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A novel therapeutic approach in asthma: depleting CD52-expressing leukocytes suppresses airway hyperreactivity and ameliorates lung inflammation

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A novel therapeutic approach in asthma: depleting CD52-expressing leukocytes suppresses airway hyperreactivity and ameliorates lung inflammation

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A novel therapeutic approach in asthma: depleting
CD52-expressing leukocytes suppresses airway
hyperreactivity and ameliorates lung inflammation

By

Pedram Shafiei Jahani

A Thesis Presented to the Faculty of

THE USC GRADUATE SCHOOL UNIVERSITY
OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
of
MASTER OF SCIENCE

in

Molecular Microbiology and Immunology

August 2019

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TABLE OF CONTENTS

ABBREVIATIONS ...………………………………………………………………………………………...……. 4
LIST OF TABLES AND FIGURES ……….……………………………………………………………..………. 5
SUMMARY ...………………………………………………………………………………………………………. 6
CHAPTER 1: INTRODUCTION ...…………………………………………………………………….…………. 7
1.1 Asthma: a rising disease of civilizations …………………………………………………………… 7
1.2 Current diagnosis and treatments …………………………………………………………….…… 7
1.3 Pathogenesis of asthma as a TH2-dependent autoinflammatory immune disorder ………….. 8
1.4 Pathogenesis of asthma as an ILC2-dependent autoinflammatory immune disorder ………. 10
1.5 Emergence of immunotherapy as a novel way to treat heterogeneous set of immune-related
disorders …………………………………………………………………………………..………… 11
1.6 Cluster of differentiation (CD52) as a promising a target ……………………………………….. 12
CHAPTER 2: MATERIALS AND METHODS ……………………………………………………………….... 14
2.1 GWAS Study Populations ………………………………………………………………………….. 14
2.2 GWAS Analyses in UK Biobank …………………………………………………………………… 14
2.3 Meta-analyses for asthma in UKBB and TAGC ………………………………………………..… 14
2.4 Validation of known asthma loci …………………………………………………………………… 14
2.5 Enrichment of novel asthma loci in epigenetic marks …………………………………………… 15
2.6 Animals …………………………………………………………………………………………….… 15
2.7 Isolation of murine pulmonary lymphocytes and ILC2s ……………………………………….… 15
2.8 Isolation of murine splenic lymphocytes ………………………………………………………...… 15
2.9 Humanized mice and Isolation of human peripheral blood mononuclear cells (PBMCs) ….... 15
2.10 Cell Culture Conditions ………………………………………………………………………….… 16
2.11 Measurement of airway hyperreactivity (AHR) and collection of bronchoalveolar lavage (BAL)
fluid ……………………………………………………………………………………………………..… 16
2.12 Flow cytometry analysis and reagents …………………………………………………………... 16
2.12.1 Murine bronchoalveolar lavage (BAL) fluid analysis via fluorescence-activated cell
sorting (FACS) ………………………………………………………………………………..… 16
2.12.2 Murine pulmonary and splenic lymphocytes analysis via fluorescence-activated cell
sorting (FACS) ……………………………………………………………………………..…… 16
2.12.3 Murine pulmonary ILC2s analysis via fluorescence-activated cell sorting (FACS).. 16
2.12.4 Human peripheral blood lymphocytes analysis via fluorescence-activated cell sorting
(FACS) ……………………………………………………………………………………...…… 17

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2.12.5 Human peripheral blood ILC2s analysis via fluorescence-activated cell sorting
(FACS) ………………………………………………………………………………………...… 17
2.13 Histological Analysis of the lungs ………………………………………………………………… 17
2.14 Statistical Analysis for AHR, FACS and Histology ……………………………………………… 17
CHAPTER 3: RESULTS …………………………………………………………………………………..……. 17
3.1 Data Harmonization and GWAS Analyses in UK Biobank ……………………………………… 17
3.2 Meta-analyses of GWAS data for asthma in UKBB and TAGC ………………………………… 18
3.3 Enrichment of novel asthma-associated variants, such as C52, in Regulatory Elements or
Pathways ………………………………………………………………………………………………… 18
3.4 CD52 is constitutively expressed on lymphocyte both at steady state and under inflammatory
conditions ………………………………………………………………………………………………… 19
3.5 Anti-CD52 treatment yields high depletion efficacy at both systemic and pulmonary levels .. 20
3.6 CD52 depletion ameliorated HDM-induced AHR by improving dynamic compliance and
abrogating airway resistance and lung inflammation ………………………………………………… 21
3.7 CD52 is constitutively expressed on murine ILC2s both at steady state and under inflammatory
conditions. Furthermore, CD52 expression on ILC2s is inducible by IL-33 ……………………..… 25
3.8 CD52 depletion ameliorates IL-33-induced AHR by improving dynamic compliance and
abrogating airway resistance and lung inflammation in wild-type mice ……………………………. 26
3.9 Amelioration of IL-33-induced AHR and lung inflammation via CD52 depletion is ILC2-
dependent ……………………………………………………………………………………..………… 28
3.10 CD52 depletion of ILC2s ameliorates Allergen-induced AHR and reduces lung inflammation
in RAG2-deficient mice ……………………………………………………………………………….… 28
3.11 CD52 is constitutively expressed on human peripheral ILC2s at steady state and under
inflammatory conditions ………………………………………………………………………………… 30
3.12 CD52 depletion ameliorates human ILC2s-mediated AHR and reduces lung inflammation in
Rag2-deficient Il2rg-deficient mice …………………………………………………………………..… 30
CHAPTER 4: DISCUSSION ……………………………………………………………….…………………… 30
ACKNOWLEDGEMENTS ……………………………………………………………………………….….….. 33
REFERENCES ………………………………………………………………………………………………...… 33

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ABBREVIATIONS

Ab Antibody
aILC2 Activated ILC2
APC Antigen-presenting cell
AHR Airway Hyperresponsiveness
BAL Bronchoalveolar Lavage
CD Cluster of Differentiation
cDyn Dynamic Compliance
CTL Cytotoxic T cell
ELISA Enzyme-linked Immunosorbent Assay
FACS Fluorescence-activated Cell Sorting
GATA3 GATA-binding Protein 3
GM-CSF Granulocyte-macrophage Colony-stimulating Factor
ICOS Inducible T cell Costimulator
IL Interleukin
ILC Innate Lymphoid Cell
ILC2 Type 2 Innate Lymphoid Cell
i.n. Intranasal
i.p. Intraperitoneal
i.v. Intravenous
nILC2 Naïve ILC2
PBS Phosphate Buffered Saline
Rag2 Recombination Activating Gene 2
TCR T-cell Receptor
Teff Effector T cell
TGF-ß Transforming Growth Factor ß
TH2 Type 2 Helper T cell
Treg Regulatory T cell
TSLP Thymic Stromal Lymphopoietin
gc Gamma-chain

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LIST OF TABLES AND FIGURES

Table 1. ILC2 biomarkers.
Table 2. Anti-CD52 as an ideal therapy.
Figure 1. Hygiene Hypothesis.
Figure 2. Narrowing of the bronchioles upon exposure to allergens.
Figure 3. Asthma as a TH2-associated disease.
Figure 4. Currently known modulatory factors of pulmonary ILC2s.
Figure 5. Asthma as an ILC2-associated disease.
Figure 6. Cluster of differentiation (CD52).
Figure 7. Mechanistic actions of depleting antibodies.
Figure 8. Manhattan plot for association of 1,983,348 SNPs with asthma.
Figure 9. Regional plot of SNPs on chromosome one.
Figure 10. Enrichment of asthma-associated loci within DNase I hypersensitive sites across various cell
types.
Figure 11. Both murine and human lymphocytes constitutively express CD52.
Figure 12. Anti-CD52 treatment yields high depletion efficacy at both systemic and pulmonary levels.
Figure 13. CD52 depletion significantly ameliorates HDM-induced airway hyperreactivity (AHR) and
abrogates inflammation.
Figure 14. CD52 depletion significantly reduces the number and percentages of inflammatory
lymphocytes in both spleen and lungs.
Figure 15. Murine ILC2s constitutively express CD52, and this expression is inducible by IL-33.
Figure 16. CD52 depletion significantly ameliorates IL-33-induced airway hyperreactivity (AHR) and
abrogates inflammation.
Figure 17. Amelioration of IL-33-induced AHR and lung inflammation via CD52 depletion is ILC2-
dependent.
Figure 18. CD52 depletion of ILC2s ameliorates Alternaria alternata-induced AHR and abrogates lung
inflammation in RAG2-deficient mice.
Figure 19. Human ILC2s constitutively express CD52.
Figure 20. CD52 depletion ameliorates human ILC2-mediated AHR and abrogates lung inflammation in
Rag2
−/−
Il2rg
−/−
mice.
Figure 21. Anti-CD52 treatment as a promising therapeutic avenue in asthma.

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SUMMARY

Autoinflammatory diseases, such as asthma, are increasing at an alarming rate. Yet, the
current steroid-based therapeutics inadequately target the clinical phenotypic features of
asthma at a pathophysiologic level and only transiently block the progression of the
inflammatory mechanisms. However, in order to better combat this immune inflammatory
disorder, more effective treatments are needed to target the fundamental immune cells that
are involved in the inflammatory response and ultimately cause the phenotypic symptoms
observed in the clinic. Recently, culmination of the advances in our understanding of the
immune system coupled with the new breakthroughs in systems biology and current
panomics have manifested into our path forward towards a new era of precision medicine. The
elegant functionality and high specificity of the immune system has opened the door for new
immunotherapeutic approaches that can target pathogenesis of diseases at either cellular or
molecular levels in highly specific and rationalized manners. Thus, such novel approaches
herald a new era of immunotherapies for autoinflammatory and autoimmune disorders where
the key factor is to target the relevant pathological components, in the case of asthma the
driving type 2 immune cells such as TH2s and ILC2s.

Here, we first elucidate the genetic architecture of asthma by performing the largest human
GWAS meta-analysis to date. We discovered 17 novel loci that are linked to the
immunopathogenesis of asthma, among which the gene CD52 was most intriguing and
exhibited significant enrichment among immune cells in the blood. CD52 gene has been
previously reported to encode for a glycosylphosphatidylinositol (GPI)-anchored cell surface
protein that consists of 12 relatively acidic amino acids. Despite limited knowledge regarding
its physiological role, CD52 has been extensively linked to various immunological disorders
ranging from multiple sclerosis (MS), graft versus host disease (GvHD), autoimmune
inflammatory neurodegenerative diseases as well as various lymphomas such chronic
lymphocytic leukemia (CLL) or acute lymphocytic leukemia (ALL). Consequently, CD52 has
been an encouraging target in many on-going clinical trials for the aforementioned disorders.
Hence, alemtuzumab–also known as Campath-1H, is an FDA approved recombinant
humanized monoclonal immunoglobulin IgG1 kappa that targets CD52 and effectively treats
and in some cases cures patients with MS or CLL. Our study reports CD52 to be significantly
associated with asthma for the first time herein.

We evaluated the therapeutic potential of anti-CD52 therapy in two different asthma models:
TH2-dependent and ILC2-dependent models. We demonstrated the CD52 is expressed on both
human and murine lymphocytes. We further established the high efficacy of a novel anti-
murine CD52 monoclonal antibody and showed that treatment via this depleting antibody
significantly ameliorates HDM-induced airway hyperreactivity (AHR) and abrogates
inflammation in a TH2-dependent manner. Moreover, we demonstrated that both murine and
human ILC2s constitutively express CD52 for the first time. We further showed that
amelioration IL-33 and Alternaria alternata induced airway hyperreactivity (AHR) and lung
inflammation is ILC2-dependent. We utilized a humanized mice model to demonstrate the
translational potential of our findings and demonstrated that the aforementioned FDA-
approved drug, alemtuzumab, can be used to treat asthma. The effectiveness of anti-CD52
treatment leads to rapid yet long-lasting depletion of CD52+ cells. Since the HSC do not
express CD52, this depletion is subsequently followed by a gradual repopulation and
reprogramming of the immune cells that arise from these stem cell precursors. Such
reprograming results in a shift of the immunological networks towards a more tolerogenic
state and restores the immune balance between mutually antagonistic cell populations. Taken
together, our results suggest that anti-CD52 treatment may serve as a novel therapeutic
avenue to treat and permanently cure both TH2 and ILC2 dependent allergic asthma.

