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Perspective
Standing on the shoulders of mice
Kwat Medetgul-Ernar2 and Mark M. Davis1,*
1Howard Hughes Medical Institute, Institute for Immunity, Transplantation and Infection, Department of Microbiology and Immunology,

Stanford University School of Medicine, Palo Alto, CA 94304, USA

2Immunology Program, Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Palo Alto, CA

94304, USA
*Correspondence: mmdavis@stanford.edu
https://doi.org/10.1016/j.immuni.2022.07.008

SUMMARY

While inbred mice have informed most of what we know about the immune system in the modern era, they
have clear limitations with respect to their ability to be informative regarding genetic heterogeneity or micro-
bial influences. They have also not been very predictive as models of human disease or vaccination results.
Although there are concerted attempts to compensate for these flaws, the rapid rise of human studies, driven
by both technical and conceptual advances, promises to fill in these gaps, as well as provide direct informa-
tion about human diseases and vaccination responses. Work on human immunity has already provided
important additional perspectives on basic immunology such as the importance of clonal deletion to self-
tolerance, and while many challenges remain, it seems inevitable that ‘‘the human model’’ will continue to
inform us about the immune system and even allow for the discovery of new mechanisms.

INTRODUCTION some of the deficiencies in the inbred mouse model, humanized

While the ‘‘Delbru €ck School’’ of bacteriophage (Judson, 1996)
and the structure of DNA were key to the rapid advancement
of molecular biology, inbred mice have long been the Rosetta
Stone for immunologists in the modern era, allowing decades
of immunological phenomena to be deciphered and reduced
to mechanistic insights, or at least workable hypotheses. Power-
ful methodologies have been developed and used to understand
antibody and T cell receptor diversity, major histocompatibility
complex (MHC) restriction, and the differences between innate
and adaptive detection of self and non-self antigens and ensuing
effector systems. Methods like monoclonal antibodies, the dis-
covery of various inflammatory pathways, and checkpoint inhibi-
tion developed in mice have been widely adopted as therapeu-
tics for cancer, infectious disease, and autoimmunity as well as
being a mainstay of ongoing basic research in many areas of
biology. But immunology is one of the least static of any area
of modern biology, and so one must always ask what’s next.
The purpose of this perspective is to suggest that human immu-
nology is the answer not as a replacement to mice, but as an
increasingly rich field in itself; scientists are taking advantage
of many new technologies and events (like the ongoing
pandemic) to expand our immunological universe into places
that mice cannot go, such as the interplay of the immune system
with thousands of human diseases, genetic and environmental
diversity, and the effects of the many medical treatments on peo-
ple’s immune systems (Pulendran and Davis, 2020). There is also
the distinct possibility that entirely new immunological mecha-
nisms will be discovered in human beings given the many unique
genes that are known and the fact that human infants have to
survive many more years than mice in sufficient numbers to allow
the species to survive. Within this context, we will discuss the
various ways immunologists have tried to compensate for
mice, collaborative cross mice, pet shop mice, etc. and how
these have definite advantages and yet significant difficulties in
terms of wide adoption and utility, which is not to say that human
immunology exists in isolation, because in most cases one must
continually go to mice to rigorously test hypotheses. An excep-
tion to this is human genetic lesions that affect the immune sys-
tem, where there is a clear cause and effect, although pheno-
types can vary widely. Human genetics has even led the way in
some cases, such as cloning the autoimmune regulator (AIRE)
gene in patients with a rare autoimmune disease (Nagamine
et al., 1997; Aaltonen et al., 1997), leading to an explosion of
work on the equivalent gene in mice and the elucidation of its
key role in immune tolerance (Mathis and Benoist, 2007). Simi-
larly, the identification of another key gene in tolerance, FoxP3,
was discovered in humans (Bennett et al., 2001) just slightly
before the mouse equivalent was identified (Brunkow et al.,
2001), and an even more prodigious amount of work occurred
in defining role of this gene (Rudensky, 2011). In any case, the
technology to analyze human immune responses is advancing
rapidly and has tremendous potential because there is so
much unexplored territory. But also important is the way human
immunology provides a different and consequently broader
perspective on basic immune mechanisms. Longer term, the
many differences seen between mice and human immune re-
sponses and the structural differences in skin and spleens high-
lighted here suggest that new mechanisms may emerge from
human work, especially as they relate to the lengths infectious
pathogens will go to evade the human immune system.
Human immunology provides a valuable alternative
perspective
The first time you visit a country and culture outside your own,
you immediately become more aware of the many normally

