Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
The role of CD99 in T cells
(USC Thesis Other)
The role of CD99 in T cells
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
Copyright 2021 Lena Keossayan
The Role of CD99 in T Cells
by
Lena Keossayan
A Thesis Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
MOLECULAR PHARMACOLOGY AND TOXICOLOGY
August 2021
ii
Dedication
With immense gratitude, I dedicate my thesis
To Karen Nunez, for inspiring me to be a scientist. You are my role model and my mentor. Thank
you for trusting and believing in me and taking me under your wing. Thank you for your
unwavering support and patience. You believed in me when no one else did, including myself.
Thank you for motivating and inspiring me to pursue graduate school, and spending countless
hours researching programs and labs with me. Thank you for challenging me and pushing me
out of my comfort zone. I am eternally grateful for you.
To Dr. Nishan Tchekmedyian, for your continuous support, encouragement, and always allowing
me to contribute to your projects.
To my mama and baba, Anie and Varoujan, for your unconditional love and pride in my
successes. Thank you for the sacrifices you have made.
To my brothers, Alan and Chris, for always being there for me and inspiring me with your strong
work ethic. You are the best siblings anyone could ask for.
iii
Acknowledgements
First and foremost, I would like to express my gratitude to my advisor, Dr. Houda Alachkar
at the University of Southern California School of Pharmacy for teaching and supporting me every
step of my Master’s. Thank you for your patience and motivating me through all the challenges I
faced. I would like to thank my committee members, Dr. Yong ‘Tiger’ Zhang and Dr. Roger
Duncan for their time and advice regarding my research project and thesis. I would like to thank
Dr. Ian Haworth and Dr. Roger Duncan, for teaching me to see the world through a scientific lens.
I am very fortunate to have been trained and assisted by senior lab members, now Doctors,
Pooja Vaikari and Lucas Gutierrez. Pooja, thank you for having infinite patience with me as I
onboarded, and helping me master almost every lab skill and technique. You are truly one of a
kind. Lucas, thank you for donating blood for my project and for teaching me PBMC isolation. I
would not have been able to complete my projects without this skill. I would like to acknowledge
my current lab mates, Atham Ali and Yang Zhao. Atham, thank you for being the best lab mate
and always helping me, supporting me, motivating me, and making every day in the lab memorable
and filled with laughter. Yang, thank you for your positivity and helping me with my project. I
consider myself lucky to have such excellent lab mates.
I would also like to thank my middle school science teacher, Mrs. Lina Arslanian. You are
the reason I fell in love with science. Your passion for science was infectious and inspired me to
pursue an education and career in science.
Most importantly, I am grateful for my parents, brothers, Berj, and friends for their constant
support, helping me to reach this milestone. I will continue to make you all proud. I love you all.
Thank you.
iv
Table of Contents
Dedication ....................................................................................................................................... ii
Acknowledgements ........................................................................................................................ iii
List of Tables ................................................................................................................................. vi
List of Figures ............................................................................................................................... vii
Abstract ........................................................................................................................................ viii
Chapter 1: Background ................................................................................................................... 1
T Cells ......................................................................................................................................... 1
T cell stimulation in vitro ............................................................................................................ 5
CD99 ........................................................................................................................................... 7
CD99 gene and protein structure ................................................................................................. 7
CD99 expression and function in normal physiology ................................................................. 9
CD99 expression in T cells ....................................................................................................... 11
The Role of CD99 in T cells ..................................................................................................... 13
Apoptosis ................................................................................................................................13
Aggregation, Adhesion and Activation ..................................................................................16
CD99 antibodies ........................................................................................................................ 20
Chapter 2: Materials and Methods ................................................................................................ 25
Healthy Subject/Donor Samples ............................................................................................... 25
Peripheral Blood Mononuclear Cell Isolation, Freezing, Storage, and Thawing ..................... 25
Cell Culture ............................................................................................................................... 26
Anti-CD99 scFv ........................................................................................................................ 26
Binding assay and flow cytometry analysis .............................................................................. 27
Proliferation Assay .................................................................................................................... 27
Stimulated and Unstimulated Cells ........................................................................................27
Stimulated Cells ......................................................................................................................28
Statistical Analysis .................................................................................................................... 28
Chapter 3: Results ......................................................................................................................... 29
Anti-CD99 scFv binding results ................................................................................................ 29
The effect of anti-CD99 scFv in stimulated versus unstimulated T cells ................................. 31
Anti-CD99 scFv does not induce proliferation of unstimulated expanded T cells ex vivo ...... 32
Anti-CD99 scFv induces proliferation of stimulated expanded T cells ex vivo ....................... 33
v
Chapter 4: Discussion ................................................................................................................... 35
References ..................................................................................................................................... 39
vi
List of Tables
Table 1. Double negative (DN) thymocyte phenotypical stages .................................................... 1
Table 2. CD4+ subsets and cytokines ............................................................................................. 4
Table 3. The main biological functions of IL-2 .............................................................................. 6
Table 4. CD99 antibody comparison ............................................................................................ 20
Table 5. Antibody fragment formats and their constituents ......................................................... 22
Table 6. Mean fluorescence value of anti-CD99 scFv binding assay ........................................... 30
vii
List of Figures
Figure 1. Overall scheme of T-cell development in the thymus ..................................................... 3
Figure 2. Antigen presentation stimulates T cells to activate "cytotoxic" CD8+ cells or
"helper" CD4+ cells ........................................................................................................................ 4
Figure 3. IL-2 biology ..................................................................................................................... 6
Figure 4. Structural characterization of CD99 gene and its isoforms ............................................. 9
Figure 5. Schematic representation of the biological processes regulated by CD99 .................... 11
Figure 6. CD99 isoform expression controls T cell fates ............................................................. 18
Figure 7. CD99 mAbs and the epitopes they recognize................................................................ 20
Figure 8. Single-chain fragment variable (scFv) format ............................................................... 23
Figure 9. Binding assay results of anti-CD99 scFv treated T cells ............................................... 30
Figure 10. Mean fluorescence intensity values of anti-CD99 scFv in cells treated with 1, 5
and 10 μM concentrations of anti-CD99 scFv .............................................................................. 31
Figure 11. Cell concentration of unstimulated and stimulated T cells ......................................... 32
Figure 12. Effect of anti-CD99 scFv on unstimulated T cells ...................................................... 33
Figure 13. Effect of anti-CD99 scFv on stimulated T cells .......................................................... 34
viii
Abstract
CD99 (MIC2, E2) is a human 32 kDa highly O-glycosylated transmembrane glycoprotein,
that is broadly expressed on both hematopoietic and non-hematopoietic cells. CD99 plays a vital
role in a large array of T lymphocyte processes, including T cell activation, adhesion,
differentiation and the selection process, apoptosis, and transmigration. In cancer, CD99 acts as
either an oncosuppressor or oncogene depending on the cancer type. In addition, it is also involved
in immune and inflammatory responses within the tumor microenvironment. The CD99 gene
encodes two isoforms, CD99 short and long, which possess different functions depending on the
level of expression and location of the two isoforms. The variable expression of CD99 in T cells
throughout different stages of maturation is well documented and supported by extensive evidence,
however the mechanisms by which CD99 participates in T cell processes is not fully understood.
Considering its expression on the surface of T cells and the recent increased interest in CD99 as a
therapeutic target in cancer, better understanding of the role of this receptor in T cells is highly
needed. Here, we extensively review and discuss research findings related to the known functions
and roles of CD99 in T cells, summarize reported CD99 monoclonal antibodies and their activities.
In addition, we have developed a new single chain antibody against CD99 and examined its activity
in healthy peripheral human T cells. Overall, we found that treatment of healthy T human
peripheral T cells with a CD99 scFv increased cell proliferation, most notably at 48 hours post
treatment. Further functional and mechanistic studies are warranted to understand whether CD99
targeting approaches can be developed into an effective form of immune therapies against cancer
cells.
1
Chapter 1: Background
T Cells
T lymphocytes are members of the adaptive immune system, more specifically, the cell-
mediated immunity, or cellular immunity branch. Adaptive immunity is the immune system’s third
line of defense, following physical and chemical barriers, and innate immunity (Marshall et al.,
2018). T cells originate from hematopoietic stem cells (HSCs) in the bone marrow. These HSCs
give rise to committed lymphoid progenitor cells that migrate to the thymus via the blood for
maturation, selection, and export to the periphery in a process called thymopoiesis (Kumar et al.,
2018). Early committed T cells do not express a T-cell receptor (TCR), CD4 and CD8, and thus
are referred to as double-negative (DN) thymocytes (Germain, 2002). These DN thymocytes either
express some combination or lack of CD markers, CD44 and CD25: CD25, functioning as the
alpha chain of interleukin-2 receptor (Waldmann, 1989) and CD44 playing a role in cell adhesion
and migration (Sneath et al., 1998). These DN thymocytes are further divided into four
phenotypically and functionally distinct subsets of differentiation (Table 1) (Godfrey et al., 1993).
Table 1. Double negative (DN) thymocyte phenotypical stages
DN Thymocyte CD44 Status CD25 Status
Combined CD
Marker
DN1 + - CD44+CD25-
DN2 + + CD44+CD25+
DN3 - + CD44-CD25+
DN4 - - CD44-CD25-
2
DN thymocyte progenitors enter the thymus at the cusp of the cortico-medullary junction
(CMJ) (Figure 1). (Dunon et al., 1997), and migrates to the subcapsular zone (SCZ), accompanied
by progressive differentiation. Progressing through the DN2 to DN4 stages, DN thymocytes begin
to express pre-TCR, comprises pre-Tα and rearranged TCR β chains (von Boehmer et al., 1997).
Successful pre-TCR expression leads to DN4 transitioning to a double positive (DP) thymocyte
(CD4+CD8+), and a complete αβ TCR. DP thymocytes engage with cortical epithelial cells
expressing MHC class I and class II molecules associated with self-peptides. Signaling mediated
by the TCR with self-peptide-MHC ligands, determines the fate of the DP thymocytes (Robey et
al., 1994) (von Boehmer et al., 1989). Too much signaling promotes negative selection, and too
little signaling results in apoptosis (death via neglect). Proper, intermediate signaling via the TCR
results in positive selection, imitating maturation. DP thymocytes that bind MHC class I become
single positive (SP) CD8+ T cells, and those that bind MHC class II become SP CD4+ T cells, and
exit to the periphery (Germain R, 2002) (Yasutomo et al., 2000). The mechanisms behind the
matching of co-receptor-defined lineage and T-cell-receptor specificity that is achieved remains
unknown to this day. The SP T cells that exit to the periphery are immunologically naïve, Th0
cells, until they encounter an MHC-peptide complex that their TCR has a high affinity for (Figure
2). In short, naïve T cells are in a state between maturation and activation. Once these naïve T cells
counter an antigen presenting cell (APC) specific to their TCR, that is when activation to effector
T cells begins (Seder et al., 2003) (Berard et al., 2002).
3
Figure 1. Overall scheme of T-cell development in the thymus. (Germain, 2002)
4
Figure 2. Antigen presentation stimulates T cells to activate "cytotoxic" CD8+ cells or "helper"
CD4+ cells (Sjef, 2008)
Depending on the stimuli, CD4+ SP naïve T cells can differentiate into various T cell subtypes,
regulatory T cells (Tregs) and the helper T cells, such as TH1 and TH2. IL-12 and interferon γ
(IFNγ) are the critical cytokines that initiate the development of TH1 cells (Trinchieri et al., 2003).