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CHAPTER 1
INTRODUCTION

1.1 Asthma: a rising disease of civilizations

Asthma is an atopic and heterogeneous disorder of
the airways that is characterized by
bronchoconstriction, bronchial hyper-
responsiveness and underlying inflammation
4
.
Mirroring the pathogenesis of other inflammatory
diseases, chronic asthma is caused by
dysregulated tolerance mechanisms of a
hyperactive immune system that affects one in
twelve people, or approximately 25 million people
in United States
5
. Worldwide, this number currently
extends beyond 300 million people, and is
projected to reach well over 400 million by 2025
6
.
Over the past few decades asthma has quickly
risen to become the most common chronic
pulmonary disorder, causing more than 250
thousand deaths annually
7
. Thus, two very
important questions arise: what is the cause of this
uprising, and more importantly how can we combat
and ameliorate it?

Many scientists and physicians have attempted to
explain the alarming growth of asthmatic
populations, particularly in developed western
countries. The leading hypothesis first published in
the New England Journal of Medicine, referred to
as the Hygiene Hypothesis (Figure 1), was
developed in 2002, and asserts that the higher
sanitation increases risks of inflammatory
dysfunctions in the developed countries
2
. This is
hypothesized to be due to the fact that exposure to
immunogenic agents early in life allows for the
establishment of tolerance mechanisms in the
innate and adaptive immunities. In other words,
when children are no longer exposed to various
environmental immunogens, their immune
tolerance mechanisms do not develop properly
2,8
.

Despite such an alarming uprising, laypeople are
not educated about asthma and the current
epidemic of this immune disorder. In fact, many
people are under the false impression that asthma
is effectively diagnosed and is remediable by
medical professionals. However, current diagnosis
and treatment methods only target the clinical
symptoms of the disease, not the underlying
causes of it
9
. As a result, there has been a
significant increase in chronic asthma prevalence.
There currently is no cure for asthma and many
patients are non-responsive to the available
treatment options
10
.

1.2 Current diagnosis and treatments

Current diagnosis of asthma, as defined by the
National Heart, Lung and Blood Institute (NHLBI) of
the National Institutes of Health (NIH), is based on
the patient’s medical history, family history and the
phenotypic symptoms that are observed in the
clinics by the physicians
4
. During routine physical
exams, physicians typically look for symptoms such
as wheezing, swollen nasal passages, and skin
conditions such as eczema. Since the absence of
such symptoms is not always indicative of a non-
asthmatic patient, spirometry to measure the
Figure 1. Hygiene Hypothesis. Over a 50-year period, an inverse association between the
incidence of prototypical Infectious diseases and the incidence of immune disorders has
manifested
2
.

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volume and speed of air flow within each inhalation
and exhalation is also used
11
. Narrowing of the
airways (Figure 2) due to underlying inflammation
in asthmatics significantly hindered air flow. Lastly,
as a final confirmation test, bronchoprovocation,
utilizes spirometry measurements after
administrations or nebulization of increasing doses
of bronchoconstrictors such as methacholine or
histamines to the patients
12
. If the spirometry
results are lower compared to unstimulated
conditions, or if the patient develops airway
hyperresponsiveness (AHR) during the
bronchoprovocation, the subject is then considered
as asthmatic and is classified on the basis of clinical
measures such as severity and persistence of the
observed phenotypic symptoms as either
intermittent, mild, moderate, or severe
13
. However,
such oversimplified empirical approaches to
classify this heterogeneous auto-inflammatory
disorder has stymied both diagnosis and treatment
because phenotypic traits such as reduction in
FEV1 (forced expired volume in 1 second) in the
clinic can be caused by multiple underlying disease
mechanisms
14-16
.

Current treatment options are mostly steroids that
only target the aforementioned clinical phenotypic
features at a pathophysiologic level and transiently
block the progression of the inflammatory
mechanisms. For example, inhaled corticosteroid is
a long-term control medication that prevents
swelling of the airways when taken in regularly
scheduled doses
17
. Other quicker relief
medications, such as an inhaled short acting β2-
agonist, act to relax the constricting smooth
muscles around the trachea, and thus dilate the
narrowed airways
18
. However, in order to better
combat this immune inflammatory disorder, more
effective treatments are needed to target the
fundamental immune cells that are involved in the
inflammatory response and ultimately cause the
phenotypic symptoms observed in the clinic
18
. Due
to such rational and auspicious potential of
precision medicine, it is now more imperative than
ever to conceptually understand the various
molecular and cellular disease endotypes.

1.3 Pathogenesis of asthma as a TH2-
dependent autoinflammatory immune
disorder

Under hemostatic conditions, naïve CD4+ T cells
can be polarized toward a type 2 phenotype via IL-
4 and STAT6 signaling
19
. In fact, TH2 cells are
characterized phenotypically based on their
transcriptional profile, particularly expression of
transcription factor GATA3, and production of type
2 cytokines such as IL-4, IL-5 and IL-13
1
. These
pluripotent cytokines can then stimulate
downstream myelocytes such as eosinophils, mast
cells and basophils in our innate immunity. The
adaptive immunity can also be ignited by these type
2 cytokines. For example, autocrine IL-4 signaling
can foster additional TH2 polarization while
paracrine IL-4 signaling can prime the humoral
immunity by initiating isotype switching and thus
production of large quantities of IgE by B
lymphocytes
20
. The constant Fc domain of IgE can
in turn bind to FcεRI receptors of aforementioned
myelocytes and cause them to degranulate their
reservoir of potent inflammatory mediators (e.g.
histamine, kinins, and proteases) in a type 2
immune response that is typically used as a part of
immunity against extracellular parasites such as
helminths in the gut
21
. Due to the potency of such
immune response, it is no surprise that any
dysregulation of the type 2 immunity can have
dramatic and dire consequences.

Traditionally, asthma has been categorized as an
TH2 associated inflammatory disorder (Figure 3)
that can be subsequently mediated by the
downstream myelocytes such as the
Figure 2. Narrowing of the bronchioles upon
exposure to allergens. This results in
symptoms such as wheezing, tightness of chest,
and difficulty breathing in asthmatics.

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aforementioned eosinophils. Broadly speaking,
initial exposure of the pulmonary system to
aeroallergen results in upstream proinflammatory
signaling near the airway epithelium
22
. More
specifically, secretion of specialized alarmins−a
group of cytokines comprised of TSLP, IL-25 and
IL-33−results in increased production of the
aforementioned type 2 cytokines that are capable
of initiating a cascade of downstream signaling
events including, but not limited to, chemotaxis of
proinflammatory effectors cells to the lungs or
remodeling of both epithelium and subepithelium
matrices along the airways
23
. Molecularly
speaking, IL-13 is a pleiotropic type 2 cytokine that
cause goblet cell hyperplasia and thus augmented
production of mucus that causes to the narrowing
of the bronchioles
24
. IL-13 can also directly cause
bronchiole smooth muscle contraction by inducing
STAT6-dependent upregulation of RhoA protein
25
.
The synergism of these two pathways manifests
into the aforementioned clinical features of asthma.
Similarly, with both paracrine and endocrine
signaling capabilities, the disulfide-linked
homodimeric IL-5 has been considered as one of
the most potent type 2 cytokines in asthma. IL-5
can bind to IL-5Rα on tissue resident effector
basophils and signal their activation and survival.
Furthermore, IL-5 is essential for recruitment of
eosinophils to the lungs from the bone marrow, as
well as their respective maturation, growth,
activation, and survival
26
. This in turn leads to one
of the hallmarks of type 2 inflammation, referred to
as eosinophilia. Due to their high granularity and
high potency of their proteases and toxins
reservoirs, long term eosinophilia causes
permanent tissue remodeling and damage in the
delicate tissues such as the lungs
27
.

Figure 3. Asthma as a TH2-associated disease. Exposure to environmental allergens results in secretion
of alarmins, such as IL-25, IL-33 and TSLP. These pro-inflammatory molecules can subsequently activate
downstream leukocytes such as tissue resident DCs. These APCs consequently migrate to the local lymph
node in order to activate the native CD4+ helper T cells and cause their polarization towards a TH2
phenotype. The newly primed TH2 cause IgE isotype switching of the B cells and migrate to the lungs
where they can perform their effector function via secretion of type 2 cytokines
2
.

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1.4 Pathogenesis of asthma as an ILC2-
dependent autoinflammatory immune
disorder

Innate lymphoid cells type 2 (ILC2s) are a newly
discovered subset of leukocytes that were first
ascribed their collective nomenclature in 2013. The
late discovery of ILCs can be attributed to the fact
that there is no specific cell marker for their
respective identification to date
28-30
. As a result,
murine and human ILC2s are defined by their lack
of previously reported immune cell lineage markers
(Table 1). ILC2s have perhaps been mostly
extensively studied for their beneficial role during
anti-helminth immunity in the intestine
31
. More
specifically, these cells induce warm expulsion by
both directly causing and further potentiating a type
2 immune response. ILC2s have similarly been
found among both murine and human adipocytes,
mainly white adipose tissue (WAT) and beige
adipose tissue (BAT)
32
. It has been well
documented that WAT causes excess lipid storage
and obesity. ILC2s, however, through production of
type 2 cytokines cause polarization of alternatively-
activated macrophages with a M2-like phenotype
that can in turn cause beiging of white adipocytes
and thus increase thermogenesis that ultimately
limits obesity
33
. In contrast to such beneficiary
roles, ILC2s have also been found to reside in
murine and human lungs where they have been
implicated to partake a more pathological role.
Under homeostatic conditions, pulmonary ILC2s
are tissue residents that are located near the
basement membrane subjacent to the epithelium
layers at a distance of less than 70 µm away from
the bronchioles
34
. Such calculated positioning
enables ILC2s to be among the first responders,
and thus the earliest inducers of type 2
inflammation in autoinflammatory airway disorders
such as asthma.

Although the adaptive TH2 response has historically
been implicated in asthma immunopathogenesis,
with the entrance of the newly discovered ILC2s
into the picture (Figure 4), the exact role of the
innate immunity in initiating and perpetuating type 2
immune responses remains to be fully elucidated.
Responding directly to pro-inflammatory cytokines
such as TSLP, IL-33 or IL-25 (Figure 5), ILC2s
have proven to be intriguing in the field as they
appear to serve as the bridge between the innate
immunity and the adaptive immunity during the
inflammatory process
24
. Although not yet fully
understood, these cells are innate cells that appear
to have memory but lack the antigen specific
receptors of the T cells that they mimic
phenotypically
24
. Specifically, ILC2s mimic Th2s in
activating transcription factors such as GATA3
35
.
Table 1. ILC2 biomarkers. Current biomarkers used
for identification of murine and human ILC2s
1
.
Figure 4. Currently known modulatory factors of
pulmonary ILC2s. Alarmins, released by the
epithelium, are the principle activators of ILC2s.
Activated ILC2s are potent producers of type 2
cytokines such as IL-4, IL-5 and IL-13. Modulation
of ILC2 activity can be achieved via contract-
independent (e.g. regularly cytokines) and contract-
dependent mechanisms (e.g. cell-surface co-
stimulatory factors)
1
.

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Moreover, these lymphoid derived cells are very
fast and proficient producers of the previously
discussed type 2 cytokines, such as IL-4, IL-5, IL-9
and IL-13. These cytokines are of course central in
pathogenesis of various inflammatory diseases
such as asthma. Due to their newly recognized
importance in both initiating and perpetuating
inflammation, there recently has been a lot of
interest and efforts in the field to identify ILC2
biomarkers that can be utilized to target and
specifically modulate their activity in future
therapeutics.
1.5 Emergence of immunotherapy as a novel
way to treat heterogeneous set of immune-
related disorders

Every scientific field may undergo certain periods of
immense progress. This is often a byproduct of
exciting advancements in both theoretical
knowledge and experimental methodologies
available. For example, the 19th century has been
considered as the golden age of physics, mainly
due to vast contributions of predominate scientists
such as Albert Einstein and Niels Bohr. The current
century, however, appears to encompass the
Figure 5. Asthma as an ILC2-associated disease. Late discovery of ILC2s has dramatically
changed the classical frameworks of asthma as an immune disorder. Highlighting the critical
role of innate immunity, ILC2s can directly respond to alarmins and thus rapidly orchestrate a
type 2 immune response via bulk production of IL-4, IL-5 and IL-13
2
. These inflammatory
cytokines subsequently lead to recruitment and/or activation of other downstream leukocytes.
Culmination of these events leads to hallmarks of asthma such as contractions of smooth
muscles, hyper mucus production by goblet cells and airway hyperreactivity.