Immunity 55, August 9, 2022 ª 2022 Published by Elsevier Inc. 1343

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‘‘hidden assumptions’’ about your own country and culture and
thus develop a more informed perspective. In a similar way,
because studies of inbred mice have been so successful and
have dominated the field for over sixty years, we have not had
much empirical input from other species to shed light on what
might be important hidden assumptions in contemporary immu-
nology. Here, human studies are an ideal complement because
they have the genetic diversity that inbred mice lack, and they
have the continuous exposure to infectious diseases that are
scrupulously avoided in modern mouse facilities. A very vivid
example of this to one of the authors of this perspective
(M.M.D.) was when Yu Wong asked what the pre-immune fre-
quency of pathogen-specific CD8+ T cells might be in blood
bank donors following the groundbreaking work on mice from
Moon et al. (2007), who used a sensitive tetramer staining enrich-
ment method (Day et al., 2003) to quantitate the pre-immune
T cell receptor (TCR) repertoire. As a control, we also analyzed
self-specific T cells and to our surprise found that the fre-
quencies were very similar (Yu et al., 2015; Su et al., 2013). We
also looked at the Y chromosome-encoded SMCY antigen-spe-
cific T cells in males and females and found a 2% to 50%–65%
reduction in the former versus the latter and almost no difference
between male and female mice (using the famous H-Y antigen).
These results were confirmed a short time later with results in
mice by Moon and colleagues (Legoux et al., 2015), who identi-
fied Cre-specific CD4+ T cells and showed that if Cre was
expressed ubiquitously, there was only a 50% reduction in fre-
quency, but in cases of organ-specific expression, there was
no detectable deletion. These results have been shocking
because of classic experiments with super antigens, and espe-
cially TCR transgenics in the late 1980s, that have shown that
self-specific T cells are massively deleted in the thymus of
mice and thus are a major component of what was called ‘‘cen-
tral tolerance’’ (Herman et al., 1991; Von Boehmer, 1990). While
there was also persistent evidence of clonal anergy (Goodnow
et al., 1988, 1989; Miller, 1993; Miller and Morahan, 1992; Pike
et al., 1982) being a factor, negative selection has been widely
accepted as the major mechanism of self-tolerance for more
than 25 years and seems to confirm Burnet’s earlier speculation
that in order to avoid autoimmunity, self-reactive immune cells
are eliminated during fetal development (Burnet, 1959).
But clearly, while negative thymic selection is a real phenom-
enon, as confirmed by the papers cited, the magnitude of its ef-
fects has been greatly exaggerated by implanting specific TCRs
into the mice, which causes the TCRs to be expressed earlier in
thymic development. For the superantigen results, this early
expression was probably due to the abundance of these partic-
ular self-antigens and the triggering of many TCRs at once.
It is important to note, though, that these experiments were the
only way this issue could be addressed at the time. Tetramers,
and the sensitivity needed, were many years in the future. But
most importantly, these results force a re-evaluation of how
important negative selection is to prevent autoimmunity. Clearly,
it’s not as important as it was thought to be because the evi-
dence shows that even healthy people have a significant fraction
of self-specific T cells, and not just of a low affinity, because
many can bind peptide-MHC tetramers perfectly well. What
this re-evaluation has led to is the fundamental question of
what shapes the TCR repertoire—if efficient negative selection
1344 Immunity 55, August 9, 2022
Perspective
is not important, what takes precedence? Following Dobzhan-
sky’s dictum that ‘‘[n]othing in biology makes sense except in
the light of evolution’’ (Dobzhansky, 1973) leads to the realization
that autoimmunity could not be a driver of TCR repertoire
because it has such a modest effect on selection in children
and young adults (<1%). In contrast, infectious diseases, before
widespread vaccination, antibiotics, and sanitation, have killed
almost half of children under 5 and many older children and
young adults as well (Casanova and Abel, 2005; Su et al.,
2013). This was the case for many generations and probably
started soon after the invention of agriculture when people
started living and trading in fixed, dense communities. This de-
gree of child mortality was even worse in the plague years. If in-
fectious diseases are the primary driver of immune repertoire,
then it makes sense to have peripheral T cells expressing as
many specificities as they can and finding other ways to hold
self-specific T cells in check, unless they are needed, as Yu
et al. (2015) provided evidence for. Indeed, a number of classic
papers in murine immunology show that this is the case—that
self-specific T cells are quiescent unless triggered by an infec-
tion that includes their cognate antigen (Ohashi et al., 1991;
Röcken et al., 1992).
There are other examples, but the lesson here is that even
when the basic immune mechanisms are the same between
mice and humans, the fact that we often must approach human
immunology in ways that are different than mice can add an
important perspective. In some cases, such as the one cited,
this can completely change our understanding of the phe-
nomenon.
Mouse models of disease
Since the development of inbred mouse strains by Snell in the late
1940s (Gorer et al., 1948), mouse models of cancer have been a
major activity by immunologists, soon joined by those interested
in autoimmunity and infectious diseases. By the 1980s, the advent
of recombinant DNA methodology and transgenics and then ge-
netic ablation, etc. made mouse models of disease the premier
route to understanding the role of the immune system in virtually
every disease where this was thought to be relevant. And it
became a given that if you were doing serious science, and espe-
cially medically relevant ‘‘translation’’ of that science, you had to
be working on a mouse model. There also arose the prejudice
that good science was ‘‘hypothesis driven and mechanistic’’
and that ‘‘descriptive’’ became an epithet to describe work that
was primitive and hopelessly old fashioned. This implicitly
discouraged work on human beings because there were few
methods that could be applied ethically to those studies; most
work could only be descriptive and correlative. But this ignores
the fact that all science starts with description, and close and thor-
ough observation is key to discovering new phenomena and
formulating useful hypotheses. A mechanistic understanding is
of course the ultimate goal, but it can take many years of observa-
tion and experiments to get there. Even then, an actual mecha-
nism can be elusive. Even in physics one often settles for an accu-
rate formula. The failure of many of these mouse translational
models was noted many years ago in autoimmunity (von Herrath
and Nepom, 2005), cancer immunology (Steinman and Mellman,
2004), and stem cell biology (Payne and Crooks, 2007). But
also, aside from these critiques, in the academic world, one rarely

