REVIEW
published: 22 February 2021
doi: 10.3389/fimmu.2021.643852
Frontiers in Immunology | www.frontiersin.org 1 February 2021 | Volume 12 | Article 643852
Edited by:
Tim Willinger,
Karolinska Institutet, Sweden
Reviewed by:
Christian Muenz,
University of Zurich, Switzerland
Hergen Spits,
University of Amsterdam, Netherlands
*Correspondence:
Anthony Rongvaux
†
These authors share first authorship
Specialty section:
This article was submitted to
Cancer Immunity and Immunotherapy,
a section of the journal
Frontiers in Immunology
Received: 18 December 2020
Accepted: 27 January 2021
Published: 22 February 2021
Citation:
Martinov T, McKenna KM, Tan WH,
Collins EJ, Kehret AR, Linton JD,
Olsen TM, Shobaki N and Rongvaux A
(2021) Building the Next Generation of
Humanized Hemato-Lymphoid
System Mice.
Front. Immunol. 12:643852.
doi: 10.3389/fimmu.2021.643852
Building the Next Generation of
Humanized Hemato-Lymphoid
System Mice
Tijana Martinov
1†
, Kelly M. McKenna
1,2,3†
, Wei Hong Tan
1†
, Emily J. Collins
1
,
Allie R. Kehret
1
, Jonathan D. Linton
1
, Tayla M. Olsen
1
, Nour Shobaki
1
and
Anthony Rongvaux
1,4
*
1
Clinical Research Division, Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA, United States,
2
Graduate Program in Molecular and Cellular Biology, University of Washington, Seattle, WA, United States,
3
Medical
Scientist Training Program, University of Washington, Seattle, WA, United States,
4
Department of Immunology, University of
Washington, Seattle, WA, United States
Since the late 1980s, mice have been repopulated with human hematopoietic cells to
study the fundamental biology of human hematopoiesis and immunity, as well as a broad
range of human diseases in vivo. Multiple mouse recipient strains have been developed
and protocols optimized to efficiently generate these “humanized” mice. Here, we review
three guiding principles that have been applied to the development of the currently
available models: (1) establishing tolerance of the mouse host for the human graft; (2)
opening hematopoietic niches so that they can be occupied by human cells; and (3)
providing necessary support for human hematopoiesis. We then discuss four remaining
challenges: (1) human hematopoietic lineages that poorly develop in mice; (2) limited
antigen-specific adaptive immunity; (3) absent tolerance of the human immune system
for its mouse host; and (4) sub-functional interactions between human immune effectors
and target mouse tissues. While major advances are still needed, the current models can
already be used to answer specific, clinically-relevant questions and hopefully inform the
development of new, life-saving therapies.
Keywords: humanized mice, immunity, hematopoiesis, infectious diseases, cancer, immunotherapies
INTRODUCTION
Biomedical research aims to provide a platform for the development and testing of new therapies
that can reduce human suffering and deaths. In vitro studies using human cells or organoids are
useful, but animal models can better elucidate th e fundamental principles of complex biological
processes in mammals. In particular, laboratory mice are often the model organism of choice,
as their small size and short generation time enable extensive genetic engineering and invasive
experimentation. Many fundamental characteristics of hematopoietic and immune systems are
shared across mice and humans. However, with 91 million years of divergent evolution, differences
exist and results from murine studies cannot always be directly translated into clinic a l appli cations,
driving the development of experimental murine platforms that faithfully model human physiology
and diseases. Specifically, mice can be transplanted with a human hemato-lymphoid system (1).
Such “humanized mice” (Box 1) have been increasingly used since the late 1980s, and have
Martinov et al. Building Next Generation Humanized Mice
BOX 1 | What is a humanized mouse?
The Wikipedia definition of a humanized mouse is “a mouse carrying
functioning human genes, cells, tissues, and/or organs.” This review focuses
on mice transplanted with human hematopoietic cells, colloquially referred
to as “humanized mice” (
6, 7). When used to study immune responses,
such mice are better designated as “human(ized) immune system” (HIS)
mice (
5, 8, 9). However, not all hematopoietic cells contribute to the immune
response, and “human(ized) hemato-lymphoid system” (HHLS) mice is a
more encompassing term, particularly when human CD34
+
HSPCs are
transplanted (
1, 10, 11).
Many models combine multiple characteristics listed in the definition of
humanized mice. In addition to human hematopoietic cell transplantation,
diverse human tissues or tumors can be co-transplanted and the genome
of the recipient mouse can be engineered to contain human genes.
contributed to major breakthroughs in several research fields,
including human hematopoiesis (2), hematologic malignancies
(3), and immunity to human-tropic pathogens (4, 5). Successful
generation of humanized mice requires: (i) a source of human
donor hematopoietic cells, (ii) an effective transplantation
protocol, and (iii) an appropriate recipient mouse strain.
Human peripheral blood mononuclear cells (PBMCs) can
be used as a source of hematopoietic cells for transplantation,
but their engraftment favors T cell maintenance, resulting in
an incomplete human immune system (
7, 12). In contrast,
hematopoietic stem and progenitor c ells (HSPCs), enriched in
the CD34
+
cell fraction, give rise to all human hematopoietic
lineages upon transplantation in mice (7, 13). Human CD34
+
HSPCs can be obtained from different sources. Fetal CD34
+
cells, abundant in the liver, very efficiently engraft and undergo
multilineage differentiation upon transplantation in mice, but
ethical concerns limit access to fetal tissues (
14). Other sources
of CD34
+
cells, such as cord blood, bone marrow (BM) or
peripheral blood following granulocyte colony-stimulating factor
(G-CSF) mobilization, are more accessible with fewer ethical
concerns. Howe ver, their stemness declines with the age of the
donor (15, 16), resulting in lower engraftment potential (17, 18).
Of note, donor cells can be obtained from patients affected by
diverse hematopoietic diseases, thereby providing small animal
models of human diseases including genetic immune disorders
or hematologica l malignancies (3, 19).
Abbreviations: AM, Alveolar macrophages; BLT, bone marrow, liver, thymus; BM,
Bone marrow; BRG, BALB/c Rag2
−/−
Il2g
−/−
; BRGF, BALB/c Rag2
−/−
Il2g
−/−
Flt3
−/−
; DCs, Dendritic cells; EBV, Epstein–Barr virus; EPO, Erythropoietin; G -
CSF, Granulocyte colony-stimulating factor; GM-CSF, Granulocyte-macrophage
colony-stimulating factor; GvHD, Graft-vs.-host disease; HCMV, Human
Cytomegalovirus; HIV-1, human immunodeficiency virus 1; HLA, Human
leukocyte antigen; HSPCs, Hematopoietic stem and progenitor cells; IL,
Interleukin; MSCs, Mesenchymal stromal cells; MHC-I, Major histocompatibility
complex class I; MHC-II, Major histocompatibility complex class II; MISTRG, M-
CSF
h/h
IL-3/GM-CSF
h/h
SIRPα
h/m
THPO
h/h
RAG2
−/−
IL2Rγ
−/−
; MISTRG6,
M-CSF
h/h
IL-3/GM-CSF
h/h
SIRPα
h/m
THPO
h/h
RAG2
−/−
IL2Rγ
−/−
IL6
h/h
;
NK, Natural killer; NOD, Non-obese diabetic; NSG, NOD/SCID/Il2rg
−/−
;
NSGW41, NOD/SCID/Il2rg
−/−
Kit
W41/W41
; PBMCs, peripheral blood
mononuclear cells; PDX, Patient derived xenograft; RBCs, Red blood cells;
SCF, Stem cell factor; SCID, severe-combined immunodeficient; TCR, T cell
receptor; xGvHD, xenogeneic graft-vs.-host disease.
