT mice (to boost human thymopoiesis), human hepatocytes to create human chimeric liver (to study human hepato-tropic viruses), and human mesenchymal stem cells (to create human stroma). An alternative approach with great potential represents the transplantation of human organoids to form intestinal, lung, or liver tissue in humanized mice. Organoids are three dimensional “mini-organs” that are derived from tissue stem cells in vitro [223, 224]. As a proof-of-concept, the transplantation of human intestinal organoids generated from ESCs or iPSCs into NSG mice resulted in the considerable expansion and maturation of different intestinal cell lineages, such as enterocytes, goblet cells, Paneth cells and tuft cells as well as enteric neurons [225, 226]. As for the lung, pseudostratified human airway organoids formed by basal cells, functional multiciliated cells, mucus-producing secretory cells, and CCL10-secreting club cells have been generated from lung tissue of healthy donors or cancer patients and used in xenotransplantation experiments in immunocompromised mice [227]. Human organoid transplantation into immunodeficient mice could be combined with the transplantation of human CD34+ cells to

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create humanized mice with a human intestinal or lung immune system. Thereby, human tissueresident immunity in non-lymphoid organs could be studied. 8.3. Mouse model with human immune system and human microbiota Another interesting area is to investigate how the microbiota regulates human immune function. The human microbiota is composed of a vast number of microorganisms that colonize the human body and regulate many physiological processes, including immune function [228]. Alterations in microbiota composition (dysbiosis) have been associated with many inflammatory diseases in humans. However, it has been difficult to determine whether dysbiosis drives pro-inflammatory immune activity or whether dysbosis is secondary to inflammation. So far, only the effect of the mouse microbiota on the human immune system has been examined in humanized mice. Using a quadruple antibiotic cocktail, Gulden et al. depleted the mouse microbiota in NSG mice engrafted with human CD34+ cells. This resulted in an increase in the frequency of human effector T cells in the intestine and consequently in the amount of circulating human IFNγ [229]. Since the microbiota differs between mice and humans and the microbiota has species-specific effects on the immune system [230], it is important to develop humanized mouse models that have a human immune system together with a matching human microbiota. Introduction of a human microbiota requires depletion of the mouse microbiota via antibiotic treatment, or better elimination of the endogenous mouse microbiota by the germfree derivation of immunodeficient mice, which are then transplantated with human CD34+ cells. Such models could be great tools to establish causality between dysbosis, immunemediated inflammatory responses, and human disease.

Concluding remarks New knowledge and concepts related to macrophages and ILCs are constantly being developed. In turn, humanized mouse models allow testing of these concepts in vivo. Undoubtedly, studies

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in humanized mice will contribute to the several important areas in the field, such as human macrophage/ILC origin and how it effects their function, defining precursors and developmental stages, examining macrophage/ILC homeostasis, tracking monocyte/ILC migration in steady-state and inflammation, as well as dissecting the role of macrophage/ILC subsets in disease (including cancer).

Acknowledgements Research in the Willinger laboratory is supported by a faculty-funded career position at Karolinska Institutet, as well as grants from the Swedish Research Council and the Center for Innovative Medicine (CIMED) financed by Region Stockholm.

Declarations of interest: none.

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Figure legends Fig. 1. Humanized mouse models to study macrophages and ILCs. After transplantation into immunodeficient mice, human hematopoietic stem and progenitor cells (HSPCs) develop into different human immune cell lineages. Provision of human cytokines in humanized mice, such as M-CSF, GM-CSF, IL-34, and IL-15, supports the development of human macrophages and ILCs. Numbers in brackets correspond to references. CILPs, common innate lymphoid progenitors; ILCPs, innate lymphoid cell precursors; NKPs, NK cell precursors.

Fig. 2. Development and differentiation of macrophages and ILCs. Macrophages and ILCs arise from precursors in fetal liver and adult bone marrow. Circulating precursors migrate to various organs, where they differentiate into mature macrophages and ILCs in response to tissuespecific local signals. The tissue microenvironment also shapes the heterogeneity and plasticity of macrophages and ILCs, thereby adapting their function to environmental challenges, such as infection and tissue injury.

