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Regulation of T cell HLA-DR by CD3 ζ signaling
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Regulation of T cell HLA-DR by CD3 ζ signaling
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Regulation of T cell HLA-DR by CD3 ζ signaling
by
Chia-Hsin Lin
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
[(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)]
May 2022
Copyright 2022 Chia-Hsin Lin
ii
Dedication
To my family for their unlimited love and support.
iii
Acknowledgments
Thesis committee members:
Weiming Yuan, Ph.D. (Chair)
Alan L. Epstein, M.D., Ph.D. (Mentor)
Harvey R. Kaslow, Ph.D.
Keigo Machida, Ph.D.
This project would not have been possible without the support of many people. I would first like
to acknowledge the enormous support from Dr.Epstein. Thanks to his patient support and the
opportunities he gave me helps me obtained a lot of skills in my master’s life. His intellectual
and emotional support always helps me keep going when I am discouraged. He certainly did
devote himself to science, and I will definitely carry this attitude with me as I move forward.
I would like to also thank my committee members, Dr. Weiming Yuan, Dr. Harvey R. Kaslow,
and Dr. Keigo Machida for inspiring my interest in immunology. Their support and suggestions
iv
helped me grow as a scientist and learn how to think differently. I want to especially thank
Dr.Kaslow for his advice and support towards my thesis and my entire master’s time. I really
enjoyed and cherished all the weekly meetings with him in his office and on zoom. In the weekly
meeting, I learned how to explain and present my data professionally. His insightful feedback
pushed me to sharpen my thinking and brought my work to higher level.
Besides my advisor and committee members, I would like to thank the co-principle investigators
of the lab Dr.Peisheng Hu and Dr. Leslie Khawli for guiding me through the project and helping
me complete my thesis.
I would also like to thank my previous colleagues Long Zheng. Long guided me on how to do
the experiments and all the skills being used in CAR production. I will never forget the times he
explained all the knowledge to me since 2019. I am so pleased I have some time to learn from
him before he graduated.
I would like to express my gratitude to all the lab members for helping me through many
difficulties and sharing their experiences with me: Luqing Ren, Tiffany Jheng, Aida Kouhi,
Vyshnavi Pachipulusu, Chumeng Chen. I really have a great time working in this lab. As an
international student, I don’t feel lonely because I have them in my daily life.
v
I would also like to acknowledge all the members in the Molecular Microbiology and
Immunology and the Pathology department in the Keck School of Medicine, for teaching,
helping, and opening my eyes to the many opportunities provided by the school and the scientific
community. In addition, I would like to offer my special thanks to my previous mentor Dr. Wei-
Ning Lin for the opportunities for conducting my own project. I learned what a scientist looks
like since I was an undergraduate student and this really helps me understand what will happened
in a master’s life.
And, as always, my appreciation to my parents and brother, who always believe in me more than
I do myself. Although they are far away in Taiwan, they always support me with their love and
cheer me on.
vi
Table of Contents
Chapter 1- Introduction ........................................................................................................... 1
1.1 MHC and its role in cancer ....................................................................................................... 2
1.1.1 MHC structure and function ................................................................................................................... 2
1.1.2 T cell HLA-DR function ........................................................................................................................ 7
1.2 Antibodies binding to HLA-DR epitopes ................................................................................. 9
1.2.1 Lym-1 Antibody ..................................................................................................................................... 9
1.2.2 L243 antibody ...................................................................................................................................... 10
1.2.3 DA2 antibody ....................................................................................................................................... 10
1.3 CAR T Cell Progress Timeline ............................................................................................... 10
Chapter 2- Hypothesis and specific aims ............................................................................. 13
2.1 Thesis Specific Aims ................................................................................................................ 14
2.1.1 Aim 1 .................................................................................................................................................... 14
Generate anti-CD19 CAR T cells with only the CD3 ζ signaling domain and use anti-CD19 CAR T cells using
DAP10-12 signaling domains as a positive control. ........................................................................................... 14
2.1.2 Aim 2 .................................................................................................................................................... 14
Determine if culturing the various anti-CD19 CAR T cell preparations with tissue culture plates coated with an
antibody (a-261) activating the CAR causes diminished detection of HLA-DR epitopes. ................................ 14
2.1.3 Aim 3 .................................................................................................................................................... 15
Determine if culturing primary T cell preparations with tissue culture plates coated with anti-CD3/CD28
causes diminished detection of HLA-DR epitopes ............................................................................................. 15
Chapter 3- Materials and Methods ....................................................................................... 16
3.1 Reagents and inhibitors ........................................................................................................... 16
3.1.1 Reagents ............................................................................................................................................... 16
3.1.2 Antibodies and Cytokines .................................................................................................................... 17
3.2 Cells and culture conditions .................................................................................................... 17
Dedication........................................................................................................................................................... ii
Acknowledgements............................................................................................................................................iii
Table of Contents.............................................................................................................................................. vi
Abstract ............................................................................................................................................................. vii
vii
3.2.1 HEK-293 LTV cell ............................................................................................................................... 17
3.2.2 CAR T cell ........................................................................................................................................... 18
3.2.3 T cell ..................................................................................................................................................... 18
3.3 CAR T cells production ........................................................................................................... 19
3.3.1 Vector Construction and Preparation of Lentivirus CAR construct .................................................... 19
3.3.2 Primary T Cell Isolation ....................................................................................................................... 20
3.3.3 Production of CAR T cells ................................................................................................................... 21
3.4 Assays ........................................................................................................................................ 21
3.4.1 Downregulation assay .......................................................................................................................... 21
3.4.2 Flow cytometry .................................................................................................................................... 22
3.4.3 Data analysis ........................................................................................................................................ 23
Chapter 4- Results .................................................................................................................. 24
4.1 Generated anti-CD19 CAR T cells with either CD3 ζ or DAP 12 signaling domain. ........ 24
4.2 Identify culture system conditions to assess downregulation of HLA-DR ......................... 26
4.2.1 Tested different doses of anti-261 antibody on CAR T cell ................................................................ 26
4.2.2 CD3/CD28 mimic TCR signaling on primary T cell ........................................................................... 37
Chapter 5- Discussion and future directions ....................................................................... 40
5.1 Discussion ................................................................................................................................. 40
5.2 Future opportunities ................................................................................................................ 42
Reference ..................................................................................................................................... 44
viii
Abstract
MHC Class II plays a crucial role in regulating the immune reaction. In recent years,
research on how HLA-DR regulates the immune response has become very popular. In a
normal immune response, the HLA-DR complex (with both 𝛼 and 𝛽 subunit) binds to T-
cell receptors (TCRs) and causes T cells activation and release cytokines to induce the
immune response.[2] In addition, cytotoxic CD4+ cells can target cells with HLA-DR and
kill the target cells in an MHC Class II-restricted fashion. [3] This thesis continues studies
involving an unreported and unexpected result. Lym-1 is an antibody recognizing an
epitope on the HLA-DR 𝛽 subunit. When an antibody bound to the chimeric antigen
receptor (CAR) of anti-CD19-DAP CAR T cells, detection of the Lym-1 epitope was lost
on the CAR T cells while detection of an epitope on the 𝛼 subunit remained (antibody
L243).
