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Development of an NFAT-GFP Jurkat T cell reporter system for acceleration of T Cell receptor affinity screening
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Development of an NFAT-GFP Jurkat T cell reporter system for acceleration of T Cell receptor affinity screening
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Content
Development of an NFAT-GFP Jurkat T Cell Reporter System for Acceleration of T Cell
Receptor Affinity Screening
by
Che-Yu Chang
A Thesis Presented to the
THE KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
December 2023
ii
Acknowledgments
I would like to express my deepest gratitude to my advisor, Dr. Rongfu Wang, for his
invaluable guidance, support, and encouragement throughout this thesis. I am also grateful to the
members of my thesis committee, Dr. Peter Mullen and Dr. Joseph D. Landolph Jr., for their
valuable insights and suggestions. I would like to thank all my lab members and colleagues, Helen
Wang, Changsheng Xing, Junjun Chu, Yang Du, Tianhao Duan, Xin Liu, Qian Chen, Yijou Chen,
and Christina Chen, for giving me advice and helping me accomplish my work. I want to thank
Dr. Rongfu Wang’s laboratory for providing me with the resources and facilities necessary to
complete this M.S. thesis. I am also grateful to my family and friends for their unwavering support
and encouragement throughout this journey. Finally, I would like to acknowledge the countless
hours of hard work, dedication, and effort I have invested in completing this thesis.
iii
Table of Contents
Acknowledgments ......................................................................................................................... ii
List of Figures .............................................................................................................................. vi
Abstract ...................................................................................................................................... viii
Introduction ................................................................................................................................... 1
T Cell Receptor and Chimeric Antigen Receptor Cell Therapy ............................................................. 1
Challenges in the Improvement of T-cell Receptor (TCR) T-cell Immunotherapy ................................ 2
T Cell Receptor (TCR) ............................................................................................................................ 2
VDJ recombination .................................................................................................................................. 3
Introduction of the NFAT-GFP Jurkat T Cells Reporter System ............................................................ 4
Clonal Selection and Functional Check of the NFAT-GFP Jurkat T Cell Reporter System .................. 5
Materials and Methods .................................................................................................................. 7
Chapter 1. Materials ...................................................................................................................... 7
Chapter 2. Methods ....................................................................................................................... 9
Construction of the CD8a-P2A-CD8b and NFAT-EGFP Gene .............................................................. 9
Construction of CD8 Transduced Jurkat T cell and CD8/NFAT Transduced Jurkat T Cells ............... 11
FACs Analysis of Functional Check of Transduced Jurkat T Cells ...................................................... 11
Fluorescent Imaging of CD8/NFAT Transduced Jurkat T Cells .......................................................... 12
Cell Sorting of CD8/NFAT-Transduced Jurkat T Cells ........................................................................ 12
CT83 and 2D2 CAR Viral Transduction of an NFAT-GFP Jurkat Reporter System ........................... 13
Functional Assay of CT83 and 2D2 CAR Transduced NFAT-GFP Jurkat Reporter System .............. 14
iv
Statical Analysis: Student's T-Test ........................................................................................................ 17
Statical Analysis: Two-Way ANOVA .................................................................................................. 17
Result .......................................................................................................................................... 18
Chapter 1. Development of an NFAT-GFP Jurkat Reporter System .......................................... 18
1-1 Construction and Functional Check of CD8 Clone-In Jurkat cells ................................................. 18
1-2 Construction of CD8/NFAT-GFP Clone-In Jurkat T cells ............................................................. 19
1-3 Functional Check of CD8/NFAT-GFP Clone-In Jurkat T cells ..................................................... 19
Chapter 2. Clone Selecting and Functional Check of NFAT-GFP Jurkat Reporter System ...... 21
2-1 Negative and Positive Selection of CD8/NFAT-GFP-Transduced Jurkat T cells .......................... 21
2-2 Functional Check of Negative and Positive Selection of CD8/NFAT-GFP Transduced Jurkat T Cells
................................................................................................................................................................ 21
2-3 Function Assay: Co-culture of the TCR transduced-Jurkat NFAT-GFP Reporter System ............ 22
2-4 Function Assay: FACs analysis of Jurkat NFAT-GFP reporter system co-culture assay .............. 23
2-5 Function Assay: FACs analysis of the CAR-transduced Jurkat NFAT-GFP reporter system co-culture
assay ...................................................................................................................................................... 24
2-6 Comparison of Jurkat NFAT-GFP reporter system and IFN-γ ELISA in the application of TCR
Screening ............................................................................................................................................... 24
Chapter 3. Application of Reporter System: Functional Improvement of H3.3K27M TCR ..... 26
3-1 Alanine Scanning of H3.3K27M Mutated TCR Library ................................................................ 26
3-2 The TCR-T Affinity Screening of Alanine-Mutated H3.3K27M TCR Library ............................. 26
3-3 The Deep Screening of A72, A74 H3.3K27M Mutated TCR Library ........................................... 27
v
Discussion ................................................................................................................................... 29
References ................................................................................................................................... 32
Figures ......................................................................................................................................... 39
Supplement ................................................................................................................................. 61
vi
List of Figures
Figure 1. The gel electrophoresis result of hCD8-alpha-P2A and P2A-hCD8-beta and pFU3W fixMCS (+) vector ........................................................................................................................... 39
Figure 2. The result of enzyme cutting CD8a-P2A-CD8b plasmid ............................................ 40
Figure 3. FACs Analysis of CD8 expression of clone-in CD8 Jurkat T cells ............................ 41
Figure 4. DNA plasmid of pSIRV-NFAT-EGFP ....................................................................... 42
Figure 5 Image of CD8 Jurkat T cells and CD8/NFAT Jurkat T cells ....................................... 43
Figure 6. The FACs analysis result of CD3, CD8, and GFP expression of CD8 Jurkat T cells and
CD8/NFAT Jurkat T cells before and after the OKT3 stimulation ............................................ 44
Figure 7. The FACs analysis result of CD3, CD8, and GFP expression of CD8 Jurkat T cells and
CD8/NFAT Jurkat T cells before and after the thapsigargin (TG) stimulation .......................... 45
Figure 8. Cell sorting data of unstained NFAT-GFP Jurkat T cell and zombie aqua dye stained
NFAT-GFP Jurkat T cell from BD FACSAria IIu Cell Sorter ................................................... 46
Figure 9. FACs analysis result of the negative selected CD8/NFAT-GFP Clone in Jurkat T cells
and the image before and after the T cell stimulation under the fluorescence microscope ........ 47
Figure 10. Cell sorting data of unstained Jurkat/NFAT T cell and zombie aqua dye stained
Jurkat/NFAT T cell from BD FACSAria IIu Cell Sorter ........................................................... 48
Figure 11. Fluorescence Image and FACs Analysis of GFP expression of the stimulated
Jurkat/NFAT T cells ................................................................................................................... 49
Figure 12. Fluorescence Image of control T cells and CT83 TCR-transduced T cells ................ 50
Figure 13. FACs Analysis of GFP expression of the TCR-transduced T cell co-culture assay . 51
Figure 14. FACs analysis of GFP expression of the CAR-transduced T cell co-culture assay .. 52
vii
Figure 15. TCR Affinity Screening of H3.3K27M Alanine-Mutated TCR Library with NFAT-GFP
Jurkat Reporter System ............................................................................................................... 53
Figure 16. TCR Affinity Screening of H3.3K27M Alanine-Mutated TCR Library A. IFN-γ ELISA
Assay of mutated H3.3K27M TCR Library affinity screening .................................................. 54
Figure 17. Comparison of B69, A72, and A74 TCR specificity in IFN-γ release ELISA assay 56
Figure 18. IFN-γ release ELISA assay from TCR Affinity Screening of H3.3K27M A72, A74
Mutated TCR Library ................................................................................................................. 58
Figure 19. IFN-γ release ELISA assay from repeated co-culture functional Assay ................... 59
viii
Abstract
T-cell receptor T cell therapy and chimeric antigen receptor T cell[2] therapy are promising
cell therapy strategies for cancer treatment. TCR-T cell therapy involves genetically modifying a
patient's T cells to express a TCR that recognizes a tumor antigen and selectively targets cancer
cells expressing the antigen. Similarly, CAR-T cell therapy uses genetically modified T cells to
express a chimeric antigen receptor on their surface to target cancer cells in the body. Several
ongoing clinical trials are evaluating the safety and efficacy of these therapies in humans, but the
specificity and affinity of TCR and CAR need improvement. However, current TCR screening
methods are limited by time-consuming processes that prolong the TCR affinity screening
procedures.
To overcome the challenge and improve the TCR screening method, we developed an
NFAT-GFP Jurkat T cell reporter system for high-affinity TCR/CAR screening. We introduced
CD8 and NFAT-GFP genes into Jurkat T cells and confirmed their activation and GFP expression
through functional assays and cell sorting. The Jurkat/NFAT reporter system can differentiate TCR
affinity by the level of GFP expression in both CAR/TCR screening. This system is a promising
tool for accelerating the improvement of TCRs and developing TCR-T cell immunotherapy. In
parallel, the H3.3K27M Mutated TCR library was constructed and screened through with the
Jurkat/NFAT reporter system in TCR. Several of the high-specificity TCRs were found and can
be potential candidates for future TCR-T cell therapy.
1
Introduction
T Cell Receptor and Chimeric Antigen Receptor Cell Therapy
TCR T cell therapy is a type of immunotherapy for cancer treatment that involves
introducing tumor specific TCR genes encoding the α and β chains into autologous T cells through
viral transfer [3]. The modified T cells are then expanded and infused back into the patient's body
to target and destroy tumor cells[4], which are engineered to recognize specific antigens. TCRredirected T cells recognize processed intracellular tumor antigen peptides presented by
MHC/HLA molecules, allowing for the recognition of a broader array of cancer antigens. The
feature is believed to facilitate a more effective infiltration of the modified T cells into solid tumors.
[2,5] This makes TCR T cell therapy a promising approach for generating long-lasting responses
against various cancers, with more than 80 ongoing clinical trials evaluating its safety and efficacy
[6]. CAR-T therapy involves genetic modification of a patient's T cells to express a CAR on their
surface, enabling them to recognize and bind to specific proteins on cancer cells[2]. This approach
has shown promising results in treating certain types of blood cancers, with FDA-approved
therapies like Kymriah for pediatric acute lymphoblastic leukemia [7] and diffuse large B-cell
lymphoma (DLBCL). Overall, both TCR and CAR-T cell therapies have demonstrated significant
potential in cancer treatment. Ongoing research aims to optimize and improve these therapies to
enhance their effectiveness and reduce potential toxic side effects.