Human hematopoietic cells can be transplanted by systemic
intravenous delivery or by orthotopic injection into a site of
primary hematopoiesis. In adult mice, hematopoietic cells can
be implanted in the BM niche by intrafemoral injection (
20).
Intrahepatic injection has also become a common route of
CD34
+
cell implantation in newborn mice; the live r is a site of
primary hematopoiesis during embryonic life and continues to
be for several days after birth, until the h ematopoietic niche is
established in the BM and the mouse host naturally supports the
expansion and multilineage differentiation of the hematopoietic
system (
21) (Figure 1).
The array of recipient mice for hematopoietic humanization
has expanded over the past few decades, with advances in mouse
genome engineering. This review focuses on these developments,
highlighting the genetic engineering of the host, as well as
the co-transplantation of human tissues to support human
hematopoiesis. We first discuss three guiding principles that
have been employed for the development of the currently
available recipient mice (Figure 2): (i) preventing rejection of
the human graft by the mouse immune system, (ii) opening
the niche to make it accessible to human hematopoietic cells,
and (iii) supporting human hematopoiesis in the mouse. We
then consider how these principles are being applied to the
development of newer mouse strains, aiming to resolve four
remaining major challenges: (i) the development and function
of missing human hematopoietic lineages, (ii) efficient and
durable antigen-specific adaptive immunity, (iii) tolerance of
the engrafted human immune system for the mouse host and
(iv) functional cross-reactivity between the human graft and
target tissues.
PRINCIPLE #1: PREVENTING REJECTION
OF THE HUMAN GRAFT
The field of humanized mice was launched in the late
1980s, a few years after the discovery of mice with severe
combined immunodeficiency (SCID). Prkdc
scid
(protein
kinase, DNA activated, catalytic polypeptide; severe combined
immunodeficiency) is a spontaneous mutation identified in a
colony of C.B-17 mice (
22). The functional inactivation of the
PRKDC enzyme in SCID mice leads to defective DNA repair
and repair-dependent somatic V(D)J recombination of B and
T cell re ceptor-encoding genes (23). As a result, lymphocyte
development is arrested at an early stage and mature B and
T lymphocytes are absent in SCID mice. Taking advantage of
the severe immunodeficiency of these animals, several groups
successfully transplanted human PBMCs (12), human BM cells
(24), human fetal tissues (25) or human HSPCs (26) in SCID
(12, 25, 26) or equivalent recipient mice (24). Coinciding with
the ea rly years of the HIV-1/AIDS epidemic, these pioneering
models provided a much-needed tool for in vivo studies of
HIV-1 infection (27–29). Recipient mouse strains have since
undergone numerous iterative improvements, and this historical
perspective has been comprehensively reviewed previously
[e.g., (
6)]. The most notable modifications include further
preventing rejection of the human graft, through the elimination
Frontiers in Immunology | www.frontiersin.org 2 February 2021 | Volume 12 | Article 643852
Martinov et al. Building Next Generation Humanized Mice
FIGURE 1 | Protocols commonly used for the generation of humanized mice.
FIGURE 2 | Fundamental principles of mouse humanization and remaining
challenges.
of endogenous natural killer (NK) cells and the induction of
phagocytic tolerance, as discussed below.
NK cells are lymphoid cells that eliminate cells lacking major
histocompatibility complex class I (MHC-I) molecules (
30).
Engrafted human cells express human MHC-I molecules that
are not recognized by mouse NK cells. Therefore, depletion of
mouse NK cells is essential, to prevent them from recognizing
and eliminating the graft as “missing self.” The interleukin-2
receptor γ chain (IL-2Rγ, encoded by Il2rg) is shared by multiple
cytokines of the IL-2 family, including IL-15 that is essential
for NK cell development (31). Consequently, Il2rg deficiency
eliminates host NK cells and improves human hematopoietic cell
engraftment in immunodeficient recipient mice (21, 32–35).
Transplanted human cells are also rejected through
phagocytosis by mouse cells, such as monocytes and
macrophages. Because phagocytes are essential for normal
development and physiology, they cannot be easily depleted
genetically without affecting mouse health and survival (36, 37).
An alternative strategy is to alter their functional properties,
by inducing phagocytic tolerance through the signal regulatory
protein alpha (SIRPα) and CD47 axis (
38). The polymorphic
Sirpa gene in the non-obese diabetic (NOD) mouse strain
encodes a variant of the SIRPα receptor that cross-reacts with
the human CD47 ligand. As a result, human cells transplanted in
NOD mice can engage t he CD47/SIRPα “don’t eat me” signal and
are protected from phagocytosis by mouse macrophages (39).
Consequently, b ackcrossing the scid mutation, and later the Il2rg
deficiency, onto the NOD background significantly increased
the efficiency of human cell transplantation (33–35, 40, 41). The
resulting strains, NOD SCID Il2rg
−/−
(NOG and NSG), became
very popular as they combine T, B and NK cell deficiencies with
SIRPα-mediated phagocytic tolerance (
33–35), but multiple
other strains are functionally equivalent. These different strains
abrogate V(D)J recombination [Prkdc
scid
mutation (12, 25), or
deletion of recombination activating gene (RAG)-1 or RAG-2
Frontiers in Immunology | www.frontiersin.org 3 February 2021 | Volume 12 | Article 643852
Martinov et al. Building Next Generation Humanized Mice
TABLE 1 | List of immunodeficient mice used as recipients for transplantation of human hemato-lymphoid system.
Acronym Genetic T and B cell NK cell Phagocytic References
background deficiency deficiency tolerance
SCID C.B-17 Prkdc
scid
- - (12, 25, 26)
NOD-SCID NOD Prkdc
scid
- Sirpa
NOD
(
40, 41)
BRG Balb/c Rag2
−/−
Il2rg
−/−
- (
21, 32)
NOG NOD Prkdc
scid
Il2rg
−/−
(truncation) Sirpa
NOD
(
35)
NSG NOD Prkd c
scid
Il2rg
−/−
Sirpa
NOD
(
33, 34)
NRG NOD Rag1
−/−
Il2rg
−/−
Sirpa
NOD
(
42)
BRGS
NOD
Balb/c Rag2
−/−
Il2rg
−/−
Sirpa
NOD
(
43)
S
tg
RG Balb/c x 129 Rag2
−/−
Il2rg
−/−
Human SIRPA (BAC tg) (
45)
S
KI
RG Balb/c x 129 Rag2
−/−
Il2rg
−/−
Human SIRPA (KI extracellular domain) (
46)
B6RGS
NOD
C57BL/6 Rag2
−/−
Il2rg
−/−
Sirpa
NOD
(
44)
B6RGS
Human
C57BL/6 Rag2
−/−
Il2rg
−/−
Human SIRPA (KI full-length) (
47)
B6RG-CD47 C57BL/6 Rag2
−/−
Il2rg
−/−
Cd47
−/−
(
48)
SCID, NOD-SCID and BRG are historical models, that provided the basis for most currently used recipient mice. All other mice listed in this table have conceptually similar properties,
with a few exceptions, as discussed in the text.