Perspective
hears about the many failures of mouse models to "translate" into
effective clinical treatments. This is because this work is almost
entirely done by biotech or pharmaceutical companies, and fail-
ures are rarely published because no one wants to spend the
time on them, and journals or reviewers don’t welcome them.
This results in a classic "publication bias" where only the good
news gets out there. But those of us who advise companies widely
(M.M.D.) know that mouse models are viewed with great skepti-
cism by the people who actually need to find useful drugs. Earlier
on, these models were almost the only source of information, but
recent advances in technology have created other options. I
asked Frank Nestle, global head of research and chief scientific
officer at Sanofi, to comment on how mouse models are viewed
in biotech and pharma:
The translational drug discovery paradigm is changing from a
mouse-first to a human-first approach. We are in the age of hu-
man immunology, where we are tapping into unprecedented
orthogonal data sets (genetics, single cell transcriptomics, pro-
teomics, metabolomics, real world clinical data, [etc.]) to drive
our target discovery and translational research engine. While
some mechanism-based mouse models retain relevance, dis-
ease-mimicking mouse models are becoming more and more
the tools of the last decade.
Nevertheless, even though mouse models of disease have
often been disappointing as a seamless path to human treat-
ments, as noted earlier, they have been a gold mine of information
about how the immune system works and will continue to be of
great value, especially as more details of human diseases are un-
covered, and we can determine exactly where the systems
converge. Progress on finding these convergences informatically
has been reported by Shen-Orr and colleagues (Normand et al.,
2018). Even better would be a broad program of comparing hu-
man immune deficiencies and their detail phenotypes with mice
with the same genetic lesions; while a number of discordances
have been noted, there has been no systematic program to date.
Similarities and differences
Humans and mice share many, although not all, of the genes and
cell types we know as important in human function: the same
types of V, D, and J gene segments in T cell receptors and immu-
noglobulin genes, as well as the same rearrangement apparatus
that goes back to early vertebrates, plus many of the same or
similar cytokine receptors and innate apparatus toll-like recep-
tors (TLRs), etc. and MHC molecules, along with basically all
the nuts and bolts of adaptive and innate immunology that we
know about at this time, but with some exceptions. One such
well-known example is the expression of class II human leuko-
cyte antigen (HLA) molecules on activated human T cells, which
is not seen in mice. This suggests that these human T cells can
serve as ‘‘professional antigen-presenting cells’’ and act on
CD4+ T cells to promote specific responses independently of B
cells or dendritic cells (DCs). Whether this has any major conse-
quences remains to be seen. Benoist and colleagues at the
Immunological Genome Project (IMMGEN) consortium have
done a deep dive into the thousands of genes that they and
others have identified as being expressed in immune cells
beyond those having generic functions and find that the vast ma-
jority of homologs are expressed in the same cell subsets (Shay
et al., 2013). However, a considerable number are expressed
ll
differently: at least 169 genes, using very stringent criteria. This
is not a trivial number and may contribute to the many differences
seen phenotypically between the species. Also, it appears that
there are differences in the magnitude of expression that could
make a substantial difference, as indicated by the activation of
CD4+ T cells between the two species (Shay et al., 2013). There
may also be major differences in posttranslational modifications,
but that is much more difficult to assess comprehensively. In
addition, many immunological cues in the form of cytokines
and chemokines are provided by non-immunological tissues,
so this is another potential source of divergence to be investi-
gated, which may be particularly relevant to differences in tissue
or organ architecture, as discussed below.
To this point, the structure of tissues and organs relevant to the
immune system between humans and mice suggests functional
differences. One of these is skin, a major starting point and target
of many pathogens. As shown in Figure 1 (Zomer and Trentin,
2018), human skin is much thicker than mice skin (
43) and
has 5–10 cellular layers, versus 2–3 for mice (Zomer and Trentin,
2018). The epidermal layers of skin in both species contain den-
dritic, antigen-presenting Langerhans together with CD4+ and
CD8+ T cells, but only mice have the mysterious dendritic
epidermal T cells (DETCs), with their uniform gd T cell receptors
(Allison and Havran, 1991; Johnson et al., 2020) apparently per-
forming a specific sensory function, but also lacking in ab T cells.
The dermis layer in both species contains a broader mix of im-
mune cells, ab and gd T cells, innate lymphocytic cells (ILCs),
DCs, and macrophages. The thickness of human skin also allows
a direct connection to the lymphatic system (Pasparakis et al.,
2014). Another striking difference in structure has emerged
from the analysis of human spleens compared with mice
spleens, which show some striking anatomical differences.
Figure 2 from Lewis et al. (2019), with data also drawn from Stei-
niger (2015), shows how anatomically different human spleens
are from mice spleens. While both types of spleens have red
pulp and white pulp regions, in humans, the lymphocyte-rich
areas (white) (where most of the adaptive immune response oc-
curs) have a very different distribution of T cells, which are
diffused in human germinal centers (GCs) versus discrete clus-
ters in mice; human spleens also have an absence of a clear
bridging outer layer. Whether these striking differences have
any impact on immune responses is not clear.
DeBoer and colleagues (van Hoeven et al., 2017) have also
noted a striking difference in thymic output and T cell half-life be-
tween mice and humans, with the mice having a much shorter