2
Challenges in the Improvement of T-cell Receptor (TCR) T-cell Immunotherapy
Despite the promising potential of TCR-T cell therapy in cancer treatment, several
challenges must be addressed [8].The low affinity of TCRs to their peptide-MHC targets and the
risks associated with TCR cross-reactivity [9] must be measured and avoided to generate safe TCR
T cell treatments. Identifying high-affinity TCRs through screening is a crucial step in TCR T cell
therapy, and multiple TCR-screening methods are available, such as the affinity-based TCRscreening method [10, Moritz, 2019 #48]. However, these methods have potential drawbacks,
including their inability to identify interactions that have low affinity between TCR and peptide
MHC. Also, TCR screening involves several complex steps such as T cell expansion, IFN-γ ELSA
assay [11-13]. Moreover, the quality of human peripheral blood mononuclear cells (PBMCs) from
different donors can be unstable[14], which will affect the outcome of the result.
We aim to develop an innovative and efficient TCR screening approach to address these
challenges and to improve the current TCR screening method. This approach will overcome the
limitations of current methods and offer a reliable and faster selection method for high-affinity
TCR screening for its use in TCR T cell therapy. Our proposed method should reduce the time and
materials required for TCR screening, making it a more cost-effective and a more practical option
for the development of TCR T cell therapy.
T Cell Receptor (TCR)
T lymphocytes have a protein complex on their surface called the T-cell receptor. The Tcell receptor is responsible for recognizing a variety of antigen fragments as peptides bound to
MHC molecules [15-18]. The T-cell receptor is composed of two chains, either α and β in αβ T cells
or γ and δ in γδ T cells. The binding site of the TCR is comprised of six loops called
3
complementarity-determining regions (CDRs), with each chain contributing three loops called
CDR1, 2, and 3. CDR2 found in the variable region, and CDR3 vary in an almost unlimited fashion
and largely dictate the TCR specificity for peptides [19]. This feature vastly increases the number
of potential antigen targets. This contrasts with antibodies, which generally only recognize a single
epitope[20]. For instance, it has been demonstrated that naturally occurring TCRs exhibit a variety
of specificity profiles that are able to identify as many as a million specific peptides. However,
TCRs can recognize the same antigen peptide, and numerous antigen peptides are recognized by
the same TCR due to the low affinity of the binding between TCR and antigen peptides[21].
VDJ recombination
VDJ recombination is a fundamental genetic process crucial for the development of T cells,
a key component of the immune system. Taking place in the thymus, this intricate molecular
mechanism involves the rearrangement of gene segments that encode the variable region of the T
cell receptor (TCR).
The TCR is responsible for recognizing specific antigens, enabling T cells to mount
targeted immune responses against pathogens and abnormal cells. Through the action of
recombination-activating genes (RAG-1 and RAG-2) and recombination signal sequences (RSS),
gene segments containing Variable (V), Diversity (D), and Joining (J) regions are cleaved, excised,
and rejoined in various combinations, resulting in a vast repertoire of unique TCRs with distinct
antigen recognition capabilities. This process of combinatorial diversity ensures that T cells can
recognize a wide range of antigens, safeguarding the body against infections and maintaining
immune surveillance throughout life. In the development of TCR-T therapy, we modified amino
4
acid sequence of CDR 2, and 3 in the variable region to see if there is any of the mutations can
affect the affinity or specificity of the transgenic-TCR.
Introduction of the NFAT-GFP Jurkat T Cells Reporter System
Based on Professor Rodrigo Vazquez Lombardi’s 2020 publication, his team developed a
TCR-accepting T cell (TnT) platform that creates transgenic T cells that can recognize and bind to
specific target cells through the use of TCRs[22]. The TnT platform allows for the generation of T
cells with customized TCRs that can be used for targeted therapies against cancer or other diseases,
which is a promising approach for developing new immunotherapies. Here, we introduced the
development of the NFAT-GFP Jurkat T cell reporter system, for the engineering, affinity profiling,
and functional display of TCRs. NFAT-GFP Jurkat T Cell reporter system is constructed by
introducing CD8 and NFAT-GFP gene into the Jurkat T cells. Cluster of Differentiation 8 (CD8)
is a protein that is expressed on the surface of cytotoxic T-lymphocytes. This protein acts as a coreceptor that binds to the T-cell receptor. The MHC class I molecules on the surface of antigenpresenting cells (APCs) recognize the CD8 during the process of T-cell activation[23,24].
Introduction of CD8 makes the NFAT-GFP Jurkat T cell reporter system become more versatile
as a tool for CD4/CD8 TCR affinity screening. NFAT (Nuclear Factor of Activated T-cells) is a
transcription factor that plays a key role in regulating immune and inflammatory responses, which
is in turn activated by the calcium signaling pathways that are triggered by T-cell receptor
activation [25,26]. Once the T cell is activated, the NFAT gene will be triggered simultaneously
along with the green fluorescent protein (GFP) gene[27]. GFP protein expression can be used as a
marker for T cell activation.
5
Clonal Selection and Functional Check of the NFAT-GFP Jurkat T Cell Reporter System
We utilized a combination of negative and positive functional cell sorting selection steps
[28] to select a specific clonal population of NFAT-GFP Jurkat T cell reporter system with the
specific characteristics of low GFP expression in the absence of activation and high GFP
expression upon activation. The functionality of transduced Jurkat T cells is confirmed by
functional assay of examining CT83 TCR with known affinity. In summary, this clonal-selected
NFAT-GFP Jurkat T cell reporter system is able to detect T cell activation by directly examining
the GFP expression using FACs, which indicates the T cell affinity. Also, the result of affinity
screening by the NFAT-GFP Jurkat reporter is identical to the IFN-γ ELISA. Furthermore, the
NFAT-GFP Jurkat T Cells reporter system can distinguish varying levels of T cell activation.
Histone H3 Variant H3.3 Lysine 27-to-Methionine Mutation (H3.3K27M)
Glioma is a type of tumor that grows in the brain and spinal cord. Symptoms of glioma in
patient with this tumor include headaches, seizures, nausea, and vision problems[29]. Gliomas are
derived from the glial cells, which are the supporting cells of the nervous system that provide
structural and metabolic support to neurons. Gliomas are categorized based on the type of glial
cell that is involved, and they can vary in severity from low-grade glioma to high-grade glioma[30].
In children, gliomas account for approximately 25% of all brain tumors [31,32]. In high-grade
pediatric glioma cases, about 60% of these have been found to contain a mutation called Lys 27-
to-methionine (K27M) in one of the two genes, H3F3A, which encodes histone H3 variant H3.3[33].
In light of the significance of the K27M mutation in H3F3A for high-grade pediatric gliomas [34],
there is a need for targeted therapeutic approaches.
6
Functional Improvement of Histone H3 Variant H3.3 Lysine 27-to-Methionine Mutation
(H3.3K27M) TCR
To achieve the development of H3.3K27M glioma TCR cell therapy that can effectively
target and destroy tumor cells [35].there is a need to screen for high specificity and high-affinity
TCR that can specifically target the H3.3K27M mutation[34]. Such TCRs could potentially be used
to develop targeted cell therapies for gliomas, which could significantly improve patient outcomes.
For our screening strategy, we first performed the alanine scanning to the screen through the
affinity of the complementarity-determining regions (CDRs), mutated H3.3K27M TCR[19,36]. We
found three amino acid positions that increased the specificity, and eleven amino acid positions
showed loss of TCR function to recognize tumor peptides. Next, a combinatorial single-mutated
Library of the H3.3K27M TCR was constructed. The result of the TCR library affinity screening
showed that H3.3K27M CDR2 alpha 72/beta 69, alpha 74/beta 69, alpha 72/ alpha 74 positions
double-alanine mutation, H3.3K27M CDR2 alpha 72 position F to M(Met), K(Lys), H(His),
L(Lys), G(Glu), V(Val) mutation enhance the specificity of H3.3K27M-specific peptide.
7
Materials and Methods
Chapter 1. Materials
QIAprep Spin Miniprep Kit, and QIAprep Maxiprep Kit were purchased from Qiagen,
Germantown, Maryland, U.S.A, Zymoclean Gel DNA Recovery Kit were purchased from ZYMO
Research, Orange, California, U.S.A. Pamifisure PCR ONE MASTER MIX (2X), HEPES solution
(10X) were purchased from GenDEPOT, Barker, Texas, U.S.A. PrimeSTAR® Max DNA
Polymerase was purchased from TAKARA, San Jose, California, U.S.A. T75/T25 Flasks, Nunc
BioAssay, Anti-human mouse antibodies, IFN gamma antibodies, CD3 monoclonal antibodies,
CD4 monoclonal Antibodies, and CD8 monoclonal antibodies were purchased from Thermo
Scientific, Waltham, Massachusetts, U.S.A.
RPMI medium 1640 (1X), DMEM medium, Trypsin-EDTA (4X), 0.25%, Phosphate
buffered saline (PBS), Penicillin-streptomycin, fetal bovine serum (FBS), Penicillin-Streptomycin
(10,000 U/mL) were purchased from Grand Island Biological Company(GIBCO), Billings,
Montana, U.S.A. Custom-designed primers were purchased from Integrated DNA Technologies,
San Diego, California, U.S.A. jetOPTIMUS® buffer, jetOPTIMUS® reagent were purchased from
Polyplus, Berkeley, California, U.S.A. The pSIRV-NFAT-eGFP plasmid was purchased from
Addgene, Watertown, Massachusetts, U.S.A. DNA ligation buffer 2x concentration (Vial 1), DNA
dilution buffer 5x concentration (Vial 2), and T4 DNA ligase (Vial 3) were purchased from Roche,
Indianapolis, Indiana, U.S.A.
Restriction enzyme EcoR1-HF, XhoI, Sal1-HF, BamHI, BgII, RsmartCut buffer, 3.1 buffer,
and NEBuilder HiFi DNA Assembly Master Mix were purchased from New England Biolabs,
Ipswich. Massachusetts, U.S.A. TMB substrate was purchased from Mabtech, Cincinnati, Ohio,
8
U.S.A. Dimethyl sulfoxide (DMSO), Bovine serum albumin (BSA) and 96-well plates were
purchased from Millipore Sigma, San Diego, California, U.S.A. The DNA ladder marker was
purchased from GeneDireX, Taoyuan, Taiwan; Zombie Aqua buffer and Avidin-HRP were
purchased from BioLegend, San Diego, California, U.S.A. TB Buffer, SOB Buffer was purchased
from Fisher Bioreagents, Waltham, Massachusetts, U.S.A. RD114 Env, RD114 Gag/Pol CT83
TCR plasmid constructed in Dr. Rongfu Wang's Lab.