(
21, 32, 42)] and IL-2Rγ [Il2rg gene deletion or truncation
(21, 32–35)]. SIRPα/CD47-dependent cross-species tolerance
can be achieved by expressing the mouse Sirpa
NOD
variant
(
43, 44) or human SIRPA (45–47), or by employing a Cd47
deficiency that produces tolerance by an unknown mechanism
(48, 49). These strains are on diverse genetic backgrounds (NOD,
BALB/c, C57BL/6), and are known by distinct acronyms, listed
in Table 1. Upon transplantation of human CD34
+
HSPCs,
all of these strains support the differentiation of high levels
of human CD45
+
cells, reaching about 80% engraftment in
the BM and 50% in the periphery. However, immune cell
differentiation is disproportionately skewed toward the B and
T lymphoid lineages (33, 35, 45). Human myelo-monocytic
and NK cells are present only at low frequencies (50, 51),
and human circulating red blood cells and platelets are barely
detectable (
52, 53). Investigators should carefully select the
background strain they use, based on their research question,
as the specific strain can impact the outcome of experiments.
For example, SCID mice are highly susceptible to DNA damage;
therefore RAG-deficient mice are the preferred recipient strain
when testing chemo- and radiotherapies (42, 54). For studies
involving complement-dependent cytotoxicity, the C57BL/6 or
BALB/c backgrounds should be preferred, since NOD mice lack
hemolytic complement C5 (41, 44).
PRINCIPLE #2: OPENING THE NICHE
Hematopoiesis is a complex and tightly regulated process
during which hematopoietic progenitors undergo expansion
and multilineage differentia tion (
2, 13). This process occurs
primarily in the BM that uniquely provides supporting factors,
such as cytokines at local physiological concentrations, and
also provides a distinct microenvironment for developing cells
(
55). Accessibility of t he transplanted human CD34
+
HSPCs
to this niche is required for efficient engraftment in the mouse
host. Reducing cellularity in the mouse BM creates the needed
physical space, described as “opening the niche.” Traditional
protocols rely on irradiation as a preconditioning regimen (
56–
60), typically achieved using sub-lethal X-ray or
137
Cs irradiation
to kill most hematopoietic cells while limiting toxicity. When no
irradiator is available, alternative pre-conditioning protocols can
be used, such as the myeloablative drug busulfan (61–63).
Recently, the requirement for radiation preconditioning has
been alleviated with recipient mice engineered to have a less
populated BM niche, thereby achieving a form of “genetic
preconditioning.” An example is provided by the Kit
W41
mutation, in a n NSG-derived mouse strain known as NSGW41
(
64) (Table 2). The receptor tyrosine kinase Kit (also known
as c-Kit or CD117, encoded by the Kit gene) is the receptor
for the cytokine stem cell factor (or SCF, also known as steel
factor or Kit ligand). SCF increases HSPC retention in the
BM niche by increasing their adhesion to neighboring stromal
cells and proteins in the extracellular matrix (
75). The Kit
W41
allelic variant encodes a protein with partially impaired kinase
activity (
76–78), producing functionally defective hematopoietic
stem cells in Kit
W41/W41
mutant mice. In addition, because SCF
is conserved between species [over 82% amino acid identity
between mouse and human (1)], mouse SCF cross-reacts on
the corresponding human Kit receptor. Consequently, when
transplanted into NSGW41 mice, human HSPCs find a partially
open BM niche and effectively compete for SCF against the
mouse HSPCs that express the impaired Kit receptor. The
impact of the Kit
W41/W41
mutation on mouse humanization
is threefold: h uman CD34
+
HSPCs efficiently engraft without
radiation preconditioning; even without radiation, engraftment
levels in NSGW41 are higher than in irradiated NSG recipients;
and better maintenance of functional human HSPCs favors
their multilineage differentiation, including into the eryt hro-
megakaryocytic lineage (64, 79).
Other genes can be inactivated to open the BM niche.
Thpo encodes the cytokine, thrombopoietin, which is essential
Frontiers in Immunology | www.frontiersin.org 4 February 2021 | Volume 12 | Article 643852
Martinov et al. Building Next Generation Humanized Mice
TABLE 2 | List of genetically engineered mice, in the order discussed in this review.
Strain Preventing graft
rejection
Opening the niche Providing support Impact References
NSGW41
Kit
W41/W41
NSG background Mouse HSPC
deficiency
- Genetic pre-conditioning (high
engraftment without irradiation)
(64)
BRG-THPO
Thpo
h/h
BRG background Mouse HSPC
deficiency
Human HSPC support by human
THPO
- Increased BM engraftment
- Thrombocytopenia
(65)
BRGF
Flt3
−/−
BRG background Mouse DC deficiency -
Only human DCs, increased by
human Flt-3 ligand treatment
(66)
BRG-IL3/GM-
CSF
Il3
h/h
Csf2
h/h
BRG background Mouse alveolar
macrophage deficiency
Human alveolar macrophage
support by human GM-CSF
-
Alveolar macrophage replacement (67)
NSG-SGM3
Human SCF,
CSF2, IL3
transgenes
NSG background Transgenic overexpression of
cytokines supporting
myelopoiesis
-
Increased myelopoiesis
- Functional mast cells
- Immature myeloid cells
- Exhaustion of HSPCs
(18, 68–70)
SRG15
Il15
h/h
S
KI
RG
background
Human NK cells and T cells by
human IL-15
- Functional human NK cells
- Intraepithelial lymphocytes in
mucosa
(46)
MISTRG
Csf1
h/h
, Il3
h/h
,
Csf2
h/h
, Thpo
h/h
S
tg
RG or S
KI
RG
background
Mouse HSPC and
alveolar macrophage
deficiency
Multiple human cytokines
supporting HPSC maintenance
and myelopoiesis
-
Genetic pre-conditioning (high
engraftment without irradiation)
- Long-term hematopoiesis
- Engraftment of patient-derived
hematologic malignancies
- Diverse and functional subsets of
myelomonocytic cells
- Functional NK cells
- Thrombocytopenia
- Hemophagocytosis
(17, 18, 71)
MISTRG6
Il6
h/h
MIS
KI
TRG
background
Support to IL-6 dependent cells -
Engraftment of patient-derived
multiple myeloma
(72)
SRG6
Il6
h/h
S
KI
RG
background
Support to IL-6 dependent cells -
Increased thymic cellularity
- Improved B cell response to
immunization
(73)
BRGST
Mouse Tslp
transgene
BRGS
NOD
background
Restore support for mouse LTi
cells and LN architecture
-
LN development
- Improved antigen-specific
adaptive immunity
(74)
Positive impacts are indicated in green, unintended detrimental effects in red. A more detailed version is provided as Supplementary Table 1, including additional details of
improvements and limitations of each model with regards to hematopoiesis, innate and adaptive immunity.
for the maintenance of quiescent and self-renewing HSPCs
(
80–83). Mouse Thpo gene inactivation reduces frequencies
of mouse HSPCs, thereby opening the niche for transplanted
human HSPCs (65). The concept of genetic preconditioning
also applies to non-HSPC cell types, and to niches other
than the BM. Fms-like tyrosine kinase 3 (Flt-3) ligand
is essential to the differentiation of dendritic cells (DCs)
(84–87), while granulocyte-macrophage colony stimulating
factor (GM-CSF) is required for the maturation of lung
alveolar macrophages (AM) (
88–90). Genetic inactivation
of Flt3 (encoding the receptor for Flt-3 ligand) or of
Csf2 (encoding GM-CSF) eliminates mouse DCs or AMs,
respectively, thereby opening the niche for the development
of the c orresponding human cell lineages (
66, 67). In these
three cases (Thpo, Ftl3, or Csf2 gene deficiencies), the
elimination of mouse cell populations was supplemented with
provision of the corresponding human cytokines (
65–67), as
discussed next.