Additional cytokines determine the type of helper T cells CD4+ SP T cells will become (Table 2).
CD8+ naïve T cells differentiate into cytotoxic T cells (CTL) (Cui et al., 2010).
Table 2. CD4+ subsets and cytokines (adapted from Luckheeram et al., 2012)
CD4+ Subset Cytokines
Th1 IL-12, IFNγ
Th2 IL-4, IL-2
Th17 IL-6, IL-21, IL-23
5
Cancer immunotherapy focused on T cells is a potent therapy against cancer because
certain studies have indicated the critical role certain T cell populations play as effectors of cancer
immunity (Acheampong et al., 2018) (Barret et al., 2010). Chimeric antigen receptors (CAR) for
T cell (CAR T) cellular therapy, is a form of immunotherapy known as adoptive cellular therapy,
which genetically alters patients’ T cells to express a specific artificial CAR, which then better
targets specific malignant cells (Sadelain et al, 2003). Another commonly used form of
immunotherapy is an immune checkpoint inhibitor, which targets the immune checkpoint
blockade, enhances T cell immunity against cancer (Darvin et al., 2018). PD1 and CTLA-4,
inhibitory receptors expressed on the surface of T cells, are the two most promising immune
checkpoint inhibitor targets currently.
T cell stimulation in vitro
The fate of T cells relies on both TCR activation and the presence of specific cytokines.
Interleukin-2 (IL-2) is a pleiotropic cytokine that was discovered in 1976 as a T cell growth factor
from the supernatant of phytohemagglutinin (PHA) -activated human peripheral blood
lymphocytes (Morgan et al., 1976). Another study found that PHA stimulation of human T cells
induced proliferation (Ceuppens et al., 1988). IL-2 is type 1 four α-helical bundle cytokine (15.5
kDa) and are primarily produced by CD4+ T cells upon antigen activation (Kim et al., 2006), and
it promotes further differentiation of activated T cells (T-helper cells, NK cells, and CTLs). IL-2
can be produced/secreted to a much lower extent by CD8+ T cells. Table 3 lists the main biological
functions of IL-2 in various cell types (Liao et al., 2011). IL-2 influences and promotes the
development of T cell lineages (Figure 3).
6
Figure 3. IL-2 biology (Ross et al., 2018)
Table 3. The main biological functions of IL-2 (adapted from Liao et al., 2011)
Cell Type Primary Activities
CD4+ T cells Induces proliferation and survival
Promotes activation-induced cell death (AICD)
Required for Th1, Th2, and Treg differentiation, but suppresses Th17
differentiation
7
CD8+ T cells Induces differentiation and expansion of effector cells
Augments cytolytic activity
Promotes generation and proliferation of memory CD8+ T cells
NK cells Promotes proliferation
Augments cytokine production
Enhances cytolytic activity
IL-2 is essentially the activator and the controller, and plays a critical role in regulating
immune responses, so it would come as no surprise that IL-2 is required in T cell culture media to
modulate proliferation and differentiation into mature peripheral T cells.
T cells can also be activated and differentiated in vitro via the crosslinking of CD3 specific
antibody with the TCR and phorbol myristate acetate (PMA) treatment, however this is a weak
and transient T cell activators (Smeets et al., 2012). IL-4 and IL-7 promote the survival of resting
T cells in vitro (Vella, et al., 1997). IL-7 has been found to promote the survival of naive T cells
and maturation of post-thymic naive CD4+ T cells in humans, but not in their differentiation
(Hassan and Reen, 1998). In in vivo activated T cells IL-4 and IL-15 were found to prevent T cell
apoptosis in vitro (Vella et al., 1998).
CD99
CD99 gene and protein structure
CD99 (Cluster of differentiation 99) was discovered in 1979 as human thymus-leukemia
antigen (Levy et al., 1979), is a highly O-glycosylated transmembrane protein encoded by the
CD99 gene, formerly known as the MIC2 gene (Goodfellow et al., 1988), or single-chain type-1
8
glycoprotein (Huijbers et al., 2019). O-linked carbohydrate chains account for 44% of its relative
molecular mass (Aubrit et al., 1989). CD99 is located in the pseudoautosomal region (PAR) of the
Y (Yp11-Ypter) and X (Xp22.33-Xpter) chromosomes in humans (Banting et al., 1989), (Aubrit
et al., 1989). CD99 shares structural homology with only the Xga protein, an X-borne allele of the
XG blood group system (Fouchet et al., 2000), suggesting these two are evolutionarily related
(Ellis et al., 1994). The CD99 gene encodes two distinct proteins: a wild-type full-length CD99
long isoform (CD99 L), also known as CD99 type I, with 185 amino acids (molecular weight of
32 kDa), and a truncated short isoform (CD99 S), also known as CD99 type II, with 161 amino
acids, as a result of alternative splicing (molecular weight of 28 kDa) of the CD99-encoding
mRNA (Hahn et al., 1997) (Figure 4). The CD99 S isoform transcript contains an 18-bp insertion
between exons 8 and 9 on the gene leading to an in-frame stop codon resulting in a truncated
polypeptide (Figure 4). CD99 L and S isoforms form hetero and homodimers beginning in the
Golgi apparatus then get translocated to the cell surface, (Scotlandi et al., 2007).
9
Figure 4. Structural characterization of CD99 gene and its isoforms (Pasello et al., 2018)
Rich in proline residues, CD99 protein is an integral membrane protein, with a 100 amino
acid extracellular domain, a transmembrane domain, and an intracellular C-terminal domain of 38
amino acids. While both CD99 isoforms contain shared motifs such as a cysteine residue in their
cytoplasmic domains, they differ in their intracytoplasmic domain. CD99 L contains two putative
phosphorylation sites on cytoplasmic domain, a serine at amino acid residue 168 (S168), and a
threonine at amino acid residue 181 (T181). NMR spectroscopy of the CD99 L isoform revealed
it has a hairpin structure secured by two flexible loops, with an unfolded protein (Kim et al., 2004).
CD99 expression and function in normal physiology
CD99 is expressed in many cell types, including immature progenitor and mature
hematopoietic stem cells, cortical thymocytes, endothelial cells, granular cells in the ovary, central
nervous system ependymal cells, pancreatic islet cells, and sertoli cells of the testes (Aussel et al.,
1993) (Dworzak et al., 1994) (Gordon et al., 1998). In hematopoietic stem cells, CD99 is
10
differentially expressed dependent on distinct maturation stages; with highest CD99 expression in
multipotent CD34+ bone marrow cells, and expression levels decreasing as cells differentiate into
more mature blood cells (Dworzak et al., 1994). CD99 is highly expressed in all leukocyte
lineages, with the highest expression in granulocytes and the most immature lymphocytes
(Dworzak et a., 1994). In normal physiology, CD99 is implicated in many different cellular
processes (Figure 5). CD99 plays a role in the diapedesis of leukocytes through homotypic
interaction of CD99 expressed on the leukocytes and at the endothelial cells (Dufour et al., 2008).
This interaction between the two cells leads to cellular migration (Dufour et al., 2008). The
expression of CD99 on endothelial cells activates protein kinase A, ultimately enabling the
migration of leukocytes across the endothelium (Watson et al., 2015) CD99 is also known to
regulate intracellular protein trafficking of MHC class 1 molecules, to the plasma membrane (Sohn
et al., 2001).
11
Figure 5. Schematic representation of the biological processes regulated by CD99 (Manara et al.,
2018)
CD99 expression in T cells
CD99 is expressed on the surface of all human T cells, though the level of expression and
isoforms control the fate and functional outcomes for T cells, Due to this variability, CD99 can
function as either the activating or inhibitory receptor in T cell regulation (Alberti et al., 2002).
In normal physiology, CD99 is known for its expression in T cells as it is involved in
numerous biological processes that affect T cell adhesion by regulating T cell rosette formation
(Bernard et al., 1988) and upregulating binding of T cells and activated peripheral blood
lymphocytes to inflamed vascular endothelial cells (Bernard et al., 2000). CD99 plays a role in
cell apoptosis and differentiation of immature thymocytes (Bernard et al., 1997).
12
T cells express different CD99 isoforms depending on their differentiation status.
Thymocytes, the most immature T cells, express both isoforms of CD99, depending on their stage
of maturation (Alberti et al., 2002). Thymocytes are classified into a number of distinct
maturational stages dependent on their expression of cell surface markers. Double positive (DP)
thymocytes, expressing both CD4 and CD8 co-receptors, express both CD99 isoforms, S and L,
and single-positive thymocytes and mature peripheral T cells, solely express CD99 L on their cell
surface (Alberti et al., 2002).
CD99 expression is found to be upregulated in both activated and memory T cells in
reactive lymph nodes. Even CD99-negative cells were found to express CD99 at very low levels
in an immunohistochemical study. In hyperplastic peri gastric lymph nodes, strong signal for CD99
positive cells were found in the superficial cortex, medullary cord and paracortex, and a small
number of these CD99 positive cells were found in the medullary sinus and germinal center cells.
Significant differences in CD99 expression in reactive lymph nodes are not observed when
adjusting for different stages of gastric carcinomas. The number of CD99 positive cells was found
to be decreased in peptic ulcer patients, but nonetheless the distribution pattern of the CD99
positive cells remained similar (Park et al., 1999). Further analysis revealed that recently activated
and/or memory CD4+ T cells express high levels of CD99, whereas naive resting CD4
+
T cells
express low levels of CD99 (Park et al., 1999).
IFN-gamma, a cytokine which stimulates the growth of cytotoxic T lymphocytes, is known
to strongly upregulate the expression of human MHC Class I molecules. CD99 and human MHC
Class I expression are positively correlated in wild type Jurkat T cells and in both CD99 positive
and negative T cells; however, upon stimulation with IFN-gamma, CD99 positive T cells display
an increase in human MHC Class I expression (Ghanekar et al., 2001), (Bremond et al., 2009).
13
Further studies revealed human MHC Class I molecules are physically linked to CD99 molecules
in Jurkat T cells (Bremond et al., 2009).
A 1994 study using a CD99 mAB that recognizes the 12E7 epitope found that strongest
density of CD99 expression is displayed by a very specific subpopulation of immature thymocytes,
carrying the antigenic profile CD34
weakor–
CD7
++
surface CD3
−
CD1a
weak
CD4
weak
CD8
- or weak
(Dworzak et al., 1994), which resemble the same profile previously reported (Terstappen et al.,
1992), suggesting that the phenotypic variation reflects the occurrence of differentiation prior to
the common thymocyte maturation stage. Further, mature peripheral T cells, CD4
+
and CD8
+
,
displayed a bimodal distribution of CD99 expression. All maturational and differentiation stages
that follow T cells express lower levels of CD99, suggesting CD99 plays a pivotal role in the early
developmental stages of thymic T cells (Dworzak et al., 1994).
The Role of CD99 in T cells
Apoptosis
Death receptor signaling plays a major role in the regulation of immune responses and
maintenance of homeostasis, particularly by triggering activation-induced cell death (AICD) of T
cells (Fas et al., 2006). These death receptors belong to the tumor necrosis factor receptor (TNFR)
superfamily (Dostert et al., 2019), and the Fas receptor, also known as CD95, and its ligand, Fas
L or CD95L mainly expressed by activated T cells (Suda et al., 1993), are the most intensively
studied members of the TNF superfamily, as the Fas/CD95 pathway is specialized in the
transduction of AICD in the immune system (Yonehara et al., 1989), (Trauth et al., 1989); namely
of chronically activated normal peripheral T cells (Lynch et al., 1995), (Trauth et al., 1989) and of
immature thymocytes during positive and negative selection in the thymus (Surh et al., 1994).