half-life than humans; this makes sense in terms of the huge dif-
ference in life spans between the species. And if other immuno-
logical properties are speeded up this way in mice, it means that
human responses might play out over a much longer timescale.
Other anomalies have emerged from the study of human immune
deficiencies in cases where they can be directly compared with
the equivalent mouse model. In the case of CD28, a key co-stim-
ulatory molecule, mice lacking this gene have very severe im-
mune dysfunction with susceptibility to multiple pathogens,
whereas humans with this deficiency are largely healthy and
apparently only susceptible to skin papilloma viruses (Béziat
et al., 2021). Another discordant gene is ISG15, an interferon-
inducible, ubiquitin-like intracellular protein that is necessary
for protection against viruses in mice; human beings deficient
Immunity 55, August 9, 2022 1345
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in this gene seem to have reasonably effective anti-viral re-
sponses, but deficient microbial defenses (Bogunovic et al.,
2012; Zhang et al., 2015).
These comparisons of the same genetic lesions in both spe-
cies give us some of the clearest evidence we have of impor-
tant functional differences between the species but with the
important caveat that humans lead much more complex lives
in terms of environmental exposure than inbred mice. But in
many cases, pursuing information about a gene from humans
to mice has been very fruitful and shed new light and context
as to its function. This is particularly true in the case of
GCN2, whose expression unexpectedly has arisen in an anal-
ysis of a human cohort exposed to a yellow fever vaccine
and correlated with an enhanced CD8+ T cell response (Querec
et al., 2009). Analyzing the effects of deleting the murine homo-
log, Pulendran and colleagues have found that this gene is
involved in the amino acid starvation response and the sup-
pression of intestinal inflammation, which leads to an enhanced
immune response (Ravindran et al., 2016) and insights into the
control of autophagy and reactive oxygen species (ROS). Thus,
the mouse system can provide much greater depth to a human
result when the systems are in alignment. Currently, there are
485 known immunodeficiency genes that have been identified
in human beings (Tangye et al., 2022). In addition, it has
become clear that a number of older individuals make autoan-
tibodies against particular cytokines, which attenuate pathogen
responses that depend on those cytokines with sometimes
fatal results, as shown in recent work on COVID-19 patients
(Bastard et al., 2020; Chang et al., 2021).
The effects of environment and genetic diversity
To reduce genetic variation, immunologists have embraced
inbred mice, originally developed by Snell et al. (Gorer et al.,
1948) in the 1950s. More recently, since mouse disease outbreaks
can wreak havoc on immunological experiments, increasingly
cleaner and more stringent mouse husbandry methods have
been introduced, up to and including ultraclean methodologies
that involve rederiving all mouse lines by cesarian delivery.
1346 Immunity 55, August 9, 2022
Perspective
Figure 1. A comparison of mouse and
human skin (Zomer and Trentin, 2018)
Human skin is much thicker and is more complex
than mouse skin. In their immune composition,
mice and humans differ in their epidermal layer,
whereas mice have the unique dendritic epidermal
T cells (DETC) cells with their uniform gd T cell
T cell receptors but share with humans Langer-
hans and CD4 and CD8 T cells. Adapted with
permission from Nature (Pasparakis et al., 2014),
copyright 2014.
These efforts make human compari-
sons problematic, especially for genetics,
where most of the many gene-manipu-
lated mice are within the same strain,
C57 BL/6. A modern fix to this is the
collaborative cross system (Welsh et al.,
2012), which consists of five inbred strains
of mice and three from wild mice that have
been crossed and then backcrossed
extensively to fix each of the scrambled genomes into a separate,
homozygous strain (Iraqi et al., 2008; Shorter et al., 2019). Over
160 separate strains have been produced; with most of these hav-
ing their genomes sequenced (Shorter et al., 2019), these strains
have been very valuable in showing how different gene polymor-
phisms in mice can alter infectious disease responses (Noll
et al., 2020; Martin et al., 2020). This system has also been used
to show how some of these strains mimic human drug responses
(Zeiss et al., 2019). These are very valuable studies that show how
the standard inbred strains give us only a very limited view of ge-
netic influences on important phenomena, but they involve
analyzing sometimes over a hundred different mice (Martin
et al., 2020), so it’s a major commitment with no guarantee of rele-
vance to human immunology. Research on these mice also
cannot benefit from the many examples of genetic ablation
models and other manipulations without a great deal of effort.
The lack of constant microbial exposure is also a problem, as
clearly demonstrated by Masopust and colleagues, who very
cleverly introduced mice obtained from pet shops to cohabitate
with C57BL/6 mice and have shown that those that survived had
acquired tissue resident T cells (Beura et al., 2016). But this
would be difficult to do in modern mouse houses, as it would
require a separate containment facility so as not to endanger
many of the users, animals, and studies.
Finally, in 1988, McCune et al. (1988) introduced severe com-
bined immunodeficiency-Human (SCID-Hu) mice, a novel effort
to reconstitute immunodeficient mice with a human immune sys-
tem in order to create ‘‘humanized’’ mice that could faithfully model
human responses. Despite many improvements over the years
(McCune, 1996; Yong et al., 2018; Rongvaux et al., 2013), there
are still issues with getting good B cell responses, although many
aspects seem to model human immune activities adequately.
Again, this approach is not able to employ the power of genetic
modifications that have made inbred mouse strains so powerful.
Do humans have novel immune mechanisms?
There is a reasonable hope that human beings might have unique
immune mechanisms because they are much less fecund and