The above result involved a CAR construct using a DAP10/DAP12 signaling domain. The
physiological signaling domain for T cell receptors is CD3 ζ. This thesis tested the
hypothesis that stimulation of a CAR with a CD3 ζ signaling domain is sufficient to cause
loss of detection of the Lym-1 epitope. The results support the hypothesis and justify future
work testing the hypothesis that epitope-stimulation of normal T cells also causes the loss of
the Lym-1 epitope which then alters the function of HLA-DR.
1
Chapter 1- Introduction
In the field of immunology, the cytotoxic cell recognizes the target cell plays an essential role in
how we understand immunology mechanism. Nowadays, many people seen MHC Class II
molecules as differentiation markers. [4] HLA-DR belongs to MHC Class II family, and it been
taken as an important role as an indicator of T cell activation.[5] Besides, some studies used
HLA-DR as a T cell activation markers to monitor immune status. [6] Recent studies
demonstrates that a CD4 T cell population will express HLA-DR and those T cell can be
recognized in human peripheral blood.[7] In 1982, CD4 CTL cells was confirmed by Dr.
Krensky from culture system.[8] Since then more and more studies demonstrated the existence of
CD4+ CTL, act physiologically via HLA-DR.[9-11] Furthermore, some studies also display
that HLA-DR restricted peptide epitopes to generate CD4+ CTL.[12] T cell can been seen as a
antigen- presenting cells(APC) and also present the exogenous protein. [13, 14] According to
these studies, epitopes presented by HLA-DR on a CD4 or CD8 T cell can make the T cell be a
target for CD4 CTL.[15] Understanding the connection between HLA-DR can be very helpful
for us to understand the immune escape and may lead to a new method for negative selection of
T cells recognizing an epitope in the future.
2
1.1 MHC and its role in cancer
1.1.1 MHC structure and function
Major histocompatibility complex (MHC), also known as major histocompatibility complex
gene, is a gene family that exists in most vertebrate genomes and consists of a group of
closely linked gene groups. These genes are highly polymorphic, and the antigens they
encoded are highly related to antigen presentation and T cell activation molecule, which
will influence immune response and immune regulation, and cause rapid and strong
rejection. MHC complexes can be separated into two classes: MHC Class I and MHC Class
II. All nucleated cells can present MHC Class I. Which in term to present to T cells peptides
derived from proteins synthesized within the cell. MHC Class II complexes present peptides
derived from extracellular proteins taken up in endosomes (Fig 1.2) Both MHC Class I and
II consist of two peptides, the 𝛼 and 𝛽units. In MHC Class I, the α chain has three separate
domains while the β has only one, β2 Macroglobulin; in MHC Class II both α and 𝛽 chains
3
have two unique domains. (Figure 1.1.1 a. Created with BioRender.com.) In MHC Class I,
the proteasome in the cytoplasm will degrade every protein into peptides. These peptides
will be transported to the endoplasmic reticulum by transporter associated with antigen
processing (TAP) and expression on the cell surface. [16] MHC Class II is normally found
on professional antigen-presenting cells such as macrophage, B cells, dendritic cells (DC).
Like MHC Class I molecules, Class II MHC also has α and β units. Unlike MHC Class I, α
and β units both have two unique domains. Different from MHC Class I, MHC Class II
Fig. 1.1.1 a. Major histocompatibility complex (MHC) has two Classes with very
similar peptide-binding sites. MHC Class I is consisted of three polypeptide domains
(α1, α2, and α3) and β2 macroglobulin; MHC Class II is consisted of two heterodimers
α and β. (Created with BioRender.com.)
4
binds to the peptides which come from endocytosis. Proteins will be ingested and degraded
after endocytosis. Due to these two classes of MHC using different mechanisms to present
the antigen, the two types of MHC molecules can represent different messages: MHC Class
I stands for the cell that has been infected[17]; and Class II means the outside part has been
infected. [18] (Figure 1.1.1 b.[1])
5
Fig. 1.1.1 b. MHC divide into two classes, and they have different ways to present
Antigen. MHC Class I antigen-presenting: Proteosomes produce peptides from
cytosolic protein, which replicates in the cell. Peptides are shipped to the
endoplasmic reticulum. After editing in ER, the MHC will express on the surface
of the nucleic cell. MHC Class II antigen show: exogenous protein is taking up
inside to the professional Antigen Presenting cell by phagocytosis. Phagosomes
meld with lysosomes, which contain proteolytic compounds that separate the
phagocytosed proteins into little peptides. Peptide-stacked MHC Class II edifices
are moved to the cell surface, permitting antigen show to CD4+ T cells. [1]
6
The MHC in humans, also called the human leukocyte antigen (HLA) system, is a complex
of cell surface proteins necessary for an appropriate acquired immune response. The HLA
gene is in chromosome 6p21.3 that includes genes encoding the HLA Class I and Class II
molecules. (Fig. 1.3[19]) HLA Class I molecules are coded from loci A, B, and C will play
a critical role in the detection and elimination of virus-infected cells, tumor cells, and
transplanted allogeneic cells. [20] The HLA-II genes encode the polymorphic HLA-DR,
Fig. 1.1.1 c. Genomic localization of the human major histocompatibility complex
(MHC) on chromosome 6p21.3 and positions of the human leukocyte antigen
(HLA) Class I (HLA-A, -B, -C) and Class II (HLA-DR, -DQ, -DP) loci.