14
Initiator caspase recruitment and subsequent activation of a caspase cascade is the main
signaling pathway for Fas/CD95 receptor death signaling, classical apoptosis; they are proteases
which cause target cell DNA fragmentation and ultimately complete disintegration of the cells
(Mcllwain et al., 2013). Both resting and activated normal mature peripheral T cells express Fas,
however only activated normal mature peripheral T cells are capable of undergoing DNA
fragmentation and cell death via Fas/CD95 induction of caspase activation (Owen-Schaub et al.,
1992). Activating these cells to be subject to Fas/CD95 death signaling takes more than 4 days
(Owen-Schaub et al., 1992), suggesting that the immune system is using non classical pathways
for T cell and immature thymocyte apoptosis - ones that do not require the activation of caspases
(Pettersen et al., 2001), or proceed independently from Fas/CD95.
A previous study found that thymocytes irradiated with dexamethasone undergoing
apoptosis expressed a translocated phosphatidylserine at the outer leaflet of their plasma
membrane (Fadok et al., 1992), a phenomenon. This phenomenon was seen once again with
thymocytes and Jurkat cells after stimulation with CD99 mAbs, O662 and L129 (Aussel et al.,
1993), posing the question of whether CD99 plays a role in the death of these cells (Bernard et al.,
1997). It was found that the only the CD99 mAb reacting with the O662 epitope induced apoptosis
of the thymocytes and Jurkat cells, albeit kinetically much slower than the Fas/CD95 pathway (18
hours vs. 4 hours) (Bernard et al., 1997). Blocking experiments revealed that this CD99-mediated
apoptosis is independent of Fas/CD95, dependent on a novel caspase signaling, as these cells
displayed mitochondrial alterations, such as membrane blebbing, rather the classical DNA
fragmentation associated with caspases, before any morphological changes commonly associated
with apoptosis, such as chromatin condensation, cell shrinkage, and karyorrhexis, occurring
(Bernard et al., 1997). This CD99-mediated apoptosis only affected a specific subpopulation of
15
thymocytes (DP [CD4
+
CD8
+
], CD3 intermediate density, and CD69+ thymocytes), and had no
effect on mature peripheral T cells, suggesting that CD99 plays an important early role in the
positive selection process of thymocyte differentiation (Bernard et al., 1997).
A study using YG32, a CD99 mAb, found that while YG32 signaling failed to induce Jurkat
death, it enhanced Fas/CD95 mediated apoptosis of the cells through homotypic aggregation of
Fas/CD95 molecules (Jung et al., 2003), independent from caspase activation (Jung et al., 2003).
Another example of nonclassical programmed cell death signaling is seen with CD47. CD47-
mediated apoptosis is observed in both CD3 activated CD4+ and CD8+ normal T cells, and Jurkat
cells, and proceeds independent of Fas/CD95 signaling or caspase activation, characterized by the
lack of DNA fragmentation (Pettersen et al., 1999).
A newer CD99 mAb, Ad20, was found to induce Jurkat and various transformed T cell
apoptosis, proceeding independently of Fas/CD95 and or caspase activation, and was more rapid
and potent when compared to O662 (Pettersen et al., 2001). CD99-deficient Jurkat cells were found
to be resistant to Ad20, directly confirming that CD99 expression is required for Ad20 mediated
T cell apoptosis, and Ad20-mediating death signaling proceeds differently from the major
signaling pathways in thymocytes and normal, mature peripheral T cells (Pettersen et al., 2001).
Ad20-mediated T cell apoptosis pathway bears a striking resemblance (Pettersen et al.,
2001) to the previously mentioned CD47-mediated death signaling pathway (Pettersen et al.,
1999); however, CD47 blocking experiments revealed Ad20-mediated apoptosis was not affected
and confirmed that CD47 and CD99 mediated cell death proceed via different pathways (Pettersen
et al., 2001).
These information suggest CD99 may have a major biological role as a nonclassical death
receptor, in that it proceeds via a different pathway than Fas/CD95, and not always caspase
16
dependent. Targeting of distinct CD99 epitopes yields different results, but ultimately suggests
CD99 plays a role in the early stages of T cell differentiation, as CD99-mediated death signaling
often proceeds independently of Fas/CD95.
Aggregation, Adhesion and Activation
CD99 was discovered to be involved in the phenomenon known as e-rosette formation
between human T cells and erythrocytes (Bernard et al., 1988) confirmed via blocking experiments
with CD99 mAbs O662 and L129. Spontaneous rosette formation has been used as a marker for T
cells and has led to the understanding of many disease states and even in developing therapy for
immunodeficiencies (Reisner et al., 1983).
Rosette formation occurs as a result of epitope recognition on the central cell by an
antibody or receptor on an erythrocyte (Ocklind 1988). The stability of the e-rosettes is dependent
on the specific T cell population; for example, cortical thymocytes form stable rosettes, while
medullary thymocytes form unstable rosettes (Bernard et al., 1982)(West et al., 1977). The
identification of CD99 as a regulator of rosette formation can help understand the mechanisms
behind T cell adhesion and activation (Bernard et al., 1988).
Previously mentioned CD99 mAbs, O662 and L129, were found to trigger the homotypic
aggregation of Jurkat cells, with aggregation observed in as little as 4 hours (Bernard et al., 1995).
Incubation with CD99 mAbs, D44 and 12E7, did not result in any aggregation. It is important to
note that mAbs involved in blocking the spontaneous formation of T cell rosettes, lead to the
homotypic aggregation. Thus, a correlation exists between the mAbs interfering with T cell
adhesion and in their ability to induce aggregation of the T cells (Bernard et al., 1995). O662 and
L129 also induced aggregation of CD4+ CD8+ (DP) thymocytes, but not in peripheral T cells
17
which express similar levels of CD99, suggesting that the aggregation phenomenon is specific to
immature thymocyte stage of differentiation (Bernard et al., 1995).
CD99 upregulates the α4β1 integrin dependent binding of resting CD4+ T cells and
activated peripheral blood lymphocytes to inflamed vascular endothelial cells, with the number of
bound T cells increasing 2-14 fold following CD99 engagement (Bernard et al., 2000). CD99wt
expression in CD99-deficient T cell lines promotes CD99-induced cell adhesion via the rapid
activation of α4β1 integrin, with co-expression of CD99wt and CD99sh inducing T cell apoptosis,
confirmed via transfection experiments (Alberti et al., 2002) (Figure 6). This cell adhesion
mechanism is possibly due to sphingomyelin degradation, which regulates T cell homeostasis, and
is essential for activating cell signaling complexes which ultimately provides T cells motility to
interact with antigen-presenting cells (Avota et al., 2019).
CD99 short isoform is restricted to localization in glycosphingolipidic rafts, whereas CD99
L isoform expressing T cells are not found to be expressed on the glycosphingolipidic rafts (Alberti
et al., 2002). This suggests that the CD99 S variant is necessary for T cell differentiation and
activation. Disrupting the glycosphingolipidic rafts via a cholesterol-depleting agent and
subsequently stimulating with CD99, does not induce sphingomyelin apoptosis, indicating that the
short CD99 isoform localization is required for T cell motility and activation (Alberti et al., 2002).
When both isoforms of CD99 are co-expressed, they form heterodimers which are covalently
bound, and localize within the glycosphingolipidic rafts, inducing sphingomyelin apoptosis,
allowing T cells to interact with antigen-presenting cells (Alberti et al., 2002).
18
Figure 6. CD99 isoform expression controls T cell fates
T cells treated with anti-CD99 antibody exhibited an upregulation in TCR and MHC class
I and II surface expression, while CD4 and CD8 expression remained unchanged (Choi et al.,
1998). The increased MHC molecules surface expression occurs as quickly as within 10 minutes
of stimulation of CD99 and anti-CD99 antibody, and it is believed to be due to the induction of
actin polymerization which mobilizes cell surface molecules (Choi et al., 1998). This effect
suggests a potential role of CD99 in antigen presentation (Peta et al., 2011). CD99 was also found
associated with MHC class I, II and tetraspanin CD81, and upon T cell activation, CD99 is
translocated into the immunologic synapse, and regulates T cell proliferation (Peta et al., 2011). It
is believed that the CD99-MHC-CD81 complex is a tetraspanin “web” that plays a critical role in
immune response (Peta et al., 2011).
Crosslinking CD99 with a CD99 mAb in the presence of a CD3 Ab in human CD4+
peripheral blood T cells, resulted in the enhanced proliferation of peripheral T cells and enhanced
19
Interleukin-2 (IL-2) receptor expression (Wingett et al., 1999). T cells were treated with two
different CD99 mAbs; CD99R (expression restricted to hematopoietic myeloid, T, and NK cells)
and unrestricted CD99 (Wingett et al., 1999). Both mAbs yielded similar levels of T cell
proliferation, suggesting that the enhanced proliferation of peripheral T cells is due to the
costimulatory effects of ligation of CD99 with CD99 mAbs, and not to a restricted cluster of the
CD99 molecule (Wingett et al., 1999).
CD99 ligated with a CD99 mAb, 3B2/TA8, and GAM-IgG, in combination with a
suboptimal TCR (T-cell receptor)/CD3 complex elicits a strong positive stimulus for peripheral
blood T cell activation, and subsequent T cell proliferation and Th1-type cytokine production
(Waclavicek et al., 1998). The strength of the activation signal is dependent on the maturation
stage for the respective T cell (Waclavicek et al., 1998). TCR/CD3 complex knockdown followed
by crosslinked CD99 treatment does not lead to increased T cell activity (Waclavicek et al., 1998).
However, when the complex is not altered, a marked increase in cells expressing the TCR/CD3
complex was observed (Waclavicek et al., 1998). These findings suggest CD99 driven signal
transduction in T cells can be disrupted by reduced expression of TCR/CD3 complex on cells,
highlighting the important role TCR/CD3 complex in CD99 co-stimulation (Waclavicel et al.,
1998). Many of the CD99 epitopes recognized by different mAbs have not yet been described,
however a few have (Figure 7). It is important to note that natural circulating ligands for human
CD99 have not yet been identified. CD99-mediated signaling activation is thought to arise from
homophilic interactions among CD99 molecules on interacting cells (Schenkel et al., 2002) and
this phenomenon reinforces the importance of CD99 expression levels for determining its
physiological functions.
20
Figure 7. CD99 mAbs and the epitopes they recognize (Pasello et al., 2018)
CD99 antibodies
Several anti-CD99 monoclonal antibodies (mAbs) have been developed and investigated
by different groups for their activities in both normal and malignant cells (Table 4). Some of these
antibodies were also investigated in animal models of different cancers. However, these mAbs
have not progressed beyond the preclinical phase of drug development, due to their high
production costs.