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golden age of immunology and
immunotherapeutics. The culmination of the
research and our understanding of the cellular and
molecular mechanisms of our immune system over
the past few decades has opened up the possibility
to design novel and exciting therapeutic
approaches that may have appeared as ‘science
fiction’ just decades ago. In fact, the most recent
Nobel Prize in Physiology or Medicine (2018) was
awarded jointly to two immunologists: James P.
Allison and Tasuku Honjo "for their discovery of
cancer therapy by inhibition of negative immune
regulation." Worth noting that this was the very first
Noble Prize ever awarded to an anti-cancer therapy
of any kind mainly due to its unparalleled and
astonishing effectiveness. Thus, an important
inquiry arises: how are immunotherapeutic
approaches fundamentally different than what
came before them?

The immune system is comprised of highly
sophisticated, powerful and specific autoregulatory
networks that serve two essential functions: i)
maintaining homeostasis in absence of any foreign
intruders; and ii) combating any potential
pathogens during an invasion. It is the elegant
functionality and high specificity of the immune
system that has enabled immunotherapeutic
approaches to specifically target pathogenesis of
diseases at either cellular or molecular levels.
Culmination of the advances in our understanding
of the immune system coupled with the new
breakthroughs in systems biology and current
panomics have manifested into our path forward
towards a new era of precision medicine. In the
context of autoinflammatory and autoimmune
diseases, we have depended on non-specific
suppressive drugs for far too long. It is now time to
apply the lessons we’ve learned from the field of
cancer immunotherapy where effective
immunomodulation is achieved in highly specific
and rationalized manners. Thus, such novel
approaches herald a new era of immunotherapies
for autoinflammatory and autoimmune disorders
where the key factor is to target the relevant
pathological components, in the case of asthma the
driving type 2 immune cells- such as TH2s and
ILC2s.

1.6 Cluster of differentiation (CD52) as a
promising a target

One of the most principal components of designing
a new immunotherapeutic treatment in any disorder
is finding the most appropriate molecular target that
is fundamentally linked to the pathogenesis of the
respective disease. Cluster of differentiation
(CD52) is a glycosylphosphatidylinositol (GPI)-
anchored cell surface protein that consists of 12
amino acids (Figure 6)
36
. Although its exact
physiological function is not yet fully understood,
CD52 has shown to be a promising target in many
on-going clinical trials for multiple sclerosis (MS),
graft versus host disease (GvHD), autoimmune
inflammatory neurodegenerative diseases as well
as various lymphomas such chronic lymphocytic
leukemia (CLL) or acute lymphocytic leukemia
(ALL)
37-41
. In fact, alemtuzumab–also known as
Campath-1H (Figure 6, right panel), is an FDA
approved recombinant humanized monoclonal
immunoglobulin IgG1 kappa that targets CD52 and
is utilized to effectively treat and potentially cure
Figure 6. Cluster of differentiation (CD52). The structure of membrane-bound human CD52 is shown in
the left panel. The peptide fragment of CD52 is shown in green. The glycan on CD52, mainly a fully sialylated
N-glycan with a bisecting GlcNAc, is shown in blue. The glycosylphosphotidylinositol (GPI) anchor and lipid
carrier are shown in red and orange, respectively
1
. The middle panel highlights the sequence of the peptide
fragment of CD52. The right panel is based on the model 1ce1 (PDB ID) and displays the binding of the anti-
CD52 antibody (The Fab domain of Alemtuzumab) and CD52
2
.

- 13 -
patients with MS or CLL
42-47
. It is worthy to note
there have been no prior reports linking any form of
association between CD52 and asthma. Previous
reports have suggested CD52 is highly expressed
on effector T and B lymphocytes with little to no
expression on innate immune cells such as
monocytes, mature natural killer (NK) cells,
macrophages and various other granulocytes. Most
importantly, CD52 is not expressed on the
hematopoietic stem cells (HSCs) and other
progenitors that give rise to leukocytes
48-50
.

Alemtuzumab (Lemtrada, Genzyme, Cambridge,
MA) was first approved for the as the first-line
therapy for treatment of MS in November 2014 by
U.S. Food and Drug Administration (FDA) following
demonstration of durable clinical efficacy in phase
II and phase III clinical trials
51-53
. Alemtuzumab has
since been approved in over 30 countries, such as
Canada, Israel, Australia and Switzerland for the
therapy of relapsing-remitting multiple sclerosis
(RRMS). The rapid approval of this monoclonal
antibody is due to its effectiveness that leads rapid
yet long-lasting depletion of CD52+ cells. Since the
HSC do not express CD52, this depletion is
subsequently followed by a gradual repopulation
and reprogramming of the immune cells that arise
from these stem cell precursors
54,55
. The exact
immunological mechanisms of such anti-CD52
induced reprogramming of immune cell
composition remains to be unraveled.

In accordance with the current labeling guidelines,
12 mg of the monoclonal antibody is infused in the
clinics for five consecutive days in an initial
regiment. After a period of 12 months, a second 3-
day long treatment course may be administered.
Although alemtuzumab is FDA approved for two
courses, there are some reports of patient
treatments for up to five separate regiments
56
.

Despite limited data regarding the physiological
functions of CD52 and its link to pathogenesis of the
aforementioned autoinflammatory disorders, the
immediate impacts of alemtuzumab is well
understood (Figure 7). The constant FC domain of
this humanized antibody is recognized by the FC
receptors (FCR) of immune cells such as CD8+
cytotoxic T cells and Natural Killer (NK) cells. This
recognition in turn will result in antibody dependent
cellular cytotoxicity
57
. Moreover, the recognition
and binding of CD52 to the antibody can further
lead to complement-dependent cytotoxicity (CDC)
as well as caspase-dependent apoptosis
57,58
. The
biochemical half-life of the alemtuzumab has been
measured in MS patients to be only 4 to 5 days
58
.
After this rapid initial depletion, repopulation of the
immune cell repertoire that arise from HSCs have
been shown to follow different kinetic patterns.
Particularly, monocytic cells have been shown to
rebound to their respective baselines within three
months post-treatment while CD8
+
CTLs and CD4
+

TH cells are fully restored after 31 and 60 months,
respectively
59
.

Interestingly, among the depleted CD4
+

lymphocytes, the frequency of CD25
high
Cd127
-
and
CD25
high
FoxP3
+
T regulatory (Treg) cells is
Figure 7. Mechanistic actions of depleting antibodies. Depleting antibodies, such as
Alemtuzumab, can potentially function through 3 different mechanisms: i) antibody-dependent cell-
mediated cytotoxicity (ADCC) via CTL and NK cells, ii) complement-dependent cytotoxicity (CDC), iii)
caspase-dependent apoptosis.

- 14 -
increased. This is rather sticking because the CD52
expression levels of these cells is equivalent to all
other CD4
+
helper T cell populations. This post-
treatment increased frequency has been suggested
to be a result of faster rebound kinetics of Tregs
60
.
Type 1, 2 or 17 T helper cells require foreign
antigen recognition in order to proliferate and
expand. In contrast, Tregs are cable of homeostatic
proliferation in response to self-antigens that are
much more abundant in their respective
environments.

CHAPTER 2
MATERIALS AND METHODS

2.1 GWAS Study Populations

Analyses in UK Biobank were based on publicly
available summary level data provided by the Neale
lab (http://www.nealelab.is/). Quality control steps
were applied to ~488,000 individuals with phased
and imputed genotype data in UK Biobank. After
retaining subjects with white British genetic
ancestry and removing closely related individuals
(or at least one of a related pair of individuals),
individuals with sex chromosome aneuploidies, and
individuals who had withdrawn consent from UK
Biobank, 337,199 were included in analyses of
~2,400 phenotypes. Participating cohorts in the
TAGC GWAS studies have been previously
described in detail 15. Briefly, TAGC includes 56
studies of European populations, 7 studies of
African populations, 2 studies of Japanese
populations and one study of a Latino population.
All participants gave written consent for
participation in genetic studies, and the protocol of
each study was approved by the corresponding
local research ethics committee or institutional
review board.

2.2 GWAS Analyses in UK Biobank

Due to mapping issues, only ~40 million autosomal
SNPs imputed from the Haplotype Reference
Consortium were used by the Neale lab for GWAS
analyses in UK Biobank. Furthermore, only SNPs
with INFO scores >0.8 (directly from UK Biobank)
and with minor allele frequencies (MAF) >0.1% and
Hardy-Weinberg equilibrium P>1x10-10 in the
337,199 individuals passing quality control filters
were included. These filters resulted in ~10.9
million SNPs being used for linear regression
analysis of both quantitative and qualitative
phenotypes. We obtained GWAS results with this
set of variants for the asthma phenotype based on
UK Biobank field code 6152_8 (doctor diagnosed
asthma). This field is a summary of the distinct
main diagnosis codes a participant has had
recorded across all their episodes in the hospital.
Diagnoses are coded according to the International
Classification of Diseases version-10 (ICD10).
Using the allele counts for the ~10.9 million SNPs
provided by the Neale lab for cases and controls
(http://www.nealelab.is/blog/2017/7/19/rapid-gwas-
of-thousands-of-phenotypes-for-337000-samples-
in-the-uk-biobank), we carried out GWAS analyses
for asthma in UK Biobank by logistic regression
under additive genetic models. The genome-wide
threshold for significant association was set at P-
value=5.0x10-8.

2.3 Meta-analyses for asthma in UKBB and
TAGC

Publicly available summary level GWAS data for
asthma with ~2.0 million SNPs in European
populations and multi-ancestry populations from
TAGC were downloaded and combined with the
results of our logistic regression-based GWAS for
asthma in UK Biobank. We used fixed-effect
inverse-variance-weighted meta-analyses with
SNPs common to both datasets assuming an
additive model, with control for genomic inflation, as
implemented in METAL 22. The genome-wide
threshold for significant association was set at P-
value=5.0x10-8. Any genome-wide significant
variant located >1Mb from the previously reported
genome-wide significant asthma and/or allergic
disease variants was considered as a new locus.
Analyses to test for the independence of the novel
loci with the nearest previous loci were performed
leveraging summary-level statistics in GCTA 23.
The lead SNP in the new locus was conditioned on
the lead SNP in the nearest known locus if they
were <3Mb apart, and a conditioned P<5.0x10-8
was used to indicate statistical significance. We
also estimated the LD between the lead variants
located at the novel loci and the nearest previous
loci to confirm they are independent with a
conservative LD threshold of r2<0.02. All analyses
were performed using R.

2.4 Validation of known asthma loci

We evaluated 51 known asthma loci using the
SNPs showing the best P values in previous
asthma GWAS or its proxies with the highest r2
available in our multi-ancestry meta-analysis 7-19.
A meta-analysis P-value<9.80x10-4 (after

- 15 -
Bonferroni correction for 51 tests) was used to
indicate statistical significance and a meta-analysis
P-value<0.05 was used to indicate
suggestive/nominal significance.

2.5 Enrichment of novel asthma loci in
epigenetic marks

To assay the enrichment of asthma associated
variants in regions of open chromatin and specific
histone marks, we utilized the GWAS Analysis of
Regulatory or Functional Information Enrichment
with LD correction (GARFIELD) method 29. Briefly,
it is a method that leverages GWAS findings with
regulatory or functional annotations (primarily from
ENCODE and Roadmap epigenomics data 30) to
find features relevant to a phenotype of interest. It
performs greedy pruning of GWAS SNPs (r2>0.1)
and then annotates them based on functional
information overlap. Next, it quantifies Odds Ratio
(OR) at GWAS significance cutoff (P-value=5.0x10-
8) and assesses them by employing a generalized
linear model framework, while matching for minor
allele frequency, distance to nearest transcription
start site and number of LD proxies (r
2
>0.8).

In addition, we investigated the 17 novel asthma
associated variants for colocalization with
regulatory elements in a wide range of human cell
types using HaploReg v4.1 24, where the
regulatory chromatin states from DNase and
histone ChIP-Seq from Roadmap Epigenomics
Consortium in 2015 were applied 30.