PRINCIPLE #3: SUPPORTING THE
DEVELOPMENT OF ENGRAFTED HUMAN
CELLS
The spatial microenvironment of the BM niche includes
diverse cell types, including endothelial cells and mesenchymal
stromal/stem cells (MSCs) that are known to release cytokines,
signaling mediators and growth factors, such as SCF, IL-3, IL-
6, THPO, and GM-CSF (
91–93). These molecules are important
for the maintenance of human HSPCs and their differentiation
into all hematopoietic and immune cell lineages (1, 2). Some
of the cytokines supporting hematopoiesis are poorly conserved
(e.g., 29% amino acid identity between human and mouse IL-
3) and do not cross-react from mouse to human, while others
are highly conserved and largely cross-reactive (e.g., SCF) (1).
However, even when amino acid identity is high, cross-reactivity
of cytokines is not always complete in local microenvironments
at physiological concentrations. To account for this incomplete
Frontiers in Immunology | www.frontiersin.org 5 February 2021 | Volume 12 | Article 643852
Martinov et al. Building Next Generation Humanized Mice
cross-reactivity, various protocols were developed to promote the
differentiation of a more complete human immune system upon
human CD34
+
cell transplantation in mice.
Exogenous Cytokine Administration
The simplest method of supplying graft-supporting factors is
the repeat ed injection of recombinant human cytokines. This
approach was used in an early-generation SCID model and the
injection of cytokines, including human SCF, IL-3, and GM-
CSF, supported enhanced engraftment levels of human BM cells
as well as their myeloid differentiation (
26). Because human
NK cell differentiation is limited in mice, humanized BRG mice
were treated with recombinant human IL-15 coupled to IL-15Rα,
which is an essential growth factor for NK cell development and
homeostasis. This treatment resulted in a significant increase in
the differentiation and homeostasis of human NK cells in mice
transplanted wit h human CD34
+
cells (94).
A second cytokine delivery approach relies on hydrodynamic
injection of a human cytokine-encoding DNA plasmid, resulting
in the in vivo “transfection” of hepatocytes. In turn, hepatocytes
produce high levels of the encoded cytokine and release it into
the circulation for up to 5 days (95, 96). This method was used
to demonstrate that human cytokines, such as GM-CSF and
M-CSF, support myeloid differentiation of human CD34
+
cells
in NSG mice, while IL-15 and Flt-3 ligand supported NK cell
differentiation (
96, 97).
The administration of human cytokines can be combined with
genetic opening of the niche. In BRGF mice, which lack the Flt3
gene, injection of human recombinant Flt-3 ligand boosts the
development of human dendritic cells, in the absence of mouse
dendritic cells (66).
Recombinant cytokine injection and hydrodynamic plasmid
delivery are easy to implement, irrespective of the recipient
mouse and protocol of human cell transplantation used.
However, they both result in transient, systemic and generally
supra-physiological cytoki ne expression.
Genetic Engineering of Human Cytokine
Expression
To circumvent the requirement for repeated cytokine
administrations, the genome of the recipient mouse can be
engineered to express human cytokines (Table 2). The initial
method relied on transgenic overexpression of a human
cytokine-encoding cDNA, under the control of a strong
promoter. Such transgenic mice are still in use, based on NSG
or similar genetic backgrounds. However, results obtained with
these mice need to be interpreted ca utiously as the systemic
overexpression of human cytokines frequently results in non-
physiological hematopoiesis. For example, the transgenic pCMV
promoter-driven overexpression of human SCF, GM-CSF, and
IL-3 results in the mobilization of CD34
+
HSPCs in the NSG-
SGM3 mouse and the loss of their functional properties (
68).
As a consequence, although high-level human hematopoietic
engraftment is achieved in NSG-SGM3 mice, hematopoietic
progenitors lose their stemness and long-term hematopoiesis is
deficient, as demonstrated by their inability to serially engraft
(
18, 69). Nevertheless, transgenic overexpression of cytokines
can support the development of specific cell lineages and
provide useful models to study those cells. In NSG-SGM3, the
overexpression of human SCF results in the development of
abundant and functional human mast cells, which are effective
in models of passive cutaneous and systemic anaphylaxis (
98).
Similarly, human IL-15 overexpression in NOG mice supports
the maintenance and function of human NK cells isolated from
human peripheral blood (99, 100). Such models can provide
useful experimental systems to evaluate the effect of candidate
drugs on specific human immune cell populations.
More physiological expression of human cytokines can be
achieved by bacterial artificial chromosome (referred to as
“BAC”) transgenesis, inserting a n entire human gene, including
its regulatory elements, into the mouse genome. This method has
been used to express human IL-6 and human IL-7 in NSG mice
(101, 102). In another strategy, the mouse gene can be eliminated
and corresponding human gene inserted in its place, including
introns and exons from the start to the stop codon (Figure 3A)
(
103). This knock-in strategy has been used for a number of
cytokine-encoding genes, including Csf1, Csf2, Il3, Il6, Il15, and
Thpo (46, 65, 67, 72, 104). A slightly different approach was used
for replacement of the Il7, Il15, and Tnfsf13b genes, t h rough
insertion of a human cDNA in frame with the start codon of the
corresponding mouse gene, followed by a poly-adenylation signal
(Figure 3B) (101, 105).
Because of its role in supporting NK cell development, human
IL-15 is highly featured in the humanized mouse literature, and
it has been delivered to recipient mice by each of the methods
described above (
46, 94, 96, 99, 101). Since these approaches
have been reported by different groups, their direct comparison
and the evaluation of respective merits and limitations are
difficult. However, the SRG-15 recipient mouse stands out as
a definitive solution to th e limited development of human NK
cells in humanized mice, owing to its simplicity of use (no
cytokine administration is needed), the physiological expression
of the cytokine by knock-in gene replacement, the comprehensive
phenotypic comparison to human periph eral blood cells and
rigorous functional in vivo characterization (46).
In addition to expressing a human cytokine, knock-in
gene replacement abrogates expression of the corresponding
mouse cytokine (Figure 3). If the human cytokine is not
fully cross-reactive, this can result in the absence of the
cytokine-dependent mouse cell population and niche opening
(
103). This is best illustrated in the case of GM-CSF
(encoded by Csf2/CSF2), where only 56% of amino acids are
shared between human and mouse (1, 67). In mice with
homozygous humanization of the Csf2 locus, the absence of
mouse GM-CSF induces alveolar macrophage deficiency in
the lung and a resulting pathology described as pulmonary
alveolar proteinosis (67), recapitulating t he phenotype of
Csf2 knockout mice (88–90). Upon human CD34
+
cell
transplantation, human GM-CSF supports the development of
human alveolar macrophages and partial rescue of the proteinosis
pathology (
67).