Table 4. CD99 antibody comparison
CD99 Antibody
Clone
Reactivity Immunogen Role/Function in T cells
YG32 Human N/A Induces homotypic
aggregation of Jurkat T
cells and enhances Fas-
mediated death
Ad20 Human N/A Rapidly induces Jurkat T
cell death signaling
O662 Human Human thymocytes Induces thymocyte
apoptosis
Inhibits T cell rosette
formation
21
Induces Jurkat T cell
apoptosis
L129 Human N/A Induces homotypic
aggregation of Jurkat T
cells
Induces aggregation of
CD4+ CD8+ (DP)
thymocytes, but not in
peripheral T cells
D44 Human N/A Does not induce homotypic
aggregation of Jurkat T
cells
12E7 Human N/A Does not induce thymocyte
apoptosis
3B2/TA8 Human Human thymocytes Enhances peripheral blood
T cells in presence of CD3
mAb
Increased IL-2 activity in
Jurkat T cells in presence
of CD3 mAb
DN16 Human N/A Co-stimulation with CD3
mAb enhances T cell
proliferation and IL-2
stimulation
Induces homotypic
aggregation of Jurkat T
cells and apoptosis
H036-1.1 Human Human
Purified E-rosette
forming cells from
human peripheral blood
lymphocytes
Induces Jurkat T cell
apoptosis
MEM-131 Human ALL peripheral blood T
cells
No death response in Jurkat
T cells
22
An alternative solution is using antibody fragments. The modular structural and functional
nature of antibodies allows for the generation of these smaller antigen binding fragments through
molecular cloning, enzymatic methods, and antibody engineering (Nelson, 2010). These antibody
fragments flaunt several advantages over the use of conventional antibodies, with the primary
being lower production costs. This is because antibody fragments are typically produced using
microbial expression systems, resulting in quick turnaround time for cultivation and higher yields
(Fernandes, 2018). Antibody fragments can access obscure epitopes because of their small size,
and they have reduced immunogenicity (Kholodenko et al., 2019). There are 3 main antibody
fragment formats; Fragment variable (Fv)-based, Fab based, and single-domain antibodies (Table
5).
Table 5. Antibody fragment formats and their constituents (adapted from Bates et al., 2019)
Antibody Fragment Formats Constituents/Members
Fragment Variable (Fv)-Based
Single-chain fragment variable (scFv)
Tandem scFv
Diabodies, DART
®
s, TandAbs
Bispecific Fv fusion antibodies with an Fc domain
Fab Based
Fab
F(ab’)2
Single-Domain Antibodies
Nanobodies® (Nbs)
Domain antibodies
23
The scFv was first described in 1988 (Bird et al., 1988), roughly 2 years following the U.S.
Food and Drug Administration’s (FDA) approval of the first monoclonal antibody for clinical use
in humans, muromonab-CD3, trade name Orthoclone™ OKT3 (Smith, 1996). The scFv comprises
the C-terminus of variable regions of light (VL) and the N-terminus variable regions of heavy
(VH) chains of an antibody, linked by a serine- and glycine-rich flexible peptide that is typically
10-25 amino acids (Sun et al., 2014) (Figure 8). scFvs retain antigen specificity of their parental
or conventional mAbs and have a molecular weight of ~27 kDa (Sun et al., 2014), a fraction of
their full-sized antibody counterpart. Their small size allows for superior orientation of targeting
ligands and increased drug delivery loading capacities, improving overall efficacy (Richards et al.,
2017).
Figure 8. Single-chain fragment variable (scFv) format (Bates et al., 2019)
scFvs are ideal candidates for large-scale production in microbial systems because of their
size (Skerra et al., 1988) (Montoliu-Gaya et al., 2017), and fragments can be produced more
rapidly, with higher yields, with substantially lower productions costs than full-sized monoclonal
antibodies, which typically require costly mammalian expression systems (Hu et al., 2007)
24
(Spadiut et al., 2014). The risk of bystander immune cell activation is mitigated due to the absence
of an Fc region on the scFv, as well as many antibody effector functions, allowing the scFv to bind
to its target without activating the host’s immune system (Kholodenko et al., 2019), which would
be beneficial for immunocompromised patients. In this study, we will use an anti-CD99 scFv.
25
Chapter 2: Materials and Methods
Healthy Subject/Donor Samples
Whole human peripheral blood was collected using BD Vacutainer® Safety-Lok™ blood
collection set in BD Vacutainer® blood collection tubes containing the anticoagulant ETDA (BD
Vacutainer, Franklin Lakes, NJ). The donors/subjects involved in this study gave their written
informed consent authorizing the use prior to collecting samples. Each subject was presented with
a California Bill of Rights and provided with a copy of their fully signed consent form. The use of
human materials was approved by the Institutional Review Boards of the University of Southern
California in accordance with the Declaration of Helsinki (Study ID: HS-16-00274, Version Date:
25Feb2019, Valid From: 02Mar2019).
Peripheral Blood Mononuclear Cell Isolation, Freezing, Storage, and Thawing
Peripheral Blood Mononuclear Cells (PBMCs), composed of lymphocytes and monocytes,
were isolated by centrifugation of phosphate-buffered saline (PBS) (Sigma, St. Louis, MO),
diluted whole blood gently and methodically layered over a Ficoll-Paque PLUS density gradient
(GE Healthcare, Uppsala, Sweden). The initial centrifugation was followed by three subsequent
washing steps with PBS. The requested number of PBMCs were resuspended in freezing media
consisting of 10% dimethyl sulfoxide (DMSO) and Roswell Park Memorial Institute (RPMI) 1640
(1X) culture medium supplemented with 20% fetal bovine serum (FBS), 1% Antibiotic-
Antimycotic (AA) (100X), and 1% L-Glutamine (Gibco, Gaithersburg, MD). Resuspended cells
were transferred into 1mL cryogenic vials and placed into a 100% isopropanol freezing container
to ensure homogenous, consistent, and reproducible cell cryopreservation, and placed into a -80°C
Ultra-Low freezer for storage.
26
Cell Culture
PBMCs were isolated and frozen as stated above. PBMCs were thawed carefully to ensure
the highest possible cell viability and yield. Thawed PBMCs were cultured in warmed RPMI 1640
(1X) culture medium supplemented with 20% FBS, 1% AA (100X), and 1% L-Glutamine. In
addition, PBMCs were split into two separate T-75 suspension culture flasks (Genesee Scientific,
San Diego, CA): Stimulated and Unstimulated. The stimulated PBMCs were cultured in 20% FBS
RPMI media supplemented with IL-2 (5 ng/mL) and Phytohemagglutinin-L (PHA-L) Solution
(500X) (1uL per mL of culture media) (Life Technologies, Carlsbad, CA), and unstimulated cells
were cultured in 20% FBS RPMI media without IL-2 and PHA. Cell cultures were allowed to
incubate at 5% CO2 at 37°C to allow for proper and adequate T cell expansion.
Anti-CD99 scFv
Anti-CD99 scFv, molecular weight 26.9 kDa, was generated by and kindly provided to us
from The Zhang Lab at University of Southern California, School of Pharmacy. The concentration
of the anti-CD99 scFv was measured using the following equation:
C scFv= (A280-A350)/ɛl
The absorbance at 280 nm (A280) and 350 nm (A350), with ɛ as the molecular extinction
coefficient at 280 nm, and l as the path length in cm. To obtain the value of ɛ, the following
equation was used:
ɛ= 125nCystine + 5500n Tryptophan + 1490nTyrosine
Assuming all pairs of cysteine residues are oxidized to form cystine, the ɛ of anti-CD99 scFv was
estimated to be 41,620 mol
-1
cm
-1
.
27
The optical absorbance at 280 and 350 nm was measured with a NanoDrop 2000
(ThermoFisher Scientific Inc., Waltham, MA) at the Translational Research Laboratory at the
University of Southern California. There is a sequence of 6 continuous histidines attached with the
target sequence of the scFv, and this allows detection by anti His Tag PE-conjugated antibodies.
Binding assay and flow cytometry analysis
For the binding study, stimulated human PBMCs allowed to expand for 3 days, were
treated with anti-CD99 scFv at 1 μM, 5 μM, and 10 μM and incubated on ice for 30 minutes.
Following the 30-minute incubation period, cells were washed with phosphate buffered saline
(Sigma, St. Louis, MO), and treated with a master mix (MM) containing His Tag PE-conjugated
Antibody (R&D Systems, Minneapolis, MN) and Human CD3 Monoclonal Antibody (OKT3),
PerCP-Cyanine5.5 (eBioscience, San Diego, CA) and were incubated at room temperature for 30
minutes prior to a final washing step with PBS. Mean fluorescence value of PerCP-Cyanine5.5
was used to quantify data. Flow cytometry was performed on the LSRII BD Fortessa X20 flow
cytometer (BD, Franklin Lakes, NJ). and data analysis was performed Data analysis was
performed using FlowJo v10 10.7.1 (FlowJo, LLC, Ashland OR).
Proliferation Assay
Stimulated and Unstimulated Cells
T cell activation and proliferation were analyzed by treating 2.0 x 10
6
cells on Day 0,
stimulated and unstimulated, with either a control/0 or anti-CD99 scFv at 1 μM, 5 μM, and 10 uM
concentrations. After a 30-minute incubation period, cells were seeded in a 24 well plate at a
concentration of 5.0 x 10
5
/mL in RPMI with 20% FBS (unstimulated) or in RPMI with 20% FBS
supplemented with IL-2 and PHA-L (stimulated). Cells were counted on Day 1, Day 2, Day 3, and
28
Day 6 using Trypan Blue Solution, 0.4% (Gibco, Gaithersburg, MD) and an Improved Neubauer
hemocytometer with a chamber depth of 100 μm. Cell concentration was obtained by summing up
and averaging the 4 outer border quadrants, multiplying by the dilution factor used to count the
cells, and multiplying one last time by 10
4
to reveal the cell concentration (cells/mL). Cell counts
were performed in triplicates. Experiment was terminated on Day 6.
Stimulated Cells
T cell viability and proliferation were analyzed by treating 1.5 x10
5
stimulated PBMCs
(allowed to expand for 5 days) daily beginning on Day 0, with either a control/0 (RPMI 1640 (1X)
culture medium supplemented with 20% FBS, 1% AA (100X), and 1% L-Glutamine) or anti-CD99
scFv at 1 μM, and 5 μM concentrations. After a 30-minute incubation period post treatment, cells
were seeded at a concentration of 5.0 x 10
5
/mL. Cells were counted prior to treatment with anti-
CD99-scFv on Day 1, Day 2, Day 3, and Day 4. Cell counts were performed in triplicates.
Statistical Analysis
Multiple t-tests were used to determine the significance in means between two groups, with
statistical significance determined using the Holm-Sidak Method. To avoid increased chance of
Type 1 errors, data was analyzed using Two-Way ANOVA with Tukey’s HSD (honestly
significant difference) or Dunnett’s post-hoc testing for multiple comparisons to determine if the
difference in mean between more than two groups was statistically significant. For all analyses, p
<0.05 was considered significant. All the data is presented as mean ± standard error (SE).
29
Chapter 3: Results
Anti-CD99 scFv binding results
Flow cytometry measurements of anti-His and anti-CD3 antibodies illustrate the binding
of anti-CD99 scFv to the cell surface and confirms anti-CD99 scFv binds to T cells at 1 μM, 5 μM,
and 10 μM concentrations (Figure 9). When analyzing the mean fluorescence intensity (MFI), we
note the highest binding occurring at 10 μM (MFI= 14310), followed by 5 μM and 1 μM (MFI=
10541 and MFI= 8545, respectively; Table 6. Figure 10).
30
Figure 9. Binding assay results of anti-CD99 scFv treated T cells. PBMC expanded in IL2 and
PHA were treated with 1 μM, 5 μM, and 10 μM concentrations of anti-CD99 scFv. A. Flow
cytometry measurements of anti-His (which binds to anti-CD99scFv) and anti-CD3 (bind to CD3
on T cells) antibodies to the cell surface. B. histogram representation of the flow cytometry
measurement of anti-His (binds to anti-CD99scFv).