7
-DQ, and -DP molecules. HLA-II molecules are central in the initiation of cellular and
humoral immune responses, but they have also been implicated as contributing factors for a
variety of autoimmune disorders. [21]
1.1.2 T cell HLA-DR function
The main function of MHC molecules is to bind peptide fragments derived from pathogens
and display them on the cell surface for recognition by the appropriate T cells so that CTL
can attack the cell which contains certain peptides. [22] CD4+ T cells, which survey MHC
Class II, play an important role in the adaptive immune system. Based on differentiation
signals, transcription factor expression, cytokine secretion, and specific functions, these
studies [22]demonstrated that CD4 T cells can develop to CD4 T helper cells. However,
growing evidence displays that CD4+ T cell can also develop to effector activity, which is
dependent on the intrinsic cytotoxic function and kill target pathogen infectious. [10]
HLA-DR can be seen as a T cell activation marker since activated T cells can express HLA-
DR and function as APCs to present epitopes from exogenous sources. [13],
[23],[24]Meanwhile, APC will capture the antigens and then process them. After the
processing, the APCs will display their fragments so that they will present MHC Class II
8
molecules and trigger Class II-restricted T cells. [14] CD4 T lymphocytes recognize the
entire complex formed by HLA-DR molecules and peptides. [25] After recognizing the cell
which expresses HLA-DR, the adaptive immune response can be adjusted. Not only APC
will express HLA-DR, but T cells can also perform the same functions and, importantly, it
could act to present epitopes indicative that the T cell has taken up a pathogen - doing so
could make it a target to be killed by a cytotoxic CD4+ T cell. Some recent papers also
demonstrate that HLA-DR can be an indicator of T cell activation in COVID studies. [14,
26-29] These paper shows that HLA-DR plays a crucial in being a T cell activation marker.
HLA downregulation is a common form of immune escape employed by cancers, in
addition to the expression of immune inhibitory molecules, and invasion of immune
suppressive cells. As previously noted, epitopes presented by HLA-DR on CD4 or CD8 T
cells can make the T cell a target for CD4+ Cytotoxic T cell. Loss of the expression of HLA
Class II molecules will lead to the loss CD4 CTL ability to bind to MHC Class II and to
lyse the target cells. Besides, CD4 CTLs target the viral peptide-MHC complex, and lead B
cells to die. Hence, CD4 CTL can be seen as immune surveillance of APC.[3] Once
cytotoxic T cells can not recognize the tumor cell effectively, the tumor is allowed to
flourish in the new immunotolerant environment.[30] Some research studies have indicated
9
the abnormal expression of HLA-Ⅱ genes in various abnormal human tissues. [31] Studies
have indicated that the expression of HLA-Ⅱ genes in tumor cells enable new cells to
present on the surface and activate Th cells, causing anti-seismic effects.[32]
1.2 Antibodies binding to HLA-DR epitopes
1.2.1 Lym-1 Antibody
In 1987, Dr. Epstein et al. investigated a murine monoclonal IgG2a antibody, Lym-1, which
was isolated from mice hyper-immunized with nuclei from Raji Burkitt’s Lymphoma cells.
[33] Lym-1 was reactive with the cell surface of B-lymphocytes and derived tumor cells.
[33] There are only 2% of Americans have HLA-DR10, but more than 80% of the
lymphoma biopsies have Lym-1 positive expression.[34] Initially, Lym-1 antibody was
thought to target a conformation epitope on the HLA-DR10 β chain when the heterodimer
interacts. This result prompted Rose et al. to determine that the Lym-1 epitope involves four
critical residues in several HLA-DRs in addition to HLA-DR10.[34]
10
1.2.2 L243 antibody
L243 is a murine IgG2a monoclonal antibody recognizing a conformational epitope in the α
chain of HLA-DR [35] The L243 epitope is seen both in tumors and circulating T cells.[36]
1.2.3 DA2 antibody
DA2, first generated in 1980, is a murine IgG1 monoclonal antibodies for analysis of the
HLA system.[37] DA2 targets to the linear epitope on HLA-DRB1 and detects the HLA-DR
β subunit in intact HLA dimers and the β chain by Western Blot. DA2 binds to all DR and
DP antigens except DR7.[37]
1.3 CAR T Cell Progress Timeline
In 1900s, scientists found that our immune cells can recognize the foreign antigens and use
different ways to kill the cancer cells. In 1992, an immunologist, Michael Sadelain wanted to use
T cell to recognize cancer. As a result, he started using genetic ways to introduce certain genes
into T cells. In the following year, the immunologist, Zelig Eshhar, investigated the first-
generation chimeric antigen receptor (CAR), which is combined with a portion of antibody and a
part of T cell receptor. This synthetic molecule contains the single chain variable region (scFv),
hinge transmembrane domain, and the TCR CD3 ζ signal domain. [37] Although some studies
11
show those first-generation CAR T cells persisted for some time, they generally failed to expand
and persist in vivo [38]. (Fig. 1.3 a.[39])
To improve CAR T cell performance, second-generation CAR constructs were investigated.
Different from the first generation, the second generation has two signal domains, the co-
stimulatory domain, and intracellular signal domain. one study used an scFv which will bind to
GD2 to replace the CD28 extracellular binding domain. With this new structure, the survival rate
and proliferation of their second-generation CAR T cells had enhanced. Later, another study that
a
Fig. 1.3 a. Both Transgenic T cell receptors (TCRs) or Chimeric antigen receptor (CAR) protein
can redirect T cells to recognize and kill a specific target. CAR protein is actually made up of 3
other proteins: one protein that recognizes antigens on the cancer cell and two proteins that
signal the T cell to activate when that first protein attaches to an antigen on the cancer cell. The
difference between first-generation and second-generation CAR is first generation only have
CD3 ζ whereas second-generation has one more co-stimulatory domain (e.g. CD28 or 4-1BB)
12
used 4-1BB as a co-stimulatory domain had also helped the CAR-T cells enhance their
proliferation and survival. Though these two second-generation CAR T cells have improved the
efficiency of proliferation and survival, none of these studies mentioned the efficiency of anti-
tumor until 2007.(Fig. 1.3. a.[39]) Our recent paper shows this DAP10-DAP12 signaling domain
may be a good candidate in second generation CAR T cells construct.[12] (Fig. 1.3.1.b. Created
with BioRender.com) DAP 12, express on many hematopoietic cells, is an adaptor protein which
contains immunoreceptor tyrosine-based activation motifs (ITAMs). Meanwhile, DAP 10 can
help PI3K activation.[40]
Fig. 1.3 b. DAP10-DAP12 signaling domain has been seen
as a good candidate in second generation CAR T cells
construct.
13
Chapter 2- Hypothesis and specific aims
Motivation
When we activate 2
nd
generation (CD19-DAP10/12) CAR T cells via the 261 tag in the
CAR construct, the expression of Lym-1 and DA2 was decreased.
Long-term goal
Test the hypothesis that T cell recognition of an MHC-presented epitope diminishes the
function of the T cell’s HLA-DR and thus protects the T cell from the cytotoxic function of
CD4+ cytotoxic lymphocytes.
HLA-DR !
HLA-DR β
T cell
TCR
CD4 CTL
Immune attack
HLA-DR !
HLA-DR β
T cell
TCR
CD4 CTL
Immune attack
T cells that have
taken up exogenous
antigen
Epitope recognition
and activation
Fig. 2 Hypothesis for T cell escape from CD4+ T cell
14
2.1 Thesis Specific Aims
Previous studies showed that when we activate 2nd generation CAR T cell, the detection of
Lym-1 and DA2 expression will decrease.[41] To determine if physiological mechanisms could
also cause this decrease, the goal of this project was to test the hypothesis that CD3 ζ signaling
domain, as found in the TCR, is sufficient to decrease detection of the HLA-DR Lym-1 epitope
on T cells.
2.1.1 Aim 1
Generate anti-CD19 CAR T cells with only the CD3 ζ signaling domain and use anti-CD19 CAR
T cells using DAP10-12 signaling domains as a positive control.
2.1.2 Aim 2
Determine if culturing the various anti-CD19 CAR T cell preparations with tissue culture plates
coated with an antibody (a-261) activating the CAR causes diminished detection of HLA-DR
epitopes.
15
2.1.3 Aim 3
Determine if culturing primary T cell preparations with tissue culture plates coated with anti-
CD3/CD28 causes diminished detection of HLA-DR epitopes
Demonstrating a CD3 ζ signaling domain is sufficient to down-regulate HLA-DR epitopes would
support the hypothesis that TCR recognition of an epitope could help the T cell escape being
killed by cytotoxic cells CD4+ T cells.
16
Chapter 3- Materials and Methods
3.1 Reagents and inhibitors
3.1.1 Reagents
RPMI-1640 (Genesee Scientific, Lot#0519108), DMEM (Genesee Scientific,
Lot#05191016), GlutaMAX (ThermoFisher, CAT#35050-061), Penicillin/Streptomycin
(Corning, CAT#30-002-CI), non-essential amino acids (Genesee Scientific, CAT#25-536),
Click’s medium (SIGMA, CAT#C5572-500ML), psPAX2 (Addgene,CAT#12260),
pMD2.G (Addgene, CAT#12259), Xfect (Clontech, CAT#631418), PolyJet ( SignaGen,
CAT# SL100688), Ficoll-Paque (Life Technologies, CAT#GE17-1440-02), EasySep
Human T cell isolation Kit (STEMCELL, CAT#19051), Lentiblast (OZBiosciences,
CAT#LB01500), 24-well G-Rex plates (Wilson Wolf ,CAT#80240M), Dynabeads human
T-activator CD3/CD28 (ThermoFisher, CAT#11131D), ImmunoCult human
CD3/CD28/CD2 T cell activator (STEM CELL, CAT#10970), UltraComp eBeads™
Compensation Bead (Invitrogen™, CAT 01-2222-42)
17
3.1.2 Antibodies and Cytokines
For flow cytometry analysis was stained with anti-chLym-1-488, anti-DA2- PE, anti-L243-
FITC, anti-CD3-APC, Chimeric Lym-1 (chLym-1) a chimeric analog of the original Lym-1
antibody, IL-7-Fc, IL-15-Fc, and Dylight 650 anti-261tag antibodies were developed and
prepared in our laboratory. For activation T cell, commercial antibody CD3 (clone OKT3,
BioXCell) and CD28 (clone 9.3, BioXCell) were used.
3.2 Cells and culture conditions
3.2.1 HEK-293 LTV cell
HEK-293 LTV cells (Cell Biolabs, CAT# LTV-100) were cultured in DMEM
(Corning, Manassas, VA) supplemented with 10% FBS, 1% GlutaMax, 1% non-
essential amino acids, and 1% Penicillin/Streptomycin were used for lentivirus
production.
18
3.2.2 CAR T cell
Primary human T cells were obtained from Leukopaks (from Gulf Coast Regional
Blood Center) by negative selection purification using T cell isolation kit (Stem Cell
Technologies, Seattle, WA). The T cell preparation was expanded by culturing for X
days with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator in T cell
medium (43% Click’s medium, 43% RPMI-1640, 10% FBS, 2% GlutaMAX, 1%
non-essential amino acids, 1% Penicillin/Streptomycin) supplemented with 50ng/mL
IL-7-Fc and 100ng/mL IL-15-Fc. T cells were frozen in (define media) for later use
either as T cells or to prepare CAR T cells.
3.2.3 T cell
Primary human T cells were enriched from Dynabeads™ Human T-Activator
CD3/CD28 (Thermo Fisher) and cultured in T cell medium (43% Click’s medium,
43% RPMI-1640, 10% FBS, 2% GlutaMAX, 1% non-essential amino acids, 1%
Penicillin/Streptomycin) supplemented with 50ng/mL IL-7-Fc and 100ng/mL IL-15-
Fc.
19
3.3 CAR T cells production
3.3.1 Vector Construction and Preparation of Lentivirus CAR
construct
For comparison purpose, CD19 CAR comprised of a CD19 specific targeting scFv derived
from antibody FMC63, a CD8α leader sequence, a (G4S)3 linker between the VH and the
VL domains, a CD8α hinge, a CD8α transmembrane domain followed by intact intracellular
CD3 ζ were cloned between the EcoRI and MluI restriction sites into the lentiviral vector
pLVX-EF1α-IRES- Zsgreen (Clontech, Mountain View, CA). another the coding genes for
CAR were synthesized by Integrated DNA Technologies (IDT) and ligated into the
lentiviral vector pLVX-EF1α-IRES-Zsgreen (Clontech) through EcoRI and MluI restriction
sites. All CAR constructs equipped with 10 amino acids epitope ‘‘AVPPQQWALS’’ (261-
tag), which derived from human placenta growth factor was inserted directly after the scFv
sequence.
To produce virus, the CD19-CD3ζ and CD19-DAP12 plasmid along with the packaging and
transfer plasmids were introduced into the HEK 293 cells. The lentiviral packaging vectors
are psPAX2 and envelope vectors are pMD2.G Briefly, mix transfer vector (the molar ratio
of plasmid and vectors is 2:1:1) and co-transfected to HEK-293LTV cells. Remove the
20
supernatant after 24 hours and collect the supernatant at 48 and 72 hours. The supernatant
which contains virus will be filtered and concentrated by ultracentrifugation at 20000 g for
1.5 hours. Resuspension the pellet virus in PBS supplemented with 1% BSA and 7%
trehalose. Aliquoted and stored at -80°C.
3.3.2 Primary T Cell Isolation
Luekopaks from healthy donors were obtained from Gulf Coast Regional Blood Center.
Primary blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque. T cells were
then isolated using a T cell negative selection kit (Stem Cell Technologies, Seattle, WA)
and cultured in T cell medium (43% Clicks, 43% RPMI 1640, 2% Glutamax (Life
Technologies, Inc.), 10% dFCS, 1% non- essential amino acid solution, 1% Pen/Strep
solution, 50ng/ml IL-7-Fc, 50ng/mL IL-15- Fc.
21
3.3.3 Production of CAR T cells
On day 0, primary T cells were thawed and activated by adding Dynabeads human T-
activator (ratio is 3:1). On day 4 primary T cells were transduced by centrifugation at 1200g
for 45 min with lentivirus and Lentiblast on a RetroNectin coated 24-well non-tissue plate.
Transferred the T cells to the 24-well G-Rex plates supplemented with fresh T cell medium
after incubated them overnight. On day 8, used Dylight 650 conjugated anti-261tag
antibody to analysis the efficiency of transduction via Flow cytometry. Reactivate the T
cells on day 8 with CD2/CD3/CD28 T cell activator with 40ul/2ml. after 2 days, replenish
the T cell medium to 6ml.
3.4 Assays
3.4.1 Downregulation assay
3.4.1.1 CAR T cells Downregulation assay
On day 0, first-generation and second-generation CAR T cells were thawed and activated by
CD3/CD28/CD2 T cell activator. Coated the plate with anti-261 antibody one day before
the experiment start. 2ml of anti-261 antibody at different doses were added to wells in the
plate. In a final volume of 2 ml one million CD19-CD3 ζ, CD19-DAP12, or CD19-
22
DAP10/12 CAR T cells were added to the 6 wells non-tissue culture plate. Three days later
the cells were harvested and Dylight 650 conjugated anti-261tag antibody, Lym-1-488, PE-
DA2, and L243-FITC were used to determine expression of HLA-DR epitopes.
3.4.1.2 Primary T cells Downregulation
On day 0, 1 vial of primary T cells were thawed and activated by adding 45 μl Dynabeads™
Human T-Activator CD3/CD28 (Thermo Fisher). One day prior to adding T cells to a 12
wells non-tissue culture were coated with anti-CD3 (clone OKT3, 10 μg/mL; BioXCell) and
anti-CD28 CD28 (clone 9.3, 2 μg/mL; BioXCell) antibody in 2ml PBS [42]. One million
primary T cells 2 ml T were seeded on an a-CD3/a-CD28 coated plate with a T cell medium
containing IL-7, Il-15. Used Dylight 650 conjugated anti-261tag antibody, PE-DA2, L243-
FITC, Lym-1-488, and CD3- APC to determine the HLA-DR in the following three days.
3.4.2 Flow cytometry
Cells seeded into 6 well plates were treated with 1mL of 261-antibody adhere 2 nights.
After 2 days, detached the cells with pipetting a few times. 0.2 million cells were transferred
into individual 12 x 75mm polystyrene tubes. Washed the cells with 4% FBS buffer,
23
centrifuged, and stained the cell with 2ul a-261 antibody, PE-DA2, L243, and Lym-1. Cell
samples were incubated with the antibodies for 30 minutes at 4
o
C. Samples were then
washed twice by centrifugation with FACS buffer and resuspended in 500ul 4% FBS-PBS
for flow cytometry analysis. We use Attune® Acoustic flow cytometer and Life
Technologies’ Attune® software (Life Technologies; Carlsbad, CA) to collect and analyzes
Flow cytometry data. Density plots of forward scatter height (FSC-H) to forward scatter
area (FSC-A) were generated to eliminate non-singlet events and help us delete ‘dead cells’.
Singlet events were then differentiated into normal structure cells versus alternate structure
cells in a separate density plot of FSC-A vs side scatter area (SSC-A) and the alternate
structure cells were excluded.
3.4.3 Data analysis
Data generated by flow cytometry was analyzed and formatted using Attune ® Flow
cytometry software.
24
Chapter 4- Results
4.1 Generated anti-CD19 CAR T cells with either CD3 ζ or DAP
12 signaling domain.
We designed two CARs comprising an anti-CD19 FMC63 ScFv generated in our laboratory,
fused to a modified a CD8α leader sequence, a (G4S)3 linker between the VH and the VL
domains, a CD8α hinge, a CD8α transmembrane domain, and a signaling domain. For the
signaling domain to these two CARs, one is followed by CD3ζ and another is by DAP12.
The coding genes for CAR were synthesized by Integrated DNA Technologies (IDT) and
ligated into the lentiviral vector pLVX-EF1a- IRES-Zsgreen (Clontech) through EcoRI and
MluI restriction sites. Both of these CARs were inserted a 10 amino acid epitope
‘‘AVPPQQWALS’’ (261-tag) derived from the human placenta growth factor. With this
tag, we can use an in-house antibody (Dylight 650 conjugated anti-261 tag antibody) to
detect the CAR expression. (Figure 4.1c) Both structures are successfully being transduced
to T cells. After enriching the transduced T cell with the a-261 antibody, cells expressing the
CAR comprised at least 60 percent of the cells. (Figure 4.1c)
25
Fig. 4.1 a. Construct design for CD19-CD3 and CD19-Dap12 CAR The CD19 first
generation CAR construct was being focused on this study. This CAR with equipped
with a CD8 leader sequence, CD19 ScFv moiety fused to a CD8 Hinge and
transmembrane domain, following by CD3 ζ or DAP12 signaling domain. b. Timeline
for first-generation CAR T production
a
b
c
Fig. 4.1 c. Result for CAR T production After enriching by a-261, the first-generation
CAR T expression goes to at least 67%. (CAR expression folder)
26
4.2 Identify culture system conditions to assess downregulation of
HLA-DR
Goal: determine culture conditions that demonstrate culturing anti-CD19 CAR T cells in
plates coated with a-261 antibody leads to less detection of HLA-DR beta.
Conclusion: 6-well plates should be coated with 2ml containing 2 ug a-261, 1M cells should
be added in 2 ml T cell medium per well.
4.2.1 Tested different doses of anti-261 antibody on CAR T cell
Thaw one vial of anti-CD19 CD3 ζ CAR T cells and culture them with CD3/CD28/CD2 T cell
activator for 4 days later, 1.5, 1, and 0.5 million of T cells will be put on the a-261 coated plate.
With a-261 stimulation, CAR T cells are activated. Meanwhile, after 2 days after stimulation the
expression of Lym-1, DA2 in the CAR positive group were significantly diminished. However,
for cells not expressing the CAR (normal T cells), the expression of Lym-1, DA2, and L243
epitopes did not change. (Figure 4.2.1 a-4.2.1 d) The acquired data were preferable for
subsequent analysis. (Figure 4.2.1 e)
27
a.
Day 0
Thaw cells
Add 30ul CD2/3/28
activator in 4mL T cell
medium
Day 3
Coat plate
With 2ug/2mL a-261
antibody
Day 4
Put cell to wells
Day 6
FACS
Fig. 4.2.1 a. Timeline for the downregulation assay (CL-Exp.021)
28
Fig. 4.2.1 b. Activation CAR T cells with 261 antibody cause Lym-1 detection
decrease. Half million, one million, and one and half million CD19-CD3 ζ, CD19-
DAP12, CD19-DAP10/12 CAR T cells were seeded on different dose of a-261 coated
plate. After 2 days, Lym-1 and CAR epitope expression were measured. The detection of
Lym-1was decreased. (CL-Exp.021)
b.
Day 4
Day 6
No addition
Day 6
1.5 million cell
In 2ug/2mL a-261
Day 6
1 million cell
In 1 ug/2mL a-261
Day 6
0.5 million cell
In 1 ug/2mL a-261
CD19-CD3z CD19-DAP12 CD19-DAP10DAP12
Lym-1
(HLA-DR !)
29
Fig. 4.2.1 c. Activation CAR T cells with 261 antibody cause DA2 detection
decrease.
Half million, one million, and one and half million CD19-CD3 ζ, CD19-DAP12, CD19-
DAP10/12 CAR T cells were seeded on different dose of a-261 coated plate. After 3
days, DA2 and CAR epitope expression were measured. The detection of DA2 was
decreased. (CL-Exp.021)
c.
Day 4
Day 6
No addition
Day 6
1.5 million cell
In 2ug/2mL a-261
Day 6
1 million cell
In 1 ug/2mL a-261
Day 6
0.5 million cell
In 1 ug/2mL a-261
CD19-CD3z CD19-DAP12 CD19-DAP10DAP12
DA2
(HLA-DR !)
a-261
(CAR)
30
d.
Day 4
Day 6
No addition
Day 6
1.5 million cell
In 2ug/2mL a-261
Day 6
1 million cell
In 1 ug/2mL a-261
Day 6
0.5 million cell
In 1 ug/2mL a-261
CD19-CD3z CD19-DAP12 CD19-DAP10DAP12
L243
(HLA-DR !)
a-261
(CAR)
Fig. 4.2.1 d. Activation CAR T cells with 261 antibody cause L243 detection
decrease.
One or one and half million CD19-CD3 ζ, CD19-DAP12, CD19-DAP10/12 CAR T cells
were seeded on different dose of a-261 coated plate. After 3 days, L243 and CAR
epitope expression were measured. The detection of L243 was increased. (CL-Exp.021)
31
Fig. 4.2.1 e. Analyzation Data
Quantification of the percentage of HLA-DR expression variation in
CD19-CD3𝜁, CD19-DAP12, and CD19-DAP10/DAP12 CAR positive
T cells was calculated by: % . Fraction is being to calculate the
detection of the antibody. Positive in CAR = Q1/(Q1+Q4) and the
Non-CAR group (seen as mock T cells group) the fraction was
calculated by Q2/ Q2+Q3. These data are not a totally repeat
experiment so we cannot see the significant result. However, the bar
chart shows detection of HLA-DR expression in different dose of
stimulation can cause similar potential.
32
According to the previous result, we repeat two groups: the one million and the one and half
million group. Meanwhile, one million of CAR T cells will be put on the 2 𝜇g a-261coated plate.
With a-261 stimulation, CAR T cells are activated. Meanwhile, after 3days after stimulation the
expression of Lym-1, DA2 in the CAR positive group were significantly being downregulated.
However, in the CAR negative group (normal T cells), the expression of Lym-1 and DA2 are the
same. Besides, the number of L243 (HLA-DR) α chain keeps the same. (Figure 4.2.1f-4.2.1I)
The acquired data were preferable for subsequent analysis. (Figure 4.2.1J)
Fig. 4.2.1 f. Time line for the downregulation assay (CL-Exp.022)
f.
33
g.
a-261
(CAR)
Day 4
Day 7
1.5 million cells
in 2 ug/2mL a-261
Day 7
No addition
CD19-CD3z
Lym-1
(HLA-DR !)
CD19-DAP12
CD19-DAP10DAP12
Day 7
1 million cells
in 2ug/2mL 2-261
Day 7
1 million cells
in 1ug/2mL 2-261
a-261
(CAR)
Fig. 4.2.1 g. Activation CAR T cells with 261 antibody cause Lym-1 detection
decrease. One million CD19-CD3 ζ, CD19-DAP12, CD19-DAP10/12 CAR T cells were
seeded on different dose of a-261 coated plate. After 3 days, Lym-1 and CAR epitope
expression were measured. The detection of Lym-1was decrease. (CL-Exp.022)
34
h.
CD19-CD3z CD19-DAP12 CD19-DAP10DAP12
Day 4
Day 7
No addition
Day 7
1.5 million cells
in 2 ug/2mL a-261
a-261
(CAR)
DA2
(HLA-DR !)
Day 7
1 million cells
in 2ug/2mL a-261
Day 7
1 million cells
in 1ug/2mL a-261
Fig.4.2.1 h. Activation CAR T cells with 261 antibody cause DA2
detection decrease.
One million CD19-CD3 ζ, CD19-DAP12, CD19-DAP10/12 CAR T cells were seeded
on different dose of a-261 coated plate. After 3 days, DA2 and CAR epitope
expression were measured. The detection of DA2 was decrease. (CL-Exp.022)
35
i.
CD19-CD3z
CD19-DAP12
CD19-DAP10DAP12
Day 4
Day 7
No addition
Day 7
1.5 million cells
in 2 ug/2mL a-261
L243
(HLA-DR α)
a-261
(CAR)
Day 7
1 million cells
in 2ug/2mL a-261
Day 7
1 million cells
in 1ug/2mL 2-261
Fig.4.2.1 i. Activation CAR T cells with 261 antibody cause L243 detection decrease.
One or one and half million CD19-CD3 ζ, CD19-DAP12, CD19-DAP10/12 CAR T cells were
seeded on different dose of a-261 coated plate. After 3 days, L243 and CAR epitope expression
were measured. The detection of L243 was increase. (CL-Exp.022)
36
j.
Lym-1
DA2
CD3z DAP12
DAP10/12
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Day 4
CD3z
Day 7
DAP10/12
1.5M cells
no addition
Day 7
DAP10/12
1M cells
1ug a-261tag
Day 7
DAP10/12
1M cells
2ug a-261tag
Day 7
DAP10/12
1.5M cells
2ug a-261tag
Fraction expressing DA2 epitope after culturing 2 days -/+ a-261
Non-CAR CD19 CAR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Day 4
CD3z
Day 7
DAP12
1.5 M cells
no addition
Day 7
DAP12
1M cells
1ug a-261tag
Day 7
DAP12
1M cells
2ug a-261tag
Day 7
DAP12
1.5M cells
2ug a-261tag
Fraction expressing DAP2 epitope after culturing 2 days -/+ a-261
Non-CAR CD19 CAR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Day 4
CD3z
Day 7
CD3z
1.5M cells
no addition
Day 7
CD3z
1M cells
1ug a-261tag
Day 7
CD3z
1M cells
2ug a-261tag
Day 7
CD3z
1.5M cells
2ug a-261tag
Fraction expressing DA2 epitope after culturing 2 days -/+ a-261
Non-CAR CD19 CAR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Day 4
CD3z
Day 7
DAP10/12
1.5M cells
no addition
Day 7
DAP10/12
1M cells
1ug a-261tag
Day 7
DAP10/12
1M cells
2ug a-261tag
Day 7
DAP10/12
1.5M cells
2ug a-261tag
Fraction expressing Lym-1 epitope after culturing 2 days -/+ a-261
Non-CAR CD19 CAR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Day 4
CD3z
Day 7
DAP12
1.5 M cells
no addition
Day 7
DAP12
1M cells
1ug a-261tag
Day 7
DAP12
1M cells
2ug a-261tag
Day 7
DAP12
1.5M cells
2ug a-261tag
Fraction expressing Lym-1 epitope after culturing 2 days -/+ a-261
Non-CAR CD19 CAR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Day 4
CD3z
Day 7
CD3z
1.5M cells
no addition
Day 7
CD3z
1M cells
1ug a-261tag
Day 7
CD3z
1M cells
2ug a-261tag
Day 7
CD3z
1.5M cells
2ug a-261tag
Fraction expressing Lym-1 epitope after culturing 2 days -/+ a-261
Non-CAR CD19 CAR
L243
0.8
0.8
0.8
0.9
0.9
0.9
0.9
0.9
1.0
1.0
1.0
Day 4
CD3z
Day 7
DAP10/12
1.5M cells
no addition
Day 7
DAP10/12
1M cells
1ug a-261tag
Day 7
DAP10/12
1M cells
2ug a-261tag
Day 7
DAP10/12
1.5M cells
2ug a-261tag
Fraction expressing L243 epitope after culturing 2 days -/+ a-261
Non-CAR CD19 CAR
0.8
0.9
0.9
0.9
0.9
0.9
1.0
1.0
1.0
Day 4
CD3z
Day 7
DAP12
1.5 M cells
no addition
Day 7
DAP12
1M cells
1ug a-261tag
Day 7
DAP12
1M cells
2ug a-261tag
Day 7
DAP12
1.5M cells
2ug a-261tag
Fraction expressing L243 epitope after culturing 2 days -/+ a-261
Non-CAR CD19 CAR
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Day 4
CD3z
Day 7
CD3z
1.5M cells
no addition
Day 7
CD3z
1M cells
1ug a-261tag
Day 7
CD3z
1M cells
2ug a-261tag
Day 7
CD3z
1.5M cells
2ug a-261tag
Fraction expressing L243 epitope after culturing 2 days -/+ a-261
Non-CAR CD19 CAR
Fig. 4.2.1 J. Analyzation Data
Quantification of the percentage of HLA-DR expression variation in CD19-
CD3𝜁, CD19-DAP12, and CD19-DAP10/DAP12 CAR positive T cells was calculated
by: % Positive in CAR = Q1/(Q1+Q4) and the Non-CAR group (seen as mock T cells
group) the fraction was calculated by Q2/ Q2+Q3. (CL-Exp.022)
37
4.2.2 CD3/CD28 mimic TCR signaling on primary T cell
Thaw one vial of isolated T cell and activate them with CD3/CD28 Dynabeads™ Human T-
Activator CD3/CD28 in day 0. 4 days later, 1 million T cells have being put on the a-CD3 and a-
CD28 coated plated. With mimic TCR signaling, T cells being activate. Detected Lym-1, DA2,
and L243 in the following days.
a
Day 1
Thaw cell
Day 0
Coat plate
Day 4
Start experiment
Day 5-7:
FACS
Fig. 4.2.2 a. Timeline for the downregulation assay
38
b.