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Fig. 3. Future application of humanized mice to study human macrophages and ILCs. The combined use of humanized mice together with powerful novel technologies, such as singlecell RNA-sequencing and CRISPR/Cas9 genome editing, will pave the way for new discoveries related to human macrophage and ILC function. Further humanization of the mouse host through the transplantation of human organoids and the human microbiota will deepen our understanding of human macrophages and ILCs. Parts of the figure have been adapted from Servier Medical Art.

58

59

Table 1 Overview of various humanized mouse models for studying human macrophages and ILCs. Factors

Human cell type

Applications

Reference

BALB/c Rag2-/- Il2rg-/- (BRG)-based mice Overall human SirpaNOD

engraftment (T cells, NK cells)

Tumor xenograft & cancer immunotherapy studies; viral infections e.g. EBV, influenza, HIV

SirpaNOD & human

Dendritic cells,

Human dendritic cell and NK

FLT3L

NK cells, ILCs

cell/ILC development & function

Overall human Human SIRPA

engraftment (T cells, NK cells)

14, 15

Tumor xenograft &cancer immunotherapy studies

60

31

11

NK cells, ILCs,

Human NK cell development &

Human SIRPA &

CD8 T cells,

function; NK & CD8 T cell-based

IL-15

intraepithelial T

cancer immunotherapy; tumor

cells

xenograft studies

Human IL-7 Human M-CSF

T cells

Human T cell development & function

Monocytes,

Human monocyte/macrophage

macrophages

development & function

Human IL-3 & GM-

Lung

CSF

macrophages

39

26, 27 18, 19, 108

Human lung macrophage development & function; viral

17

infections such as influenza

Overall high human Human SIRPA, MCSF, GM-CSF, IL-3 & TPO

engraftment; NK cells, ILCs, monocytes, macrophages (lung, liver,

Human monocyte/macrophage development & function; human NK cell/ILC development & function; innate immune response

12, 13, 35, 109-111

to human pathogens and tumors

colon, brain) NOD/SCID Il2rg-/- (NSG)-based mice Human bone marrow/liver/thymus (BLT) BLT & human IL-2 Human IL-7 & IL15 Human IL-3, GMCSF & SCF

T cells, B cells,

T cell responses to human

myeloid cells

pathogens, e.g. HIV

NK cells, regulatory T cells NK cells Monocytes, macrophages, neutrophils

7-9

Human NK cell/regulatory T cell development & function, cancer

41

immunotherapies Human NK cell development & function Human myeloid cell development & function

NOD/Shi-SCID Il2rg-/- (NOG)-based mice

61

28

20-22, 24

Development of microglia; Human IL-34

Microglia

infection-induced neuropathogenesis, e.g. HIV-1 induced

25

brain pathology Human IL-3 & GMCSF

Granulocytes (basophils), mast cells

Human basophil/mast cell development & function, allergy

23

Human NK cell development & Human IL-2

NK cells

function; NK cell-based cancer

40

immunotherapy

Table 2 Human macrophage-/ILC-specific findings in humanized mice. Mouse model

Humanized mouse model

Macrophages Differentiation of Ly6Chi into Ly6Clo blood

Differentiation of CD14+CD16lo into CD14loCD16+ blood

monocytes

monocytes via intermediate CD14+CD16+monocytes

Lung macrophages develop from fetal liver

Human lung macrophages can originate from HSPCs of diffe

monocytes

developmental age (fetal, neonatal, adult)

No organized granulomas when infected

Human-like granulomas, containing macrophages, after infec

with M. tuberculosis or BCG

with M. tuberculosis or BCG

Less replication of S. aureus

Preferential targeting of human macrophages by S. aureus

Less susceptible to cytokine storm & sepsis

Cytokine storm mediated by human macrophages leading to s

ILCs Common helper innate lymphoid progenitor (CHILP) gives rise to helper ILCs only

Multipotent ILCPs give rise to both NK cells and helper ILCs

RORt mainly expressed by ILC3s

Generalized RORt expression during ILC development

Tissue-specific ILC compartmentalization

Tissue ILCs less compartmentalized

62

ILCs mostly tissue-resident in steady-state

ILCPs found in blood and tissues

63