Table 6. Mean fluorescence value of anti-CD99 scFv binding assay
Group
Mean Fluorescence
Intensity Normalized to Untreated Group
Untreated 2640 1.000
CD99 scFv 1 uM 8545 3.237
CD99 scFv 5 uM 10541 3.992
CD99 scFv 10 uM 14310 5.420
31
Figure 10. Mean fluorescence intensity values of anti-CD99 scFv in cells treated with 1, 5 and 10
μM concentrations of anti-CD99 scFv. The values were normalized to the untreated group.
The effect of anti-CD99 scFv in stimulated versus unstimulated T cells
To examine the effect of anti-CD99 scFv on T cell proliferation, both unstimulated and
stimulated T cells were treated under identical conditions with anti-CD99-scFv. When comparing
unstimulated and stimulated controls, there was a statistically significant finding, with an increase
in stimulated cells (p=0.002823) (Figure 11).
32
Figure 11. Cell concentration of unstimulated and stimulated T cells. PBMC were thawed and T
cells were expanded the presence of IL-2 and PHA. Four days later, cells were washed to remove
the IL-2 and PHA; cells were counted daily for three days and then on day 6.
Anti-CD99 scFv does not induce proliferation of unstimulated expanded T cells ex vivo
Anti-CD99 scFv does not induce the proliferation of unstimulated T cells, as revealed in
our ex vivo experiment (Figure 12). Multiple comparisons between groups revealed that the anti-
CD99 scFv appears to significantly decrease cell viability of unstimulated cells (cells/mL) on days
2, 3 and 6 when comparing the 5 μM treatment group to the control (day 2, p=0.0012, fold-change=
0.510; day 3, p=0.0477, fold-change= 0.478; day 6, p=0.0043, fold-change= 0.266; Figure 12),
rather than induce proliferation. There were no statistically significant changes in cell counts per
mL when comparing the 1μM treatment group with the control throughout the duration of the
experiment.
33
Figure 12. Effect of anti-CD99 scFv on unstimulated T cells. PBMC were thawed and T cells were
expanded the presence of IL-2 and PHA. Four days later, cells were washed to remove the IL-2
and PHA and treated with either 1 or 5uM of anti-CD99 scFv in the absence of IL-2 and PHA;
cells were counted daily for three days and then on day 6. Cell count was compared with the
untreated cells.
Anti-CD99 scFv induces proliferation of stimulated expanded T cells ex vivo
Anti-CD99 scFv induces the proliferation of stimulated T cells, as revealed in our ex vivo
experiment (Figure 13). Multiple comparisons between groups revealed additional findings. When
comparing the 5 μM treatment group to the control (Figure 13), we observed significant increase
in cell counts (cells/mL) on Day 1 and Day 2 (p=0.0012, fold-change= 1.7; and p=0.0031, fold-
change= 1.8, respectively), however on day 3 and day 4, there is no significant difference in cell
counts between these two groups (day 3, p=0.6845, fold-change= 1.1; day 4, p=0.8715, fold-
change= 0.96; Figure 13). When comparing the 1 μM treatment group to the 5 μM treatment group,
we observe a significant difference in average cell counts on day 1, day 2, day 3, and day 4, with
the 5 μM treatment group having higher average cell counts per mL (day 1, p=0.0134, fold-
34
change= 1.5; day 2, p<0.0001, fold-change= 1.5; day 3, p=0.0090, fold-change= 1.4; day 4,
p=0.0027, fold-change= 1.3; Figure 13). While cell counts were increased on day 1 and day 2 for
the 1 μM treatment group, when compared to the control, the effect was not statistically different
(day 1, p=0.5755, fold-change= 1.2; day 2, p=0.3532, fold-change = 1.2; Figure 13). However, we
observed a slight but significant decrease in cell counts in the 1 μM treatment group compared
with the control on day 3 and 4 post treatment (day 3, p=0.0359, fold-change= 0.74; day 4,
p=0.0147, fold-change= 0.72; Figure 13).
Day 1 Day 2 Day 3 Day 4
0
1×10
6
2×10
6
3×10
6
4×10
6
5×10
6
Time (Days)
Cell Count (/mL)
0/Control
1uM
5 uM
✱✱
✱✱
✱
✱
Figure 13. Effect of anti-CD99 scFv on stimulated T cells. PBMC were thawed and T cells were
expanded the presence of IL-2 and PHA. Four days later, cells were treated with either 1 or 5uM
of anti-CD99 scFv in the presence of IL-2 and PHA; cells were counted daily for four days.
35
Chapter 4: Discussion
With the increased interest in the role of CD99 in various solid tumor and hematological
malignancies, and the knowledge related to it is expression and function in normal T cells, it is
crucial to investigate the developed therapeutic approaches against CD99 in T cells, and whether
observed effects can be leveraged in the development of innovative immunotherapies.
CD99 has been implicated in many biological processes related to normal cell physiology. CD99
has been shown to play a role in T cell activation, proliferation, thymocyte selection process,
aggregation and adhesion, and apoptosis. CD99 is expressed on almost all human cell types at low
levels, it is expressed at significantly high levels for specific cell types, including cortical
thymocytes, hematopoietic cells, and most notably having the highest level of expression in
immature thymic cells. This information, coupled with reports of CD99 being involved in T cell
activation (Dufour et al., 2008) (Takheaw et al., 2019), we wanted to determine if our newly
developed anti-CD99 scFv would activate human peripheral T cells leading to increased T cell
viability and proliferation. scFvs are known as antibody fragments, and they are derived from
conventional antibodies or easily manipulated through recombinant protein engineering
(Fernandes et al., 2018). The anti-CD99 scFv designed to target human CD99 was generated using
a mammalian expression system. This provides a great advantage because this antibody fragment
will be as close to the native structure as possible, including all the post translational modifications,
allowing for higher possibility of proper binding.
Data obtained regarding the treatment of stimulated and unstimulated cells, shows that
stimulation with PHA and IL-2 does lead to T cell expansion. The purpose of stimulating PBMCs
with IL-2 and PHA in particular is to strictly expand T cells, and no other cells found in PBMCs,
such as monocytes and dendritic cells. Anti-CD99 scFv treatment of unstimulated cells resulted in
no statistically significant increase in cell proliferation at either concentration, but rather a decline
36
in cell viability with the 5 μM treatment group. As a result, we proceeded to treat stimulated cells
for the rest of our experiments. We found that anti-CD99 scFv treatment at the 5 μM concentration
for the stimulated cells resulted in a statistically significant increase in T cell proliferation on Day
1 and 2.
Cells were in culture and treated with PHA and IL-2 to allow for T cell expansion. The
cells were not being stimulated anymore after they were seeded in culture plates. This may be a
possibility for the decline in the number of cells. IL-2 controls T cell function by regulating the
activity of cytosolic tyrosine kinases, and it is independent from Lck, which was the previously
proposed hypothesis (Ross et al., 2016). PHA binds to sugars on glycosylated surface proteins as
well as the TCR, and cross links them (Movafagh et al., 2011). This crosslinking leads to nuclear
factor of activated T cells (NFAT) activation via calcium-dependent signaling pathways (Kinoshita
et al., 1997).
Although using an scFv has many advantages, it also has several disadvantages. Its small
size results in their rapid glomerular filtration in the kidneys, leading to a short half-life (Hutt et
al., 2012) which can require higher doses or more frequent dosing regimens in vivo (Cumber et
al., 1992) (Sanz et al., 2005). More frequent dosing or higher dosing can be managed by coupling
the scFv to polyethylene glycol (PEG) chains or fusing them to human serum albumin to improve
half-life, but this goes against the very advantages that an scFv holds over a monoclonal antibody,
production costs and size increase (Jain et al., 2008). We previously engineered a fusion protein
composed of the same anti-CD99 scFv sequence conjugated with a high molecular weight elastin-
like polypeptide (ELP), A192: anti-CD99-A192, using a bacterial expression system (E. coli). Our
data using this fusion protein did not have any effect on healthy PBMCs, but it did result in
decreased cell viability of AML cell lines and primary AML cells (Vaikari et al., 2020). It is
37
important to note that these PBMCs were not stimulated with any T cell specific cytokines or
mitogens.
Future experiments should delve further into the functional role of CD99 on T cell
activation, including ELISpot assays to determine the activation of T cells by looking into specific
cytokines that are secreted by activated T cells and CD markers, such as IL-2, IL-7, CD3, CD8,
CD4, and CD25. Additionally, further investigations to determine which populations of T cells are
more or less affected by anti-CD99 antibody treatment will be crucial to expand the understanding
of its role in T cell function. Assessment of the effect of anti-CD99 scFV on T cells metabolic
activity using colorimetric assays may add some insights into the phenotypic changes induced by
CD99 targeting therapies. Experiments dissecting the dynamic changes of cell proliferation with
longer time points and different treatment concentrations may help explain the early expansion
followed by decline in cell counts observed in our experiments. Further, mechanistic studies should
be done to determine the regulatory role of anti-CD99 scFv on the T cell activation pathway. This
can be done by investigating a kinase, for example, Lck, and determining the effect of anti-CD99-
scFv on this pathway, and the downstream effect on the T cell signaling pathway.
A popular antibody fragment being used in clinical settings, namely in oncology, are
Bispecific T-cell engagers (BiTE®s). BiTE®s are a class of tandem scFv used to redirect cytotoxic
T cells to the site of tumors, resulting in a highly selective response, more powerful than can be
achieved with chemotherapy or radiotherapy (Huehls et al., 2015). Tandem scFvs can link two or
more different scFvs, forming the simplest bispecific antibody (Brinkmann et al., 2017). If CD99
proves to be a potential therapeutic marker for T cells, BiTE®s can be developed to target CD99
rather than CD3. Additionally, a tandem scFv can be used to target albumin, combatting the
relatively short half-life of scFvs (Holt et al., 2008). These therapies can be a great alternative to
38
targeting T cells solely using an scFv and seem like an ideal candidate to mitigate the downsides
of using an scFv, while remaining an affordable option in the antibody fragment class of therapies.
There are numerous CD99 antibodies as previously summarized, many focusing on Jurkat
T cells. These CD99 antibodies appear to have contradictory roles in the same cell lines: For
example, L129 inducing the homotypic aggregation of Jurkat T cells and D44 not inducing the
homotypic aggregation of Jurkat T cells. It was difficult to find the effect and role of many CD99
antibodies in human peripheral T cells. It is clear that these antibodies each target a specific epitope
on CD99, and it would be beneficial to determine the effects of these antibody clones on peripheral
human T cells and the specific epitopes it targets.
There are numerous reports characterizing CD99 and its expression and function in
different malignancies, yet many questions remain unanswered regarding the role of CD99 in T
cells and the mechanisms of action for T cell inhibition, activation, apoptosis during
differentiation, and so much more. CD99 had been largely neglected until recently. Ultimately, the
molecular mechanisms and signaling pathways by which CD99 participates in T cell function
remains largely unclear. With the increased interest and use of T cells for cancer immunotherapy,
we believe a fine-tuned understanding of the role of CD99 in regulating T cells will present a huge
therapeutic application potential, for cancer, autoimmune diseases, and numerous other ailments
and malignancies.
39
References
Acheampong, D. O., C. K. Adokoh, D. B. Asante, E. A. Asiamah, P. A. Barnie, D. O. M. Bonsu
& F. Kyei (2018) Immunotherapy for acute myeloid leukemia (AML): a potent alternative
therapy. Biomed Pharmacother, 97, 225-232.