Day 4
Day 5
No addition
Day 5
a-CD3/CD28
stimulated
Lym-1 L243 DA2
Z
Day 6
No addition
Day 6
a-CD3/CD28
stimulated
6
Day 7
No addition
Day 7
a-CD3/CD28
stimulated
6
Fig .4.2.2 b Raw Data One million T cells were seeded on a-CD3/CD28 coated plate. In
following 3 days, Lym-1, DA2, and L243 epitope expression were measured.
39
c.
0
10
20
30
40
50
60
70
Day 0 Day 1 Day 2 Day 3
No Addition
Lym-1 DA2 L243
0
10
20
30
40
50
60
70
Day 0 Day 1 Day 2 Day 3
+ a-CD3, a-CD28
Lym-1 DA2 L243
% cells staining with antibody
Fig. 4.2.2 c. Analyzing Data
The expression percentage was being comparison through the bar chart.
40
Chapter 5- Discussion and future directions
5.1 Discussion
Publications frequently refer to the detection of HLA-DR using an antibody such as L243 that
detects only the HLA-DR alpha subunit. The previous studies have demonstrate that the epitope
on the alpha subunit recognized by L243 can be present while epitopes on the beta subunit are
not. One possibility is that the beta subunit is no longer present. Another is that the beta subunit
is present, but its conformation or location is altered. Regardless of what caused the loss the
epitopes it is possible that the function of HLA-DR is altered and it may not be an appropriate
detection method relying on only the alpha subunit epitope to conclude that a functional HLA-
DR protein is present.
This report demonstrates that activation of a CAR comprised of only a CD3 ζ signaling domain
was sufficient to diminish detection of beta subunit epitopes. We generated the first-generation
CAR T cells which only have a signaling domain (CD3 ζ or DAP12). After using different doses
(0.5ug/mL or 1ug/mL) of a-261 to activate the different amount of CAR T cells (0.5 million, 1
million, or 1.5 million) , we saw that 1ug/mL of a-261 shows best stimulation of 1 million CAR
T cells. The detection of Lym-1 (conformational HLA-DR β chain), and DA2 (linear HLA-DR β
chain) was decreased with above concentration. However, the detection of L243 (HLA-DR α
41
chain) increased. In other words, the CD3 ζ signaling in CAR T cells suggests the HLA-DR β
chain detection decrease. This result supports the hypothesis that activation of normal TCRs
would do the same.
To prove this hypothesis, another experiment was done with primary T cells. Primary T cells
which were activated with CD3/CD28 antibodies showed the low expression of HLA-DR
epitopes. However, T cells cultured in the absence of the antibodies showed increased expression
of the epitopes. To sum up, CD3/CD28 antibody as a mimic TCR signaling can prevent primary
T cell from the HLA-DR detection.
In conclusion, screening by flow cytometry, provided convincing evidence of activation of
normal TCRs would diminish detection of HLA-DR beta subunit epitopes. This evidence can
urges us to rethink the conformation of HLA-DR and how immune system uses HLA-DR to
escape from being killing by other T cells.
42
5.2 Future opportunities
Our experiments provide preliminary results for the activation of T cell HLA-DR β chain
detection decrease. However, current studies provide little information on will the activation of
the cells can influence HLA-DR β chain expression. Several questions remain to be answered.
In our experiments, we did not look for apoptosis or activation markers. So, in the future, we can
compare detection of CD137 or some apoptosis (like BLC2) markers to loss of Lym-1-E as
markers of epitope stimulation of T cells. Besides, we can also detect the expression of the
invariant chain (CD74) in the cell to see if the detection decrease happens in the protein take-up
process.
A more important question to ask than about changes in expression of epitopes is whether these
changes indicate changes in the function of HLA-DR. HLA-DR expressed by professional
antigen-presenting cells can send signals to T cells to regulate T cell function regarding the
epitope presented by HLA-DR. An alternative function for HLA-DR on T cells is that cytotoxic
CD4+ T cells could recognize an HLA-DR presented epitope and kill the T cell presenting the
epitope. The data reported here prompt the hypothesis that when a T cell expresses HLA-DR it is
a risk of being killed, but when the T cell recognizes its target epitope the ability of its HLA-DR
43
to present epitopes is lost and the T cell is shielded from the killing potential of CD4+ CTLs.
The results reported here justify testing this hypothesis.
44
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Abstract (if available)
Abstract
MHC Class II plays a crucial role in regulating the immune reaction. In recent years, research on how HLA-DR regulates the immune response has become very popular. In a normal immune response, the HLA-DR complex (with both α and β subunit) binds to T- cell receptors (TCRs) and causes T cells activation and release cytokines to induce the immune response.[2] In addition, cytotoxic CD4+ cells can target cells with HLA-DR and kill the target cells in an MHC Class II-restricted fashion. [3] This thesis continues studies involving an unreported and unexpected result. Lym-1 is an antibody recognizing an epitope on the HLA-DR β subunit. When an antibody bound to the chimeric antigen receptor (CAR) of anti-CD19-DAP CAR T cells, detection of the Lym-1 epitope was lost on the CAR T cells while detection of an epitope on the α subunit remained (antibody L243).
The above result involved a CAR construct using a DAP10/DAP12 signaling domain. The physiological signaling domain for T cell receptors is CD3 ζ. This thesis tested the hypothesis that stimulation of a CAR with a CD3 ζ signaling domain is sufficient to cause loss of detection of the Lym-1 epitope. The results support the hypothesis and justify future work testing the hypothesis that epitope-stimulation of normal T cells also causes the loss of the Lym-1 epitope which then alters the function of HLA-DR.
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University of Southern California Dissertations and Theses
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Asset Metadata
Creator
Lin, Chia-Hsin
(author)
Core Title
Regulation of T cell HLA-DR by CD3 ζ signaling
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Degree Conferral Date
2022-05
Publication Date
04/27/2024
Defense Date
01/26/2022
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
CD3 ζ signaling,HLA-DR,OAI-PMH Harvest,T cell
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Yuan, Weiming (
committee chair
), Epstein, Alan (
committee member
), Kaslow, Harvey (
committee member
), Machida, Keigo (
committee member
)
Creator Email
clin1273@usc.edu,nana50622@hotmail.com.tw
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-oUC111136635
Unique identifier
UC111136635
Document Type
Thesis
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application/pdf (imt)
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Lin, Chia-Hsin
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texts
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20220428-usctheses-batch-934
(batch),
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
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Tags
CD3 ζ signaling
HLA-DR
T cell