9
Chapter 2. Methods
Cell lines and Cell Culture
Cell culturing is the process of growing cells in a controlled environment. This involves
providing the cells with the appropriate nutrients, growth factors, and physical conditions
necessary for their survival and proliferation. Human Embryonic Kidney (HEK) 293T cells and
All cell lines were cultured at 37 °C, 5% CO2, in Forma™ Steri-Cycle™ i160 CO2 165 L Incubator
(Thermo Scientific, Waltham, Massachusetts, U.S.A.). HEK 293T cells and Jurkat leukemia T
cells were purchased from ATCC, Manassas, Virginia, U.S.A.[38] Peripheral blood mononuclear
cells (PBMCs) were collected from Donors of Norris Comprehensive Cancer Hospital. HEK 293T
cells were cultured in DMEM medium. The Jurkat leukemia T cells were cultured in RPMI-1640
medium. PBMCs were cultured in TCM medium with 100 U ml-1 Interleukin-2 (IL-2). All media
were supplemented with 10% fetal bovine Serum (FBS), 50 U ml-1 penicillin, and 50 μg ml-1
streptomycin.
Construction of the CD8a-P2A-CD8b and NFAT-EGFP Gene
To construct the CD8 gene plasmid. A Postdoc Fellow in our laboratory, Dr. Yijou Chan,
extracted RNA from Human PBMC and produced cDNA by performing RT-PCR [42,43]. 1µl of
the 10ng/µl samples, 1µl of each forward and reverse primer, 12µl of water, and 15µl of
PrimeSTAR® Max DNA Polymerase were combined and PCR was performed. The program was
started up with the following steps: 98o
c for 3 min; 30 cycles of 98o
c for 15sec, 57 o
c for 15sec,
72o
c for 60sec; 72o
c for 5min. The CD8 gene was used as the template to construct the hCD8-
alpha-P2A and P2A-hCD8-beta (fig1.). The pFU3W fix-MCS (+) vector was prepared by
restriction enzyme digestion. The restriction enzyme digestion system was comprised of 2µg of
10
DNA of pFU3W fix-MCS vector, 0.5 µl of Hpal, 0.5 µl of EcoRI restriction enzyme (10,000
units/ml), 3µl of CutSmart® buffer, and 27µl of water. The sample mixture was incubated for 2
hrs at 37°C. I performed gel electrophoresis to examine the PCR and enzyme digestion product
(fig1.). Then, I performed a DNA recovery and determined the DNA yield by using nanodrop
procedure[44].
Next on, the hCD8-alpha-P2A, P2A-hCD8-beta DNA, and pFU3W fix-MCS vector
concentrations were determined by Nanodrop and used for DNA recombination. Vector (100ng),
insert (12ng), water (4.2µl), and NEBuilder HiFi DNA assembly master mix (5µl) were mixed
(From NEB) in a 10 µl volume system on ice. The sample was next incubated in a thermocycler
at 50°C for 1 hr. Next, competent bacterial cells were incubated on ice. 5 µl of the DNA assembly
product was next added to a tube of competent cells and waited for 30 mins on ice. The sample
was heat shocked at 43o
C for 90 sec and then placed on ice for 3 mins. 600 µl of the super optimal
broth (SOB medium) was added into an Eppendorf tube and incubated for 1 hr at 37°C in a shaker
at 300 rpm. The sample was then centrifuged and distributed on the surface of pre-warmed
ampicillin-selective Luria broth [45] plates for 1 hour of incubation at 37o
C. Colonies were picked
from LB plate and incubated at a starter culture of 5 ml terrific broth (TB) medium containing the
appropriate selective antibiotic for 15 hours of incubation in a shaker at 37°C.
The bacterial cells were then harvested by centrifugation at 600 G for 5 mins. Next,
miniprep was performed to purify plasmid DNA from bacterial cultures [46]. The yield was
determined by using a nanodrop procedure [44] to ensure the quality of the result [44]. The CD8aP2A-CD8b plasmid was then successfully verified by restriction enzyme cutting check and
sequencing. The NFAT-EGFP gene plasmid was purified from bacterial transformants using the
11
pSIRV-NFAT-EGFP colony slate, and the QIAprep Spin Maxiprep kit, and verified by enzyme
digestion check.
Construction of CD8 Transduced Jurkat T cell and CD8/NFAT Transduced Jurkat T Cells
10 cm tissue culture cell plates (Thermo Scientific, Waltham, Massachusetts, U.S.A.) were
coated with poly-L-lysine phosphate-buffered saline (PBS) and incubated for 10 mins at room
temperature. 4x106 of HEK 293T cells were seeded into a 10 cm tissue culture plate one day prior
to transfection. CD8a-P2A-CD8b or pSIRV-NFAT-EGFP plasmid (3.75 µg), RD114 Env (2.5 µg),
RD114 Gag/Pol (3.75 µg), 1000 μl of jetOPTIMUS® buffer (from Polyplus), and 10 μl of the
jetOPTIMUS® reagent were used to transfect 1x106 293T cells for virus packaging. After 48 hr
of transfection, the viral supernatant was then collected for T cell Transduction 0.5 x106 of the
Jurkat NFAT reporter system in retronectin-coated 24-Well plates. The viral transduction step was
repeated on the next day. The transduced Jurkat T cells were then incubated for 24 hours and
prepared for the functional check; The yield was determined by using nanodrop to ensure the
quality of the result.[44]
FACs Analysis of Functional Check of Transduced Jurkat T Cells
For transduced Jurkat T cell FACs analysis. The samples were collected from the cell plate
and washed with 1ml 1% BSA PBS. 0.5ml 5X diluted EDTA (0.02%)-trypsin (0.25%) was next
used to detach cells from the culture dish, and DMEM medium was used to neutralize the trypsin.
The collected samples were washed with 1ml PBS containing 1% Bovine Serum Albumin (1%
BSA in PBS) and centrifuged at 500 G for 5 mins at 4o
C. The supernatant was removed, and 50µl
of 1 μg ml-1 CD3, hCD4, and CD8 monoclonal antibodies were used to stain the sample T cells.
12
The samples were incubated for 30 min at room temperature avoiding light and repeating the cell
wash step. Then, 400µl 1% BSA PBS was used to resuspend the cell pellet. I started up the FACs
machine and set up a program for cell gating. I SSC and FSC-A were selected for live-cell gating,
FSC-H and FSC-A were selected for single-cell gating, histogram and FITC for GFP expression,
and a histogram and APC for CD3 expression. A histogram and FITC for CD4 expression, a
histogram, and efluor 450 for CD8 expression. Next, the control unstained cells were used for
adjusting the voltage of FSC and SSC to set the cell population to match the gating area. The Xaxis level of the baseline was set to 102-3 for all panels[47,48]. Next, I loaded the samples onto the
machine and started the analysis[45,49].
Fluorescent Imaging of CD8/NFAT Transduced Jurkat T Cells
For fluorescent microscope imaging, cell plates containing samples were placed under the
ZOETM fluorescent cell imager. The cell imager was started up and switched from white light
source to a fluorescent green light source. Then, the focus was adjusted until the cell images were
clear. Next, the samples were observed, and I used the imager to record the imaging data.
Cell Sorting of CD8/NFAT-Transduced Jurkat T Cells
For negative selection, 40x106 of the CD8/NFAT Transduced-T cells were collected and
resuspended in 400 µl of RPMI medium supplemented with 10% FBS and 1% PenicillinStreptomycin [33] and then stained with zombie aqua dye for 15 mins, avoiding light by being
covered in aluminum foil. Then, the cells were washed and resuspended with 1ml of 10% FBS
1%PS RPMI medium. The cell sorting was performed, and I selected 5% of the least GFPexpressing cells by a BD FACSAria IIu Cell Sorter (Fig 8.). SSC and FSC-A were selected for Tcell shape gating. FSC-H and FSC-A were selected for singles cells gating, DAPI-A and FSC-A
13
were selected for GFP expression gating. FSC-H and FITC-A were selected for 5% of the least
GFP expression. 3x105 of selected T cells were collected [28]. The collected T cells were then
washed with 10% FBS and 2% PS RPMI medium, and I proceeded to expand the sorted T Cells
for the future functional check. For position selection, the T cells were activated by seeding 2.0x106
of the CD8/NFAT Transduced-T cells into an OKT3-coated 24-well cell plate at 37°C overnight.
40x106 of the activated T cells were collected and resuspended in 400µl of 10% FBS 1% PS RPMI
medium and stained with zombie aqua dye for 15mins, avoiding light. Then, the cells were washed
and resuspended into 1ml of 10%FBS 1%PS RPMI medium. The cell sorting was then performed,
and I selected 50% of the highest GFP expression cells by BD FACSAria IIu Cell Sorter (Fig. 8)
[28]
OKT3 and Thapsigargin T cell stimulation of CD8/NFAT-Transduced Jurkat T Cells
For OKT3 T cell stimulation, a 24-well tissue culture plate was coated with 1:1000 diluted
OKT3 monoclonal antibody and incubated for 24 hrs at 4°C. Then, 1.0x106 of CD8/NFATTransduced T cells were seeded into an OKT3-coated plate and incubated at 37°C overnight [50,51].
For Thapsigargin (TG) T cell stimulation, a 24-well tissue culture plate was coated with 1:1000
diluted OKT3 monoclonal antibody and incubated for 4 hrs at 37°C Then, 1.0 x106 of CD8/NFATTransduced T cells were seeded into TG-coated plate and incubated at 37°C overnight [52,53].
CT83 and 2D2 CAR Viral Transduction of an NFAT-GFP Jurkat Reporter System
10 cm tissue culture cell plates were coated with poly-L-lysine PBS. 4x106 of 293T cells
were then seeded into each cell plate one day ahead of transfection. CT83 & 2D2 plasmid (750ng),
RD114 Env (500ng), RD114 Gag/Pol (750ng), 200µl of jetOPTIMUS® buffer and 10µl of
14
jetOPTIMUS® reagent were used to transfect 1x106 293T cells for virus packaging. After 48 hrs
of transfection, the viral supernatant was then collected for T cell transduction of 0.5 x106 Jurkat
NFAT reporter system in 10 ng/µl retronectin coated 24-well plate. The viral transduction step
was repeated on the next day. The transduced Jurkat T cells were then incubated for 24 hours and
prepared for the functional check.
Functional Assay of CT83 and 2D2 CAR Transduced NFAT-GFP Jurkat Reporter System
0.1x106 HEK 293T cells were co-cultured with 0.05x106 HEK 293T cells and 0.01µg/ml
diluted A2/CT83-specific peptide overnight in 200µl of TCM medium in 96-well U-bottom cell
plate. For functional assay of 2D2 CAR transduced NFAT-GFP Jurkat reporter system, 0.1x106
HEK 293T cells were co-cultured in 1:2000 diluted A2/Eso-specific peptide overnight in 200µl of
TCM medium in 96-well U-bottom cell plate[54]. The following day, the samples were collected
and washed twice with 1% BSA PBS. The samples were then analyzed by FACs for GFP
expression to determine T cell activation and affinity.