Alberti, I., Bernard, G., Rouquette-Jazdanian, A. K., Pelassy, C., Pourtein, M., Aussel, C., &
Bernard, A. (2002). CD99 isoforms expression dictates T cell functional outcomes. FASEB
journal : official publication of the Federation of American Societies for Experimental Biology,
16(14), 1946–1948. https://doi.org/10.1096/fj.02-0049fje
Aubrit, F., Gelin, C., Pham, D., Raynal, B., & Bernard, A. (1989). The biochemical
characterization of E2, a T cell surface molecule involved in rosettes. European journal of
immunology, 19(8), 1431–1436. https://doi.org/10.1002/eji.1830190813
Aussel, C., Bernard, G., Breittmayer, J. P., Pelassy, C., Zoccola, D., & Bernard, A. (1993).
Monoclonal antibodies directed against the E2 protein (MIC2 gene product) induce exposure of
phosphatidylserine at the thymocyte cell surface. Biochemistry, 32(38), 10096–10101.
https://doi.org/10.1021/bi00089a027
Avota, E., de Lira, M. N., & Schneider-Schaulies, S. (2019). Sphingomyelin Breakdown in T
Cells: Role of Membrane Compartmentalization in T Cell Signaling and Interference by a
Pathogen. Frontiers in cell and developmental biology, 7, 152.
https://doi.org/10.3389/fcell.2019.00152
Banting, G. S., Pym, B., Darling, S. M., & Goodfellow, P. N. (1989). The MIC2 gene product:
epitope mapping and structural prediction analysis define an integral membrane protein.
Molecular immunology, 26(2), 181–188. https://doi.org/10.1016/0161-5890(89)90100-4
Barrett, A. J. & K. Le Blanc (2010) Immunotherapy prospects for acute myeloid leukaemia. Clin
Exp Immunol, 161, 223-32
Bates, A., & Power, C. A. (2019). David vs. Goliath: The Structure, Function, and Clinical
Prospects of Antibody Fragments. Antibodies (Basel, Switzerland), 8(2), 28.
https://doi.org/10.3390/antib8020028
Berard, M., & Tough, D. F. (2002). Qualitative differences between naïve and memory T cells.
Immunology, 106(2), 127–138. https://doi.org/10.1046/j.1365-2567.2002.01447.x
Bernard, A., Aubrit, F., Raynal, B., Pham, D., & Boumsell, L. (1988). A T cell surface molecule
different from CD2 is involved in spontaneous rosette formation with erythrocytes. Journal of
immunology (Baltimore, Md. : 1950), 140(6), 1802–1807.
40
Bernard, A., Gelin, C., Raynal, B., Pham, D., Gosse, C., & Boumsell, L. (1982). Phenomenon of
human T cells rosetting with sheep erythrocytes analyzed with monoclonal antibodies.
"Modulation" of a partially hidden epitope determining the conditions of interaction between T
cells and erythrocytes. The Journal of experimental medicine, 155(5), 1317–1333.
https://doi.org/10.1084/jem.155.5.1317
Bernard, G., Breittmayer, J. P., de Matteis, M., Trampont, P., Hofman, P., Senik, A., & Bernard,
A. (1997). Apoptosis of immature thymocytes mediated by E2/CD99. Journal of immunology
(Baltimore, Md. : 1950), 158(6), 2543–2550.
Bernard, G., Raimondi, V., Alberti, I., Pourtein, M., Widjenes, J., Ticchioni, M., & Bernard, A.
(2000). CD99 (E2) up-regulates alpha4beta1-dependent T cell adhesion to inflamed vascular
endothelium under flow conditions. European journal of immunology, 30(10), 3061–3065.
https://doi.org/10.1002/1521-4141(200010)30:10<3061::AID-IMMU3061>3.0.CO;2-M
Bernard, G., Zoccola, D., Deckert, M., Breittmayer, J. P., Aussel, C., & Bernard, A. (1995). The
E2 molecule (CD99) specifically triggers homotypic aggregation of CD4+ CD8+ thymocytes.
Journal of immunology (Baltimore, Md. : 1950), 154(1), 26–32.
Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson, S., Kaufman, B. M., Lee, S.-M., Lee, T.,
Pope, S. H., Riordan, G. S., & Whitlow, M. (1988). Single-chain antigen-binding proteins.
Science, 242(4877), 423+.
Brémond, A., Meynet, O., Mahiddine, K., Coito, S., Tichet, M., Scotlandi, K., Breittmayer, J. P.,
Gounon, P., Gleeson, P. A., Bernard, A., & Bernard, G. (2009). Regulation of HLA class I
surface expression requires CD99 and p230/golgin-245 interaction. Blood, 113(2), 347–357.
https://doi.org/10.1182/blood-2008-02-137745
Brinkmann, U., & Kontermann, R. E. (2017). The making of bispecific antibodies. mAbs, 9(2),
182–212. https://doi.org/10.1080/19420862.2016.1268307
Ceuppens, J. L., Baroja, M. L., Lorre, K., Van Damme, J., & Billiau, A. (1988). Human T cell
activation with phytohemagglutinin. The function of IL-6 as an accessory signal. Journal of
immunology (Baltimore, Md. : 1950), 141(11), 3868–3874.
Choi, E. Y., Park, W. S., Jung, K. C., Kim, S. H., Kim, Y. Y., Lee, W. J., & Park, S. H. (1998).
Engagement of CD99 induces up-regulation of TCR and MHC class I and II molecules on the
surface of human thymocytes. Journal of immunology (Baltimore, Md. : 1950), 161(2), 749–754.
Cui, W., & Kaech, S. M. (2010). Generation of effector CD8+ T cells and their conversion to
memory T cells. Immunological reviews, 236, 151–166. https://doi.org/10.1111/j.1600-
065X.2010.00926.x
41
Cumber, A. J., Ward, E. S., Winter, G., Parnell, G. D., & Wawrzynczak, E. J. (1992).
Comparative stabilities in vitro and in vivo of a recombinant mouse antibody FvCys fragment
and a bisFvCys conjugate. Journal of immunology (Baltimore, Md. : 1950), 149(1), 120–126.
Darvin, P., Toor, S.M., Sasidharan Nair, V. et al. Immune checkpoint inhibitors: recent progress
and potential biomarkers. Exp Mol Med 50, 1–11 (2018). https://doi.org/10.1038/s12276-018-
0191-1
Dostert, C., Grusdat, M., Letellier, E., & Brenner, D. (2019). The TNF Family of Ligands and
Receptors: Communication Modules in the Immune System and Beyond. Physiological reviews,
99(1), 115–160. https://doi.org/10.1152/physrev.00045.2017
Dufour, E. M., Deroche, A., Bae, Y., & Muller, W. A. (2008). CD99 is essential for leukocyte
diapedesis in vivo. Cell communication & adhesion, 15(4), 351–363.
https://doi.org/10.1080/15419060802442191
Dunon, D., Courtois, D., Vainio, O., Six, A., Chen, C. H., Cooper, M. D., Dangy, J. P., & Imhof,
B. A. (1997). Ontogeny of the immune system: gamma/delta and alpha/beta T cells migrate from
thymus to the periphery in alternating waves. The Journal of experimental medicine, 186(7),
977–988. https://doi.org/10.1084/jem.186.7.977
Dworzak, M. N., Fritsch, G., Buchinger, P., Fleischer, C., Printz, D., Zellner, A., Schöllhammer,
A., Steiner, G., Ambros, P. F., & Gadner, H. (1994). Flow cytometric assessment of human
MIC2 expression in bone marrow, thymus, and peripheral blood. Blood, 83(2), 415–425.
Ellis, N.A., Tippett, P., Petty, A., Reid, M., Weller, P.A., Ye, T.Z., German, J., Goodfellow,
P.N., Thomas, S., and Banting, G. (1994). PBDX is the XG blood group gene. Nat Genet 8, 285-
290.
Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., & Henson, P. M.
(1992). Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific
recognition and removal by macrophages. Journal of immunology (Baltimore, Md. : 1950),
148(7), 2207–2216.
Fas, S. C., Fritzsching, B., Suri-Payer, E., & Krammer, P. H. (2006). Death receptor signaling
and its function in the immune system. Current directions in autoimmunity, 9, 1–17.
https://doi.org/10.1159/000090767
Fernandes J. C. (2018). Therapeutic application of antibody fragments in autoimmune diseases:
current state and prospects. Drug discovery today, 23(12), 1996–2002.
https://doi.org/10.1016/j.drudis.2018.06.003
42
Fernandes J. C. (2018). Therapeutic application of antibody fragments in autoimmune diseases:
current state and prospects. Drug discovery today, 23(12), 1996–2002.
https://doi.org/10.1016/j.drudis.2018.06.003
Fouchet, C., Gane, P., Huet, M., Fellous, M., Rouger, P., Banting, G., Cartron, J. P., & Lopez, C.
(2000). A study of the coregulation and tissue specificity of XG and MIC2 gene expression in
eukaryotic cells. Blood, 95(5), 1819–1826.
Germain R. N. (2002). T-cell development and the CD4-CD8 lineage decision. Nature reviews.
Immunology, 2(5), 309–322. https://doi.org/10.1038/nri798
Ghanekar, S. A., Nomura, L. E., Suni, M. A., Picker, L. J., Maecker, H. T., & Maino, V. C.
(2001). Gamma interferon expression in CD8(+) T cells is a marker for circulating cytotoxic T
lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus
phosphoprotein pp65. Clinical and diagnostic laboratory immunology, 8(3), 628–631.
https://doi.org/10.1128/CDLI.8.3.628-631.2001
Godfrey, D. I., Kennedy, J., Suda, T., & Zlotnik, A. (1993). A developmental pathway involving
four phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative adult
mouse thymocytes defined by CD44 and CD25 expression. Journal of immunology (Baltimore,
Md. : 1950), 150(10), 4244–4252.
Goodfellow, P. N., Pym, B., Pritchard, C., Ellis, N., Palmer, M., Smith, M., & Goodfellow, P. J.
(1988). MIC2: a human pseudoautosomal gene. Philosophical transactions of the Royal Society
of London. Series B, Biological sciences, 322(1208), 145–154.
https://doi.org/10.1098/rstb.1988.0122
Gordon MD, Corless C, Renshaw AA, Beckstead J. CD99, keratin, and vimentin staining of sex
cord-stromal tumors, normal ovary, and testis. Mod Pathol. 1998;11:769–73
Hahn, J. H., Kim, M. K., Choi, E. Y., Kim, S. H., Sohn, H. W., Ham, D. I., Chung, D. H., Kim,
T. J., Lee, W. J., Park, C. K., Ree, H. J., & Park, S. H. (1997). CD99 (MIC2) regulates the LFA-
1/ICAM-1-mediated adhesion of lymphocytes, and its gene encodes both positive and negative
regulators of cellular adhesion. Journal of immunology (Baltimore, Md. : 1950), 159(5), 2250–
2258.
Hassan, J., & Reen, D. J. (1998). IL-7 promotes the survival and maturation but not
differentiation of human post-thymic CD4+ T cells. European journal of immunology, 28(10),
3057–3065. https://doi.org/10.1002/(SICI)1521-4141(199810)28:10<3057::AID-
IMMU3057>3.0.CO;2-Z
Holt, L. J., Basran, A., Jones, K., Chorlton, J., Jespers, L. S., Brewis, N. D., & Tomlinson, I. M.
(2008). Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs.