2.6 Animals

Wild-type (WT) BALB/cByJ, recombination-
activating gene 2-deficient (Rag2)
-/-
, recombination-
activating gene 2-deficient gamma-chain-deficient
(Rag2
-/-
γc
-/-
) mice were purchased from the
Jackson Laboratory (Bar Harbor, Me) and bred in
our animal facility at the University of Southern
California. We used 5- to 8-week-old age-matched
female mice in our studies. All the described studies
were approved by the Institutional Animal Care and
Use Committee of the University of Southern
California.

2.7 Isolation of murine pulmonary lymphocytes
and ILC2s

Utilizing fine surgical scissors, murine lungs were
surgically removed and minced in a sterile
environment subsequently incubated in type IV
collagenase (1.6 mg/mL; Worthington
Biochemicals, Lakewood, NJ) at 37°C for 60
minutes. After digestion, murine lung fragments
were then pressed through a 70 μm nylon cell
strainer, using the rubber end of a sterile 10 mL
syringe plunger, in order to create a single cell
suspension. In order to terminate the enzymatic
reaction of collagenase, the cells were washed with
1x phosphate buffered saline (PBS) by
centrifugation at 400x g for 7 minutes at 4°C. In
order to exclude and lyse the red blood cells
(RBCs), the cell pellet was subsequently
resuspended in 1x RBC lysis buffer (Biolegend®,
San Diego, CA) and incubated at room temperature
(RT) for 5 minutes. In order to terminate the
chemical reaction, the cells were subsequently
washed and centrifuged—at 400x g for 7 minutes
at 4°C—with 1x PBS. The remaining pellet was
then further prepared for flow cytometry.

2.8 Isolation of murine splenic lymphocytes

Murine spleen was surgically removed and minced
in a sterile via fine surgical scissors under steriles
environment and subsequently pressed through a
70 μm nylon cell strainer, using the end of a sterile
10 mL syringe plunger, in order to create a single
cell suspension. The cells were then washed with
1x phosphate buffered saline (PBS) by
centrifugation at 400x g for 7 minutes at 4°C. In
order to exclude and lyse the red blood cells
(RBCs), the cell pellet was subsequently
resuspended in 1x RBC lysis buffer (Biolegend®,
San Diego, CA) and incubated at room temperature
(RT) for 5 minutes. In order to terminate the
chemical reaction, the cells were subsequently
washed and centrifuged—at 400x g for 7 minutes
at 4°C—with 1x PBS. The remaining pellet was
then further prepared for flow cytometry.

2.9 Humanized mice and Isolation of human
peripheral blood mononuclear cells (PBMCs)

For human peripheral ILC2, peripheral blood
mononuclear cells (PBMCs) were first isolated from
human fresh blood by diluting the blood 1:1 in PBS
then adding to SepMateTM-50 separation tubes
(STEMCELL Technologies Inc, Vancuver, Canada)
prefilled with 15-ml LymphoprepTM each (Axis-
Shield, Oslo, Norway) and centrifugation at 1200 xg
for 15 minutes. Human PBMCs were then stained
and purified using BD FACS ARIA III (BD
Biosciences, San Jose, CA) with a purity of 495%.
Purified human ILC2s were cultured with rh-IL2 (20
ng ml
-1
) and rh-IL-7 (20 ng ml
-1
) for 48 hours, then
adoptively transferred to Rag2
−/−
Il2rg
−/−
mice (2.5 x

- 16 -
10
5
cells per mouse) followed by i.n. administration
of recombinant human IL-33 (1 µg per mouse) with
or without Alemtuzumab (Hu116) i.p. on day 1 and
i.v. on days 2–4. On day 5, lung function was
measured, and BAL was performed and analyzed.

2.10 Cell Culture Conditions

All cells were purified by flow cytometry using BD
FACS ARIA III (BD biosciences, San Jose, CA) with
a purity of >95%. The isolated ILC2s were cultured
(at least 5 x 10
3
cells/well) in 96-well round-bottom
plates with Gibco
™
Roswell Park Memorial Institute
(RPMI) 1640 medium (Thermo Fisher Scientific,
Waltham, MA) that was supplemented with 10%
fetal bovine serum (FBS), 2% antibiotics (penicillin
and streptomycin), and 0.05 mM b-
mercaptoethanol. The cells were maintained in a
37°C incubator with 5% CO2. All murine ILC2s
were cultured in the presence of recombinant
mouse rmIL-2 (10 ng/mL), rmIL-7 (10 ng/mL),
and/or rmIL-33 (10 ng/mL). All human ILC2s were
cultured in the presence of recombinant mouse
rhIL-2 (10 ng/mL), rhIL-7 (10 ng/mL), and/or rhIL-33
(10 ng/mL).

2.11 Measurement of airway hyperreactivity
(AHR) and collection of bronchoalveolar lavage
(BAL) fluid

Lung function was measured by direct
measurement of lung resistance and dynamic
compliance in anesthetized tracheostomized mice,
in which mice were mechanically ventilated via the
FinePointe RC system (Buxco Research Systems,
Wilmington, NC) and sequentially challenged with
aerosolized increasing doses of methacholine.
After measurements of AHR, the trachea was
cannulated and the bronchial alveolar lavage (BAL)
fluid was collected as described before
61
. Briefly,
we tracheostomized and intubated the mice and
then washed the airways 3 times with 1 mL of PBS
each time, followed by centrifuging at 400g for 7
minutes and harvesting the cells.

2.12 Flow cytometry analysis and reagents

2.12.1 Murine bronchoalveolar lavage (BAL) fluid
analysis via fluorescence-activated cell sorting
(FACS)

Relative and absolute cell numbers in BAL fluid
were calculated by means of flow cytometry by
staining the cells with phycoerythrin (PE)–anti–
Siglec-F (BD Biosciences, San Jose, Calif),
fluorescein isothiocyanate(FITC)–anti-CD19,
peridinin-chlorophyll-protein complex
(PerCP)/Cy5.5–anti-CD3ε, allophycocyanin
(APC)–anti–Gr-1, PE/Cy7–anti-CD45, APC/Cy7–
anti-CD11c (BioLegend, San Diego, Calif), and
eFluor450–anti-CD11b (eBioscience, San Diego,
Calif) in the presence of anti-mouse FC-block
(BioXcell, West Lebanon, NH). We used
CountBright Absolute Count Beads (Thermo Fisher
Scientific, Waltham, Mass), according to the
manufacturer’s instructions. At least 10
5
CD45
+

cells were acquired on a BD FACSCanto II (BD
Biosciences). Data were analyzed with FlowJo
software (TreeStar, Ashland, Ore).

2.12.2 Murine pulmonary and splenic lymphocytes
analysis via fluorescence-activated cell sorting
(FACS)

Relative and absolute cell numbers in lung and
spleen were calculated by means of flow cytometry
by staining the cells with phycoerythrin (PE)–anti–
CD52 (MBL International, Woburn,
Massachusetts), fluorescein isothiocyanate(FITC)–
anti-CD19, peridinin-chlorophyll-protein complex
(PerCP)/Cy5.5–anti-CD3ε, brilliant violet 421™
(BV) –anti-CD4, PE/Cy7–anti-CD8, APC/Cy7–anti-
CD45 (BioLegend, San Diego, Calif) in the
presence of anti-mouse FC-block (BioXcell, West
Lebanon, NH). We used CountBright Absolute
Count Beads (Thermo Fisher Scientific, Waltham,
Mass), according to the manufacturer’s
instructions. At least 10
5
CD45
+
cells were acquired
on a BD FACSCanto II (BD Biosciences). Data
were analyzed with FlowJo software (TreeStar,
Ashland, Ore).

2.12.3 Murine pulmonary ILC2s analysis via
fluorescence-activated cell sorting (FACS)

Relative and absolute cell numbers in lung tissue
were calculated by means of flow cytometry. Murine
ILC2s were defined based on the lack of expression
of classical lineage markers (CD3e, CD45R, Gr-1,
CD11c, CD11b, Ter119, NK1.1, TCR-gd and
FCeRI) and expression of CD45, ST2, and CD117.
The cells were stained with Biotinylated anti-mouse
lineage (CD3e (145-2C11), CD45R (RA3-3B2), Gr-
1 (RB6-8C5), CD11c (N418), CD11b (M1/70),
Ter119 (TER-119), NK1.1 (PK136), TCR-b (H57
597), TCR-gd (GL3), and FceRIa (MAR-1)),
fluorescein isothiocyanate(FITC)–anti-streptavidin,
phycoerythrin (PE)–anti–CD52 (MBL International,

- 17 -
Woburn, Massachusetts), APC/Cy7–anti-CD45,
PE/Cy7–anti-CD127 (BioLegend, San Diego,
Calif), and peridinin-chlorophyll-protein complex
(PerCP)/Cy5.5–anti-ST2 (Thermo Fisher Scientific,
Waltham, Mass). We used CountBright Absolute
Count Beads (Thermo Fisher Scientific, Waltham,
Mass), according to the manufacturer’s
instructions. At least 10
5
CD45
+
cells were acquired
on a BD FACSCanto II (BD Biosciences). Data
were analyzed with FlowJo software (TreeStar,
Ashland, Ore).

2.12.4 Human peripheral blood lymphocytes
analysis via fluorescence-activated cell sorting
(FACS)

Relative and absolute cell numbers in from human
peripheral blood were calculated by means of flow
cytometry by staining the cells with peridinin-
chlorophyll-protein complex (PerCP)/Cy5.5–anti-
CD45, APC/Cy7–anti-CD8 (eBioscience, San
Diego, Calif), fluorescein isothiocyanate(FITC)–
anti-CD3, phycoerythrin (PE)–anti–CD19, brilliant
violet 421™ (BV) –anti-CD4 and allophycocyanin
(APC)–anti–CD52 (BioLegend, San Diego, Calif).
We used CountBright Absolute Count Beads
(Thermo Fisher Scientific, Waltham, Mass),
according to the manufacturer’s instructions. At
least 10
5
CD45
+
cells were acquired on a BD
FACSCanto II (BD Biosciences). Data were
analyzed with FlowJo software (TreeStar, Ashland,
Ore).

2.12.5 Human peripheral blood ILC2s analysis via
fluorescence-activated cell sorting (FACS)

Human ILC2s were defined based on the lack of
expression of classical human lineage markers and
expression of CRTH2, CD127 and CD45. The cells
were stained with fluorescein
isothiocyanate(FITC)–anti-human lineage (CD3,
CD14, CD16, CD19, CD20, CD56, CD235a, CD1a,
CD123), APC/Cy7–anti-CD45, phycoerythrin (PE)–
anti–CD294 (CRTH2), PE/Cy7–anti-CD127,
allophycocyanin (APC)–anti–CD52 (BioLegend,
San Diego, Calif). We used CountBright Absolute
Count Beads (Thermo Fisher Scientific, Waltham,
Mass), according to the manufacturer’s
instructions. At least 10
5
CD45
+
cells were acquired
on a BD FACSCanto II (BD Biosciences). Data
were analyzed with FlowJo software (TreeStar,
Ashland, Ore).

2.13 Histological Analysis of the lungs

After euthanizing the mice, lungs were harvested
and fixed immediately in with 4% paraformaldehyde
in PBS. After overnight fixation, the lungs were
processed for histology. The lung tissue was
embedded in paraffin, cut into 4 µm sections and
stained with H&E according to standard protocols.
Sections were scanned using light microscope for
inflammation. Images of hematoxylin and eosin–
stained tissue slides were acquired with a
KeyenceBZ-9000 microscope (Keyence, Itasca, Ill)
and assembled into multipanel figures using Adobe
Illustrator software (version 22.1). Histologic
images were analyzed with the ImageJ Analysis
Application (NIH & LOCI, University of Wisconsin).

2.14 Statistical Analysis for AHR, FACS and
Histology

All data are expressed as mean ± standard error of
the mean (SEM). Comparisons between study
groups were analyzed by Student’s t-tests. P values
of <0.05 were considered to be statistically
significant: *p < 0.05, **p < 0.01, ***p < 0.001.
Statistical analyses were performed using the
GraphPad Prism 7 software (La Jolla, CA).