Importantly, because hematopoiesis is a stepwise process in
which stem cells gradually differentiate toward more committed
progenitors and multiple lineages of mature cells (
1, 2),
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Martinov et al. Building Next Generation Humanized Mice
FIGURE 3 | Methods of knock-in gene humanization by (A) gene replacement or (B) cDNA insertion.
humanization of several cytokines is required to support the
maintenance and multilineage differentiation of human HSPCs.
This is exemplified in the MISTRG mouse in which M-CSF, I L-
3, GM-CSF and THPO are humanized by knock-in replacement,
on the SRG genetic background (17, 71). Niche opening and four
human cytokines synergize to support the engraftment of human
CD34
+
cells without radiation preconditioning (17, 18). The
long-term maintenance of functional HSPCs is demonstrated
by their serial transplantation for up to four generations of
MISTRG re ci pients (
18). These mice also support multilineage
differentiation of human B, T and dendritic cells (similarly to
NSG mice) as well as different subsets of functional myelo-
monocytic cells in lymphoid and non-lymphoid tissues (17, 18).
The myelo-monocytic cells themselves express human IL-15/IL-
15Rα, which in turn supports the development and function
of human NK cells (17). Overall, MISTRG mice emphasize the
benefits of combining multiple strategies for th e provision of
human cytokines in the development of improved recipient
mice by: niche opening by elimination of the corresponding
mouse cytokines; phys iological expression of each cytokine;
synergy between multiple cytokines; and, indirectly, cross-
support between different human immune cell lineages.
Co-transplantation of Supporting Human
Cells or Tissues
Although providing specific cytokines can be effective, it
would be a long and arduous journey to humanize the
entire spectrum of hematopoiesis-supporting cytokines (and
possibly other required factors). Therefore, co-transplantation of
supporting human cells or tissues, naturally found in the BM
niche or in oth er primary lymphoid organs, can be used to
provide all required factors and more fully recapitulate human
hematopoietic development and homeostasis.
A well-established protocol of such co-transplantation is the
BM, liver, thymus (BLT) humanized mouse model (
106, 107).
BLT mice are generated by co-transplanting human fetal liver
and thymus under the murine renal capsule in preconditioned
adult immunodeficient mice, before injecting syngeneic CD34
+
HSPCs intravenously. The liver and thymus implants form a
liver-thymus organoid that contains stromal microenvironments
and provides cytokines at local physiological concentrations,
requisite for the differentiation of functional B, T, and NK cells,
dendritic cells a nd monocytes/macrophages, as well as long-
term maintenance of human hematopoiesis and lymphopoiesis
(
106–108). Because human T cells develop in the context of
thymic epithelial cells, the human thymus organoid contains
significantly higher absolute numbers of thymocytes, compared
to mouse thymus (109) and a repertoire of immunocompetent
human leukocyte antigen (HLA) class I- and class II-restricted
T lymphocytes is selected (106, 107, 110). The immune cell
distribution in BLT humanized mice is well-described for both
primary and secondary lymphoid organs, including BM, human
and mouse thymus, intestines (lamina propria), mesenteric
lymph nodes, vaginal tissues, liver and lungs (
106, 107, 109, 111–
113). Although the BLT protocol was first described using the
NOD-SCID background (106, 10 7), this technique can be appli ed
to any recipient mouse, and has been used successfully in NSG
(109, 113), NSG-SGM3 (98), and B6RG-CD47 (48) mice. Thus,
investigators c an use the BLT protocol to enhance human T cell
development in a mouse strain with immune characteristics that
are appropriate for their study.
Intravenous or intrafemoral co-transplantation of human
BM-derived MSCs along with CD34
+
cord blood cells is another
strategy to improve engraftment of CD34
+
and CD45
+
cells in
immunodeficient mice (
114–117). Overall, co-transplantation of
the BM-derived MSCs into humanized mice can improve human
HSPC maintenance and expansion, and improve reconstitution
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Martinov et al. Building Next Generation Humanized Mice
of the human hemato-lymphoid system in humanized mice.
Furthermore, recent studies have used genetically engineered
MSCs t o deliver additional human factors to support human
hematopoiesis, or to facilitate the expansion and maintenance of
HSPCs to allow serial transplantation and generation of larger
quantities of humanized mice (
118, 119).
Recent studies have used human bone organoid (ossicles) as
a method to add human BM microenvironment for engrafted
HSPCs to oc cupy (120, 121). Ossicles are often generated
by seeding 3D polymer scaffolds with human BM-derived
MSCs. These are then implanted subcutaneously into NSG
mice and become colonized with subsequently injected human
HSPCs, enabling their expansion and differentiation (122–128).
Humanized ossicles contain increased human immature and
mature hematopoietic cells as compared to the bones of host mice
implanted with only human CD34
+
cells, indicating homing
of HSPCs (
122, 128). Additionally, self-renewing HSPCs from
humanized ossicles can reconstitute hematopoiesis in secondary
recipient mice, demonstrating maintenance of their functional
properties (129). Most importantly, high engraftment levels of
hCD45
+
cells were measured in the blood, spleen and mouse
bone (130, 131). High numbers of human erythroid lineage cells
and robust differentiation of mature myeloid cells were also
detected (132).
With the development of MISTRG and MISTRG6 mouse
strains that express essential human cytokines, and protocols
for co-transplantation of human fetal bone chips or ossicles,
major progress has been made in transplanting patient-
derived hematologic malignancies into humanized BM niches.
Samples from patients with myelodysplastic syndromes,
myeloproliferative neoplasms, low risk acute myeloid leukemia,
diffuse large B cell lymphoma or multiple myeloma have been
successfully engrafted, using diverse recipient mice and protocols
(72, 123, 125, 133–136).
FUTURE CHALLENGE #1: THE MISSING
LINEAGES
Provision of specific human cytokines significantly improved the
development, homeostasis and function of human NK cells and
myelo-monocytic cells in humanized mice. But se veral lineages
remain defective. For example, human neutrophils are generally
present in the BM of humanized mice, but their frequency
is negligible in t he periphery (
50). Cytokine overexpression in
NSG-SGM3 mice increases the frequency of granulocytic CD33
+
cells in the BM and the periphery (
18, 69, 70), but these cells
display t h e morphology and cell surface phenotype of immature
cells (18). Human cytokine knock-in MISTRG mice also do
not have improved mature neutrophil numbers in the periphery
(17, 18). Therefore, the differentiation, egress, maturation and/or
survival of human neutrophils likely requires additional factors.
Human red blood cells (RB Cs) and platelets are probably
the most challenging hematopoietic cells to develop in mice.
They illustrat e that additional strategies, beyond the provision of
human cytokines, will likely be needed to support the complete
spectrum of human hematopoietic line ages in mice. In the BM
of humanized NSG mice, human erythroid (CD235a
+
) and
megakaryocytic (CD41
+
CD61
+
) progenitors are extremely rare.
Their frequency is increased by at least an order of magnitude
in NSGW41 and MISTRG recipient mice but surprisingly,
overexpression of human erythropoietin (EPO) did not further
improve human erythropoiesis (
79, 133). The increase in
genetically pre conditioned mice is likely due to t he better
competition of human progenitors against mouse progenitors in
the open hematopoietic niche, and/or support from knocked-
in human f actors. However, erythropoiesis is arrested at an
immature (CD71
+
CD235a
+
) stage. As a result, few mature
CD71
−
CD235a
+
human reticulocytes are detectable in the BM
and human RBCs rarely exceed 1% in peripheral blood (79).