Perspective
take much longer to reach sexual maturity. Thus, it’s fine if the
average mouse lives six months or so, but it’s a major problem
for survival of the species if enough humans don’t make it to
25–30 years. This means that if the risk of a fatal infectious dis-
ease is relatively constant, then humans are
503 more vulner-
able over the average lifetime. Thus, we may find additional
‘‘immunological defense’’ mechanisms in human beings if we
look hard enough. How might we find these if they exist? One
way might be the repurposing of genes that are shared between
mice and humans, as discussed above. And thus, those 169
genes with different expression patterns between mice and hu-
mans might mediate novel functions (Shay et al., 2013) cited
earlier. But another way to look for human or primate-specific
mechanisms would be to identify human-specific genes versus
mouse-specific genes. Here we have used the Ensemble
Program to search for likely genes that are unique to humans
versus mice (Table 1).
The program compares sequence homologies to determine
whether a human gene is sufficiently different from mice to be
considered unique. The list is not long, and while it may just
reflect divergent evolution and minor tweaking of functions that
are well known from the many intensive studies of mouse immu-
nology, given the many apparent differences seen for years be-
tween mouse models and clinical outcomes, these genes and
those identified by Shay and colleagues (Shay et al., 2013)
may be well worth following up on for clues regarding possible
novel immunological mechanisms.
Although the above examples show that even with respect
to the same molecules and organs, humans and mice exhibit
clear differences, another way to ask the question is to identify
genes that are unique in humans because these may lead to
even more profound differences and mechanisms. Thus, we
have taken advantage of a rich dataset of RNA sequencing
data for 29 different human immune cell subsets that have
been isolated and analyzed for gene expression (Monaco
et al., 2019).
The table listing all human genes based on Genome Refer-
ence Consortium human build (GRCh38.p13) was downloaded
from Ensembl BioMart (Kinsella et al., 2011). Genes whose
‘‘gene type’’ is ‘‘protein coding’’ were selected from the table.
Next, the table listing mouse genes based on GRCm39 and
their human homologs were downloaded from Ensembl
BioMart. Human genes from the former table that do not
have any mouse homologs in the latter were identified as
candidate-unique human protein-coding genes. In addition,
the National Heart, Lung, and Blood Institute (NHLBI) website
(https://www.nhlbi.nih.gov/) collates a very extensive collection
of species data on all known genes, and with this resource we
have been able to eliminate many genes that did have identified
homologs in mice and other species. We also used the UniProt
blastp program, using the protein sequence to search the
mouse genome to make sure there were not any mouse
sequence homologies of significance (Bateman et al., 2021).
The resulting summary of this work is presented in Table 1
together with some of the information about cell-type distribu-
tion and other species that had this particular gene. Many of
these genes have rat homologs, suggesting that their lack in
mice was not due to very different functionality. But a few
were exclusively in primates, which would fit with novel immune
ll
functions related to increased longevity and low fecundity. Also
noteworthy is the large number of unique genes in human DCs,
neutrophils, and basophils, which may correlate with the diffi-
culty encountered in modeling allergy and other human pathol-
ogies involving those cells in mice.
Also, this list should not be considered the last word; we in fact
missed intercellular adhesion molecule-3 (ICAM3) and TLR10 in
this survey, which came up anecdotally, so there are likely
others.
The entire workflow of identifying the human genes, matching
with immune population genes, and running blastp can be
found in GitHub repositories (https://github.com/KwatMDPhD/
immune_populations.pro and https://github.com/KwatMDPhD/
human_genes.pro).
It was also beneficial to vet candidate genes on the very helpful
NHLBI website, which compares particular genes across many
species.
In the table, it is interesting to note the relatively large num-
ber of human genes in DCs, neutrophils, and basophils. This
may be an explanation for why many of the anomalies be-
tween mice and humans occur in the innate immune mecha-
nisms that these cells mediate. Specifically, DCs play a pivotal
role in gathering and presenting antigens to T cells and have
long been thought to be somewhat different between humans
and mice, but exactly how and what the consequences might
be are unclear. For example, in humans, TLR7 and 9 are
mostly expressed in plasmacytoid DCs, and thus, agonists
for these TLRs can only activate these cells, whereas in
mice TLRs7 and 9 are more broadly expressed on other sub-
sets of DCs. The power of recent advances in single-cell tran-
scriptional analysis has great potential in illuminating human-
mouse differences, but the limitations are also evident in a
recent analysis of DCs in the two species by Rudensky and
colleagues. They note clear differences in subsets, but
because a functional readout is not readily available, whether
these differences are consequential is not clear, which is a
general problem for all of these genes that don’t have a mouse
homolog, pointing to the need for a complete human system
that can be analyzed. Here, humanized mice or immune orga-
noids are possible answers.
The number of unique neutrophil genes is also interesting and
may underlie some of the observed discordances between mice
and humans, especially given the very different composition of
these cells in circulating white blood cells (60%–70% in humans
versus only
30% in inbred mice) (Junhee et al., 2013; Nauseef,
2014, 2019). But again, until we can probe more deeply into a
human assay system, it remains a mystery how significant these
are.
Lastly, the number of basophil genes is interesting and poten-
tially explains the difficulties in modeling human allergic reac-
tions in mice.
Our main hope in presenting these results is not to resolve
questions about their importance but to tantalize readers with
the possibility that some of these genes might mediate novel im-
mune mechanisms. We also caution about thinking about indi-
vidual genes in isolation as they always work in pathways where
they can be influenced by polymorphisms in other genes or by
different environmental factors such as pathogens or micro-
biome components.
Immunity 55, August 9, 2022 1347
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Another way we are likely to discover additional mechanisms
in immunology is by taking advantage of the vast number of hu-
man pathogens and learning what their strategies are for avoid-
ing or distracting the immune system. These mechanisms may
not be unique to humans, but the sheer number of human path-
ogens compared with the few commonly used in mouse
models suggests that there is a wealth of insight to be gained
and, enabled with recently developed technologies, important
discoveries to be expected. Indeed, Rolf Zinkernagel (Zinkerna-
gel, 1996) entitled a famous review (‘‘Immunology taught by vi-
ruses’’) to make this point years ago, but only now do we have
the ability to analyze human responses to pathogens in depth.
Some pathogen strategies are well known, like HIV for
example, which uses an error-prone polymerase to produce
an extreme degree of genomic diversity, producing thousands
of quasispecies in each infected individual, eventually wearing
down the immune system’s ability to keep up (Bbosa
et al., 2019).
T. brucei, a parasite that causes sleeping sickness, is well
known for employing thousands of possible surface proteins
where immune pressure against one type leads to another one
becoming dominant (Cross, 1990). But these strategies are
very simple compared with the complexities of other pathogens.
Cytomegalovirus (CMV) is remarkable in both its lifelong staying
power and its ability to enhance immune responses against other
viruses in both mice and humans (Furman et al., 2015). Addition-
ally, it has a number of mechanisms to subvert the immune sys-
tem, as reviewed by Goodrun et al. (2021).
1348 Immunity 55, August 9, 2022
Perspective
Figure 2. Mouse and human splenic
immune cellular architecture at steady state
There are structural differences between the mu-
rine (left) and human (right) splenic immune sys-
tem, most notably the organization of T cell zone
([TC], turquoise; also known as pediatric advanced
life support [PALS]) and B cell zone (BCZ) follicles
(gray and shades of blue, shown with light zone
[LZ] and dark zone [DZ], organization in mouse
spleen) within the WP and the border between the
WP and RP, the marginal zone (MZ) in mouse, or
perifollicular zone (PFZ) in human (dark blue outer
ring). Because applications of advanced imaging
techniques to the human spleen have been
limited, the extent to which the mouse MZ and
human PFZ are analogous remains unknown. For