Polymerase chain reaction (PCR)
PrimeSTAR Max DNA polymerase and custom-designed primers were used to perform
PCRs for generating amplicons and creating TCR libraries. The cycling conditions were as follows:
98°C for 3 min; 35 cycles of 98°C for 15 s, 55°C for 15 s, 72°C for 60 s per kb; final extension
72°C for 5 min per kb. For genotyping of bacterial colonies from transformation, PCRs were
performed using custom-designed primers and the PamfiSure PCR Master Mix with the following
cycling conditions: 98°C for 3 min; 30 cycles of 98°C for 15 s, 58°C for 15 s, 72°C for 60 s per
kb; final extension 72°C for 5 min per kb.
15
Generation of H3.3K27M Combinatorial TCR Library
This procedure is based on the T cell receptor alpha joining 43 (TRAJ43) protein-coding
gene. There are 27 amino acid positions, which are at CDR2α position 71-74, CDR3α position
114-116, CD2β position 67-74, and CD3β position 110-122. For library construction, ssDNA
oligonucleotides containing a 25 nt complementary overlap were designed and purchased as
custom primers (From IDT). The forward primer encoded wild-type TCRA codons, while the
reverse primer contained the reverse complement with a mutated codon[55]. Both 200 Pmol of
each primer were mixed and subjected to PCR using the following conditions: 98°C for 3 min; 35
cycles of 98°C for 15 s, 55°C for 15 s, 72°C for 60 s per kb; final extension 72°C for 5 min per kb.
The resulting dsDNA product was gel-purified and was utilized as a DNA fragment insert for DNA
assembly. In parallel, pMSGV-A2 H3.3K27M TCR plasmid was digested with XhoI and BamHI
(For CDR2α, CDR3α mutation) or BamHI and SalI (For CD2β, CD3β mutation). PCR product
and plasmid were assembled in a 20 μl reaction containing 10ml NEBuilder® HiFi DNA Assembly
Master Mix for 1 hr at 50°C. Next, the DNA assembly mix was transformed into 100 μL of
competent E. coli cells. The transformed cells were plated on ampicillin LB agar in VWR Petri
10cm dish(). Plasmid libraries were purified from bacterial transformants using the QIAprep Spin
Miniprep kit. Plasmid libraries were verified by enzyme digestion check and sequencing.
H3.3K27M Combinatorial TCR Library Screening
Six-well tissue culture cell plates were coated with poly-L-lysine PBS. 1x106 of HEK 293T
cells were seeded into each cell plate one day ahead of transfection. Mutated TCR plasmid(750ng),
RD114 Env(500ng), RD114 Gag/Pol(750ng), 200µl of jetOPTIMUS® buffer and 10µl of
16
jetOPTIMUS® reagent were used to transfect 1x106 293T cells for virus packaging. The viral
supernatant was then collected for T cell transduction of 0.5 x106 Jurkat NFAT reporter system
cells or human T cells in retronectin coated 24-well plates. PBMC required OKT3 activation for
T cell expansion two days prior to transfection. The viral transduction step was repeated the next
day. Transduced TCR-T cells were then maintained at least 24 hrs for further functional check.
Co-culture of Transduced TCR-T cells and IFN-γ ELISA
For peptide co-culture group, 0.1x106 of transgenic TCR-T cells were washed twice with
RPMI medium lacking IL-2 and overnight co-culture with 0.05x106 HEK 293T cells and 0.1 µg/ml
or 0.01µg/ml H3.3K27M specific-peptide. For the tumor cells co-culture group, 0.1x106 of
transduced T cells were co-cultured with 0.05x106 of tumor cells overnight. After co-culture, the
co-culture medium was collected. IFN-γ ELISA assays were performed using the anti-human
mouse antibody, IFN gamma antibodies, 96-well plates, Avidin-HRP, DMSO, and precipitating
TMB substrate[13]. Wells were coated with 100µl 2 μg/ml-1 IFN gamma antibody (in 1%BSA PBS)
at 4°C overnight. Wells were then blocked with 300µl of 2%BSA PBS at room temperature for
2hrs and washed three times with PBS. After incubation, wells were blocked with 50µl culture
medium and incubated at room temperature for 30mins. Then, wells were blocked with 1 μg/ml-1
Anti-human mouse antibody biotin 50µl(in 1%BSA PBS) for 1 hr, and the cells were washed
three times with PBS. 50µl 1:5000 Avidin-HRP (in 1%BSA PBS) was added for 30 min at room
temperature in the dark by being covered in aluminum foil. Wells were washed three times with
PBS, followed by development with 100µl TMB substrate for 3-10 mins at room temperature.
Development was stopped by blocking with 50µl 2.5N H2SO4, and plates were to analysis using
an ELISA reader [56,57].
17
Statical Analysis: Student's T-Test
Student's t-test is vital when the research design involves comparing the means of two independent
groups or conditions. Student’s t-test is employed to determine whether there are statistically
significant differences between the means of these groups. It assumes that the data is continuous
and follows a normal distribution, and it tests the null hypothesis that the means are equal. The
independent nature of the groups makes Student's t-test a suitable choice when studying variables
with two distinct conditions, such as experimental and control groups. By utilizing this test, we
aim to assess whether there is empirical evidence to support the presence of meaningful
distinctions between the two groups, which is instrumental in addressing our research objectives.
Statical Analysis: Two-Way ANOVA
Two-way ANOVA (Analysis of Variance) is statistical method when the research design
involves two or more independent categorical variables, and the aim is to investigate the potential
influence of these variables on a continuous dependent variable. Two-way ANOVA is employed
to determine whether there are statistically significant main effects associated with the factors and
whether there is an interaction effect between these factors. As part of this analysis, we assumed
that the data met the underlying assumptions of normality and homoscedasticity. Two-way
ANOVA is a powerful statistical tool that can reveal insights into the interplay of multiple factors
on the study's dependent variable, making it a good choice for our research investigation.
18
Result
Chapter 1. Development of an NFAT-GFP Jurkat Reporter System
1-1 Construction and Functional Check of CD8 Clone-In Jurkat cells
CD8 is the co-receptor of the TCR, expressed by cytotoxic T cells, and forms the TCRCD8 complex[58]. It helps recognize MHC class I molecules [59]. Introducing the CD8 gene into
NFAT-GFP Jurkat reporter system allows future use of CD8 cytotoxic T-cell TCR screening,
which makes the NFAT-GFP Jurkat reporter system become more versatile as a tool for CD4/CD8
TCR affinity screening.
To construct a CD8 clone in Jurkat cells, Postdoctoral fellow Dr. Yijou Chan extracted
RNA from Human PBMC and produced cDNA by performing RT-PCR. Then, she performed PCR
to generate the hCD8-alpha-P2A and P2A-hCD8-beta. (Fig 1.A) Then, the pFU3W fix-MCS (+)
vector (fig 1.B) was prepared with restriction enzyme cutting and combined hCD8-alpha-P2A and
P2A-hCD8-beta DNA by DNA assembly method [60,61]. Later, plasmids were purified from
bacterial transformants, and the CD8a-P2A-CD8b plasmid was successfully verified by restriction
enzyme cutting check and sequencing (Fig 2.A, B).
Next, the CD8a-P2A-CD8b plasmid was used for introducing the CD8 gene into the Jurkat
T cell by viral transduction. To ensure the success of the clone-in of the CD8 gene into Jurkat T
cells. The CD8 clone-in Jurkat cells were stained with anti-CD8 antibody, and I performed a FACs
analysis to determine the difference between the control T cell group and the CD8 clone-in Jurkat
cells group. Based on the result (Fig 3.A), the untransduced-Jurkat T cells, as a control, showed
no CD8 expression. For CD8-transduced-Jurkat T cells, the unstained CD8-transduced-Jurkat T
19
cell group showed no GFP expression. The stained CD8 transduced-Jurkat T cell group shows
99.7% of the cell population with CD8 expression compared to the control group, showing the
success of introducing the CD8 gene into Jurkat cells (Fig 3.B).
1-2 Construction of CD8/NFAT-GFP Clone-In Jurkat T cells
The nuclear factor of activated T-cells (NFAT) is a transcription factor that plays an
important role in the immune response[25]. NFAT proteins are activated by the phosphatase
calcineurin and are involved in the regulation of gene expression in response to immune stimuli
such as antigens[25,62,63]. Green fluorescent protein (GFP) is a common indicator for studying
signal transduction pathways or cell development [27,64]which represents the TCR affinity. The
NFAT-GFP gene containing 4xNFAT with EGFP right next to the NFAT sequence was chosen as
the plasmid for the NFAT-GFP gene (Fig 4.A). Maxiprep was performed for constructing pSIRVNFAT-EGFP plasmid, and the quality was verified by gel electrophoresis (Fig 4.B). Next, the
pSIRV-NFAT-EGFP gene was introduced into CD8 Jurkat T cells by viral transduction.
1-3 Functional Check of CD8/NFAT-GFP Clone-In Jurkat T cells
To ensure the success of the clone-in of the NFAT-GFP gene into CD8 Jurkat T cells, a Tcell stimulation test was performed to determine whether the CD8/NFAT transduced Jurkat T cell
could be activated and showed GFP expression. One of the T cell stimuli used in T cell stimulation
is OKT3. OKT3 is a CD3 monoclonal antibody that has a distinct affinity for human T cells and
induces a mitogenic response, which is essential for the T cell activation [51].
Another one is T cell stimulus is thapsigargin (TG). TG is an inhibitor of sarcoendoplasmic
reticulum Ca2+ ATPase (SERCA)(Ali, 1985 #52). It pumps calcium ions from the cytoplasm into
20
the lumen of the endoplasmic reticulum and thapsigargin [65]. This process will cause an increase
in the cytoplasmic calcium levels while also depleting ER stores, which leads to T cell activation
[65].The purpose of T cell stimulation is to induce activation of T cells, The transgenic Jurkat T
cell was expected to express a green fluorescent protein (GFP), which is used as an indicator for
T cell affinity. CD8/NFAT transduced Jurkat T cells were seeded into 10µM OKT3 or 20µM TGcoated plates and incubated 24 hrs for T cell activation and observed GFP expression. Based on
the fluorescent image data, the CD8 Jurkat control group showed no GFP expression (Fig 5.A).