CHAPTER 3
RESULTS

3.1 Data Harmonization and GWAS Analyses in
UK Biobank

To further elucidate the genetic architecture of
asthma, we leveraged publicly available GWAS
data from the TAGC and in subjects of European
ancestry from the UK Biobank
62
. Since the
summary level data available to us in UK Biobank
for asthma as a binary trait was based on linear
regression analyses, we first re-analyzed the data
to match the summary level data provided by
TAGC
62
. Specifically, we used allele counts for
10,894,596 SNPs in 38,791 asthma cases, defined
as doctor-diagnosed asthma, and 297,991 controls
to carry out a GWAS analysis based on logistic
regression in UK Biobank. These analyses yielded
a genomic inflation factor of 1.11 and, as expected,
the P-values calculated by logistic regression for
the ~11 million SNPs were highly correlated with
those obtained by linear regression (r=0.98; P<10
-
300
).

- 18 -
Our GWAS analysis for asthma in UK Biobank by
logistic regression identified 13,833 significantly
associated SNPs distributed among 69 loci. Of
these loci, 33 overlapped with the 51 known asthma
loci identified previously in adults, children, or
subjects from multiple ancestries
62-67
and 11
overlapped with 66 loci previously identified for
allergic disease, defined as a combined phenotype
of asthma, eczema, and hay fever
68
. The remaining
25 loci were novel and have not been previously
reported in GWAS for either asthma or allergic
disease. In addition, the effect sizes in UK Biobank
for 41 known adult asthma loci were highly
correlated with those reported by either TAGC
62
or
other previous GWAS in subjects of European
ancestry
63-65,67
(r=0.80, P=5.4x10
-10
). These
concordant results thus validated both our selection
of the asthma phenotype in UK Biobank and the
results of our GWAS analysis by logistic regression.

3.2 Meta-analyses of GWAS data for asthma in
UKBB and TAGC

We next used the harmonized GWAS results to
perform a meta-analysis for asthma in European
subjects from UK Biobank and TAGC, controlling
for genomic inflation, and identified 17 loci novel
that were significantly associated with asthma for
the first time herein. We next carried out a meta-
analysis with UK Biobank and all multi-ancestry
populations from TAGC (total n=479,268) with
1,983,348 SNPs common to both datasets. This
analysis yielded a total of 4,954 variants distributed
across 68 regions that were significantly associated
with asthma (Figure 8). In addition to yielding either
equivalent or more significant association signals at
the 17 novel loci identified in Europeans, three
additional loci were significantly associated with
asthma in the meta-analysis with all subjects. The
lead SNPs at these 17 loci yielded moderate and
consistent odds ratios (ORs) for asthma (1.04-1.07)
in both UK Biobank and TAGC, with no evidence
for heterogeneity. Of the remaining 51 significantly
associated regions, 38 were part of the 51 known
asthma loci
62-67
and 13 were previously identified
for allergic disease
68
. Of the 17 novel loci
discovered, the lead SNP rs7541333 at
chromosome 1p36.11 was most intriguing due to its
strong statistical significance (P<5.0x10
-8
). As
shown in Figure 9, the lead SNP rs7541333 maps
directly to a gene, referred to as CD52, on the first
chromosome.

3.3 Enrichment of novel asthma-associated
variants, such as C52, in Regulatory Elements
or Pathways

In order to investigate in which tissue and cell-types
these novel asthma-associated variants play an
important role, we next carried out with all asthma-
associated variants identified, such as the
aforementioned SNP rs7541333 mapping to CD52,
in our meta-analysis with P<5.0x10
-8
(n=4,954) or
1.0x10
-8
(n=4,291). There was ~2.5-fold enrichment
Figure 8. Manhattan plot for association of 1,983,348 SNPs with asthma. The plot highlights significant
associations at total of 68 total loci throughout the human genome. Of the aforementioned loci, 38 (blue) have
been previously reported to be associated with asthma and 12 (green) loci have been previously reported to
be linked to other allergic disorders such as hay fever or eczema. Due to the large number of SNPs in our
analysis, we were able to identify 17 novel loci (red) that were significantly associated with asthma.

- 19 -
of asthma-associated variants colocalizing to
DNase I hypersensitive sites in blood but not in
other tissues (Figure 10A). Moreover, this
enrichment was primarily in immune cells such as
CD20
+
B lymphocytes and CD3
+
, CD4
+
, or CD8
+
T
lymphocytes (Figure 10B). Taken together, these
results further highlight importance both of the
immune system component as well as the
identified genes, such as CD52, in the
pathophysiology of asthma.

3.4 CD52 is constitutively expressed on
lymphocyte both at steady state and under
inflammatory conditions

Next, we examined whether murine CD52 is
expressed at a protein level on the
aforementioned T and B lymphocytes. A cohort of
BALB/c BYJ mice were intraperitoneally (i.p)
immunized with 100µg of house dust mite (HDM)
in 2 mg of aluminum hydroxide and challenged
with HDM (50µg) or PBS intranasally (i.n.) on days
8, 9 and 10. 1 day after the first challenge the lungs
and spleen were isolated for FACS analysis
(Figure 11A). The CD19+ B cells, CD4+ helper T
cells, CD8+ cytotoxic T cells were examined as
shown in Figure 11B. Further analysis of the cell
surface phenotype of the aforementioned cells
showed that both naïve and HDM-induced murine
lymphocytes had high expression of CD52 (Figure
11C and 11D). Furthermore, human lymphocytes
show similarly high expression of CD52 (Figure
Figure 9. Regional plot of SNPs on chromosome
one. Strongly asthma-associated SNP (rs7541333)
is positioned on chromosome 1 within a small LD
and maps to the CD52 gene (indicated by small
rectangle). An <1 Mb region is shown, and the lead
SNP is represented by a purple diamond within the
regional plot. The blue line indicates estimated
recombination rates.
Figure 10. Enrichment of asthma-associated loci
within DNase I hypersensitive sites across
various cell types. A, Enrichment tests were
performed independently, and the radial axis shows
fold enrichment calculated at each of the eight GWAS
thresholds, each indicated by a different color, for
different cell types. Cell types are sorted by tissues,
represented along the outside edge of the plot. Font
size is proportional to the number of cell types from
that tissue. Among all of the tissues examined, the
strongest enrichment was seen for DNaseI
hypersensitivity sites in the blood. B, Enrichment of
asthma-associated loci within various cell types in the
blood. The immune cells, particularly lymphocytes
such as CD4+ helper T cell, CD8+ cytotoxic T cells
and CD20+ B cells display the most significant and
strongest enrichment.

- 20 -
11E and 11F). Taken together, these results
suggest that CD52 is highly expressed on
lymphocytes at both steady state and under
inflammatory conditions.

3.5 Anti-CD52 treatment yields high depletion
efficacy at both systemic and pulmonary levels

Since murine lymphocytes show high expression
levels of CD52, we next asked whether a novel anti-
murine CD52 monoclonal antibody (αCD52, BTG-
2G) from MBL laboratories can efficiently deplete
these cells in our AHR model. As shown in Figure
12A, a cohort of BALB/c BYJ mice were
intraperitoneally (i.p) treated with 500µg of αCD52
i.p. or isotype control 12 hours before three
subsequent intravenous (i.v.) administrations of
250µg of αCD52 or isotype control days 2, 3 and 4.
On the fifth day, the mice were euthanized, and the
lungs and spleen were isolated and the depletion
efficacy was analyzed by flow cytometry. As shown
in Figure 12B-E, anti-CD52 treatment resulted in
significant decrease in both number and
percentages of pulmonary and splenic CD45+
leukocytes. More specifically, the absolute number
and percentages of CD3+CD4+ and CD3+CD8+ T
cells, as well as CD19+ B cells decreased
dramatically at the end of the treatment course.
Figure 11. Both murine and human lymphocytes constitutively express CD52. A, A cohort of
BALB/c BYJ mice were intraperitoneally (i.p) immunized with 100µg of house dust mite (HDM) in 2 mg
of aluminum hydroxide (alum) and challenged with HDM (50µg) or PBS intranasally (i.n.) on days 8, 9
and 10. The mice were euthanized on day 11 and the lung and spleen were isolated, as shown in the
timeline. B, The gating strategy of CD45+C19+ B cells, CD45+CD3+CD4+ helper T cells (TH cells)
and CD45+CD3+CD8+ cytotoxic T cells (CTLs). C and D, Expression levels of CD52 in both naïve
(PBS) and HDM-activated murine B and T lymphocytes in the lungs and spleen. Corresponding
quantitation of CD52 expression shown as MFI+/− SEM, n = 6. E, 10 mL of peripheral blood was
collected from 5 donors and analyzed via flow cytometry. F, Expression of CD52 on human peripheral
blood B and T lymphocytes. Student’s t-test, *P < .05, **P < .01, and n.s., non-significant.

- 21 -
Taken together, these results demonstrate that the
novel anti-murine CD52 monoclonal antibody
(αCD52, BTG-2G) from MBL laboratories can
efficiently deplete CD52+ murine lymphocytes.
Moreover, the depletion effects of this monoclonal
antibody are observed both systemically, as well as
in the pulmonary lymphocytes at steady state.

3.6 CD52 depletion ameliorated HDM-induced
AHR by improving dynamic compliance and
abrogating airway resistance and lung
inflammation

Next, we investigated whether CD52 depletion
affects HDM-induced airway hyperreactivity (AHR)
and lung inflammation by comparing anti-CD52
Figure 12. Anti-CD52 treatment yields high depletion efficacy at both systemic and
pulmonary levels. A, A cohort of naïve BALB/c BYJ mice were treated with 500µg of anti-CD52
depleting antibody (αCD52) i.p. or isotype control on day 1, and subsequently treated with 250µg
of αCD52 antibody or isotype control intravenously (i.v.) on days 2, 3 and 4. On day 5, the mice
were dissected, as shown in the timeline. B and C, the number and percentage of pulmonary
lymphocytes, respectively. D and E, the number and percentage of splenic lymphocytes,
respectively. Data are shown as means ± SEMs and are representative of 2 individual
experiments. Error bars are the mean ± SEM. Student’s t-test, *P < .05, **P < .01, ***P < .005,
and ****P < .0005.

- 22 -
depleting antibody (αCD52) with isotype treated
control in a cohort of BALB/c BYJ mice. As shown
in Figure 13A, mice were intraperitoneally (i.p)
immunized with 100µg of house dust mite (HDM) in
2 mg of aluminum hydroxide and challenged with
HDM (50µg) or PBS intranasally (i.n.) on days 8, 9
and 10. Additionally, mice were treated with 500µg
of αCD52 i.p. or isotype control on day 7, and
subsequently treated with 250µg of αCD52 or
isotype control intravenously (i.v.) on days 8, 9 and
10. 1 day after the last i.n. challenge, lung function
was measured by direct measurement of lung
resistance and dynamic compliance in
anesthetized tracheostomized mice, in which mice
were mechanically ventilated and sequentially
challenged with aerosolized increasing doses of
methacholine. After measurements of AHR, the
trachea was cannulated and the bronchial alveolar
lavage (BAL) fluid was collected and analyzed by
flow cytometry. As expected, i.n. administration of
Figure 13. CD52 depletion significantly ameliorates HDM-induced airway hyperreactivity (AHR)
and abrogates inflammation. A, A cohort of BALB/c BYJ mice were intraperitoneally (i.p) immunized
with 100µg of house dust mite (HDM) in 2 mg of aluminum hydroxide (alum) and challenged with HDM
(50µg) or PBS intranasally (i.n.) on days 8, 9 and 10. Additionally, mice were treated with 500µg of
anti-CD52 depleting antibody (αCD52) i.p. or isotype control on day 7, and subsequently treated with
250µg of αCD52 antibody or isotype control intravenously (i.v.) on days 8, 9 and 10. On day 11, we
assessed the lung function, as shown in the timeline. B and C, Line graph show lung resistance and
dynamic compliance (cDyn) in response to increasing doses of methacholine. D, Total numbers of
CD45+ cells, eosinophils, T cells and neutrophils in Bronchoalveolar lavage (BAL) fluid have been
demonstrated in the bar graphs. E, Representative images (left panel) and quantification (right panel)
of hematoxylin and eosin–stained histologic sections of the lungs of mice. Scale bars=50 μm. Data are
shown as means ± SEMs and are representative of 2 individual experiments (n=10-11 each). Error
bars are the mean ± SEM. Student’s t-test, *P < .05, ***P < .005, and ****P < .0005.