43
Protein engineering, design & selection : PEDS, 21(5), 283–288.
https://doi.org/10.1093/protein/gzm067
Hu, X., O'Hara, L., White, S., Magner, E., Kane, M., & Wall, J. G. (2007). Optimisation of
production of a domoic acid-binding scFv antibody fragment in Escherichia coli using molecular
chaperones and functional immobilisation on a mesoporous silicate support. Protein expression
and purification, 52(1), 194–201. https://doi.org/10.1016/j.pep.2006.08.009
Huehls, A. M., Coupet, T. A., & Sentman, C. L. (2015). Bispecific T-cell engagers for cancer
immunotherapy. Immunology and cell biology, 93(3), 290–296.
https://doi.org/10.1038/icb.2014.93
Huijbers, E., van der Werf, I. M., Faber, L. D., Sialino, L. D., van der Laan, P., Holland, H. A.,
Cimpean, A. M., Thijssen, V., van Beijnum, J. R., & Griffioen, A. W. (2019). Targeting Tumor
Vascular CD99 Inhibits Tumor Growth. Frontiers in immunology, 10, 651.
https://doi.org/10.3389/fimmu.2019.00651
Hutt, M., Färber-Schwarz, A., Unverdorben, F., Richter, F., & Kontermann, R. E. (2012). Plasma
half-life extension of small recombinant antibodies by fusion to immunoglobulin-binding
domains. The Journal of biological chemistry, 287(7), 4462–4469.
https://doi.org/10.1074/jbc.M111.311522
Jain, A., & Jain, S. K. (2008). PEGylation: an approach for drug delivery. A review. Critical
reviews in therapeutic drug carrier systems, 25(5), 403–447.
https://doi.org/10.1615/critrevtherdrugcarriersyst.v25.i5.10
Jung, K. C., Kim, N. H., Park, W. S., Park, S. H., & Bae, Y. (2003). The CD99 signal enhances
Fas-mediated apoptosis in the human leukemic cell line, Jurkat. FEBS letters, 554(3), 478–484.
https://doi.org/10.1016/s0014-5793(03)01224-9
Kholodenko, R. V., Kalinovsky, D. V., Doronin, I. I., Ponomarev, E. D., & Kholodenko, I. V.
(2019). Antibody Fragments as Potential Biopharmaceuticals for Cancer Therapy: Success and
Limitations. Current medicinal chemistry, 26(3), 396–426.
https://doi.org/10.2174/0929867324666170817152554
Kim, H. P., Imbert, J., & Leonard, W. J. (2006). Both integrated and differential regulation of
components of the IL-2/IL-2 receptor system. Cytokine & growth factor reviews, 17(5), 349–
366. https://doi.org/10.1016/j.cytogfr.2006.07.003
Kim, H. Y., Kim, Y. M., Shin, Y. K., Park, S. H., & Lee, W. (2004). Solution structure of the
cytoplasmic domain of human CD99 type I. Molecules and cells, 18(1), 24–29.
44
Kinoshita, S., Su, L., Amano, M., Timmerman, L. A., Kaneshima, H., & Nolan, G. P. (1997).
The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene expression
in T cells. Immunity, 6(3), 235–244. https://doi.org/10.1016/s1074-7613(00)80326-x
Kumar, B. V., Connors, T. J., & Farber, D. L. (2018). Human T Cell Development, Localization,
and Function throughout Life. Immunity, 48(2), 202–213.
https://doi.org/10.1016/j.immuni.2018.01.007
Levy, R., Dilley, J., Fox, R. I., & Warnke, R. (1979). A human thymus-leukemia antigen defined
by hybridoma monoclonal antibodies. Proceedings of the National Academy of Sciences of the
United States of America, 76(12), 6552–6556. https://doi.org/10.1073/pnas.76.12.6552
Liao, W., Lin, J. X., & Leonard, W. J. (2011). IL-2 family cytokines: new insights into the
complex roles of IL-2 as a broad regulator of T helper cell differentiation. Current opinion in
immunology, 23(5), 598–604. https://doi.org/10.1016/j.coi.2011.08.003
Luckheeram, R. V., Zhou, R., Verma, A. D., & Xia, B. (2012). CD4⁺T cells: differentiation and
functions. Clinical & developmental immunology, 2012, 925135.
https://doi.org/10.1155/2012/925135
Lynch, D. H., Ramsdell, F., & Alderson, M. R. (1995). Fas and FasL in the homeostatic
regulation of immune responses. Immunology today, 16(12), 569–574.
https://doi.org/10.1016/0167-5699(95)80079-4
Manara, M. C., Pasello, M., & Scotlandi, K. (2018). CD99: A Cell Surface Protein with an
Oncojanus Role in Tumors. Genes, 9(3), 159. https://doi.org/10.3390/genes9030159
MANN, J. D., CAHAN, A., GELB, A. G., FISHER, N., HAMPER, J., TIPPETT, P., SANGER,
R., & RACE, R. R. (1962). A sex-linked blood group. Lancet (London, England), 1(7219), 8–10.
https://doi.org/10.1016/s0140-6736(62)92637-5
Marshall, J. S., Warrington, R., Watson, W., & Kim, H. L. (2018). An introduction to
immunology and immunopathology. Allergy, asthma, and clinical immunology : official journal
of the Canadian Society of Allergy and Clinical Immunology, 14(Suppl 2), 49.
https://doi.org/10.1186/s13223-018-0278-1
McIlwain, D. R., Berger, T., & Mak, T. W. (2013). Caspase functions in cell death and disease.
Cold Spring Harbor perspectives in biology, 5(4), a008656.
https://doi.org/10.1101/cshperspect.a008656
Montoliu-Gaya, L., Esquerda-Canals, G., Bronsoms, S., & Villegas, S. (2017). Production of an
anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic
effect. PloS one, 12(8), e0181480. https://doi.org/10.1371/journal.pone.0181480
45
Morgan, D. A., Ruscetti, F. W., & Gallo, R. (1976). Selective in vitro growth of T lymphocytes
from normal human bone marrows. Science (New York, N.Y.), 193(4257), 1007–1008.
https://doi.org/10.1126/science.181845
Movafagh, A., Heydary, H., Mortazavi-Tabatabaei, S. A., & Azargashb, E. (2011). The
Significance Application of Indigenous Phytohemagglutinin (PHA) Mitogen on Metaphase and
Cell Culture Procedure. Iranian journal of pharmaceutical research : IJPR, 10(4), 895–903.
Nelson A. L. (2010). Antibody fragments: hope and hype. mAbs, 2(1), 77–83.
https://doi.org/10.4161/mabs.2.1.10786
Ocklind G. (1988). Activation of human T lymphocytes through CD3 and CD2 (T11) with anti-
CD3-coupled sheep erythrocytes. Scandinavian journal of immunology, 27(5), 609–613.
https://doi.org/10.1111/j.1365-3083.1988.tb02389.x
Owen-Schaub, L. B., Yonehara, S., Crump, W. L., 3rd, & Grimm, E. A. (1992). DNA
fragmentation and cell death is selectively triggered in activated human lymphocytes by Fas
antigen engagement. Cellular immunology, 140(1), 197–205. https://doi.org/10.1016/0008-
8749(92)90187-t
Park, C. K., Shin, Y. K., Kim, T. J., Park, S. H., & Ahn, G. H. (1999). High CD99 expression in
memory T and B cells in reactive lymph nodes. Journal of Korean medical science, 14(6), 600–
606. https://doi.org/10.3346/jkms.1999.14.6.600
Pasello, M., Manara, M. C., & Scotlandi, K. (2018). CD99 at the crossroads of physiology and
pathology. Journal of cell communication and signaling, 12(1), 55–68.
https://doi.org/10.1007/s12079-017-0445-z
Pata, S., Otáhal, P., Brdička, T., Laopajon, W., Mahasongkram, K., & Kasinrerk, W. (2011).
Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin
CD81 and recruitment into immunological synapses. BMC research notes, 4, 293.
https://doi.org/10.1186/1756-0500-4-293
Pettersen, R. D., Bernard, G., Olafsen, M. K., Pourtein, M., & Lie, S. O. (2001). CD99 signals
caspase-independent T cell death. Journal of immunology (Baltimore, Md. : 1950), 166(8),
4931–4942. https://doi.org/10.4049/jimmunol.166.8.4931
Pettersen, R. D., Hestdal, K., Olafsen, M. K., Lie, S. O., & Lindberg, F. P. (1999). CD47 signals
T cell death. Journal of immunology (Baltimore, Md. : 1950), 162(12), 7031–7040.
Race, R.R. & Sanger, R. Blood groups in man 578-593 (Blackwell Scientific, Oxford, 1950)
46
Reisner, Y., Kapoor, N., Kirkpatrick, D., Pollack, M. S., Cunningham-Rundles, S., Dupont, B.,
Hodes, M. Z., Good, R. A., & O'Reilly, R. J. (1983). Transplantation for severe combined
immunodeficiency with HLA-A,B,D,DR incompatible parental marrow cells fractionated by
soybean agglutinin and sheep red blood cells. Blood, 61(2), 341–348.
Richards, D., Maruani, A., & Chudasama, V. (2016). Antibody fragments as nanoparticle
targeting ligands: a step in the right direction. Chemical Science (Cambridge), 8(1), 63–77.
https://doi.org/10.1039/c6sc02403c
Robey, E., & Fowlkes, B. J. (1994). Selective events in T cell development. Annual review of
immunology, 12, 675–705. https://doi.org/10.1146/annurev.iy.12.040194.003331
Ross, S. H., & Cantrell, D. A. (2018). Signaling and Function of Interleukin-2 in T
Lymphocytes. Annual review of immunology, 36, 411–433. https://doi.org/10.1146/annurev-
immunol-042617-053352
Ross, S. H., Rollings, C., Anderson, K. E., Hawkins, P. T., Stephens, L. R., & Cantrell, D. A.
(2016). Phosphoproteomic Analyses of Interleukin 2 Signaling Reveal Integrated JAK Kinase-
Dependent and -Independent Networks in CD8(+) T Cells. Immunity, 45(3), 685–700.
https://doi.org/10.1016/j.immuni.2016.07.022
Sadelain M, Riviere I, Brentjens R. Targeting tumours with genetically enhanced T lymphocytes.
Nat Rev Cancer 2003; 3: 35–45.