The CD8/NFAT transduced Jurkat T cells sample group showed significant green fluorescent light
after the T cell activation. (Fig 5.B) Then, activated T cells were collected for FACs analysis. The
FACs result also shows identical outcomes when compared to the fluorescent image data (Fig 6.A
Fig 7.A). The CD8/NFAT transduced Jurkat T cells group with T cell activation showed a
significant shift of GFP expression compared to the CD8 Jurkat control group (Fig 6.B, C Fig
7.B,C), which indicates the success of NFAT-GFP cloning into Jurkat T cells.
21
Chapter 2. Clone Selecting and Functional Check of NFAT-GFP Jurkat Reporter System
2-1 Negative and Positive Selection of CD8/NFAT-GFP-Transduced Jurkat T cells
Although the majority cell population of the CD8/NFAT-GFP Clone in Jurkat T cells have
GFP expression after the T cell stimulation, there is a different level of GFP expression among the
cell populations. Therefore, the specific cell population, with the ideal characteristic that it has no
GFP expression without T cell activation,should be selected by conducting cell sorting. To achieve
this goal, the selected T cells were stained with zombie aqua dye. The cell sorting was then
performed, and the P3 population is the live/dead population. The P4 population is 5% cells that
express the lowest GFP. I selected 5% of the Lowest GFP expression cells (Fig 8.). Selected T
cells were collected. Next, positive selection was performed to select the specific cell population
with characteristics of the high population of GFP expression after T-cell activation. To achieve
the goal, The negatively selected CD8/NFAT-GFP-Transduced Jurkat T cells were activated by T
cell stimulation before cell sorting. The cell sorting was then performed. I selected 50% of the
highest GFP expression cells with BD FACSAria IIu Cell Sorter. (Fig 10.) The P3 population is
the GFP-expressed population. The P4 population is 50% of the highest GFP-expressing cells. The
P5 population is the cell population that has no GFP population. Selected T cells were collected
and proceeded to expand sorted T Cells for further functional check.
2-2 Functional Check of Negative and Positive Selection of CD8/NFAT-GFP Transduced
Jurkat T Cells
To ensure the success of the negative selection of the CD8/NFAT-GFP-Transduced Jurkat
T cells, we conducted a T cell stimulation test to make sure the T cell population has no GFP
expression before the stimulation and can be activated after the stimulation. Based on the
22
fluorescent microscope data, before the stimulation, there was no green fluorescent light emission
for both the control and sample group (Fig 9.A). After 24 hours of stimulation, the control
remained with no fluorescent light emission, but the sample group showed significant green
fluorescent light after stimulation. Then, the T cells were collected, and I performed a FACs
analysis. The FACs analysis showed the correlated result with fluorescent microscope data that
the cell population showed a significant shift in GFP expression (fig 9.B). This negative selection
sorted the cell population with the ideal characteristic that has no GFP expression before
stimulation but can still be activated after the stimulation. Next on, to ensure the success of the
positive selection of clone-in of CD8/NFAT-GFP Transduced Jurkat T cells, a T cell stimulation
test was conducted to activate T cells for ensuring a high population of GFP expression. The
negative selected T cells were stimulated with OKT3 monoclonal antibody and incubated for 24
hours. Based on the fluorescent microscope data, selected T cells showed significant green
fluorescent light after stimulation. (Fig 11.) Then, the T cells were collected, and I conducted the
FACs analysis. The result showed that 82.8% of the selected T cell population have GFP
expression (Fig11.). This positive selection sorted the cell population with the ideal characteristic
that has high GFP expression after stimulation.
2-3 Function Assay: Co-culture of the TCR transduced-Jurkat NFAT-GFP Reporter System
After the cell selection steps, a functional check was conducted to evaluate the functionality
of the Jurkat NFAT-GFP reporter system. In this assay, we aimed to use the Jurkat NFAT-GFP
reporter system to screen for TCR or CAR with high affinity for tumor peptides for future
application in TCR-T / CAR-T Cell therapy. To stimulate the recognition ability of TCR-T/CART cells toward cancer cells in the human body, the transgenic Jurkat NFAT-GFP reporter system
was co-cultured with tumor-specific peptides and HEK 293T cells. For the experiment, there are
23
three sample groups of TCR-transgenic Jurkat cells, which have different levels of known affinity
that recognize tumor-specific antigens. TCR-transgenic Jurkat cells are expected to be activated
after the co-culturing, and the Jurkat NFAT-GFP reporter system would be able to distinguish the
different affinity among the group by detecting the GFP expression. In the assay, the transgenic T
cells were then co-cultured with A2/CT83 peptide and 293T cells for 24 hrs.
Human leukocyte antigen-A2 (HLA-A2) is a type of protein found on the surface of cells
in the body. HLA molecules play a crucial role in the immune system by presenting antigens to T
cells, which are a type of immune cell that helps to recognize and respond to foreign substances in
the body [66]. HLA-A2 is a specific allele of the HLA-A gene. CT83 is part of cancer/testis antigen
(CTA) family, a group of proteins that are overexpressed in certain types of cancers. It is expressed
in a wide range of tumors, including testicular cancer, lung cancer, melanoma, and sarcoma [67].
CT83 has been the subject of research in the field of cancer immunotherapy [67,68].
2-4 Function Assay: FACs analysis of transduced-Jurkat NFAT-GFP reporter system coculture assay
Three groups of transduced T cells were collected for fluorescence imaging and FACs
analysis (Fig 12.). Based on the FACs result. The data from the sample group showed that the Low
affinity CT83 T cell group had less than 1% of GFP expression. The intermediate affinity CT83 T
cell group had 34% of GFP expression. The High-affinity CT83 T cell group had 74% of GFP
expression. (Fig 13.) In summary, the TCR-transgenic Jurkat/NFAT reporter system was proved
able to be activated after co-culturing with 293T cells and A2/CT83-specific peptides. Also,
Jurkat/NFAT reporter system can differentiate the level of GFP expression between the groups.
24
2-5 Function Assay: FACs analysis of the CAR-transduced Jurkat NFAT-GFP reporter
system co-culture assay
We would like to determine whether the Jurkat/NFAT reporter system can also work in the
CAR-T cell screening systems. In this assay, we aimed to determine whether the 2D2 transduced
CAR-T cells are able to recognize the tumor-specific peptide. For the functional assay, the sample
group of Jurkat/NFAT-GFP T cells was virally transduced with 2D2(Clone Plate 2, D2 position) -
CAR plasmid. 2D2-CAR to demonstrate activity against an A2/ESO-specific peptide. The control
group was virally transduced with F5-CAR plasmid. F5 is a mesothelin antibody being developed
as a form of cancer immunotherapy, and it is unspecific to the A2/ESO peptide.
After viral transduction, the transduced T cells were co-cultured with 293T cells and
A2/NY-ESO-specific peptides for 24 hrs. Based on the FACs result, the F5 CAR-T control group
showed 2% of GFP expression in the T cell population. The 2D2 sample group showed 40% of
GFP expression in the T cell population (Fig 14.). The CAR-transgenic Jurkat T-cells can be
activated and showed GFP expression after co-culturing with 293T cells and A2/ NY-ESO-specific
peptides. Also, there is a significant difference in GFP expression between the groups. In summary,
the functionality of the NFAT-GFP Jurkat reporter system has been demonstrated in the screening
of CAR-T affinities.
2-6 Comparison of Jurkat NFAT-GFP reporter system and IFN-γ ELISA in the application
of TCR Screening
In the search for the functional-critical positions in the CDR2 and CDR3 regions for
H3.3K27M TCR. the Jurkat NFAT-GFP reporter system was used for TCR screening compared
to the IFN-γ ELISA assays to determine whether the result can be correlative. Eleven Alanine
25
mutated TCRs plasmid were used to transfect the Jurkat NFAT reporter system. NFAT-GFP Jurkat
T cells were then co-cultured with HEK 293T cells and H3.3K27M-specific peptide. Based on the
result, The control group TCR-T cells showed same low GFP expression compared to H3.3
Transduced TCR, which indicates TCR-T cells showed no recognition to H3.3 peptide (Fig 15. B).
In parallel, the sample group co-cultured with HEK 293T cells and H3.3K27M peptide, B113,
B114, B115, B116, B117, B118, B119, and A114 mutated transduced TCRs showed loss of GFP
expression compared to the H3.3 Transduced TCR. Loss of GFP expression indicated loss of TCR
ability against the tumor antigen. In summary, these amino acid positions are highly correlated to
the affinity of TCR due to the substitution of alanine (Fig 15.).
In the IFN-γ release ELISA assay, both the sample and control group B113, B114, B115,
B116, B117, B118, B119, and A114 mutated transduced TCRs showed low IFN-γ release
compared to H3.3 Transduced TCR. The result of the NFAT-GFP reporter system group is
identical to the samples in the IFN-γ ELISA group (Fig 16.). The experiments demonstrated that
the TCR affinity screening using the NFAT-GFP reporter system can produce results consistent
with the IFN-γ ELISA. Also, NFAT-GFP reporter systems are able to differentiate low
affinity/specificity of mutated TCRs, which identifies interactions that have low affinity between
TCR and peptide MHC (Fig 15.).
26
Chapter 3. Application of Reporter System: Functional Improvement of H3.3K27M TCR
3-1 Alanine Scanning of H3.3K27M Mutated TCR Library
To develop effective H3.3K27M glioma TCR cell therapy, it is crucial to screen for TCRs
that are high specific or high affinity for targeting the H3.3K27M mutation[34]. As the first step of
the screening strategy, an alanine scanning was performed to reduce the scope of the targeted
amino acid position associated with the TCR affinity/specificity. Alanine scanning is a widely used
mutagenesis approach that substitutes targeted amino acid for alanine at selected positions by sitedirected mutagenesis to examine function[35,36]. Alanine is an amino acid without any functional
group. Substituting other amino acids with alanine eliminates side-chain interactions without
altering the main-chain conformation or introducing steric or electrostatic effects. Therefore,
alanine scanning is often the preferred choice for testing the contribution of specific side-chains
while preserving native protein structure[36].
3-2 The TCR-T Affinity Screening of Alanine-Mutated H3.3K27M TCR Library
The target of the alanine scanning is the complementarity-determining regions (CDRs) of
the H3.3K27M TCR. Complementarity-determining regions (CDRs) are amino acid sequences
within the variable regions of T-cell receptors (TCRs) that play a vital role in recognizing and
binding to antigens[19]. These regions are responsible for the antigen-binding specificity and
diversity of these molecules, allowing for a wide range of targets to be recognized. CDRs are found
in the variable regions of the alpha and beta chains of TCRs. Understanding the function of CDRs
is crucial for developing immunotherapies for various diseases, including cancer and autoimmune
27
disorders. Alanine-mutated TCR library is constructed based on CDR2 and CDR3 of the
H3.3K27M TCR, which is at CDR2α position 71-74, CDR3α position 114-116, CD2β position
67-74, CD3β position 110-122. Alanine-mutated TCR plasmids were used to transfect the HEK
293T cells for viral transduction of the Jurkat NFAT reporter system and human T cells. Next,
Jurkat NFAT-GFP Jurkat T cells were co-cultured with HEK 293T cells and H3.3K27M-specific
peptide. Based on the IFN-γ ELISA result, Alanine-mutated TCR B70, B74, B110, B113, B114,
B115, B116, B117, B118, B119, A114 showed a trend of low IFN-γ release, which indicated loss
of TCR ability against tumor antigen compared to H3.3-TCR (Fig 16.A). In the replicate peptide
co-culturing experiment, each sample was repeated in triplicate. The results showed B74, B110,
B113, B117, B119, and A114 showed statically significance compared to H3.3-TCR (Fig 16.A, B,
C). In parallel, Alanine-mutated TCR A72, A74, and B69 samples had higher IFN-γ release in
group co-culture with H3.3K27M peptide compared H3.3 peptide group (Fig 17 A, B, C).
(Fig16.B). In summary, these amino acid positions are potential functional critical positions
associated with TCR affinity and specificity.
3-3 The Deep Screening of A72, A74 H3.3K27M Mutated TCR Library
We started to conduct further deep screening on the potential functional critical positions.
CD2 alpha regions 72, 74, and beta region 69 were chosen as our targets, which improves the
specificity of TCR recognition toward H3.3K27M specific peptide based on the result of the
alanine scanning results. CDR2 A72, A74 H3.3K27M Combinatorial TCR libraries were
constructed. Amino-acid mutated TCR plasmids were used to transfect the HEK 293T cells for
viral transduction of human T cells. Next, transgenic human T cells were co-cultured with HEK
293T cells and H3.3K27M-specific peptide. Based on the IFN-γ ELISA result, H3.3K27M CDR2
Alpha Region N 72 to M, V, S mutation, H3.3K27M CDR2 Alpha Region 74 Phenylalanine to M,
28
K, H, L, V mutation, CDR2 alpha region 72 Asparagine and alpha region 74 Phenylalanine to
Alanine double mutation and CDR2 alpha region 72 Phenylalanine, Beta Region 69 Phenylalanine
to alanine double mutation had higher IFN-γ release in group co-culture with H3.3K27M peptide
compared H3.3 peptide group. The experiment was repeated in triplicate to see if there was a
statically difference. In addition, to understand these high H3.3K27M-specific peptide specificity
TCR has the same function in real tumor cells, overexpressed H3.3K27M full-length cDNA 293T
cells and A2 Epitope transfected 293T cells were added into the sample groups. H3.3K27M fulllength cDNA 293T cells and Epitope transfected 293T Cells were transfected with H3.3K27M
full-length cDNA or A2 epitope gene cDNA to mimic the intracellular process and antigen
presentation of real tumor cells[69]. SF8628-A2 diffuse intrinsic pontine gliomas (DIPGs) cells
were also included into the sample group. SF8628-A2 DIPG cells is a human cancer cell line that
harbors the histone H3.3 K27M mutation. It is a suitable candidate to study the efficacy of DIPGrelated TCR therapy in real-life situations[69]. Based on the result, there are statical differences
among the groups (K, M, H, L, E, CDR2 alpha region 72 F alpha region 74 F to alanine double
mutation. CDR2 alpha region 72 F Beta Region 69 F to alanine double mutation) (Fig.19 A, B, C).
H3.3K27M CDR2 Alpha Region 74 Phenylalanine to M, K, H, L, E mutation, CDR2 alpha region
72 F alpha region 74 F to alanine double mutation, CDR2 alpha region 72 F Beta Region 69 F to
alanine double mutation and CDR2 alpha region 74 F Beta Region 69 F TCR-T cell all show a
trend of higher IFN-γ release compared to the control group. In summary, these mutated TCRs
showed enhancement of H3.3K27M peptide specificity compared to K27M-TCR. (Fig 18. 19.)
29
Discussion
Here, we present the development of the NFAT-GFP Jurkat reporter system, an efficient
tool for screening T cell receptor (TCR) affinity. This system facilitates the identification of TCRs
that recognize specific antigens presented by major histocompatibility complex (MHC) molecules
[71]. The NFAT-GFP Jurkat reporter system involves the genomic modification of human Jurkat
cells by introducing CD8 and NFAT-GFP genes[25]. The Jurkat NFAT-GFP reporter system was
chosen for its low GFP expression without T cell activation, and high GFP expression with T cell
activation, which was validated through cell sorting and functional testing. Later on, we used the
alanine scanning approach to identify the amino acid positions responsible for affinity and
specificity in H3.3K27M TCRs, and the Jurkat NFAT-GFP reporter system was employed for
affinity screening. Mutated transduced TCRs demonstrated a loss of TCR ability against tumor
antigens, indicating a high correlation between these amino acid positions and TCR affinity due to
the substitution of alanine. The NFAT-GFP reporter system results were consistent with the IFNγ ELISA group, confirming its reliability for TCR affinity screening.
The use of the NFAT-GFP reporter system as a TCR affinity tool offers several advantages.
NFAT-GFP reporter systems are able to differentiate low affinity/specificity of transgenic TCRs,
which identifies interactions that have low affinity between TCR and peptide MHC (Fig 15.).
Unlike the IFN-γ ELISA, which employs color reaction or antibody staining for FACs analysis,
the NFAT-GFP Jurkat reporter system directly examines GFP expression from activated T cells,
providing a faster, simpler, and more direct approach for TCR screening [72]. Additionally, it is a
more economical method for TCR screening since there is no need for reagents and antibodies.
The NFAT-GFP Jurkat reporter system is more stable than using PBMC from donors because it
utilizes an immortalized line of human T cells. Moreover, there is no need for T cell expansion
30
when performing TCR screening with the NFAT-GFP Jurkat reporter system, unlike the use of
human T cells, which typically requires Interleukin-2 (IL-2) expansion before CD8 TCR screening
[73]. The NFAT-GFP Jurkat T Cells reporter system is capable of directly screening CD4 and CD8
T cell TCRs.
Next on, we wished to improve the functionality of H3.3K27M TCR for H3.3K27M glioma
TCR cell therapy. The first step of TCR screening is performing alanine scanning on the CDR2
and CDR3 of the H3.3K27M TCR. These regions are the second and third hypervariable regions
that form the antigen-binding site and determine the specificity and affinity of TCR-MHC
interactions. In the context of cancer immunotherapy, CDR2 and CDR3 can be engineered to target
specific antigens on the surface of cancer cells [19]. By modifying these regions, researchers can
develop new TCRs that have improved specificity and affinity for cancer antigens, which can be
used to target and kill cancer cells more effectively.
Alanine scanning is a mutagenesis approach used to study the role of individual amino
acids in protein function. This technique involves systematically replacing each amino acid residue
in a protein of interest with alanine, a neutral, non-polar amino acid, and analyzing the effects of
these mutations on protein function [36]. By screening the amino acid sequences of the CDR2 and
CD3, we can identify the functional critical positions correlated with TCR affinity, which can
narrow down the task of TCR screening. Next, we designed custom primers to create point
mutations on the functional critical positions of CDR2 and CDR3 of the TCR. By constructing a
point-mutated TCR library and conducting TCR affinity screening, we identified H3.3K27M
CDR2 Alpha Region N 72 to M, V, S mutation, H3.3K27M CDR2 Alpha Region F 74 to M, K,
H, L, E, mutation, CDR2 alpha region 72 F alpha region 74 F to alanine double mutation, CDR2
alpha region 72 F Beta Region 69 F to alanine double mutation and CDR2 alpha region 74 F Beta
31
Region 69 F, that showed trend of enhancement recognition to H3.3K27M-specific peptide. In
Summary, these mutated TCRs are potential candidates for future TCR-T cell immunotherapy.
In future work, we can produce H3.3K27M high-specificity mutated TCR-T cells and
conduct in vivo experiments in tumor mouse model to determine if the high-specificity TCRs can
recognize tumor cells in mouse model and destroy the tumor cells. To assess the potential
effectiveness of high-specificity TCRs as a cancer treatment, we will conduct in vivo experiments
using a mouse model with tumors. Our study will involve administering the TCR-T cells treatment
to mice with implanted tumors and monitoring the tumor size over time. This type of preclinical
experiment is frequently performed to evaluate the safety and efficacy of a treatment before it is
tested in humans [8,74].
Overall, the NFAT-Jurkat GFP reporter system is an excellent tool for more efficient TCR
screening, which streamlines the search for high-affinity/specificity TCRs for developing TCR T
cell therapy. NFAT-Jurkat GFP reporter system TCR affinity screening is likely to become a
standard tool for the identification of TCRs for use in adoptive cell therapies, both for autologous
and allogenic therapies. However, there are still some technical and biological challenges that need
to be addressed, such as the optimization of TCR screening protocols, the identification of suitable
target antigens, and the development of methods to generate T cells with high affinity/specificity
TCR. Despite these challenges, the future of affinity based TCR screening looks promising and
has the potential to accumulate the development of cancer immunotherapy significantly.
32
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Figures
Fig 1. The gel electrophoresis result of hCD8-alpha-P2A and P2A-hCD8-beta and pFU3W fixMCS (+) vector A. The gel electrophoresis result of hCD8-alpha-P2A and P2A-hCD8-beta. The
expected band size for hCD8-alpha-P2A is 772bp, and for P2A-hCD8-beta it is 692bp. B. The gel
electrophoresis result of the pFU3W fix-MCS (+) vector treated with enzyme digestion. C. DNA
map of hCD8-alpha-P2A- P2A-hCD8-beta
A B
CD8a P2A CD8b
C
Figure 1. The gel electrophoresis result of hCD8-alphaP2A and P2A-hCD8-beta and pFU3W fix-MCS (+) vector
40
Figure 2. The result of enzyme cutting CD8a-P2A-CD8b plasmid.
Fig 2. The result of enzyme cutting CD8a-P2A-CD8b plasmid. A. The gel electrophoresis result
is the miniprep product of CD8a-P2A-CD8b. The expected DNA size of CD8a-P2A-CD8b is
1425bp. B. The gel electrophoresis result of the PCR product of CD8a-P2A-CD8b
A B
41
Figure 3. FACs Analysis of CD8 expression of clone-in CD8 Jurkat T cells.
Fig 3. FACs Analysis of CD8 expression of clone-in CD8 Jurkat T cells. A. The unstained
Jurkat/CD8 T cells, as a control, showed no CD8 expression. B. The stained Jurkat/CD8 T cells
showed 99.7% of the cell population with CD8 expression.
A
B
42
Figure 4. DNA plasmid of pSIRV-NFAT-EGFP. A.DNA map of pSIRV-NFAT-EGFP from Addgene. B. The gel electrophoresis
result and DNA Concentration of the maxiprep result of pSIRV-NFAT-EGFP is 1900ng/µl, and the O.D value is 1.91.
Fig 4. DNA plasmid of pSIRV-NFAT-EGFP. A.DNA map of pSIRV-NFAT-EGFP from Addgene.
B. The gel electrophoresis result and DNA Concentration of the maxiprep result of pSIRV-NFATEGFP is 1900ng/µl, and the O.D value is 1.91.
A
B
43
Figure 5. Image of CD8 Jurkat T cells and CD8/NFAT Jurkat T cells before and after T cell stimulation under the fluorescence
microscope
Fig 5. Image of CD8 Jurkat T cells and CD8/NFAT Jurkat T cells before and after T cell
stimulation under the fluorescence microscope A. Image of Jurkat T cells before and after
thapsigargin (TG) treatment. B. Image of Jurkat T cells before and after OKT3 monoclonal
antibody treatment.
A
B
44
Figure 6. The FACs analysis result of CD3, CD8, and GFP expression of CD8 Jurkat T cells and CD8/NFAT Jurkat T cells before
and after the OKT3 stimulation.
Fig 6. The FACs analysis result of CD3, CD8, and GFP expression of CD8 Jurkat T cells and
CD8/NFAT Jurkat T cells before and after the OKT3 stimulation. A. CD3 and CD8 expression of
the control group of CD8+ Jurkat T cell and CD8+/ NFAT+ Jurkat T cell. B. GFP expression of
Control group of CD8+ Jurkat T cell and CD8+/ NFAT+ Jurkat T cell C. The comparison before
and after the OKT3 stimulation of the control group and the sample group T cells.
A
B C
45
Figure 7. The FACs analysis result of CD3, CD8, and GFP expression of CD8 Jurkat T cells and CD8/NFAT Jurkat T cells before
and after the thapsigargin (TG) stimulation.
Fig 7. The FACs analysis result of CD3, CD8, and GFP expression of CD8 Jurkat T cells and
CD8/NFAT Jurkat T cells before and after the thapsigargin (TG) stimulation. A. Control group of
CD8+ Jurkat T cell before and after TG stimulation. B. CD8+/ NFAT+ Jurkat T cell before and after
TG stimulation. C. The comparison before and after the TG stimulation of the control group and
the sample group T cells
A
B C
NFAT+ CD8+ CD8 Jurkat + Jurkat
46
Figure 8. Cell sorting data of unstained NFAT-GFP Jurkat T cell and zombie aqua dye stained NFAT-GFP Jurkat T cell from BD
FACSAria IIu Cell Sorter.
Fig 8. Cell sorting data of unstained NFAT-GFP Jurkat T cell and zombie aqua dye stained NFATGFP Jurkat T cell from BD FACSAria IIu Cell Sorter. The first gate is for live cell sorting. The
second gate is for single cells sorting. The third gate is for GFP-expression sorting. The P3
population is the GFP-expressed population. The P4 population is 5% of the least GFP-expressed
cells. A. Cell sorting data of unstained Jurkat/NFAT T cell. B. Cell sorting data of zombie aqua
dye stained Jurkat/NFAT T cell
A B
47
Figure 9. FACs analysis result of the negative selected CD8/NFAT-GFP Clone in Jurkat T cells and the image before and after the
T cell stimulation under the fluorescence microscope
Fig 9. FACs analysis result of the negative selected CD8/NFAT-GFP Clone in Jurkat T cells and
the image before and after the T cell stimulation under the fluorescence microscope. A. The FACs
analysis and fluorescence microscope image of untransduced T cells. B. The FACs analysis and
fluorescence microscope image of NFAT-transduced T cells.
A
B
48
Figure 10. Cell sorting data of unstained Jurkat/NFAT T cell and zombie aqua dye stained Jurkat/NFAT T cell from BD FACSAria
IIu Cell Sorter.
Fig 10. Cell sorting data of unstained Jurkat/NFAT T cell and zombie aqua dye stained
Jurkat/NFAT T cell from BD FACSAria IIu Cell Sorter. The first gate is for live cell sorting. The
second gate is for single cells sorting. The third gate is for GFP-expression sorting. The P3
population is the GFP-expressed population. The P4 population is 50% of the highest GFPexpressed cells. The P5 population is the cell population that has no GFP population. A. Cell
sorting data of unstained Jurkat/NFAT T cell. B. Cell sorting data of zombie aqua dye stained
Jurkat/NFAT T cell
A B
49
Figure 11. Fluorescence Image and FACs Analysis of GFP expression of the stimulated Jurkat/NFAT T cells
Fig 11. Fluorescence Image and FACs Analysis of GFP expression of the stimulated Jurkat/NFAT
T cells A. fluorescence Image of the Jurkat/NFAT T cells before and after the OKT3 stimulation.
B. FACs Analysis of the stimulated Jurkat/NFAT T cells. The red peak is the untransduced T cell.
The orange peak is the two times selected Jurkat/NFAT T cells without T cell stimulation. The
blue peak is the unselected Jurkat/NFAT T cells. The green peak is the two times selected
Jurkat/NFAT T cells with 24hr T cell stimulation. The population of GFP-expressed selected
Jurkat/NFAT T cells after T cell stimulation is 82.8%.
50
Figure 12. Fluorescence Image of control T cells and different CT83 TCR-transduced T cells
Fig 12. Fluorescence Image of control T cells and different CT83 TCR-transduced T cells
51
Figure 13. FACs Analysis of GFP expression of the TCR-transduced T cell co-culture assay. A. The T-cells only group and T-cells
co-cultured with HEK 293T cells group as control
Fig 13. FACs Analysis of GFP expression of the TCR-transduced T cell co-culture assay. A. The
T-cells only group and T-cells co-cultured with HEK 293T cells group as control B. The sample
group of transduced T-cells co-cultured with 293T cells and A2/CT83-specific peptides.
A
B
B
52
Figure 14. FACs analysis of GFP expression of the CAR-transduced T cell co-culture assay.
Fig 14. FACs analysis of GFP expression of the CAR-transduced T cell co-culture assay. The
control groups are the T-cell only group and T-cell co-cultured with 293T cells group. The sample
group is transduced T-cell co-cultured with 293T cells and A2/ESO-specific peptides.
53
Figure 15. TCR Affinity Screening of H3.3K27M Alanine-Mutated TCR Library with NFAT-GFP Jurkat Reporter System
Fig 15. TCR Affinity Screening of H3.3K27M Alanine-Mutated TCR Library with NFAT-GFP
Jurkat Reporter System A. Transgenic TCR-T cells co-culture with HEK 293T cells and 0.01µg/ml
of H3.3K27M Peptide B. Transgenic TCR-T cells co-culture with HEK 293T cells and 0.01µg/ml
of H3.3Peptide
A B
54
Figure 16. TCR Affinity Screening of H3.3K27M Alanine-Mutated TCR Library A. IFN-γ ELISA Assay of mutated H3.3K27M
TCR Library affinity screening
Fig 16. TCR Affinity Screening of H3.3K27M Alanine-Mutated TCR Library A. IFN-γ ELISA
Assay of mutated H3.3K27M TCR Library affinity screening B. Repeated IFN-γ release ELISA
assay with 0.1µg/ml of peptide co-culture in triplicate C. Repeated IFN-γ ELISA release assay
with 0.01µg/ml of peptide co-culture in triplicate
B
C
A
55
Table 1
Student T Test
0.1µg/ ml H3.3K27M peptide 0.1µg/ ml H3.3 peptide 0.01µg/ ml H3.3K27M peptide 0.01 µg/ mlH3.3 peptide
P Value Significance P Value Significance P Value Significance P Value Significance
WT Control vs. B69 0.0002 *** 0.005 ** <0.0001 **** 0.0005 ***
WT Control vs. B70 <0.0001 **** 0.0001 *** <0.0001 **** 0.0002 ***
WT Control vs. B74 <0.0001 **** 0.0001 *** <0.0001 **** 0.0002 ***
WT Control vs. B110 <0.0001 **** 0.0001 *** <0.0001 **** 0.0002 ***
WT Control vs. B113 <0.0001 **** 0.0001 *** <0.0001 **** 0.0002 ***
WT Control vs. B114 <0.0001 **** 0.0012 ** 0.0008 *** 0.0003 ***
WT Control vs. B115 <0.0001 **** 0.0014 ** <0.0001 **** 0.001 ***
WT Control vs. B116 <0.0001 **** 0.0001 *** <0.0001 **** 0.0003 ***
WT Control vs. B117 <0.0001 **** 0.0001 *** <0.0001 **** 0.0002 ***
WT Control vs. B118 <0.0001 **** 0.006 ** 0.0003 *** 0.0027 **
WT Control vs. B119 <0.0001 **** <0.0001 **** <0.0001 **** 0.0002 ***
WT Control vs. A72 0.0149 * 0.03 * <0.0001 **** 0.0016 **
WT Control vs. A74 0.0003 *** 0.0017 ** <0.0001 **** 0.0004 ***
WT Control vs. A114 <0.0001 **** 0.0001 *** <0.0001 **** 0.0002 ***
Table 1. Statical Analysis Result of TCR Affinity Screening of H3.3K27M Alanine-Mutated TCR
56
Figure 17. Comparison of B69, A72, and A74 TCR specificity in IFN-γ release ELISA assay.
Fig 17. Comparison of B69, A72, and A74 TCR specificity in IFN-γ release ELISA assay. A.
Comparison of B69, A72, and A74 TCR specificity (0.1µg/ml of peptide co-culture) B. Repeated
Comparison of B69, A72, and A74 TCR specificity (0.1 and 0.01µg/ml of peptide co-culture) in
in triplicate
A.
B.
57
Table 2
0.1µg/ml Peptide 0.01µg/ml Peptide
Two-way ANOVA P Value Significance P Value Significance
K27M B69 vs. H3.3 B69 <0.0001 **** 0.016 *
K27M B72 vs. H3.3 B72 <0.0001 **** 0.0019 **
K27M B74 vs. H3.3 B74 0.0001 *** 0.0446 *
Table 2. Statical Analysis Result of B69, A72, and A74 TCR specificity in IFN-γ release assay
58
Figure 18. IFN-γ release ELISA assay from TCR Affinity Screening of H3.3K27M A72, A74 Mutated TCR Library
Fig 18. IFN-γ release ELISA assay from TCR Affinity Screening of H3.3K27M A72, A74 Mutated
TCR Library A. A72/Double/Triple Library mutated TCR affinity screening B. A74 library
mutated TCR affinity screening
A.
B.