- 23 -
HDM significantly increased lung resistance in
isotype treated cohort (Figure 13B); however, lung
resistance in αCD52 treated cohort was
significantly reduced, indicating that CD52
depletion ameliorates HDM-induced AHR. In
agreement with lung-resistance findings, the results
of dynamic compliance showed an improved
response in αCD52 treated cohort of mice
compared to isotype treated group, but they
showed lower dynamic compliance than their PBS-
treated counterparts (Figure 13C). HDM treatment
significantly increased the total numbers of CD45+
cells, eosinophils, T cells, as well as, neutrophils in
the BAL of isotype treated control. However, BAL
analysis in the αCD52 treated cohort revealed
significant reduction in the number of these
inflammatory cells, indicating that HDM-induced
inflammation is impaired upon CD52 depletion
(Figure 13D). Histological analyses further
revealed that CD52 depletion prevented airway wall
thickness and infiltrated cells (Figure 13E). These
findings concur with the reduction of AHR and
eosinophilia in BAL fluid.

Figure 14. CD52 depletion significantly reduces the number and percentages of inflammatory
lymphocytes in both spleen and lungs. A, A cohort of BALB/c BYJ mice were intraperitoneally (i.p)
immunized with 100µg of house dust mite (HDM) in 2 mg of aluminum hydroxide (alum) and challenged
with HDM (50µg) or PBS intranasally (i.n.) on days 8, 9 and 10. Additionally, mice were treated with 500µg
of anti-CD52 depleting antibody (αCD52) i.p. or isotype control on day 7, and subsequently treated with
250µg of αCD52 antibody or isotype control intravenously (i.v.) on days 8, 9 and 10. On day 11, we
dissected the mice, as shown in the timeline. B and C, the number and percentage of pulmonary
lymphocytes, respectively. D and E, the number and percentage of splenic lymphocytes, respectively.
Data are shown as means ± SEMs and are representative of 2 individual experiments (n=10-11 each).
Error bars are the mean ± SEM. Student’s t-test, *P < .05, **P < .01, ***P < .005, and ****P < .0005.

- 24 -
We also collected and quantified the number and
frequency of pulmonary and splenic lymphocytes
(Figure 14A). In agreement with the
aforementioned findings in the BAL, there was a
significant reduction of inflammatory cells in the
lungs (Figure 14B and 14C). Furthermore, analysis
of splenocytes demonstrated CD52 depletion
systematically reduced the number and frequency
of these lymphocytic populations (Figure 14D and
14E). Thus, these results demonstrate that effective
CD52 depletion significantly reduce HDM-
dependent lung inflammation and AHR. Taken
together, our results suggest that anti-CD52
treatment may serve as a novel therapeutic avenue
to treat allergic asthma.

Figure 15. Murine ILC2s constitutively express CD52, and this expression is inducible by IL-
33. A, A cohort of BALB/c BYJ mice were challenged with recombinant mouse (rm) IL-33 (0.5 µg in
50 µL) or PBS intranasally (i.n.) on days 1, 2 and 3. The mice were euthanized on day 4 and the
lung was isolated, as shown in the timeline. B, The gating strategy of Lin-CD45+CD127+ST2+ ILC2
cells. C, Expression levels of CD52 in both naïve (PBS) and IL-33-activated ILC2s in the lungs.
Corresponding quantitation of CD52 expression shown as MFI+/− SEM, n = 6. D, percentage of
CD52+ ILC2s under steady state (PBS) and inflammatory conditions (IL-33). E, Naïve pulmonary
ILC2s were sorted and subsequently cultured with rmIL-2 and rmIL-7 and rmIL-33 for 12, 24, and 48
hours. Freshly isolated ILC2 at 0 hours and ex vivo activated ILC2s were analyzed by flow cytometry
as indicated in the scheme and the kinetics of CD52 induction by IL-33 is shown. Error bars are the
mean ± SEM. Student’s t-test, **P < .01, and ***P < .005.

- 25 -
3.7 CD52 is constitutively
expressed on murine
ILC2s both at steady state
and under inflammatory
conditions. Furthermore,
CD52 expression on ILC2s
is inducible by IL-33

Since ILC2s have been
recently recognized for their
central role in both initiating
and perpetuating
inflammation, there recently
has been a lot of interest and
efforts in the field to identify
ILC2 biomarkers that can
serve as targets in future
therapeutics. As a result, we
next asked whether ILC2s
express CD52.

A cohort of BALB/c BYJ
mice were challenged with
IL-33 (0.5 µg) or PBS
intranasally (i.n.) on days 1,
2 and 3 (Figure 15A). On
day four, ILC2s from the
lungs were isolated and
analyzed by flow cytometry
and gated as lineage-
CD45+ CD127+ and ST2+
(Figure 15B). Further
analysis of the cell surface
phenotype of ILC2s
demonstrated both naïve
and IL-33-activated ILC2s
had high expression levels
of CD52. Furthermore, this
expression appears to be
inducible in vivo by IL-33,
and the percentages of
CD52+ ILC2s increase
under inflammatory
conditions (Figure 15C and
D). In order to determine the
kinetics of CD52 induction
by IL-33, we next sorted
naïve ILC2s from a cohort of
BALB/c BYJ mice and
cultured them in vitro in
presence of IL-2 and IL-7.
The pulmonary ILC2s were
subsequently ex-vivo
stimulated with IL-33, and
CD52 expression was
Figure 16. CD52 depletion significantly ameliorates IL-33-induced
airway hyperreactivity (AHR) and abrogates inflammation. A, A cohort of
BALB/c BYJ mice were treated with 500µg of anti-CD52 depleting antibody
(αCD52) i.p. or isotype control on day 1, and subsequently treated with 250µg
of αCD52 antibody or isotype control intravenously (i.v.) on days 2, 3 and 4.
Additionally, the mice were challenged with recombinant mouse (rm) IL-33
(0.5 µg) or PBS intranasally (i.n.) on days 2, 3 and 4. On day 5, we assessed
the lung function, as shown in the timeline. B and C, Line graph show lung
resistance and dynamic compliance (cDyn) in response to increasing doses
of methacholine. D−F, Total numbers of CD45+ cells, CD3+ T cells and
neutrophils in Bronchoalveolar lavage (BAL) fluid have been demonstrated in
the bar graphs. G, the percentage and absolute numbers of eosinophils in the
BAL. H, the percentage and absolute numbers of ILC2s in the lungs. E,
Representative images (left panel) and quantification (right panel) of
hematoxylin and eosin–stained histologic sections of the lungs of mice. Scale
bars=50 μm. Data are shown as means ± SEMs and are representative of 3
individual experiments. Error bars are the mean ± SEM. Student’s t-test, *P <
.05, **P < .01, ***P < .005, and n.s., non-significant.

- 26 -
analyzed over time at 0, 12, 24,
and 48 hours (Figure 15E). We
observed that CD52 expression
was increased over time and
reached statistical significance
after 48 hours of ex vivo IL-33
stimulation. Overall, these results
demonstrate pulmonary murine
ILC2s highly express CD52 at
both steady-state and under
inflammatory conditions.

3.8 CD52 depletion ameliorates
IL-33-induced AHR by
improving dynamic
compliance
and abrogating airway
resistance and lung
inflammation in wild-type mice

Next, we explored whether CD52
depletion affects IL-33-induced
airway hyperreactivity (AHR) and
lung inflammation by comparing
anti-CD52 depleting antibody
(αCD52) with isotype treated
control in a cohort of BALB/c BYJ
mice.
As shown in Figure 16A, the
mice were challenged with IL-33
(0.5 µg) or PBS intranasally (i.n.)
on days 2, 3 and 4. Additionally,
mice were treated with 500µg of
αCD52 i.p. or isotype control on
day 1, and subsequently treated
with 250µg of αCD52 or isotype
control intravenously (i.v.) on
days 2, 3 and 4. On the firth day
lung function was measured by
direct measurement of lung
resistance and dynamic
compliance in anesthetized
tracheostomized mice, in which
mice were mechanically
ventilated and sequentially
challenged with aerosolized
increasing doses of
methacholine. After
measurements of AHR, the
trachea was cannulated and the
bronchial alveolar lavage (BAL)
fluid was collected and analyzed
by flow cytometry.

Figure 17. Amelioration of IL-33-induced AHR and lung
inflammation via CD52 depletion is ILC2-dependent. A, A cohort
of Rag2
−/−
mice were treated with 500µg of anti-CD52 depleting
antibody (αCD52) i.p. or isotype control on day 1, and subsequently
treated with 250µg of αCD52 antibody or isotype control
intravenously (i.v.) on days 2, 3 and 4. The mice were also challenged
with recombinant mouse (rm) IL-33 (0.5 µg) or PBS intranasally (i.n.)
on days 2, 3 and 4. On day 5, we assessed the lung function, as
shown in the timeline. B and C, Line graph show lung resistance and
dynamic compliance (cDyn) in response to increasing doses of
methacholine. D, the percentage and absolute numbers of
eosinophils in the BAL. E, the percentage and absolute numbers of
ILC2s in the lungs. F, Representative images (left panel) and
quantification (right panel) of hematoxylin and eosin–stained
histologic sections of the lungs of mice. Scale bars=50 μm. Data are
shown as means ± SEMs and are representative of 3 individual
experiments. Error bars are the mean ± SEM. Student’s t-test, *P <
.05, **P < .01, and ***P < .005.

- 27 -
As anticipated, i.n.
administration of IL-33
significantly increased lung
resistance in isotype treated
cohort (Figure 16B);
however, lung resistance in
αCD52 treated cohort was
significantly reduced,
indicating that CD52
depletion ameliorates IL-33-
induced AHR. In agreement
with these findings, the
results of dynamic
compliance showed an
amended response in αCD52
treated cohort of mice
compared to isotype control,
but they showed lower
dynamic compliance than
their PBS-treated
counterparts (Figure 16C).
IL-33 treatment significantly
increased the total numbers
of CD45+ cells, eosinophils,
CD3+ T cells, as well as,
neutrophils in the BAL of
isotype treated control.
However, BAL analysis in the
αCD52 treated cohort
revealed significant reduction
in the number of these
inflammatory cells. Moreover,
the number of ILC2s is
similarly abated after αCD52
treatment compared to the
positive control, indicating
that IL-33-induced
inflammation is abridged
upon CD52 depletion
(Figures 16D-G). Further
histological analyses also
revealed that CD52 depletion
averted airway wall thickness
and infiltrated cells (Figure
16E). These findings concur
with the decline in AHR and
eosinophilia in BAL fluid.
Taken together, our results
indicate that anti-CD52
treatment significantly
ameliorates IL-33-induced
AHR by improving dynamic
compliance and abrogating
airway resistance and lung
inflammation.
Figure 18. CD52 depletion of ILC2s ameliorates Alternaria
alternata-induced AHR and abrogates lung inflammation in RAG2-
deficient mice. A, A cohort of Rag2
−/−
mice were treated with 500µg
of anti-CD52 depleting antibody (αCD52) i.p. or isotype control on day
1, and subsequently treated with 250µg of αCD52 antibody or isotype
control intravenously (i.v.) on days 2, 3 and 4. The mice were
challenged with Alternaria alternata (100 µg in 50 µL) or PBS
intranasally (i.n.) on days 2, 3 and 4. On day 5, we assessed the lung
function, as shown in the timeline. B and C, Line graph show lung
resistance and dynamic compliance (cDyn) in response to increasing
doses of methacholine. D, the percentage and absolute numbers of
eosinophils in the BAL. E, the percentage and absolute numbers of
ILC2s in the lungs. F, Representative images (left panel) and
quantification (right panel) of hematoxylin and eosin–stained histologic
sections of the lungs of mice. Scale bars=50 μm. Data are shown as
means ± SEMs and are representative of 3 individual experiments.
Error bars are the mean ± SEM. Student’s t-test, **P < .01, and ***P <
.005.