Sanz, L., Cuesta, A. M., Compte, M., & Alvarez-Vallina, L. (2005). Antibody engineering:
facing new challenges in cancer therapy. Acta pharmacologica Sinica, 26(6), 641–648.
https://doi.org/10.1111/j.1745-7254.2005.00135.x
Schenkel, A. R., Mamdouh, Z., Chen, X., Liebman, R. M., & Muller, W. A. (2002). CD99 plays
a major role in the migration of monocytes through endothelial junctions. Nature immunology,
3(2), 143–150. https://doi.org/10.1038/ni749
Scotlandi, K., Zuntini, M., Manara, M. C., Sciandra, M., Rocchi, A., Benini, S., Nicoletti, G.,
Bernard, G., Nanni, P., Lollini, P. L., Bernard, A., & Picci, P. (2007). CD99 isoforms dictate
opposite functions in tumour malignancy and metastases by activating or repressing c-Src kinase
activity. Oncogene, 26(46), 6604–6618. https://doi.org/10.1038/sj.onc.1210481
Seder, R. A., & Ahmed, R. (2003). Similarities and differences in CD4+ and CD8+ effector and
memory T cell generation. Nature immunology, 4(9), 835–842. https://doi.org/10.1038/ni969
Skerra, A., & Plückthun, A. (1988). Assembly of a functional immunoglobulin Fv fragment in
Escherichia coli. Science (New York, N.Y.), 240(4855), 1038–1041.
https://doi.org/10.1126/science.3285470
47
Smeets, R.L., Fleuren, W.W., He, X. et al. Molecular pathway profiling of T lymphocyte signal
transduction pathways; Th1 and Th2 genomic fingerprints are defined by TCR and CD28-
mediated signaling. BMC Immunol 13, 12 (2012). https://doi.org/10.1186/1471-2172-13-12
Smith S. L. (1996). Ten years of Orthoclone OKT3 (muromonab-CD3): a review. Journal of
transplant coordination : official publication of the North American Transplant Coordinators
Organization (NATCO), 6(3), 109–121. https://doi.org/10.7182/prtr.1.6.3.8145l3u185493182
Sneath, R. J., & Mangham, D. C. (1998). The normal structure and function of CD44 and its role
in neoplasia. Molecular pathology : MP, 51(4), 191–200. https://doi.org/10.1136/mp.51.4.191
Sohn, H. W., Shin, Y. K., Lee, I. S., Bae, Y. M., Suh, Y. H., Kim, M. K., Kim, T. J., Jung, K. C.,
Park, W. S., Park, C. S., Chung, D. H., Ahn, K., Kim, I. S., Ko, Y. H., Bang, Y. J., Kim, C. W.,
& Park, S. H. (2001). CD99 regulates the transport of MHC class I molecules from the Golgi
complex to the cell surface. Journal of immunology (Baltimore, Md. : 1950), 166(2), 787–794.
https://doi.org/10.4049/jimmunol.166.2.787
Spadiut, O., Capone, S., Krainer, F., Glieder, A., & Herwig, C. (2014). Microbials for the
production of monoclonal antibodies and antibody fragments. Trends in biotechnology, 32(1),
54–60. https://doi.org/10.1016/j.tibtech.2013.10.002
Suda, T., Takahashi, T., Golstein, P., & Nagata, S. (1993). Molecular cloning and expression of
the Fas ligand, a novel member of the tumor necrosis factor family. Cell, 75(6), 1169–1178.
https://doi.org/10.1016/0092-8674(93)90326-l
Sun, T., Zhang, Y. S., Pang, B., Hyun, D. C., Yang, M., & Xia, Y. (2014). Engineered
nanoparticles for drug delivery in cancer therapy. Angewandte Chemie (International ed. in
English), 53(46), 12320–12364. https://doi.org/10.1002/anie.201403036
Surh, C. D., & Sprent, J. (1994). T-cell apoptosis detected in situ during positive and negative
selection in the thymus. Nature, 372(6501), 100–103. https://doi.org/10.1038/372100a0
Takheaw, N., Earwong, P., Laopajon, W., Pata, S., & Kasinrerk, W. (2019). Interaction of CD99
and its ligand upregulates IL-6 and TNF-α upon T cell activation. PloS one, 14(5), e0217393.
https://doi.org/10.1371/journal.pone.0217393
Terstappen, L. W., Huang, S., & Picker, L. J. (1992). Flow cytometric assessment of human T-
cell differentiation in thymus and bone marrow. Blood, 79(3), 666–677.
Trauth, B. C., Klas, C., Peters, A. M., Matzku, S., Möller, P., Falk, W., Debatin, K. M., &
Krammer, P. H. (1989). Monoclonal antibody-mediated tumor regression by induction of
apoptosis. Science (New York, N.Y.), 245(4915), 301–305.
https://doi.org/10.1126/science.2787530
48
Trinchieri, G., Pflanz, S., & Kastelein, R. A. (2003). The IL-12 family of heterodimeric
cytokines: new players in the regulation of T cell responses. Immunity, 19(5), 641–644.
https://doi.org/10.1016/s1074-7613(03)00296-6
Vaikari, V. P., Park, M., Keossayan, L., MacKay, J. A., & Alachkar, H. (2020). Anti-CD99
scFv-ELP nanoworms for the treatment of acute myeloid leukemia. Nanomedicine :
nanotechnology, biology, and medicine, 29, 102236. https://doi.org/10.1016/j.nano.2020.102236
Vella, A. T., Dow, S., Potter, T. A., Kappler, J., & Marrack, P. (1998). Cytokine-induced
survival of activated T cells in vitro and in vivo. Proceedings of the National Academy of
Sciences of the United States of America, 95(7), 3810–3815.
https://doi.org/10.1073/pnas.95.7.3810
Vella, A., Teague, T. K., Ihle, J., Kappler, J., & Marrack, P. (1997). Interleukin 4 (IL-4) or IL-7
prevents the death of resting T cells: stat6 is probably not required for the effect of IL-4. The
Journal of experimental medicine, 186(2), 325–330. https://doi.org/10.1084/jem.186.2.325
von Boehmer, H., & Fehling, H. J. (1997). Structure and function of the pre-T cell receptor.
Annual review of immunology, 15, 433–452. https://doi.org/10.1146/annurev.immunol.15.1.433
von Boehmer, H., Teh, H. S. & Kisielow, P. The thymus selects the useful, neglects the useless
and destroys the harmful. Immunol. Today 10, 57–61 (1989)
Waclavicek, M., Majdic, O., Stulnig, T., Berger, M., Sunder-Plassmann, R., Zlabinger, G. J.,
Baumruker, T., Stöckl, J., Ebner, C., Knapp, W., & Pickl, W. F. (1998). CD99 engagement on
human peripheral blood T cells results in TCR/CD3-dependent cellular activation and allows for
Th1-restricted cytokine production. Journal of immunology (Baltimore, Md. : 1950), 161(9),
4671–4678.
Waldmann T. A. (1989). The multi-subunit interleukin-2 receptor. Annual review of
biochemistry, 58, 875–911. https://doi.org/10.1146/annurev.bi.58.070189.004303
Watson, R.L., Buck, J., Levin, L.R., Winger, R.C., Wang, J., Arase, H., and Muller, W.A.
(2015). Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate
leukocyte transendothelial migration. J Exp Med 212, 1021-1041.
West, W. H., Payne, S. M., Weese, J. L., & Herberman, R. B. (1977). Human T lymphocyte
subpopulations: correlation between E-rosette-forming affinity and expression of the Fc receptor.
Journal of immunology (Baltimore, Md. : 1950), 119(2), 548–554.
Wingett, D., Forcier, K., & Nielson, C. P. (1999). A role for CD99 in T cell activation. Cellular
immunology, 193(1), 17–23. https://doi.org/10.1006/cimm.1999.1470
49
Yasutomo, K., Doyle, C., Miele, L., Fuchs, C., & Germain, R. N. (2000). The duration of antigen
receptor signalling determines CD4+ versus CD8+ T-cell lineage fate. Nature, 404(6777), 506–
510. https://doi.org/10.1038/35006664
Yonehara, S., Ishii, A., & Yonehara, M. (1989). A cell-killing monoclonal antibody (anti-Fas) to
a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. The Journal
of experimental medicine, 169(5), 1747–1756. https://doi.org/10.1084/jem.169.5.1747
Abstract (if available)
Abstract
CD99 (MIC2, E2) is a human 32 kDa highly O-glycosylated transmembrane glycoprotein, that is broadly expressed on both hematopoietic and non-hematopoietic cells. CD99 plays a vital role in a large array of T lymphocyte processes, including T cell activation, adhesion, differentiation and the selection process, apoptosis, and transmigration. In cancer, CD99 acts as either an oncosuppressor or oncogene depending on the cancer type. In addition, it is also involved in immune and inflammatory responses within the tumor microenvironment. The CD99 gene encodes two isoforms, CD99 short and long, which possess different functions depending on the level of expression and location of the two isoforms. The variable expression of CD99 in T cells throughout different stages of maturation is well documented and supported by extensive evidence, however the mechanisms by which CD99 participates in T cell processes is not fully understood. Considering its expression on the surface of T cells and the recent increased interest in CD99 as a therapeutic target in cancer, a better understanding of the role of this receptor in T cells is highly needed. Here, we extensively review and discuss research findings related to the known functions and roles of CD99 in T cells, summarize reported CD99 monoclonal antibodies and their activities. In addition, we have developed a new single chain antibody against CD99 and examined its activity in healthy peripheral human T cells. Overall, we found that treatment of healthy T human peripheral T cells with a CD99 scFv increased cell proliferation, most notably at 48 hours post treatment. Further functional and mechanistic studies are warranted to understand whether CD99 targeting approaches can be developed into an effective form of immune therapies against cancer cells.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Investigating CD99 as a therapeutic target in acute myeloid leukemia
PDF
Investigating the effects of targeting CD99 on T cells to enhance their antileukemia activity
PDF
Clinical, functional and therapeutic analysis of CD99 in acute myeloid leukemia
PDF
Development of engineered antibodies as novel anti-cancer agents
PDF
Generation and characterization of fully human anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
PDF
Antibodies and elastin-like polypeptides: cellular and biophysical characterization of an anti-ELP monoclonal and an anti-CD3 single-chain-ELP fusion
PDF
FLT3/CD99 bispecific antibody-based nanoparticles (BiAbs) for acute myeloid leukemia
PDF
Deregulation of CD36 expression in cancer presents a potential targeting therapeutic opportunity
PDF
Structure-based computational analysis and prediction of TCR CDR3 loops in the TCR-peptide-MHC complex using solvation parameters and peptide molecular dynamics.
PDF
The immunomodulatory effects of midostaurin on T cells
PDF
Novel approaches of mobilizing human iNKT cells for cancer immunotherapies
PDF
Employing engineered exosomes for combating colon cancer and engineering CD38 as an optimized drug carrier
PDF
Immunotherapy of cancer
PDF
Investigating the effects of T cell mediated anti-leukemia activity in FLT3-ITD positive acute myeloid leukemia
PDF
Investigation of the synergistic effect of midostaurin, a tyrosine kinase inhibitor, with anti FLT3 antibodies-based therapies for acute myeloid leukemia
PDF
Enhancing the anti-cancer specificity of chimeric antigen receptor T cells through targeting HLA loss
PDF
Development of cyclized bispecific antibodies for enhanced serum stability
PDF
T cell regulation of HLA-DR
PDF
Generation of monoclonal antibodies via phage display and in vitro affinity maturation using activation induced deoxycytidine deaminase and DNA polymerase eta
PDF
Generation and characterization of humanized anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies
Asset Metadata
Creator
Keossayan, Lena
(author)
Core Title
The role of CD99 in T cells
School
School of Pharmacy
Degree
Master of Science
Degree Program
Molecular Pharmacology and Toxicology
Degree Conferral Date
2021-08
Publication Date
07/23/2021
Defense Date
06/18/2021
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
antibody fragment,cancer,CD99,cytokine,IL-2,immunology,MIC2,monoclonal antibody,OAI-PMH Harvest,scFv,single-chain fragment variable,T cells,T-cell,thymocyte
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Alachkar, Houda (
committee chair
), Duncan, Roger (
committee member
), Zhang, Yong (
committee member
)
Creator Email
keossaya@usc.edu,LKeossayan@gmail.com
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-oUC15619462
Unique identifier
UC15619462
Legacy Identifier
etd-KeossayanL-9850
Document Type
Thesis
Format
application/pdf (imt)
Rights
Keossayan, Lena
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 author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.
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
Repository Email
cisadmin@lib.usc.edu
Tags
antibody fragment
CD99
cytokine
IL-2
immunology
MIC2
monoclonal antibody
scFv
single-chain fragment variable
T cells
T-cell
thymocyte