example, the precise layering and composition of
macrophage subsets in the MZ is known for mice
(see bottom left box). CD169+ MMMs (dark blue)
form a concentric ring around the WP with MZMs
(light blue) and MB cells (darker blue), but not for
humans. In humans, MZB cells surround activated
B cells, containing a GC (light blue in the human
spleen on the right) and corona (gray, "Cor"). The
homeostatic location of DC subsets in mice is
shown (with cDC2s in the bridging channel [BC]
and cDC1s in the TCZ, MZ, and red pulp [RP]).
Release of blood into the MZ of the WP from a
central arteriole (CA) is shown. From Science
Immunology (Lewis et al., 2019). Adapted with
permission from AAAS.
But recent work has shown even
greater subtlety, where work by Picker
and colleagues has shown that the vac-
cine vector that they made was remark-
ably effective against both simian immunodeficiency virus (SIV)
and tuberculosis (TB) in monkeys (Hansen et al., 2010, 2016; Ma-
louli et al., 2021; Verweij et al., 2021), and this was accompanied
by a novel HLA-E-restricted T cell response and class II MHC-
restricted CD8+ T cells. Just last year, it was reported that the
class II-restricted CD8+ T cells were not important for these
remarkable vaccine responses, indicating that the HLA-E ones
were. Also telling was the finding that no fewer than seven
CMV genes seemed to be engaged in diverting T cell responses
away from HLA-E (Yang et al., 2021). Thus, while T cell re-
sponses against CMV antigens are quite active, accounting for
an estimated 10%–20% of all CD8+ T cells in carriers, there
are no known cases of the virus being cleared from anyone.
Another interesting fact is that in 16 monozygotic twins discor-
dant for CMV seropositivity, there have been massive changes
in immune system variables in the positives versus the negatives
(Brodin et al., 2015). The implications of all this are not very clear
other than that the virus has things under control, and we have a
lot to learn about what it is up to. The same is probably true for
many of our other pathogens as well.
The way forward
Almost 14 years ago, one of us (M.M.D.) wrote in this journal a
plea for the immunology community to not just pay lip service
to human immunity, but to put serious effort into welcoming it
to the fold as a distinct, but vitally important, complement to
the juggernaut that is mouse immunology (Davis, 2008). There
is still a long way to go to catch up to that standard, but human
 Perspective
Table 1. Shows genes in human immune cells that are not found in inbred mice and thus may mediate novel mechanisms. But note that many are shared with rats and thus are
likely not related to longevity, but nonetheless may mediate important functions in human beings
The human protein atlas tissue
Gene Immune population specificity Group Top blastp against mouse
GLYATL1B B naive breast, lymphoid tissue glycine-N-acyltransferase-like 1B 0.412 primates – – –
ZNF860 B naive + memory lymphoid tissue zinc fingers C2H2-type 0.438 primates – – –
PLEKHG7 B naive + memory cervix, epididymis, fallopian tube Pleckstrin homology domain 0.258 mammals rats sea urchins –
containing/Dbl family Rho GEFs
PLAAT2 plasmablasts intestine phospholipase A and primates – – –
acyltransferase family
ZNF683 T CD8 TE testis zinc fingers C2H2-type 0.56 mammals rats – –
TRABD2A T naive intestine, lymphoid tissue, ovary TraB domain containing 2A 0.648 broad fish – –
FAAH2 Tfh + Th multiple tissues including lymphoid fatty acid amide hydrolase 2 0.304 broad fish Drosph xeno
GNLY T, NK bone marrow, lymphoid tissue granulysin, cytotoxic T cells, 0.292 broad – – –
NK cells
KIR2DP1 NK lymphoid tissue killer cell immunoglobulin like 0.401 mammals – – –
receptors
KLRF1 NK bone marrow, lymphoid tissue killer cell lectin like receptors/C-type 0.303 mammals – – –
lectin domain containing
LGALS9B NK esophagus, intestine, stomach 1 galectins 0.685 – – – –
LGALS9C NK esophagus, intestine, stomach 1 galectins 0.688 – – – –
CD1B mDCs lymphoid tissue CD molecules/C1-set domain 0.489 mammals – – –
containing
CD1E mDCs lymphoid tissue CD molecules/C1-set domain 0.521 mammals bats chinchilla –
containing
CLIC2 mDCs multiple tissues including lymphoid chloride intracellular channels 0.662 broad rats – –
KCNK17 pDCs lung, thyroid gland potassium two pore domain channel 0.42 broad Drosophila – –
subfamily K
NLRP7 pDCs testis NLR family/pyrin domain containing 0.408 mammals – – –
CTSV pDCs lymphoid tissue cathepsins 0.792 mammals mole rat – –
PROC pDCs liver receptor ligands/Gla domain 0.695 mammals rats – –
containing
Immunity 55, August 9, 2022 1349

SLC9C2 pDCs brain, choroid plexus, testis solute carriers 0.292 mammals rats – –
ADGRE3 granulocytes LD bone marrow, lymphoid tissue adhesion G protein-coupled 0.459 mammals – – –
receptors, subfamily E
NT5DC4 monocytes I testis 50 -nucleotidase domain containing 4 0.654 broad – – –
CASP5 monocytes NC brain, intestine, lymphoid tissue caspases|caspase recruitment 0.636 broad – – –
domain containing
LILRA1 monocytes NC + I lymphoid tissue CD molecules/activating leukocyte 0.507 primates – – –

ll
immunoglobulin like receptors
(Continued on next page)
1350 Immunity 55, August 9, 2022

Table 1. Continued
The human protein atlas tissue
Gene Immune population specificity Group Top blastp against mouse

ll
LILRB2 monocytes NC + I bone marrow, lung, lymphoid tissue CD molecules/inhibitory leukocyte 0.452 mammals rats – –
immunoglobulin-like receptors/Ig-
like cell adhesion molecule family
LILRA2 myeloid bone marrow, lymphoid tissue CD molecules/activating leukocyte 0.513 primates – – –
immunoglobulin like receptors
SIGLEC14 myeloid bone marrow, lymphoid tissue V-set domain containing/sialic acid 0.498 mammals
binding Ig-like lectins
– – lung, lymphoid tissue – – – – – –
OR14L1P basophils LD brain olfactory receptors, family 14 0.53 mammals reptiles – –
OR6K3 basophils LD bone marrow olfactory receptors, family 6 0.784 mammals rats – –
OXER1 basophils LD liver leukotriene receptors 0.418 mammals – – –
TRIM49D1 basophils LD undetected ring finger proteins/tripartite motif 0.364 primates – – –
containing
TRIM49D2 basophils LD undetected ring finger proteins/tripartite motif 0.364 primates – – –
containing
TRIM51 basophils LD undetected ring finger proteins/tripartite motif 0.367 – – – –
containing
TRIM64 basophils LD placenta ring finger proteins/tripartite motif 0.348 – – – –
containing
TRIM64B basophils LD placenta ring finger proteins/tripartite motif 0.348 – – – –
containing
FCAR neutrophils LD bone marrow, lung, lymphoid tissue CD molecules/immunoglobulin-like 0.327 – – – –
domain containing/Fc receptors
H2AC19 neutrophils LD multiple tissues including lymphoid H2A histones 1 – – – –
CSNK1A1L neutrophils LD testis casein kinase 1 family 0.91 broad Drosophila – –
LINC02218 neutrophils LD undetected long intergenic non-protein 0.441 – – – –
coding RNAs
PEAK3 neutrophils LD bone marrow, lymphoid tissue PEAK family member 3 0.268 broad fish – –
PI3 neutrophils LD esophagus, lymphoid tissue, vagina WAP four-disulfide core domain 0.941 broad chkns – –
containing
S100A12 neutrophils LD bone marrow S100 calcium binding proteins/EF- 0.388 mammals rats – –
hand domain containing
SIRPB2 neutrophils LD lymphoid tissue V-set domain containing/signal 0.348 mammals rats – –

Perspective
regulatory proteins
SIRPD neutrophils LD testis V-set domain containing/signal 0.48 mammals rats – –
regulatory proteins
ST20 neutrophils LD multiple tissues including lymphoid suppressor of tumorigenicity 20 0.724 ? – – –
TMEM272 neutrophils LD brain transmembrane protein 272 0.368 mammals – – –

Perspective
immunology is definitely getting there. And it isn’t just conceptual
or abstract anymore, because in the last 2+ years, we have all
been affected by this horrendous pandemic and it has had
such a wide range of health effects, from negligible symptoms
to painful death. It has shown that serious, life-threatening infec-
tions are not just something that happen to unfortunate countries
far away from most of us; it can also be close and very real. And
as immunologists, this has to hit close to home and make clear
the urgency of why understanding the human side of immu-
nology, with its complexity and wide variations, is critical. And
even as this pandemic fades, as it might, another is brewing
somewhere.
Clearly, inbred mice and the amazing technology built up
over the past 60+ years are such a powerful and useful system
that it will continue to be a mainstay of the field for the fore-
seeable future. Even when investigating new human phenom-
ena, being able to go to mice to investigate mechanisms and/
or confirm hypotheses has been invaluable to my group and
many others. But it is also important not to ignore the many
variances, whether genetic, anatomical, or in gene expres-
sion. It is unlikely that all of these represent minor tweaks in
the human immune system, and so we could be missing
important aspects of human immunity. While humanized
mice might represent part of the answer in exploring these
anomalies, there will always be questions about how faithfully
these hybrid animals can represent human immunity, espe-
cially given the constraints of mouse husbandry. Work on
monkeys is one solution, which has been used to great effect
in many studies, but they are extremely expensive and in short
supply. Thus, the advent of human immune organoids using
discarded tonsils or spleens may be the key to being to be
able to do mechanistic and hypothesis-driven experiments
that will be needed (Wagar et al., 2021). The advent of gene
modifications using CRIPR-Cas9 is also introducing powerful
ways to screen for important genes such as the genome-
wide screen of CD8 T cells by Marson and colleagues or early
work on immune and intestinal organoids (Shifrut et al., 2018;
Fujii et al., 2015; Wu et al., 2018).
In conclusion, while human immunology is still very much a
work in progress, there is so much potential and uncharted ter-
ritory to explore, and with such a strong foundation from mouse
immunology and continued interactivity between the two sys-
tems, one can’t help but to be optimistic about what we will learn
in the coming decades.
ACKNOWLEDGMENTS
We thank J.L. Casanova, Bali Pulendran, Paul Kubes, Stephanie Eisenbarth,
Karan Kathuria, Juliana Idoyaga, Guangbo Chen, Chen Wang, Xin Chen, Ed
Mocarski, and John Boothroyd. We thank the Howard Hughes Medical Insti-
tute, the Open Philantropy Foundation, and NIAID AI057229 for support.
DECLARATION OF INTERESTS
The authors declare no competing interests.
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