- 28 -

3.9 Amelioration of IL-33-induced AHR and lung
inflammation via CD52 depletion is ILC2-
dependent

Next, we questioned whether the amelioration of IL-
33-induced AHR and lung inflammation via CD52
depletion is ILC2-dependent. We examined the
effects of anti-CD52 depleting antibody on IL-33-
induced AHR and lung inflammation in Rag2
−/−
mice that lacked any mature B and T cells. A
cohort of Rag2
−/−
mice received either anti-CD52 or
isotype-matched control antibody (500µg per
mouse) intraperitoneally on day 1, and each cohort
also received either αCD52 or isotype control
(500µg per mouse) intravenously on days 2–4. The
mice were challenged with IL-33 (0.5 µg) or PBS
intranasally on days 2, 3 and 4.
Measurements of lung function
and sample acquisition followed
on day 5 (Figure 17A). Lung-
function data show that i.n. IL-33
administration increases lung
resistance (Figure 17B) and
decreases dynamic compliance
(Figure 17C) in Rag2
−/−
mice. In
contrast, lung resistance in IL-33-
treated cohort that received
αCD52 was significantly lower,
and dynamic compliance higher,
compared to the IL-33-treated
isotype-control-receiving mice
(Figures 17B and 17C).
Furthermore, IL-33 challenge led
to eosinophilia (Figure 17D),
increased ILC2 numbers and
thus lung inflammation in the
isotype-treated group. However,
αCD52 treated cohort showed
significant abrogation of
inflammatory eosinophil
recruitment, and thus reduced
eosinophilia in BALF (Figure
17D). Moreover, αCD52 treated
cohort showed significant
depletion and decreased ILC2
numbers in the lungs (Figure
17E). Histological analysis of the
lungs further demonstrated that
IL-33 challenge led to a
thickening of the epithelium and
increased inflammatory cells in
isotype control but not anti-CD52
treated mice (Figure 17H).
Taken together, these results
indicate that amelioration of IL-33-induced AHR
and lung inflammation via CD52 depletion is ILC2-
dependent.

3.10 CD52 depletion of ILC2s ameliorates
Allergen-induced AHR and reduces lung infl-
ammation in RAG2-deficient mice

We next investigated whether targeting CD52 on
ILC2s ameliorates induction of AHR and lung
inflammation induced by a clinically relevant
allergen. Rag2
−/−
mice received either αCD52 or
isotype control antibody intraperitoneally on day 1
(500 µg per mouse), and intravenously (250 µg per
mouse) on days 2, 4, and 6. The mice were
challenged intranasally with extract of Alternaria
Figure 19. Human ILC2s constitutively express CD52. A, Human
peripheral-blood ILC2s were freshly sorted and cultured with 10 ng of
recombinant human (rh) IL-2 and rhIL-7 in presence or absence of
rhIL-33 (10 ng). CD52 mRNA expression levels were analyzed after 9
hours and CD52 protein expression levels were analyzed after 48
hours. B, The gating strategy of Lin-CD45+CD127+CRTH2+ ILC2
cells. C, Expression levels of CD52 at a protein level in both naïve
(PBS) and IL-33-activated human ILC2s. Corresponding quantitation
of CD52 expression shown as MFI+/− SEM, n = 6. Student’s t-test,
****P < .0005, n.s., non-significant.

- 29 -
alternata (100 µg per
mouse) or PBS on days
2–5, followed by
subsequent
measurements of lung
function and sample
withdraw on day 7
(Figure 18A). Intranasal
administration of
Alternaria induced AHR,
as evidenced by both
increase in both lung
resistance (Figure 18B)
and decreased in
dynamic compliance
(Figure 18C), in
isotype-treated mice but
not αCD52 treated
group. The number of
eosinophils was
increased in the BAL of
Alternaria-treated mice,
but it was significantly
ablated in αCD52-
treated compared to the
isotype-treated control
(Figure 18D). Similarly,
the total number of
ILC2s was significantly
lowered in αCD52-
receiving mice
compared to the
isotype-receiving cohort
(Figure 18E). In
agreement with the
aforementioned results,
lung histology
demonstrated an
increased thickening of
the epithelium layer, as
well as an increased
number of inflammatory
cells in Alternaria-
treated, isotype-
receiving mice but not in
Alternaria-treated,
αCD52-receiving mice
(Figure 18F).
Collectively these
results anti-CD52
therapy can ameliorate
ILC2-dependent asthma
in response to clinically
relevant allergens.

Figure 20. CD52 depletion ameliorates human ILC2-mediated AHR and
abrogates lung inflammation in Rag2
−/−
Il2rg
−/−
mice. A, human peripheral
ILC2s were purified via FACS and cultured with rh-IL2 (20 ng/ml) and rh-IL-7 (20
ng/ml) for 48 hours, and then adoptively transferred into Rag2
−/−
Il2rg
−/−
mice that
were challenged with either recombinant human (rh) IL-33 (1 µg) or PBS
intranasally (i.n.) on days 2, 3 and 4. Additionally, mice were treated with
500µg of alemtuzumab i.p. or isotype control on day 1, and subsequently
treated with 250µg of alemtuzumab or isotype control intravenously (i.v.) on
days 2, 3 and 4. Measurement of lung function and analysis of BALF followed
on day 5, as shown in the timeline. B and C, Line graph show lung resistance
and dynamic compliance (cDyn) in response to increasing doses of
methacholine. D, the percentage and absolute numbers of eosinophils in the
BAL. E, the gating and absolute numbers of human ILC2s in the lungs. Data are
shown as means ± SEMs and are representative of 3 individual experiments.
Error bars are the mean ± SEM. Student’s t-test, *P < .05, **P < .01, and ***P <
.005.

- 30 -
3.11 CD52 is constitutively expressed on human
peripheral ILC2s at steady state and under
inflammatory conditions

In order to determine the translation potential of our
findings, we next investigated whether human
ILC2s express CD52. Human ILC2s were freshly
purified from human peripheral blood monocular
cells and cultured in the presence of both
recombinant human IL-2 (rhIL-2; 10 ng) and IL-2
(rhIL-7; 10 ng) in the presence or absence of rhIL-
33 (10 ng) for further analysis (Figure 19A). CD52
mRNA expression levels were analyzed after 9
hours of ex vivo stimulation and demonstrate CD52
is constitutively expressed at a mRNA level (Figure
19B). Peripheral blood from healthy donors was
collected and gated for ILC2s on the basis of the
lack of expression of human lineage markers (CD3,
CD14, CD16, CD19, CD20, CD56, CD235a, CD1a,
and CD123) and expression of CD45, CRTH2, and
CD127 (Figure 19C), and subsequently analyzed
for expression of CD52 after 48 hours of ex vivo
stimulation. In agreement with aforementioned
results, CD52 is also constitutively expressed at a
protein level (Figure 19D). However, IL-33
stimulation does not affect the expression of CD52
by human ILC2s.

3.12 CD52 depletion ameliorates human ILC2s-
mediated AHR and reduces lung inflammation
in Rag2-deficient Il2rg-deficient mice

In order to further confirm translation potential of
our findings, we next examined efficacy of anti-
CD52 treatment in human ILC2-mediated AHR. We
purified ILC2s form human PBMCs and, after 48
hours of in vitro culture in the presence of rh-IL-2
(20 ng/ml) and rh-IL-7 (20 ng/ml), adoptively
transferred ILC2s through the tail vain to
Rag2
−/−
Il2rg
−/−
mice, which lack T, B, and NK cells
and ILCs. As shown in Figure 20A, the mice were
challenged with recombinant human (rh) IL-33 (1
µg) or PBS intranasally (i.n.) on days 2, 3 and 4.
Additionally, mice were treated with 500µg of the
FDA-approved anti-human CD52 antibody
alemtuzumab (Hu116) i.p. or isotype control on day
1, and subsequently treated with 250µg of
alemtuzumab or isotype control intravenously (i.v.)
on days 2, 3 and 4. On the fifth day we measured
lung function as described above and assessed the
BAL fluid.

Lung-function demonstrate that i.n. rhIL-33
administration increases lung resistance (Figure
20B) and decreases dynamic compliance (Figure
20C) in Rag2
−/−
Il2rg
−/−
mice. In contrast, lung
resistance in rhIL-33-treated cohort that received
alemtuzumab was significantly lower, and dynamic
compliance higher, compared to the rhIL-33-treated
isotype-control-receiving mice (Figures 20B and
20C). Furthermore, rhIL-33 challenge led to
eosinophilia, increased human ILC2 numbers and
thus lung inflammation in the recipients of isotype
control. However, alemtuzumab treated cohort
showed significant abrogation of eosinophilia in
BAL fluid (Figure 20D). Moreover, alemtuzumab
treated cohort showed significant depletion and
decreased human ILC2 numbers in the lungs
(Figure 20E). Taken together, these results
indicate that human peripheral ILC2s alemtuzumab
depletes human ILC2s and may serve as a novel
potential therapeutic strategy for ameliorating
human ILC2-mediated lung inflammatory diseases.

CHAPTER 4
DISCUSSION

In this report, we performed the largest meta-
analysis for asthma to date by leveraging GWAS
case/control data in both multi-ancestry populations
and European populations. Our study validated
previously identified asthma loci in multi-ancestry
populations and identified 17 novel asthma loci at a
level of genome-wide significance, which explained
additional 4.4% of the overall SNP-based asthma
heritability. Importantly, the newly identified loci are
underlying asthma rather than allergic diseases.
The enrichment of known and novel asthma
variants in regulatory elements among blood and
immunologically related cells improves the
understanding of the GWAS results.

Among the 17 novel loci discovered, the lead SNP
rs7541333 at chromosome 1p36.11 was
captivating owing to its strong statistical
significance (P<5.0x10
-8
) and significant
enrichment for enhancer and promoter marks in
various asthma related tissues. This lead SNP
maps directly to a gene, referred to as cluster of
differentiation 52 (CD52), on the first chromosome.
CD52 gene has been previously reported to encode
for a glycosylphosphatidylinositol (GPI)-anchored
cell surface protein that consists of 12 relatively
acidic amino acids
36
. Despite limited knowledge
regarding its physiological role, CD52 has been
extensively linked to various immunological
disorders ranging from multiple sclerosis (MS), graft
versus host disease (GvHD), autoimmune
inflammatory neurodegenerative diseases as well

- 31 -
as various lymphomas such chronic lymphocytic
leukemia (CLL) or acute lymphocytic leukemia
(ALL)
37-41
. Consequently, CD52 has been an
encouraging target in many on-going clinical trials
for the aforementioned disorders. Hence,
alemtuzumab–also known as Campath-1H, is an
FDA approved recombinant humanized
monoclonal immunoglobulin IgG1 kappa that
targets CD52 and effectively treats and in some
cases cures patients with MS or CLL
42-47
. Our study
reports CD52 to be significantly associated with
asthma for the first time herein.

Numerous previous studies have implicated
asthma as a heterogeneous disease that involves
both the innate and adaptive immunity
18,69
. The
immunopathogenesis of asthma is derived by type
2 cytokine producing TH2 and ILC2 cells
70-72
. In this
study, we demonstrate that these key pathogenic
cell populations express CD52 and their depletion
significantly ameliorates airway hyperreactivity
(AHR) and abrogates lung inflammation.

We choose to evaluate the potential of anti-CD52
therapy in a house dust mite (HDM) induced AHR
model because HDM has been previously shown to
trigger a potent TH2 response and generate
pathogenic memory TH2 cells
73,74
. Our results
demonstrate that CD52 depletion drastically
reduces the number and percentages of
inflammatory T cells both systemically, as well as in
the pulmonary tissues. Depletion of the
inflammatory TH2 cells in the lung results
abrogation of lung resistance and improved
dynamic compliance. Furthermore, depletion of
lymphocytes at a systemic level ensures elimination
of any pathogenic memory TH2 cells. Previous
studies have reported that certain memory T cells
populations may leave the lungs and recirculate
75,76
.