Several lines of evidence demonstrate that this deficiency
is due to a developmental defect as well as impaired survival.
Indeed, human RBCs are highly susceptible to destruction in
mice. Expression of human SIRPα in BRG mice, or depletion
of macrophages by clodronate treatment in NOD SCID mice,
extends the lifespan of adoptively transferred human RBCs
(45). However, in both cases, the half-life of human RBCs
does not exceed ∼16 h, which is much shorter than the 10–20-
days half-life of mouse RBCs in similar transfer experiments
(137). Accordingly, clodronate treatment results in significant but
transient and incomplete increase in human RBCs and platelets
in mice humanized by CD34
+
cell transplantation (52, 53, 79).
In those conditions, injection of the human cytokines, IL-3 and
EPO, promotes an increase in peripheral RBC counts (52). But,
because clodronate targets both mouse and human cells, the
treatment results in a humanized mouse entirely lacking human
phagocytic cells, which limits the applicability of th e model for
studies of human immunity.
Therefore, the entire panel of mechanisms limiting the half-
life of human RBCs and platelets in mice will need to be
identified and resolved. In addition, the adequate combination
of human cytokines will have to be provided, to enable the
mice to live with primarily human platelets and RBCs. Such
a model would be highly useful for studying diseases caused
by pathogens with exclusive tropism for human RBCs [e.g.,
malaria caused by Plasmodium falciparum (
138)] or diseases in
which platelets contribute to pathogenesis [e.g., dengue fever,
autoimmune thrombocytopenia (
139, 140)]. In the meantime,
current models (such as MISTRG) have already demonstrated
their utility for modeling the early BM stages of human
erythropoiesis and thrombopoiesis, as well as for studying drug
responses in pathologies, including myelodysplastic syndromes
and myeloproliferative neoplasms (133, 134).
FUTURE CHALLENGE #2: ADAPTIVE
IMMUNITY
As a result of advances discussed above, we now have humanized
mice in which both cellular and humoral adaptive immune
responses can be elicited. However, these responses are largely
modest in magnitude, quality and duration, as outlined below.
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Martinov et al. Building Next Generation Humanized Mice
Antigen-Specific Adaptive Immunity
Humanized mice generated by transplantation of CD34
+
cells,
or following the BLT protocol, can mount antigen-specific
adaptive immune responses (
21, 107, 141, 142). Evidence
of protective immunity, capable of controlling pathogen
replication and resulting disease, was provided in t h e context
of Epstein-Barr virus (EBV), dengue virus (DENV), and
human immunodeficiency virus-1 (HIV-1) infection. Indeed,
in CD34
+
-humanized NSG mice, antibody-mediated depletion
of T cells led to the development of EBV-associated tumors,
suggesting that T cell-mediated immunity can control EBV
infection in this setting (143). Robust CD4
+
and CD8
+
T
cell responses were also detected after DENV and HIV-1
infection. Notably, in HIV-1-infected BLT mice, CD8
+
T
cell responses were HLA-restricted and directed against
epitopes previously described as immunogenic in humans.
Furthermore, CD8
+
T cell-mediated viral recognition
led to viral epitope evolution, closely resembling clinical
obser vations (142).
Despite having detectable anti-viral T cells, CD34
+
-
humanized and BLT mice generally exhibit weak humoral
responses after viral infection (144, 145). While neutralizing
antibodies were detected in a subset of DENV-infected mice,
in both CD34
+
-only and BLT humanized mice, the anti-
EBV or anti-HIV-1 antibody responses were either weak,
equivocal or delayed (
111, 141). This was also the case in
humanized MISTRG mice, where enhanced innate immune
cell engraftment led to improved T cell function after Listeria
monocytogenes infection, but humoral immunity remained
weak (17). Across these models, immunoglobulin class-
switching and somatic hypermutation are rarely achieved, likely
because most B cells fail to reach a fully mature phenotype
in the periphery, T cell repertoire is selected on mouse and
human MHC and is suboptimal, and recipient mice have
disorganized lymph nodes and little capacity for germinal center
formation (146).
While human B cells are detectable in high frequencies
in humanized mice, most exhibit an immature phenotype,
and remain blocked at the transitional stage of B cell
development (1, 146). Since immature B cells have a reduced
capacity to respond to antigen (147), several groups sought
to improve B cell maturation and by extension, their
function by knocking-in human cytokines that are known
to support B cell development. Surprisingly, h uman B cell
activation factor (BAFF, encoded by TNFSF13B) knock-in
resulted in reduced numbers of mature naïve B cells, and
reduced antibody production after immunization (105).
Human IL-7 knock-in had no effect on B cell numbers
and any effect on B cell function remains to be determined
(101). In contrast, human IL-6 positively impacted B cell
differentiation into plasmablasts and memory cells after
immunization with model antigen, and promoted somatic
hypermutation and class switching, albeit to a lower level than
that observed in humans (73). IL-6 knock-in mice also had
improved T cell development; therefore, it is possible that
enhanced B cell responses were in part due to increased T
cell help.
Thymic Lymphopoiesis and Repertoire
Selection
Thymic lymphopoiesis is suboptimal in mice humanized by
transplantation of CD34
+
HSPCs, in part due to a lack of
thymopoiesis-supporting human cytokines. As a result, thymic
cellularity is extremely low (only a few million cells) and
CD4
+
CD8
+
double positive cells, which represent the vast
majority of thymocytes in a healthy thy mus, are frequently
underrepresented. Human IL-6 knock-in produces increased
thymic cellularity (
73), but additional cytokines are likely
required to restore the normal size of a mouse t hymus, populated
by human thymocytes. While a human IL-7 knock-in recipient
mouse has been developed, a thorough characterization of thymic
cellularity upon HSPC engraftment has not been reported to
date (
101).
Human T cell repertoire selection in the mouse thymus
involves interactions with both mouse MHC and human
HLA-expressing cells-expressing cells (1, 10, 21). Specifically,
developing human T cells are positively selected by mouse
epithelial cells and negatively selected by both mouse epithelial
and mouse a nd human hematopoietic cells (148). The resulting T
cell receptor (TCR) repertoire is weakly reactive to autologous
human leukocyte antigen (HLA) class I and II, and tolerant
to mouse MHC. Indeed, in in vitro re-stimulation assays, T
cells from humanized mice were shown to proliferate better
in response to allogeneic human D Cs compared to autologous
human DCs or mouse DCs (
21). To improve the repertoire and
function of human T cells, several rec ipient strains have been
engineered to express HLA class I and class II (143, 144, 149–
154). HLA-restricted T cell responses have been reported in these
mice (143, 149). Additional characterization and comparison of
these mice are required to rigorously evaluate how transgenic
HLA expression in recipient HSPC-humanized mice impacts
human T cell selection and function.
In BLT mice, T cells develop in the human thymus organoid
and are positively and negatively selected on human autologous
HLA molecules (
106, 107, 155). Consequently, BLT mice are
generally accepted as having a more diverse T cell repertoire,
capable of mounting more robust adaptive immune responses, as
discussed above. However, because the induction of tolerance for
mouse MHC and mouse tissue-restricted antigens is incomplete,
BLT mice are prone to t he development of xenogeneic graft-vs.-
host dise ase (xGvHD) (156, 157), as dis cussed below.
Few direct comparisons between BLT and HSPC-engrafted
mice have been reported to date (144, 145). In both models, it is
apparent that thymic lymphopoiesis and the selection of a diverse
and tolerant T cell repertoire is a complex issue, and additional
work is needed.
Secondary Lymphoid Organs
Secondary lymphoid organs including lymph nodes (LNs),
spleen, Peyer’s patches, and mucosa-associated lymphoid tissues
normally provide niche microenvironments where T and B
cells interact with hematopoietic antigen-presenting cells and
stromal follicular dendritic cells (FDCs) to initiate the adaptive
immune response (
158, 159). LN formation requires IL-7- and
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Martinov et al. Building Next Generation Humanized Mice
IL-2Rγ-dependent lymphoid tissue inducer (LTi) cells (160).
However, because most immunodeficient recipient mice lack
Il2rg to promote tolerance to the human graft through NK cell
depletion, they also lack LTi cells and defined LN structures.
This represents a major obstacle in recapitulating the human
adaptive immune response in mice. While engraftment with
human CD34
+
cells can partially rescue the LN anlagen, T and B
cell zones remain poorly organized compared to those in normal
human or normal mouse LNs. Furthermore, the germinal center
formation is impaired, in part bec ause it involves human B cells
interacting with stromal FDCs of mouse origin (1, 159).
Given that the germinal center is the primary site of B
cell-T cell collaboration, antibody affinity maturation and class
switching, multiple groups sought to improve LN formation
and germinal center development (158). Adoptive transfer
of autologous human DCs lentivirally transduced to express
GM-CSF, IFNα, and human cy tomegalovirus (HCMV) viral
antigen enhances LN development in humanized NRG mice,
and by extension promotes T cell-B cell collaboration a nd
the production of neutralizing anti-HCMV antibodies (
161,
162). Human fetal organ co-transplantation also partially
rescues secondary lymphoid organ development. BLT mice
have improved lymphoid structure development in the spleen
and lymph nodes compared to CD34
+
-only engrafted mice,
and as a result, they also have more robust antigen-specific
adaptive immunity (106, 107). However, a comparison of IL-
2Rγ-sufficient NOD-SCID BLT humanized mice and IL-2Rγ-
deficient NSG BLT humanized mice showed that only NOD-
SCID BLT mice contain mucosal tissue-associated T cells (
109).
This result emphasizes the critical nature of IL-2Rγ in the
development of LN and other se cond ary lymphoid organs
that are essential for adaptive immune responses and mucosal
immunity. The importance of structured secondary lymphoid
organs for adaptive immunity is further demonstrated by the
addition of fetal spleen to BLT mice (163). Human fetal spleen
implants grow into spleen organoids with prominent follicular
lymphoid structure; implanted mice have improved B cell and
T cell engraftment, compared to control mice without human
spleen implants, and can mount antigen-specific responses to
immunization (163).
Another strategy to overcome the near-absence of LNs in
Il2rg
−/−
humanized mice involves transgenic overexpression of
murine thymic stromal lymphopoietin (TSLP). TSLP is another
cytokine of the IL-2 family, its receptor is independent of
the IL-2Rγ chain and, when overexpressed under a keratin 14
promoter (specific for epithelial/mesenchymal cells), it rescues
LN development in Il7 deficient mice (
164, 165). Crossing the
transgene to BRGS resulted in the BRGST model, in which
mouse LTi cells and LN structures are restored (74). Upon
transplantation of CD34
+
HSPCs, BRGST mice support the
development of a human immune system, including sizeable
LNs with compartmentalized human T and B cell zones. BRGST
mice also have more mature B cells and IL-21 producing
follicular helper T cells, essential to promoting adaptive
immunity. Consequently, BRGST mice mounted enhanced
antigen-specific humoral immune responses upon immunization
with an experimental antigen (
74). Overall, this novel model
successfully addresses a major limitation that had hampered
immune function in most humanized mouse models to date.
FUTURE CHALLENGE #3:
GRAFT-TO-HOST TOLERANCE
The transplant ation of human cells into recipient mice is feasible
because the recipient mice used are immunologically tolerant to
the graft. As these mice support a n increasingly functional human
immune system, immunologic tolerance of the graft for its host
can become an issue.
Xenogeneic Graft-vs.-Host Disease
Adoptive transfer of human T cells from donor PBMCs into
immunodeficient recipient mice results in xGvHD, thereby
limiting the potential duration of experiments with these mice.
To prevent xGvHD, two strains of immunodeficient mice
lacking mouse MHC-I and MHC-II have been developed, which
prevented the onset of xGvHD, while retaining t he functional
properties of human T cells (
166, 167). But, as discussed above,
PBMC transfer results in an incomplete human hematopoietic
system in the mice.
When mice are humanized by transplantation of CD34
+
HSPCs, developing human T cells undergo positive and negative
selection in the mouse thymus. Consequently, they are tolerized
for mouse MHC-I and MHC-II, and xGvHD is not a limitation
in th is model (
1, 10, 21).
Finally, in BLT mice, T cells are educated in the human
thymus organoid and once they reach the periphery, they
are allogeneic to the mouse MHC molecules and can
induce xGvHD (
156, 157). Different levels of xGvHD are
reported by different groups, suggesting that the disease
could be affected by subtle differences in protocols, the
microbiota of the mice, or the recipient strain used (48).
The development of xGvHD in the BLT model is attributed
to residual mature T cells present in the fetal human
thymus grafts, and these passenger T cells can be removed
by treating th e thymic implants with 2
′
-deoxyguanosine,
or by treating the mice with anti-human CD2 antibody
post-surgery (168).
Xenogeneic Hemophagocytosis
The efficient development of human phagocytic cells of the
myelo-monocytic lineage in MISTRG creates a new challenge;
i.e., the absence of phagocytic tolerance from the human graft
toward the mouse host. Mouse red blood cells are particularly
susceptible to destruction by human phagocytic cells, and
highly engrafted MISTRG mice develop lethal anemia (
17).
Consequently, the transplanta tion protocol in MISTRG needs to
be optimized so that engraftment allows mouse eryt hropoiesis
in the BM (or extramedullary erythropoiesis) to sufficiently
compensate for the loss of mouse red blood cells by phagocytosis,
as long as a specific experiment requires (17, 18, 58, 133).
Long-term solutions will need to be implemented, either to
establish human-to-mouse phagocytic tolerance or to enable
human erythropoiesis to reach healthy human RB C counts and
maintain mouse homeostasis, as discussed above.
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Martinov et al. Building Next Generation Humanized Mice
Xenogeneic GvHD and hemophagocytosis remind us t hat
activation and tolerance are two equally important features of the
immune system and are inseparable. As human immune function
improves in mouse recipients, it is likely that parallel strategies
will need to be developed to maintain tolerance of the human
immune graft for the mouse host.
FUTURE CHALLENGE #4: INTERACTIONS
BETWEEN IMMUNE CELLS AND TARGET
TISSUES OR TUMORS
Immune responses require functional i nteractions between
immune cells and their target, either through direct cell-cell
contact or via soluble factors. In humanized mice generated by
transplantation of human HSPCs, this requirement is fulfilled
BOX 2 | Which humanized mouse model is best for your studies?
There is no one-size-fits-all model. Here are a few practical considerations:
(1) Which mice and human cells/tissue do you have access to?
(2) What question are you trying to answer?
(3) Which cell types are important to answer your question?
In addition to considering these practical questions, it is important to
know what has been done before you. Take time to read the literature
and critically analyze the experiments. If you are able to talk to the people
who developed the model or to someone who has recently published on
the model of interest, you may receive invaluable unpublished data and
advice.
Finally, applying these criteria, we provide our own selection, sub jective
and probably slightly biased, of the models that we consider most suitable
for specific applications.
Hematopoiesis
• HSPC biology: NSGW41, MISTRG, MISTRG6
• Erythropoiesis and thrombopoiesis: NSGW41, MISTRG, MISTRG6 (BM
only, limited)
• Myelopoiesis: MISTRG, MISTRG6
• Hematologic malignancies: MISTRG, MISTRG6
Innate immunity
• Monocytes/macrophages: MISTRG, MISTRG6
• DCs: NSG (or any other strain, depending on additional requirements)
• Mast cells: NSG-SGM3
• Neutrophils: no suitable model available
• NK cells: SRG15, MISTRG
• Innate lymphoid cells: NSG (or any other strain, depending on
additional requirements)
Adaptive immunity
BLT mice are generally considered as supporting more robust adaptive
immunity than mice transplanted with HSPCs only. However, few direct
comparisons have been reported and antigen-specific immune responses
remain relatively weak or delayed, even in BLT mice. Importantly, the BLT
protocol can be applied with any recipient mice.
Expression of HLA molecules by the mouse host qualitatively favors HLA-
restricted immune responses. But the impact on the amplitude of responses
remains to be rigorously quantified.
• B cells: BLT, SRG6, MISTRG6
• T cells: BLT, BRGST.
when the target of the immune response is a lso a human
hematopoietic cell. Accordingly, T and B cell-mediated immunity
have been demonstrated in the context of infection by pathogens
with tropism for human hematopoietic c ells, such as EBV, HIV-1,
or dengue virus (141, 143, 169). But, for pathogens that infect
non-hematopoietic tissues, or for inflammatory mediators that
induce systemic responses, the cross-reactivity between human
immune effec tor mechanisms and mouse target tissues may be
incomplete. Humanizing cytokine receptors or other factors,
such as adhesion molecules, could improve the responsiveness of
mouse tissues to human immune cells and soluble mediators.
Co-transplantation of the human target tissue along with the
human hematopoietic system has been performed in the context
of cancer and infectious diseases. Implantation of human tumors,
either from an established cell line or from a “patient-derived
xenograft” (PDX), in mice alre ady repopulated with a human
immune sy stem, provides useful models for immuno-oncology
and immunotherapy studies (170–173). However, developing
such “immuno-PDX” models ca n be challenging as each patient-
derived tumor or cell line has different growth characteristics
in mice, and matching patient’s HLA to the HLA of the HSPC
donor is not always feasible. Additionally, many components of
the tumor microenvironment (e.g., vasculature) remain of mouse
origin. Consequently, antitumoral immunity, or t he response to
immunotherapies, can be highly variable from experiment to
experiment (171). In the case of hematologic malignancies, new
strains of recipient mice (i.e., MISTRG and MISTRG6) extend
the range of transplantable diseases (72, 133–136) and provide
new opportunities to evaluate candid a te immunotherapies, such
as adoptive T cell therapies (
174, 175).
Transplantation of human liver tissue enables infection
of the mouse host by human hepatotropic viruses. Because
implantation of a liver fragment at an ectopic site does not
fully recapitulate t he architecture and function of the liver,
protocols of orthotopic implantation have been developed. These
methods follow the same principles as for the transplantation
of human hematopoiesis (176): an immunodeficient strain to
prevent immune rejection of the graft; engineering of the mouse
to induce mouse liver injury and open the niche for human
hepatocytes; and, in some instances, support from human growth
factors (177). Several methods have been developed to eliminate
mouse hepatocytes, relying on the expression of cytolytic proteins
[e.g., overexpression of urokinase plasminogen activator under
an albumin promoter in uPA mice (178, 179)] and/or inactivation
of metabolic enzymes essential for hepatocyte homeostasis [e.g.,
inactivation of the fumarylacetoacetate hydrolase in Fah
−/−
mice
(180, 181)]. Upon injection of human hepatocytes, these mice
support high levels of liver chimerism and are permissive for
infection by the human hepat otropic viruses, HBV and HCV. To
study human immune responses to these pathogens, mice can be
dually transplanted with human hepatocytes and CD34
+
HSPCs;
human immune cells are recruited to the human liver in these
chimeric mice and immune responses are triggered upon viral
infections (177, 182, 183).
Finally, subcutaneous implantation of a fragment of human
fetal lung cont ains all cell types naturally present in this tissue,
and extends the permissiveness of the host mouse to respiratory
pathogens with human tropism, including respiratory syncytial
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Martinov et al. Building Next Generation Humanized Mice
virus, Middle East respiratory syndrome coronavirus, and
HCMV. This was recently accomplished by surgical implant a tion
of a fragment of human lung tissue in BLT mice, resulting
in the “BLT-L” model (184). Upon infection with HCMV, by
direct inject ion in the lung implant, BLT-L mounted an antigen
specific adaptive immune response, c a pable of controlling virus
replication (
184).
Thus, co-transplantation models greatly broaden the
spectrum of human immune responses that can be studied in
humanized mice. However, current approaches rely on highly
specialized protocols and recipient mice, and should still be
considered as prototypes under development.
CONCLUSION
Tremendous progress has been accomplished since pioneering
humanized mouse models were developed in the late 1980s. In
the past decade, a flurry of new opportunities have been enabled
by the optimization of recipient mouse strains and humanization
protocols. The most advanced models support long-term
multilineage human hematopoiesis, and recapitulate essential
aspects of innate immunity a nd antigen-specific adaptive
immunity. Furthermore, numerous hematologic diseases can
now be modeled by xenotransplantation of primary patient-
derived samples.
Despite this progress, limitations remain. Rigorous evaluation
and comparison of the new models is needed, while supporting
additional innovations that might drive transformative advances.
Until one or more humanized mouse model fully recapitulates
human immunity, we can ent husiastically but critically use the
currently available strains to answer specific, clinically-relevant
questions and hopefully inform the development of new, life-
saving therapies (Box 2).
AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual
contribution to the work, and approved it for publication.
FUNDING
Research in our lab was supported by the NIH/NCI CA234720,
NIH/NIAID AI138011, The Hartwell Foundation, The Emerson
Collective, the Seattle Translational Tumor Research, the
University of Washington Center for AIDS Research, and the
Immunotherapy Integrated Research Center at Fred Hutch. TM
was supported by a Parker Institute for Cancer Immunotherapy
Scholar Award. EC was supported by a postdoctoral fellowship
from Fred Hutch’s Immunotherapy Integrated Rese arch Center.
ACKNOWLEDGMENTS
We thank Deborah Banker for manuscript editing, and the Guest
Editors for inviting us to submit this manuscript, which we wrote
as an educational work from home activity. Figures created with
BioRender.com.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fimmu.
2021.643852/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2021 Martinov, McKenna, Tan, Collins, Kehret, Linton, Olsen, Shobaki
and Rongvaux. This is an open-access article distributed under the terms of
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Frontiers in Immunology | www.frontiersin.org 17 February 2021 | Volume 12 | Article 643852