59
Figure 19. IFN-γ release ELISA assay from repeated co-culture functional Assay
Fig 19. IFN-γ release ELISA assay from repeated co-culture functional Assay A. IFN-γ release
ELISA assay from co-culture functional Assay of transduced TCR-T cells and epitope transfected
293T cells. B. IFN-γ release ELISA assay from co-culture functional Assay of Transduced TCRT cells and cDNA-expressed 293T cells. C. IFN-γ release ELISA assay from co-culture functional
Assay of Transduced TCR-T cells and SF-8628-A2 Cells
A.
B.
C.
60
Table 3. Statical Analysis Result of repeated IFN-γ release ELISA assay
Table 3
Epi-Transfected Group cDNA-expressed Group SF8628 Group
Two-way ANOVA P Value Significance P Value Significance P Value Significance
Control vs. A74-M 0.0562 ns 0.0029 ** 0.0739 ns
Control vs. A74-K 0.0068 ** 0.1243 ns 0.067 ns
Control vs. A74-H 0.014 * 0.0158 * 0.0691 ns
Control vs. A74-L 0.049 * 0.2343 ns 0.1118 ns
Control vs. A74-E 0.007 ** 0.1159 ns 0.0255 *
Control vs. B69 A74 0.0457 * 0.0409 * 0.1028 ns
Control vs. B69 A72 74 0.0576 ns 0.261 ns 0.7056 ns
Control vs. A72 74 0.0507 ns 0.0147 * 0.072 ns
Control vs. B69 A72 0.035 * 0.0088 ** 0.0829 ns
61
Supplement
Table of Primers:
Primer Name
Orientatio
n of
Primer
Primer Sequence
CDR2 B67 F Forward GGCTTAAGGCAGATCTACGCATCAATGAATGTTGAG
CDR2 B68 F Forward TTAAGGCAGATCTACTATGCAATGAATGTTGAGGTG
CDR2 B69 F Forward AGGCAGATCTACTATTCAGCAAATGTTGAGGTGACT
CDR2 B70 F Forward CAGATCTACTATTCAATGGCAGTTGAGGTGACTGAT
CDR2 B71 F Forward ATCTACTATTCAATGAATGCAGAGGTGACTGATAAG
CDR2 B72 F Forward TACTATTCAATGAATGTTGCAGTGACTGATAAGGGA
CDR2 B73 F Forward TATTCAATGAATGTTGAGGCAACTGATAAGGGAGAT
CDR2 B74 F Forward TCAATGAATGTTGAGGTGGCAGATAAGGGAGATGTT
CDR2 B67 R Reverse CTCAACATTCATTGATGCGTAGATCT
CDR2 B68 R Reverse CACCTCAACATTCATTGCATAGTAGA
CDR2 B69 R Reverse AGTCACCTCAACATTTGCTGAATAGT
CDR2 B70 R Reverse ATCAGTCACCTCAACTGCCATTGAAT
CDR2 B71 R Reverse CTTATCAGTCACCTCTGCATTCATTG
CDR2 B72 R Reverse TCACTGCAACATTCATTGAATAGTA
CDR2 B73 R Reverse CAGTTGCCTCAACATTCATTGAAT
CDR2 B74 R Reverse TATCTGCCACCTCAACATTCATTG
CDR3 B110 F Forward CAGACCTCTCTGTACTTCGCAGCCAGCGGCTGGGGT
CDR3 B112 F Forward TCTCTGTACTTCTGTGCCGCGGGCTGGGGTGGTCCA
CDR3 B113 F Forward CTGTACTTCTGTGCCAGCGCATGGGGTGGTCCATTC
CDR3 B114 F Forward TACTTCTGTGCCAGCGGCGCAGGTGGTCCATTCTAC
CDR3 B115 F Forward TTCTGTGCCAGCGGCTGGGCAGGTCCATTCTACGAG
CDR3 B116 F Forward TGTGCCAGCGGCTGGGGTGCACCATTCTACGAGCAG
CDR3 B117 F Forward GCCAGCGGCTGGGGTGGTGCATTCTACGAGCAGTAC
CDR3 B118 F Forward AGCGGCTGGGGTGGTCCAGCATACGAGCAGTACTTC
CDR3 B119 F Forward GGCTGGGGTGGTCCATTCGCAGAGCAGTACTTCGGG
CDR3 B120 F Forward TGGGGTGGTCCATTCTACGCACAGTACTTCGGGCCG
CDR3 B121 F Forward GGTGGTCCATTCTACGAGGCATACTTCGGGCCGGGC
CDR3 B122 F Forward GGTCCATTCTACGAGCAGGCATTCGGGCCGGGCACC
CDR3 B110 R Reverse CCGCTGGCTGCGAAGTACAGAGAGGT
CDR3 B112 R Reverse TGGACCACCCCAGCCCGCGGCACAGA
CDR3 B113 R Reverse GAATGGACCACCCCATGCGCTGGCAC
CDR3 B114 R Reverse GTAGAATGGACCACCTGCGCCGCTGG
CDR3 B115 R Reverse CTCGTAGAATGGACCTGCCCAGCCGC
62
CDR3 B116 R Reverse CTGCTCGTAGAATGGTGCACCCCAGC
CDR3 B117 R Reverse GTACTGCTCGTAGAATGCACCACCCC
CDR3 B118 R Reverse GAAGTACTGCTCGTATGCTGGACCAC
CDR3 B119 R Reverse CCCGAAGTACTGCTCTGCGAATGGAC
CDR3 B120 R Reverse CGGCCCGAAGTACTGTGCGTAGAATG
CDR3 B121 R Reverse GCCCGGCCCGAAGTATGCCTCGTAGA
CDR3 B122 R Reverse GGTGCCCGGCCCGAATGCCTGCTCGT
CDR2 A71 R Reverse AGAGTTTGCACGAATAAGGAAAACCAA
CDR2 A72 R Reverse AAAAGATGCCCGACGAATAAGGAAAAC
CDR2 A73 R Reverse ATCAAATGCGTTCCGACGAATAAGGAA
CDR2 A74 R Reverse CTCATCTGCAGAGTTCCGACGAATAAG
CDR3 A114 R Reverse CTCACTTGCAGCACAGAAGTATACTGC
CDR3 A115 R Reverse CTCCTCTGCCAGAGCACAGAAGTATAC
CDR3 A116 R Reverse TTCTCTGCACTCAGAGCACAGAAGTA
CDR2 A71 F Forward TTGGTTTTCCTTATTCGTGCAAACTCTTTTGATGAG
CDR2 A72 F Forward GTTTTCCTTATTCGTCGGGCATCTTTTGATGAGCAA
CDR2 A73 F Forward TTCCTTATTCGTCGGAACGCATTTGATGAGCAAAAT
CDR2 A74 F Forward CTTATTCGTCGGAACTCTGCAGATGAGCAAAATGAA
CDR3 A114 F Forward GCAGTATACTTCTGTGCTGCAAGTGAGGAGAATGAC
CDR3 A115 F Forward GTATACTTCTGTGCTCTGGCAGAGGAGAATGACATG
CDR3 A116 F Forward TACTTCTGTGCTCTGAGTGCAGAGAATGACATGCGC
CDR2 A72F Random Forward AGTGGAGAATTGGTTTTCCTTATTCGTCGGNNKTCTTTTGATGAGCAAAATGAA
ATAA
CDR2 A72R Random Reverse CCGACGAATAAGGAAAACCAATTCTCC
CDR2 A74F Random Forward GAATTGGTTTTCCTTATTCGTCGGAACTCTNNKGATGAGCAAAATGAAATAAGT
GGTC
CDR2 A72, A74F Ala Ala Forward AGTGGAGAATTGGTTTTCCTTATTCGTCGGGCATCTGCAGATGAGCAAAATGAA
ATA
CDR2 A74R Random Reverse AGAGTTCCGACGAATAAGGAAAACCAA
CDR2 B69F Random Forward GGGCTGGGCTTAAGGCAGATCTACTATTCANNKAATGTTGAGGTGACTGATAA
GGGAG
CDR2 B69R Random Reverse TGAATAGTAGATCTGCCTTAAGCCCAG
Abstract (if available)
Abstract
T-cell receptor T cell therapy and chimeric antigen receptor T cell[2] therapy are promising cell therapy strategies for cancer treatment. TCR-T cell therapy involves genetically modifying a patient's T cells to express a TCR that recognizes a tumor antigen and selectively targets cancer cells expressing the antigen. Similarly, CAR-T cell therapy uses genetically modified T cells to express a chimeric antigen receptor on their surface to target cancer cells in the body. Several ongoing clinical trials are evaluating the safety and efficacy of these therapies in humans, but the specificity and affinity of TCR and CAR need improvement. However, current TCR screening methods are limited by time-consuming processes that prolong the TCR affinity screening procedures.
To overcome the challenge and improve the TCR screening method, we developed an NFAT-GFP Jurkat T cell reporter system for high-affinity TCR/CAR screening. We introduced CD8 and NFAT-GFP genes into Jurkat T cells and confirmed their activation and GFP expression through functional assays and cell sorting. The Jurkat/NFAT reporter system can differentiate TCR affinity by the level of GFP expression in both CAR/TCR screening. This system is a promising tool for accelerating the improvement of TCRs and developing TCR-T cell immunotherapy. In parallel, the H3.3K27M Mutated TCR library was constructed and screened through with the Jurkat/NFAT reporter system in TCR. Several of the high-specificity TCRs were found and can be potential candidates for future TCR-T cell therapy.
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Asset Metadata
Creator
Chang, Che-Yu
(filename)
Core Title
Development of an NFAT-GFP Jurkat T cell reporter system for acceleration of T Cell receptor affinity screening
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Degree Conferral Date
2023-12
Publication Date
12/08/2023
Defense Date
12/07/2023
Publisher
Los Angeles, California
(original),
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
functional improvement of H3.3K27M TCR,H3.3K27M mutation,Jurkat T Cell Reporter System,OAI-PMH Harvest,T-cell therapy,TCR therapy,TCR-Screening
Format
theses
(aat)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Wang, Rongfu (
committee chair
), Landolph, Joseph (
committee member
), Muller, Peter (
committee member
)
Creator Email
cheyucha@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-oUC113787815
Unique identifier
UC113787815
Identifier
etd-ChangCheYu-12534.pdf (filename)
Legacy Identifier
etd-ChangCheYu-12534
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Thesis
Format
theses (aat)
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Chang, Che-Yu
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texts
Source
20231211-usctheses-batch-1113
(batch),
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
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Repository Email
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Tags
functional improvement of H3.3K27M TCR
H3.3K27M mutation
Jurkat T Cell Reporter System
T-cell therapy
TCR therapy
TCR-Screening