Since ILC2s have been recently recognized for their
central role in both initiating and perpetuating type
2 inflammation, there recently has been a lot of
interest and efforts in the field to identify ILC2
biomarkers that can serve as targets in future
therapeutics
1
. For the first time we have
demonstrated that CD52 is constitutively expressed
by murine and human ILC2s at steady state and
under inflammatory conditions. Importantly, we
establish the high efficacy of CD52 depletion for
amelioration of IL-33-induced AHR. We utilize the
IL-33-based model because IL-33 and IL-25 had
been previously shown to induce ILC2-mediated
AHR and lung inflammation; IL-33 has been found
to be more potent than IL-25
77
. In order to exclude
the effect of the adaptive immunity in amelioration
of AHR, we choose to utilize Rag2
−/−
mice that
lacked any mature B and T cells
78
. Our results
clearly demonstrate that amelioration of AHR and
lung inflammation is ILC2-dependent. Since
alarmins, such as IL-33, are not naturally found in
the environment, we subsequently choose to
examine the potential of anti-CD52 treatment on
lung inflammation and AHR induced by a clinically
relevant allergen. We utilized Alternaria alternata
because it is a common fungus in the environment
and a common allergen in humans
79
. Moreover,
Alternaria alternata has been reported to cause
allergic inflammation in mice independently of
adaptive immunity, making it an ideal model to
study ILC2-dependent asthma
80,81
. Our result
display CD52 depletion of ILC2s improves lung
function and curtails lung inflammation following
exposure to principal allergenic agents. In order to
further assess the clinical relevance of our findings,
we introduce a humanized mouse model in which
human peripheral ILC2s are adoptively transferred
to Rag2
−/−
Il2rg
−/−
mice and intranasal administration
of recombinant human IL-33 causes AHR and
inflammation. Importantly, human IL-5 has been
reported to activate murine eosinophils,
underscoring the feasibility of using humanized
mice in eosinophilic inflammatory studies
82,83
. As a
result, this humanized mouse model provides a
unique platform for investigating the contribution of
ILC2s to human asthma and assessing the efficacy
of potential therapeutic targets in preclinical
studies. Utilizing this model, we demonstrate that
Table 2. Anti-CD52 as an ideal therapy. Anti-
CD52 drug, Alemtuzumab has been shown to meet
almost all of the criteria for an ideal therapy
3
.

- 32 -
the aforementioned FDA-approved anti-CD52
monoclonal antibody Alemtuzumab efficiently
ameliorates AHR by improving dynamic
compliance, and abrogating airway resistance and
lung inflammation. These results further emphasize
the therapeutic potential and translatability of our
findings to future clinical studies.

Current standard asthma therapeutics transiently
manage the clinical symptoms of asthma and do
not target the fundamental TH2 and ILC2 immune
cells that drive the pathogenesis of the disease.
The standard asthma care currently involved
steroid-based treatments target the clinical
symptoms at a pathophysiologic level and only
transiently stymie the progression of the underlying
inflammatory mechanisms. For instance, inhaled
corticosteroid prevents swelling of the airways
when taken in regularly scheduled daily doses
17
.
Other relief medications, such as an inhaled short
acting β2-agonist, act to relax the constricting
smooth muscles around the trachea, and thus
temporary dilate the narrowed airways
18
. In order
to improve the quality of life in asthmatic patients,
there has been a lot of efforts in the field to find
more effective treatments
1
. One potential
therapeutic approach has made use of blocking
antibody against type 2 cytokines such as IL-4, IL-
5 or IL-13
84
. However, treating asthma by targeting
these pluripotent cytokines will likely have
unwanted adverse effects on numerous other
pathways. On the hand, directly targeting and
modulating the function of the TH2 and ILC2
immune cell population that drive the pathogenesis
of the disease has shown to be promising
85-89
.

In this study, we demonstrate the effectiveness of
anti-CD52 treatment in asthma. Depletion of
CD52+ immune cells leads to rapid yet long-lasting
effect. Our results fall in line with previous studies
that explored the efficacy of anti-CD52 therapy in
other inflammatory disorders such as MS
42,90,91
. In
Figure 21. Anti-CD52 treatment as a promising therapeutic avenue in asthma. 1, utilizing the largest
human GWAS analysis to date, we’ve determined CD52 has a fundamental link to immunopathogenesis
of asthma. 2, anti-CD52 treatment results in rapid depletion of type 1 immune cells such as TH1 and ILC1s
(blue), type 2 immune cells such as TH2 and ILC2s (orange), and type 3 immune cells such as TH17 and
ILC3s (green), but not other unrelated immune cell populations such as macrophages (white) or HSCs
(grey). As a result, the risk of infections has been shown to be minimized compared current
immunosuppressive drugs, such as corticosteroids and antiproliferative agents, in phase II and III clinical
trials for other autoinflammatory diseases. 3, reprogramming of the immune cell composition and
polarization towards a tolerogenic state. 4, restoration of the tolerogenic networks and immune balance
between mutually antagonistic populations. The regulatory and effector branches of the immune system
can keep each other at bay, and the system reaches equilibrium.

- 33 -
fact, anti-CD52 treatments appears to meet almost
all the criteria that are expected from an ideal
disease-modifying immunosuppressive or
immunomodulatory drug for asthma (Table 2)
3
.

CD52 is expressed at a high level on both T cells
and ILC2s. It is also expressed on other asthma
related immune cells such as eosinophils,
neutrophils, mast cells and basophils. Importantly,
hematological stem cells (HSCs) do not express
CD52
48,49,92,93
. As a result, CD52 targeted depletion
has been shown to be subsequently followed by a
gradual repopulation and reprogramming of the
immune cells that arise from these stem cell
precursors
54,55
. Such reprograming ultimately
results in a shift of the immunological networks
towards a more tolerogenic state. This is because
among the depleted CD4
+
T cell population, the
frequency of CD25
high
CD127
-
and CD25
high
FoxP3
+

T regulatory (Treg) cells is increased. This is rather
striking because CD52 expression levels of these
cells is equivalent to all other CD4
+
helper T cell
populations. This post-treatment increased
frequency has been suggested to be a result of
faster rebound kinetics of Tregs
60
. Type 1, 2 or 17
T helper cells require foreign antigen recognition in
order to proliferate and expand. However, Tregs
are cable of homeostatic proliferation in response
to self-antigens that are much more abundant in
their respective microenvironments. As a result, the
immune balance between mutually antagonistic
immune cell populations is restored (Figure 21).

There are currently several other therapeutic
strategies that aim to reboot the immune system in
order to induce a tolerogenic state. One popular
method for treatment of MS involves collection and
in vitro storage of the hematological stem cells from
each patient
94
. Each patient is subsequently
subjected to extensive radiation and chemotherapy
in order to deplete the individual’s immune cells,
including the HSCs. The stored hematological stem
cells are then transplanted back into the patient,
migrate to the bone marrow and over time allow for
reconstitution of the immune system
94
. Although
such aggressive approaches have shown
promising results in on-going phase 3 clinical trials,
anti-CD52 treatment offers similar efficacy while
minimizing all of the toxic side effects associated
with radiation and chemotherapy regiments
3,42
.
These regiments nonspecifically target a board
range of cells, including non-asthma related
immune cells and HSCs in the bone marrow
94
. This
results in extreme toxicity of these regiments and
the additional need for a bone marrow
transplantation procedure. However, anti-CD52
treatment in less invasive as it mainly targets the
aforementioned asthma related immune cells
without affecting the HSCs
95
. As a result, there are
no toxic side effects and no requirement for any
additional invasive procedures.

Taken together, our results suggest that anti-CD52
treatment may serve as a novel therapeutic avenue
to treat and permanently cure both TH2 and ILC2
dependent allergic asthma.

ACKNOWLEDGEMENTS

I would like to thank my mentor and chair of
committee Dr. Omid Akbari for all of his guidance
and support over the past 2 years. I would also like
to express my deepest gratitude to my committee
members Dr. Jae U. Jung and Dr. Si-Yi Chen. I
have the highest level of admiration for their
research and greatly appreciate their time.

I would like to thank our postdocs Drs. Benjamin P.
Hurrell

and Lauriane Galle-Treger for all of their
support, including helping me troubleshoot and go
back to the drawing board after long experiments
sometimes at 3 am or even on Christmas and New
Year’s Eve. I would also like to thank our graduate
students Emily Howard, Richard Lo, and Swetha
Santosh for making lab a truly fun and productive
work environment.

Fight on!

This article was financially supported by National
Institutes of Health Public Health Service grants
R01 ES025786, R01 ES021801, R21 ES024707,
R21 AI109059 (O. A.).

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Abstract (if available)

Abstract Autoinflammatory diseases, such as asthma, are increasing at an alarming rate. Yet, the current steroid-based therapeutics inadequately target the clinical phenotypic features of asthma at a pathophysiologic level and only transiently block the progression of the inflammatory mechanisms. However, in order to better combat this immune inflammatory disorder, more effective treatments are needed to target the fundamental immune cells that are involved in the inflammatory response and ultimately cause the phenotypic symptoms observed in the clinic. Recently, culmination of the advances in our understanding of the immune system coupled with the new breakthroughs in systems biology and current panomics have manifested into our path forward towards a new era of precision medicine. The elegant functionality and high specificity of the immune system has opened the door for new immunotherapeutic approaches that can target pathogenesis of diseases at either cellular or molecular levels in highly specific and rationalized manners. Thus, such novel approaches herald a new era of immunotherapies for autoinflammatory and autoimmune disorders where the key factor is to target the relevant pathological components, in the case of asthma the driving type 2 immune cells such as TH2s and ILC2s. In this work, we first elucidate the genetic architecture of asthma by performing the largest human GWAS meta-analysis to date. We discovered 17 novel loci that are linked to the immunopathogenesis of asthma, among which the gene CD52 was most intriguing and exhibited significant enrichment among immune cells in the blood. CD52 gene has been previously reported to encode for a glycosylphosphatidylinositol (GPI)-anchored cell surface protein that consists of 12 relatively acidic amino acids. Despite limited knowledge regarding its physiological role, CD52 has been extensively linked to various immunological disorders ranging from multiple sclerosis (MS), graft versus host disease (GvHD), autoimmune inflammatory neurodegenerative diseases as well as various lymphomas such chronic lymphocytic leukemia (CLL) or acute lymphocytic leukemia (ALL). Consequently, CD52 has been an encouraging target in many on-going clinical trials for the aforementioned disorders. Hence, alemtuzumab—also known as Campath-1H—is an FDA approved recombinant humanized monoclonal immunoglobulin IgG1 kappa that targets CD52 and effectively treats and in some cases cures patients with MS or CLL. Our study reports CD52 to be significantly associated with asthma for the first time herein. We evaluated the therapeutic potential of anti-CD52 therapy in two different asthma models: TH2-dependent and ILC2-dependent models. We demonstrated the CD52 is expressed on both human and murine lymphocytes. We further established the high efficacy of a novel anti-murine CD52 monoclonal antibody and showed that treatment via this depleting antibody significantly ameliorates HDM-induced airway hyperreactivity (AHR) and abrogates inflammation in a TH2-dependent manner. Moreover, we demonstrated that both murine and human ILC2s constitutively express CD52 for the first time. We further showed that amelioration IL-33 and Alternaria alternata induced airway hyperreactivity (AHR) and lung inflammation is ILC2-dependent. We utilized a humanized mice model to demonstrate the translational potential of our findings and demonstrated that the aforementioned FDA-approved drug, alemtuzumab, can be used to treat asthma. The effectiveness of anti-CD52 treatment leads to rapid yet long-lasting depletion of CD52+ cells. Since the HSC do not express CD52, this depletion is subsequently followed by a gradual repopulation and reprogramming of the immune cells that arise from these stem cell precursors. Such reprograming results in a shift of the immunological networks towards a more tolerogenic state and restores the immune balance between mutually antagonistic cell populations. Taken together, our results suggest that anti-CD52 treatment may serve as a novel therapeutic avenue to treat and permanently cure both TH2 and ILC2 dependent allergic asthma.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Shafiei Jahani, Pedram (author)

Core Title A novel therapeutic approach in asthma: depleting CD52-expressing leukocytes suppresses airway hyperreactivity and ameliorates lung inflammation

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 06/13/2021

Defense Date 05/15/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag airway hyperreactivity (AHR),antigen-presenting cells (APCs),bronchoalveolar lavage (BAL),cytotoxic T cell (CTL),innate lymphoid cell,interleukin (IL),OAI-PMH Harvest,T helper cell,type 2 (ILC2),type 2 (TH2)

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Akbari, Omid (committee chair), Chen, Si-Yi (committee member), Jung, Jae (committee member)

Creator Email pshafieijahani@gmail.com,shafieij@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-172409

Unique identifier UC11660610

Identifier etd-ShafieiJah-7474.pdf (filename),usctheses-c89-172409 (legacy record id)

Legacy Identifier etd-ShafieiJah-7474.pdf

Dmrecord 172409

Document Type Thesis

Format application/pdf (imt)

Rights Shafiei Jahani, Pedram

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA