University of Southern California Dissertations and Theses

T cell regulation of HLA-DR

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T cell regulation of HLA-DR

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Content T CELL REGULATION OF HLA-DR
by
Olivia Hart
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
(MEDICAL PHYSIOLOGY)
May 2024
Copyright 2024 Olivia Hart

ii
Table of Contents
List of Figures.................................................................................................................................iv
Chapter I: Introduction ....................................................................................................................1
HLA-DR Epitopes.....................................................................................................................................4
L243...................................................................................................................................................................... 5
LN3....................................................................................................................................................................... 5
Tu36...................................................................................................................................................................... 5
Lym-1 ................................................................................................................................................................... 5
CAR-T Cell Immunotherapy.....................................................................................................................6
Lym-1 CAR-T Cells............................................................................................................................................. 7
Chapter II: Motivation, Hypothesis and Goals................................................................................9
Motivation .................................................................................................................................................9
Initial Hypothesis ....................................................................................................................................10
Thesis Goals............................................................................................................................................13
Chapter III: Results........................................................................................................................14
Preferential expansion of huLym-1-B-DAP CAR-T cells over non-transduced cells is reproducible
in other donors.........................................................................................................................................14
a-CD19-DAP CAR-T cells do not expand preferentially over non-transduced T cells after the
addition of T Cell Activator. ...................................................................................................................17
Lym-1-E is downregulated in epitope-stimulated huLym-1-B-DAP CAR-T cells. ...............................18
Tu36-E is downregulated in epitope-stimulated huLym-1-B-DAP CAR-T cells and identifies
possible functional relevance. .................................................................................................................20
a-261 serves as a surrogate for CAR epitope-stimulation.......................................................................21
Lym-1-E and Tu36-E are downregulated in epitope-stimulated a-CD19-CD3z CAR-T cells...............24
Chapter IV: Discussion..................................................................................................................29
Future Directions.....................................................................................................................................30
Translational Opportunities.....................................................................................................................31
Chapter V: Materials and Methods................................................................................................34
Cell Culture .............................................................................................................................................34
T cells ................................................................................................................................................................. 34
Karpas299 cells................................................................................................................................................... 35
CAR-T Cell Production...........................................................................................................................35
Flow Cytometry.......................................................................................................................................36
Antibodies and Isotype Controls ........................................................................................................................ 36
Flow Cytometry Protocol ................................................................................................................................... 37
Antibody Interference......................................................................................................................................... 38
T Cell Activation.....................................................................................................................................41
Surrogate Epitope-Stimulation of CAR-T cells......................................................................................41
CAR-T Cell Expansion ...........................................................................................................................41

iii
References .....................................................................................................................................43
Supplemental Data.........................................................................................................................46
Appendices....................................................................................................................................50
Appendix A: OCH-Exp-022 Protocol.....................................................................................................50
Appendix B: OCH-Exp-023 Protocol .....................................................................................................56
Appendix C: OCH-Exp-025 Protocol .....................................................................................................61

iv
List of Figures
Figure I.1 MHC Class I and Class II Pathways............................................................................................2
Figure I.2 M1- and M2- paired HLA-DR.....................................................................................................3
Figure I.3 HLA-DR Molecule Structure and Measurable Epitopes (Created with BioRender.com)...........4
Figure I.4 A physiological T Cell Receptor (TCR) and three generations of CAR constructs [26]. ...........6
Figure I.5 CAR constructs of huLym-1-B-BB3z and huLym-1-B-DAP CAR-T cells [24]. .......................8
Figure II.1 Preferential expansion of huLym-1-B-DAP CAR T cell population over non-transduced
population after addition of a-CD2/3/28 T Cell Activator. ...........................................................................9
Figure II.2 Visual representation of Initial Hypothesis..............................................................................10
Figure II.3 Data from Chia-Hsin Lin M.S. Thesis (2022)..........................................................................11
Figure II.4 Visual representation of hypothesized mechanism for T cell protection from CD4+ CTL
(Created with BioRender.com)....................................................................................................................12
Figure III.1 Timeline for the analysis of huLym-1-B-DAP CAR-T cell expansion and flow
cytometric detection of HLA-DR epitopes (OCH-Exp-022). .....................................................................15
Figure III.2 Expansion of Donor 6 huLym-1-DAP CAR-T cells and non-transduced T cell cultures
after addition of T cell activator on Day 0 and Day 7 (OCH-Exp-022)......................................................16
Figure III.3 huLym-1-B DAP10/12 CAR T cells expand preferentially over non transduced T cells......18
Figure III.4 Flow cytometry dot plots from Day 7 showing expression of LN3-E vs. L243-E and
CD137 vs. L243-E, LN3-E, and Lym-1-E in huLym-1-B-DAP CAR-T cells (OCH-Exp-022). ...............20
Figure III.5 Tu36-E is downregulated in epitope-stimulated CAR-T cells with a similar pattern to
Lym-1-E (OCH-Exp-022). ..........................................................................................................................21
Figure III.6 CD137 expression in a-CD19-DAP CAR-T cells and non-transduced cells, cultured in
wells with or without an a-261-coating for 48 hours (OCH-Exp-011). ......................................................22
Figure III.7 Experimental timeline to test whether epitope-stimulated a-CD19-CD3z CAR-T cells
downregulate Lym-1-E and Tu36-E (OCH-Exp-023).................................................................................24
Figure III.8 a-CD19-CD3z CAR-T cells downregulate Lym-1-E and Tu36-E when epitopestimulated (OCH-Exp-023). CD137 expression is specific to epitope-stimulated cells. ............................25
Figure III.9 CD137 vs. Lym-1-E expression (A) and CD137 vs. Tu36-E expression (B) in the nontransduced T cells (a-261-) in an a-CD19-CD3z CAR-T cell prep 2 days after two days of culture
with a-261 antibody (OCH-Exp-023)..........................................................................................................28
Figure IV.1 Expression of HLA-DR epitopes in various T cell lymphoma cell lines (OCH-Exp-014). ...33
Figure V.1 Antibodies used in flow cytometry and their respective Isotype Controls...............................37
Figure V.2 Antibody Interference Experimental Design (OCH-Exp-006, -007, -014, -018). ...................39
Figure V.3 MFI Data from Antibody Interference Experiments (OCH-Exp-006, -007, -014, -018),........40
Figure 0.1 HLA-DR epitope expression in huLym-1-DAP CAR-T cells on Day 0 of (OCH-Exp022)..............................................................................................................................................................46
Figure 0.2 HLA-DR epitope expression in non-transduced T cells on Day 0 of (OCH-Exp-022). ...........46
Figure 0.3 HLA-DR epitope expression in non-transduced T cells on Day 7 of (OCH-Exp-022). ...........47
Figure 0.4 HLA-DR Epitope Expression of Donor 3 and Donor 8 huLym-1-DAP CAR-T cells on
Day 7 of (OCH-Exp-025)............................................................................................................................47
Figure 0.5 Gating strategy for flow cytometry plots of huLym-1-B-DAP CAR-T cells using isotype
control staining (OCH-Exp-025).................................................................................................................48
Figure 0.6 Gating strategy for flow cytometry plots of a-CD19-DAP CAR-T cell prep using isotype
control staining (OCH-Exp-025).................................................................................................................48
Figure 0.7 Gating strategy for flow cytometry plots of a-CD19-CD3z CAR-T cell prep using isotype
control staining (OCH-Exp-023).................................................................................................................49
Figure 0.8 Isotype control gating and a-261 Expression in a-CD19-CD3z CAR-T cell preps (OCHExp-023). .....................................................................................................................................................49

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Abstract
HLA-DR is a well-recognized marker of activated T cells. However, the mechanism and
functional relevance behind HLA-DR upregulation in T cells are not well understood.
HLA-DR is a Class II MHC molecule expressed on the surface of antigen-presenting
cells (APCs). HLA-DR presents peptides derived from exogenous antigens to CD4+ T cells,
allowing the CD4+ T cells to regulate immune responses against the presented antigen and/or kill
the cell presenting the peptide.
The Lym-1 antibody recognizes an epitope (Lym-1-E) on the beta subunit of HLA-DR. It
had been previously discovered that epitope-stimulation of CAR-T cells utilizing either
DAP10/12 or CD3z signaling domains downregulated the expression of Lym-1-E. This thesis
further characterizes changes in the HLA-DR structure caused by epitope-stimulation and
discusses its possible functional relevance.
Epitope-stimulation of a T cell was found to also lead to the downregulation of Tu36-E,
an epitope expressed on the beta subunit of “M1” HLA-DR but not “M2” HLA-DR. M1 HLADR is a conformation considered more likely to present peptides to T cells than the M2
conformation. The downregulation of Tu36-E on epitope-stimulated T cells therefore prompts
the hypothesis that epitope-stimulation leads to a conformational change in HLA-DR from M1 to
M2, making the molecule less likely to present peptides and protecting the T cell from killing by
a CD4 cytotoxic lymphocyte.
These results prompt further investigation into the regulation of HLA-DR function in T
cells, as well as the possibility of treating T cell lymphomas that express M1 HLA-DR with
CAR-T cells targeting its epitopes.

1
Chapter I: Introduction
HLA-DR (Human Leukocyte Antigen–DR Subtype) is a cell surface molecule belonging
to the MHC Class II family. MHC molecules, or major histocompatibility complex molecules,
function by presenting peptides derived from antigens on the cell surface for recognition by T
cells, allowing the immune system to recognize and fight pathogens [1].
MHC molecules fall into two subcategories, MHC Class I and MHC Class II. MHC Class
I molecules can be found on the surface of all nucleated cells, and specifically present on the cell
surface peptides derived from endogenous antigens: antigens synthesized within the cell, such as
from viruses replicating in an infected the cell. If a CD8+ cytotoxic T cell (CTL) recognizes an
MHC I-presented peptide, the CTL can kill the cell and thus destroy the virus. [2].
MHC Class II molecules are expressed on the cell surface of professional APCs (antigenpresenting cells) such as dendritic cells, macrophages, and B cells. MHC Class II molecules,
including HLA-DR, specifically present peptides from exogenous antigens on the cell surface to
CD4+ T cells [1]. Upon recognition of an antigen’s peptide presented by an MHC Class II
molecule, CD4+ T cells can differentiate into specific subtypes based upon the cytokine milieu
in the surrounding environment [3]. These subtypes include CD4+ T helper cells and T
regulatory cells, which can stimulate or inhibit actions of other immune cells to regulate immune
responses [3]. In addition, CD4+ T cells can also exhibit cytotoxic functions: these cells are
known as CD4+ cytotoxic lymphocytes (CD4+ CTL) [4].

2
T cells use their T cell receptor (TCR) to recognize peptides from antigens presented by
MHC molecules [5]. This interaction leads the T cell to upregulate expression of CD137 on its
surface. CD137 expression is thus used as a marker for T cells that have been epitope-stimulated
by recognition of a presented peptide [6]. Along with TCR signaling, CD137 binding to its
ligand, CD137L, provides co-stimulation that increases T cell proliferation, survival, and
function [7].
Figure 0.1 MHC Class I and Class II Pathways. MHC Class I molecules present peptides from
endogenous antigens on the cell surface for recognition by CD8+ T cells. Epitope-stimulation of the CD8
TCR leads the T cell to upregulate CD137 and kill the infected cell. MHC Class II molecules present
exogenous antigens to CD4 T cells, allowing the CD4 T cells to regulate other T cells or kill the infected
cell (Created with BioRender.com).
HLA-DR and other MHC Class II molecules are expressed on professional APCs.
However, HLA-DR can also be expressed on T cells, allowing them to act as APCs to present
peptides from extracellular antigens on their cell surface [8]. HLA-DR expression in T cells is
widely recognized as a marker of T cell activation and can lead T cells to become targets for

3
CD4+ CTL [9, 10] [11]. The external signals and internal mechanisms leading to T cell
upregulation of HLA-DR are not well-understood, and its functional significance is unknown
[12].
HLA-DR is a heterodimeric surface molecule made up of two polypeptide chains, the
alpha and beta chains. Each chain contains two domains: a transmembrane domain and an
external domain that together form the molecule’s peptide-binding cleft (Figure 1.3). The
transmembrane domains of HLA-DR alpha and beta chains contain multiple GxxxG
dimerization motifs, and different GxxxG motif pairings lead to two conformers of HLA-DR:
M1-paired and M2-paired (Figure 1.2). M1-paired HLA-DR is thought to be more likely to
present peptides on the cell surface than M2-paired HLA-DR [13, 14].
Figure 0.2 M1- and M2- paired HLA-DR. M1-paired and M2-paired HLA-DR conformers are
differentiated by different pairing of GxxxG dimerization motifs between the transmembrane alpha and
beta chains of HLA-DR [13].

4
HLA-DR molecules are encoded by the HLA system of genes located on band 6p21.3 of
chromosome 6. The HLA-DRB gene, which encodes the beta chain of HLA-DR, is highly
polymorphic [15]. The beta chain of HLA-DR therefore has many different subtypes between
different people. In contrast, the HLA-DRA gene encoding the alpha chain does not show
considerable polymorphism [16].
HLA-DR Epitopes
Figure 0.3 HLA-DR Molecule Structure and Measurable Epitopes (Created with BioRender.com).
Several antibodies have been developed that bind to different epitopes within the HLADR molecule.

5
L243
Murine IgG2a monoclonal antibody L243 recognizes a conformational epitope on the
alpha chain of HLA-DR (Figure 1.3) [17]. Binding of the L243 antibody to L243-E requires
proper association of the alpha and beta chains of HLA-DR [18].
LN3
The LN3 antibody recognizes an epitope on the beta chain of HLA-DR (Figure 1.3) [19].
The LN3 antibody can bind to LN3-E in flow cytometry analysis as well as
immunohistochemical staining of frozen sections and formalin-fixed paraffin sections [20].
Tu36
The Tu36 antibody recognizes an epitope on the beta chain of HLA-DR, and its binding
is specific to M1-paired HLA-DR (Figure 1.3) [13].
Lym-1
The murine IgG2a monoclonal antibody Lym-1 was isolated in 1987 by Epstein et al,
from mice immunized with nuclei from Burkitt’s Lymphoma cell line Raji [21]. Lym-1
recognizes a discontinuous conformational epitope (Lym-1-E) on the beta chain of several
subtypes of HLA-DR (Figure 1.3) [22]. CAR-T cell immunotherapy targeting the Lym-1 epitope
has shown promise in the treatment of B cell lymphomas [23, 24].

6
CAR-T Cell Immunotherapy
Chimeric Antigen Receptor (CAR) T cell therapy involves the genetic alteration of T
cells, allowing them to detect specific epitopes on cancer cells to kill them. This can be
accomplished by transducing T cells with a viral vector, causing the T cells to express the CAR
construct on their surface. The “first generation” CAR constructs contained three domains: 1) the
extracellular domain derived from the single-fragment variable chain (scFv) of an antibody,
responsible for epitope recognition, 2) the transmembrane domain, and 3) the intracellular CD3z
signaling domain derived from the signaling domain of a T cell receptor (TCR) (Figure 1.4) [25].
Signaling from first generation CAR-T cells resembles physiologic signaling from TCRs.
Recognition of a specific epitope on a cancerous cell by the CAR extracellular domain induces
CD3z signaling within the CAR-T cell, allowing it to elicit an immune response against the
malignant cell.
Figure 0.4 A physiological T Cell Receptor (TCR) and three generations of CAR constructs [26].

7
Although first generation CAR-T cells showed initial promise for cancer treatment, they
lacked persistence in vivo during clinical trials [25]. In normal T cell physiology, to be cytotoxic
and persist, T cells must receive co-stimulation from other cell surface molecules, such as CD28
or 4-1BB (another term for CD137), in addition to CD3z signaling. Thus “second generation”
CAR constructs were developed containing both the CD3z signaling domain and a costimulatory
domain (Figure 1.4) [25]. Second-generation CAR-T cells have shown success in the treatment
of B cell malignancies, particularly with the use of an anti-CD19 (a-CD19) extracellular domain
[27]. CD19 (Cluster of Differentiation 19) is a protein expressed on the surface of cells of B-cell
lineage [25]. Although initial response rates of certain B cell malignancies to a-CD19 CAR-T
cells are high, over time they lose efficacy in some patients and the malignancy returns. This
mechanism of tumor cell escape is thought in some cases to be due to CD19 downregulation
upon crosslinking [28].
Lym-1 CAR-T Cells
The Lym-1 epitope on HLA-DR has shown promise as a target for CAR-T cell
immunotherapy against B cell lymphomas [23, 24]. The Lym-1 antibody binds Lym-1-E on
malignant B cells with a higher avidity than on normal B cells. Lym-1-E also does not
downregulate upon crosslinking like CD19, an issue leading to diminished efficacy in a-CD19
CAR-T cell therapy [24].
In 2020, Zheng et al created second generation CAR-T cells using the scFv of a
humanized Lym-1 antibody in the extracellular domain [24]. The cells utilized a 4-1BB
costimulatory domain along with the CD3z intracellular signaling domain (Figure 1.5). A 10-

8
amino acid tag derived from human placental growth factor, termed “261”, was inserted into the
extracellular domain of the CAR construct to enable detection of the construct using an in-house
antibody (a-261). These cells (huLym-1-B-BB3z CAR-T cells) successfully killed Raji tumors in
vivo, however they failed to proliferate in vitro apparently due to tonic signaling. For clinical
applications, CAR-T cells must be able to expand in vitro during their production, therefore
alterations were needed to eliminate tonic signaling [24].
To enable in vitro expansion, Zheng et al replaced 4-1BB and CD3z with a novel
DAP10/DAP12 signaling domain (Figure 1.5) which contains one immunoreceptor tyrosinebased activation motif (ITAM) and one immunoreceptor tyrosine-based inhibitory motif (ITIM).
The ITIM, which the CD3z signaling domain lacks, diminishes tonic signaling [24]. DAP12 can
initiate cytotoxic activity in T cells but requires the DAP10 signaling domain to activate PI3K
pathways for proliferation [29]. Using this novel signaling domain, huLym-1-B-DAP CAR-T
cells showed significant expansion in vitro and success against B cell lymphomas in vivo [24].
Figure 0.5 CAR constructs of huLym-1-B-BB3z and huLym-1-B-DAP CAR-T cells [24].

9
Chapter II: Motivation, Hypothesis and Goals
Motivation
Typically, to expand CAR-T cell preparations, which contain both transduced and nontransduced T cells, an anti-CD2/3/28 T Cell Activator is added into the culture system. The
activator normally expands both populations of cells: successfully transduced CAR-T cells and
non-transduced T cells. However, in 2020, Zheng (unpublished) observed that huLym-1-B-DAP
CAR-T cells showed significant preferential expansion over non-transduced T cells after the
addition of T cell activator. (Figure II.1). The laboratory thus sought to determine the mechanism
underlying this observation.
Figure 0.1 Preferential expansion of huLym-1-B-DAP CAR T cell population over non-transduced
population after addition of a-CD2/3/28 T Cell Activator. The x-axis represents the cells’ expression of
the “261” tag on the CAR construct: the peak on the left represents non-transduced T cells in the
preparation and the peak on the right represents huLym-1-B-DAP CAR-T cells. The number in each plot
indicates the percentage of CAR-T cells in the preparation (Unpublished data from Zheng 2020).

10
Following the addition of anti-CD2/3/28 T Cell Activator, it is plausible that the culture
system could produce an environment that would activate T cells which would then express
HLA-DR. If the HLA-DR contained Lym-1-E, then the huLym-1-B-DAP CAR-T cells in the
preparation would recognize and kill the T cells expressing Lym-1-E. This notion prompts this
question: what prevented the huLym-1-B-DAP CAR-T cells from committing fratricide? The
hypothesis arose that signaling from epitope-stimulation of the CAR construct caused the
huLym-1-B-DAP CAR-T cells to downregulate expression of Lym-1-E.
Initial Hypothesis
Figure 0.2 Visual representation of Initial Hypothesis. When both non-transduced T cells and Lym-1
CAR-T cells are activated with a-CD2/3/28 T Cell Activator, both cell types upregulate activation marker
HLA-DR. After a few days the Activator is degraded but Lym-1 CAR-T cells continue to receive epitopestimulation through the CAR construct recognizing Lym-1-E. This signaling causes the Lym-1 CAR-T
cells to kill non-transduced T cells while downregulating Lym-1-E on their own surface, protecting
themselves from fratricide (Created with BioRender.com).

11
The question then arose whether this signal required the novel DAP signaling domain in
the CAR construct, or if it could arise from a CD3z signaling domain, indicating that the
mechanism could operate physiologically in normal T cells. In 2022, Chia-Hsin Lin in the
Epstein lab demonstrated that simulating epitope-stimulation in first-generation a-CD19 CAR-T
cells led to downregulation of Lym-1-E (Figure II.3). First-generation CAR-T cells contain only
the CD3z signaling domain, as do TCRs in normal T cell physiology. This discovery therefore
prompted the idea that the downregulation of Lym-1-E upon epitope-stimulation may represent a
mechanism within normal T cell physiology.
Figure 0.3 Data from Chia-Hsin Lin M.S. Thesis (2022). Fraction Lym-1-E+ in non-transduced T cells
and first-generation CAR-T cells before and after simulating epitope-stimulation shows the
downregulation of Lym-1-E in CAR-T cells, but not in non-transduced T cells. Epitope-stimulation was
simulated using a plate coated with antibody directed against the “261” tag in the extracellular domain of
the CAR construct to elicit epitope-specific CD3z signaling.

12
Long-Term Goal
Test the hypothesis that epitope stimulation of a T cell leads to a structural change in its
HLA-DR, diminishing its function and protecting it from recognition and killing by a CD4+
CTL.
Figure 0.4 Visual representation of hypothesized mechanism for T cell protection from CD4+ CTL
(Created with BioRender.com). T Cells are activated by the “activating milieu” in an immune response,
leading them to upregulate HLA-DR expression. This makes the T cell a target for killing by a CD4+
CTL if the HLA-DR presents an epitope recognized by a CD4+ CTL. When a T cell receives epitopestimulation to its TCR, this causes a structural change in HLA-DR, protecting the T cell from recognition
and killing by a CD4+ CTL.

13
Thesis Goals
1. Determine whether the preferential expansion of huLym-1-DAP CAR-T cells over nontransduced T cells is reproducible in other donors and assess changes in expression of
CD137 and HLA-DR epitopes during this expansion.
2. Determine whether a-CD19-DAP CAR-T cells expand preferentially over non-transduced
T cells to assess whether the DAP signaling domain can cause a loss of Lym-1-E
expression in the absence of epitope-stimulation.
3. Determine whether a surrogate for epitope-stimulation in first-generation (a-CD19-CD3z)
CAR-T cells downregulates Lym-1-E in other donors and assess expression of CD137
and other HLA-DR epitopes.

14
Chapter III: Results
Preferential expansion of huLym-1-B-DAP CAR-T cells over nontransduced cells is reproducible in other donors.
In 2020, Zheng (unpublished) found that huLym-1-B-DAP CAR-T cells showed
preferential expansion over non-transduced T cells in a culture system after the addition of T cell
activator (Figure II.1). This prompted the hypothesis that while activation leads to the
upregulation of HLA-DR on T cells, epitope-stimulation of the CAR-T cells by Lym-1-E leads
them to downregulate their own Lym-1-E. This system allows the CAR-T cells to kill nontransduced T cells with upregulated HLA-DR (and therefore Lym-1-E) but spares the CAR-T
cells from fratricide (Figure II.2).
To establish that this phenomenon is seen in huLym-1-B-DAP CAR-T from other donors,
CAR T cell preparations from three separate donors were thawed and cultured in T cell media.
Because the percentage of non-transduced cells in these preparations was low, autologous nontransduced T cells were added on Day 0. ImmunoCult™ Human CD3/CD28/CD2 T Cell
Activator was added to the combined cell preparation on Day 0 and Day 7. Flow cytometry was
conducted on Days 0 and 7 for all three donors (3, 6, 8) and in addition on Days 14 and 21 for
one donor (Donor 6). Expression of the 261 tag (CAR), CD137 (epitope-stimulation marker),
LN3-E (HLA-DR-B), L243-E (HLA-DR-A), Lym-1-E (HLA-DR-B), and Tu36-E (HLA-DR-B,
M1-paired) were assessed by flow cytometry on Days 0, 7, 14, and 21.

15
Figure 0.1 Timeline for the analysis of huLym-1-B-DAP CAR-T cell expansion and flow cytometric
detection of HLA-DR epitopes (OCH-Exp-022).
For the expansion assessed for 21 days, on Day 0, CAR-T cells comprised 29.4% of the
cell preparation. The T cell activator was added on Day 0, and by Day 7, the percentage of CART cells increased to 86.5% (Figure III.2A). Similar results were obtained for CAR T cells from
the other two donors (Figure 0.3) These experiments replicate the phenomenon Zheng previously
observed that huLym-1-B-DAP CAR-T cells expand preferentially over non-transduced T cells
after activation (Figure II.1).
As a control, the Activator was added to a separate culture containing only nontransduced T cells. There was significant proliferation of non-transduced T cells when they were
not in the presence of CAR-T cells, indicating the CAR-T cells likely killed the non-transduced
T cells in the co-culture due to the non-transduced cells’ expression of Lym-1-E (Figure III.2B).
For data and a detailed protocol see “OCH-Exp-022-Protocol and data” in the Appendix.
OCH-Exp-022

16
Figure 0.2 Expansion of Donor 6 huLym-1-DAP CAR-T cells and non-transduced T cell cultures after
addition of T cell activator on Day 0 and Day 7 (OCH-Exp-022). huLym-1-B-DAP CAR-T cells expand
preferentially over non-transduced T cells in a co-culture upon activation with ImmunoCult™ Human
CD3/CD28/CD2 T Cell Activator. B) Non-transduced T cells received the same treatment as the CART/non-transduced cell co-culture and showed significant expansion upon activation when not in the
presence of huLym-1-B DAP CAR-T cells.
OCH-Exp-022
OCH-Exp-022

17
a-CD19-DAP CAR-T cells do not expand preferentially over nontransduced T cells after the addition of T Cell Activator.
To test whether the preferential expansion of huLym-1-DAP CAR-T cells over nontransduced T cells required the portion of the CAR construct recognizing Lym-1-E or whether
the DAP10/12 signaling domain alone was sufficient, a-CD19 CAR-T cells were made using the
DAP10/12 signaling domain. The percentage of a-CD19 DAP10/12 CAR-T cells (a-261+, those
expressing the “261” tag) within each culture system was assessed using flow cytometry on Day
0 (2 days after transduction) and Day 7. Immunocult T Cell Activator was added into each
culture system on Day 0.
huLym-1-DAP CAR-T cells from each of three donors (Donors 3, 6, and 8) all showed
preferential expansion over non-transduced T cells after the addition of ImmunoCult T Cell
Activator on Day 0 (Figure III.3). a-CD19-DAP CAR-T cells did not, indicating that the nontransduced T cells’ failure to expand in culture with huLym-1-DAP CAR-T cells required the
portion of the CAR construct recognizing Lym-1-E (Figure III.3).
For a detailed experimental protocol, see “OCH-Exp-025-Protocol” in the Appendix.

18
Figure 0.3 Expansion of (A) huLym-1-B-DAP CAR-T cells and (B) a-CD19-DAP CAR-T cells vs. nontransduced T cells after the addition of a-CD2/3/28 T Cell Activator on Day 0. A) huLym-1-B DAP10/12
CAR T cells from multiple donors expand preferentially over non-transduced T cells. B) a-CD19-DAP
CAR-T cells do not show preferential expansion of the CAR-T cell population after addition of aCD2/3/28 T Cell Activator (OCH-Exp-022, OCH-Exp-025).
Lym-1-E is downregulated in epitope-stimulated huLym-1-B-DAP CART cells.
7 days after the addition of T cell activator to a culture of huLym-1-B-DAP CAR-T cells
and non-transduced T cells, flow cytometric analysis was performed to test for the expression of
the 261 tag (CAR), CD137 (epitope-stimulation marker), LN3-E (HLA-DR-B), L243-E (HLADR-A), Lym-1-E (HLA-DR-B), and Tu36-E (HLA-DR-B, M1-paired) (Figure III.4). Expression
of the 261 tag was used to separately assess HLA-DR epitopes on the huLym-1-B-DAP CAR-T
cells and non-transduced T cells in the preparation.
OCH-Exp-022
OCH-Exp-025

19
On Day 7, in the Donor 6 huLym-1-B-DAP CAR-T cell population, 34.03% of cells
expressed CD137, indicating that these cells received epitope-stimulation from Lym-1-E (Figure
III.4B/C/D). CD137 expression peaks between 12-24 hours after epitope-stimulation [30] The
low count of non-transduced T cells left in the culture by Day 7 makes CAR-T cells less likely to
encounter Lym-1-E, thereby reducing the expression of CD137.
81.2% of CAR-T cells expressed both L243-E and LN3-E on Day 7, indicating that most
of the CAR-T cells express HLA-DR on their surface (Figure III.4A). Of the epitope-stimulated
(CD137+) cells, 86.5% express L243-E and 98.4% express LN3-E (Figure III.4B/C). Binding of
the L243 antibody to the alpha subunit of HLA-DR requires proper assembly of the heterodimer,
whereas LN3 binding to the beta subunit does not, likely explaining the disparity between L243-
E and LN3-E expression.
In contrast, only 20% of the CD137+ CAR T cells expressed Lym-1-E (Figure III.4D).
This data supports the hypothesis that although HLA-DR is present on the surface of
epitope-stimulated T cells in some form, the conformation of HLA-DR is such that most of these
cells lack Lym-1-E. Taken together, this data suggests that while activation of T cells leads to the
upregulation of HLA-DR, epitope-stimulation leads to a change in its structure which could
impact its function.
For a detailed experimental protocol, see “OCH-Exp-022-Protocol” in the Appendix.

20
Figure 0.4 Flow cytometry dot plots from Day 7 showing expression of LN3-E vs. L243-E and CD137
vs. L243-E, LN3-E, and Lym-1-E in huLym-1-B-DAP CAR-T cells (OCH-Exp-022). Quadrant gates
were created based on samples stained with isotype controls for their respective antibodies.
Tu36-E is downregulated in epitope-stimulated huLym-1-B-DAP CAR-T
cells and identifies possible functional relevance.
Using a B cell model, Drake et al reported that the antibody Tu36 bound to HLA-DR-B
in the M1 conformation, but not the M2 conformation. Of the epitope-stimulated (CD137+)
CAR-T cells from Donor 6, only 19.7% expressed Tu36-E (Figure III.5B) on Day 7. This
epitope shows a similar expression pattern to that of Lym-1-E, which was expressed on only
20% of epitope-stimulated (CD137+) cells. It has been suggested that M1-paired HLA-DR is
OCH-Exp-022

21
more likely to present peptides on the cell surface than M2-paired HLA-DR [13]. The results
here support the hypothesis that epitope-stimulation leads to the transition of HLA-DR from the
M1-paired to M2-paired conformation. This change may decrease the molecule’s ability to
present peptides on the surface of the cell, leading to resistance from killing by CD4+ CTL.
Figure 0.5 Tu36-E is downregulated in epitope-stimulated CAR-T cells with a similar pattern to Lym-1-E
(OCH-Exp-022).
a-261 serves as a surrogate for CAR epitope-stimulation
The above experiments assessed epitope-stimulation arising from the huLym-1-B DAP10/12 CAR construct recognizing Lym-1-E. The “261” tag incorporated into the CAR construct
allows plates coated with the a-261 antibody to bind to the CAR and be a surrogate for epitope
stimulation. To demonstrate this stimulation, an a-CD19-DAP CAR-T cell preparation
containing transduced CAR-T cells and non-transduced T cells was thawed and activated with
ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator. After five days in culture, the cells
were counted and diluted to 1 x 106 cells/mL in T cell media. 500 µL (0.5 x 106 cells) from the
cell preparation were added to triplicate wells either coated with a-261 or not coated with a-261.
OCH-Exp-022

22
After 48 hours, a-261 (CAR) and CD137 expression were measured for cells from each
well using flow cytometry. Cells staining positively for a-261 contain the “261” tag specific to
the CAR construct, allowing for the distinction between CAR-T cells and non-transduced cells
during flow cytometric analysis. CD137 expression was assessed in both the a-CD19-DAP CART cells and the non-transduced cells.
In the a-CD19-DAP CAR-T cells from the non-coated wells, the average percentage of
CD137+ cells was 1.97%. For CAR-T cells from the a-261-coated plate, CD137 expression was
63.19%. In non-transduced cells, cells expressing CD137 was less than 5% whether or not wells
were coated with a-261 (Figure III.6).
Figure 0.6 CD137 expression in a-CD19-DAP CAR-T cells and non-transduced cells, cultured in wells
with or without an a-261-coating for 48 hours (OCH-Exp-011). The upregulation of CD137 in CAR-T
cells cultured with an a-261-coated plate shows that a-261 can simulate epitope-stimulation in CAR-T
cells with the “261” tag in their CAR construct.
OCH-Exp-011

23
CD137 is a marker specifically expressed on the cell surface upon epitope-stimulation of
T cells [30]. The increased expression of CD137 in a-CD19-DAP CAR-T cells after culture in an
a-261-coated plate therefore shows that a-261 simulates epitope-stimulation in CAR-T cells
made with the “261” tag. Non-transduced T cells lack the CAR construct, and therefore do not
receive epitope-stimulation from a-261 or upregulate CD137 in response to it.
As a part of the extracellular domain of the CAR construct, stimulation of the “261” tag
through binding its antibody allows the CAR to mimic signaling pathways normally induced by
epitope recognition of the scFv. This mechanism allows for an experimental system in which
epitope-stimulation can be simulated in CAR-T cells containing the “261” tag, and the response
of the CAR-T cells be distinguished from non-transduced cells in the preparation.

24
Lym-1-E and Tu36-E are downregulated in epitope-stimulated a-CD19-
CD3z CAR-T cells.
Figure 0.7 Experimental timeline to test whether epitope-stimulated a-CD19-CD3z CAR-T cells
downregulate Lym-1-E and Tu36-E (OCH-Exp-023).
Former member of the Epstein Lab, Chia-Hsin Lin, previously showed that Lym-1-E
expression was downregulated in a-CD19-CD3z CAR-T cells when the cells were cultured in a
plate coated with a-261 (Figure II.3). To determine whether this phenomenon could be
replicated, the experiment was repeated with additional assessment for CD137 and other HLADR epitopes in addition to Lym-1-E
For unknown reasons, the a-261-coated plate caused only modest induction of CD137 in
a-CD19-CD3z CAR-T cells. Nonetheless, most CAR-T cells expressing CD137 (CD137+) did
not express Lym-1-E and Tu36-E (Figure III.8).
OCH-Exp-023

25
Figure 0.8 a-CD19-CD3z CAR-T cells downregulate Lym-1-E and Tu36-E when epitope-stimulated
(OCH-Exp-023). CD137 expression is specific to epitope-stimulated cells. When a-CD19-CD3z CAR-T
cells receive epitope-stimulation from the a-261 coated plate and upregulate CD137, most of these cells
lack Lym-1-E and Tu36-E.
OCH-Exp-023

26
a-CD19-CD3z CAR-T cells staining positively for CD137 appear to downregulate Lym1-E expression, confirming that this downregulation is a result of epitope-stimulation of the
CAR-T cells (Figure III.8A). Additionally, Tu36-E appears to downregulate in epitopestimulated cells, suggesting that epitope-stimulation of these cells may lead to a structural
transition in HLA-DR from M1-paired to M2-paired (Figure III.8B). The CD3z signaling domain
utilized in these a-CD19-CD3z CAR-T cells is the same signaling domain used physiologically
in a normal T cell receptor. This result therefore suggests that the structural change in HLA-DR
due to epitope-stimulation may represent a physiological phenomenon in normal T cells.
Although Lym-1-E was downregulated in the epitope-stimulated a-CD19-CD3z CAR-T
cell population, the non-transduced T cells did not reproduce the upregulation of Lym-1-E shown
in Figure II.3 (Figure III.9). The results in Figure II.3 had prompted a previous hypothesis: that
signaling from epitope-stimulated CAR-T cells created the cytokine milieu necessary for the
upregulation of HLA-DR in non-transduced T cells. The cytokine leading to this physiological
response is currently unknown [12]. However, this phenomenon was not reproduced, therefore
further experimentation is needed to determine the signaling leading to expression of HLA-DR
with Lym-1-E in “activated” T cells.
In the previous experimentation by Chia-Hsin Lin (Figure II.3), ImmunoCult™ Human
CD3/CD28/CD2 T Cell Activator was added to the a-CD19-CD3z CAR-T cell preparation four
days prior to beginning co-culture of the cell preparation in an a-261-coated plate. In the
experiment yielding Figures III.8 and III.9 (OCH-Exp-023), the same T Cell Activator was
added to the a-CD19-CD3z CAR-T cell preparation six days prior to beginning the co-culture. It
is therefore possible that the minimal CD137 upregulation in CAR-T cells and lack of
upregulation of Lym-1-E in non-transduced T cells is due to a lack of co-stimulation in the

27
culture system. It has been previously reported that T cells require co-stimulation, such as by
CD28 recognition, to elicit significant signaling upon epitope-recognition by their TCR [31]. It is
therefore plausible that while first-generation CAR-T cells showed a modest response to the a261-coated plate, the downregulation of Lym-1-E and Tu36-E in epitope-stimulated CAR-T cells
would be more significant with the addition of co-stimulation into the culture system. It is also
possible that this stronger signaling would create the milieu leading to the upregulation of HLADR (and therefore Lym-1-E and Tu36-E) in non-transduced cells in the culture system.
For a detailed experimental protocol, see “OCH-Exp-023 Protocol” in the Appendix.

28
Figure 0.9 CD137 vs. Lym-1-E expression (A) and CD137 vs. Tu36-E expression (B) in the nontransduced T cells (a-261-) in an a-CD19-CD3z CAR-T cell prep 2 days after two days of culture with a261 antibody (OCH-Exp-023).
OCH-Exp-023

29
Chapter IV: Discussion
HLA-DR is well-recognized as a marker of T cell activation; however, the regulation and
functional implications of this system are not well understood [12]. This report investigates a
hypothesized regulatory mechanism of HLA-DR on T cells and reveals a possible functional
significance of the mechanism.
In 2020, Zheng et al discovered that huLym-1-B-DAP CAR-T cells expand preferentially
over non-transduced T cells after activation, igniting the hypothesis that epitope-stimulation
leads to a loss of Lym-1-E, protecting Lym-1 CAR-T cells from fratricide. This hypothesis
prompted the work of Chia-Hsin Lin in the Epstein Lab, which revealed that downregulation of
Lym-1-E occurs upon epitope-stimulation of a first-generation CAR-T cell containing only a
CD3z signaling domain. This work demonstrated that epitope-stimulation-induced changes in the
structure of HLA-DR were not a function of DAP signaling and may occur in normal T cell
physiology.
When CD137 expression was tested alongside the expression of different HLA-DR
epitopes, epitope-stimulated CAR-T cells were shown to upregulate HLA-DR (L243-E and LN3-
E) but downregulate specific HLA-DR epitopes (Lym-1-E and Tu36-E). These findings replicate
the previously observed phenomenon and advance the understanding of structural changes within
HLA-DR upon epitope-stimulation.
The downregulation of Tu36-E in epitope-stimulated CAR-T cells reveals a possible
functional purpose behind the previously hypothesized epitope-stimulation-induced structural
change of HLA-DR and subsequent loss of Lym-1-E. The loss of Tu36-E in epitope-stimulated
CAR-T cells may represent the transition of HLA-DR from an M1-paired to M2-paired
conformation, reducing the molecule’s ability to present peptides on the cell surface.

30
The transition of HLA-DR on T cells from M1-paired to M2-paired may provide a
protective mechanism against CD4+ CTL. HLA-DR and its presented epitopes are specifically
recognized by CD4+ T cells, and despite their reputation for developing into helper T cells,
CD4+ T cells can also exhibit cytotoxic effects [4]. When a T cell receives epitope stimulation
through its TCR, this T cell gains the ability to elicit an immune response against the recognized
antigen. These epitope-stimulated T cells therefore require protection from being killed by CD4+
CTL so that they can contribute to the immune response. The transition of HLA-DR on epitopestimulated T cells from M1-paired to M2-paired would make the HLA-DR less likely to present
peptides, and therefore protect the cell from recognition and killing by a CD4+ CTL.
Future Directions
Further experimentation is needed to gain a better understanding of the physiological
applicability and relevance of the structural changes in HLA-DR upon epitope-stimulation of T
cells. Most T cells are “αβ T cells”, a part of the adaptive immune response in which the αβ TCR
must receive epitope-stimulation from an MHC molecule to elicit an immune response [32]. γδ
(gamma-delta) T cells are a unique subset of T cells which are often considered a part of the
innate immune response, for the γδ TCR can be activated without recognition of a peptide
presented by an MHC molecule. Upon stimulation of the γδ TCR, γδ T cells can produce
cytokines and chemokines as well as exert cytotoxic effects on recognized cells [32]. However,
ligands for the γδ TCR are not well-understood. Deseke et al recently identified a naturally
occurring γδ TCR, termed “TCR05”, that recognizes HLA-DR as its target. In the studies leading
to this identification another γδ TCR was identified, TCR04, caused by a PCR error that led to a
substitution of valine for a glycine. The TCR04 sequence was not found in datasets from other

31
individuals or public databases. The TCR04 γδ TCR had higher affinity and produced stronger
signaling than the TCR05 γδ TCR [33]. If TCR05 γδ T cells can kill T cells expressing M1-
paired HLA-DR, then the phenomenon reported here could be a physiologic mechanism that
regulates this cytotoxic function. To test this hypothesis, T cells transduced to express TCR04 or
TCR05 could be cultured with control and epitope-stimulated T cells. If epitope-stimulated T
cells are protected against being killed by a T cell expressing TCR05, the result would support
the hypothesis that the alteration of the HLA-DR structure upon epitope-stimulation may serve
as a physiological protective mechanism for T cells.
Most of the U.S. population today is immunized against diphtheria toxoid (DT) [34].
Culture of donor T cells with diphtheria toxoid, therefore, can lead T cells to act as APCs and
present diphtheria toxoid peptides on HLA-DR. Enrichment of a CD4+ CTL cell population
recognizing HLA-DR diphtheria peptides and their subsequent co-culture with DT-exposed T
cells would also test the hypothesis presented here, that epitope-stimulation downregulates Lym1-E and Tu36-E and protects T cells from CD4+ CTLs. These opportunities for future
experiments could provide insight into the true physiological applicability and function of the
structural alteration of T cell HLA-DR upon epitope-stimulation.
Translational Opportunities
The Lym-1 epitope (Lym-1-E) has previously shown promise as a target for CAR-T cell
immunotherapy against B cell malignancies [23, 24]. huLym-1-B CAR-T cells utilizing a novel
DAP10/12 signaling domain successfully killed B cell lymphoma tumors in mice, as Lym-1-E is
upregulated on certain B cell lymphomas [24].

32
Lym-1 CAR-T cells have not been previously investigated in the treatment of T cell
malignancies. Treating T cell malignancies with CAR-T cell immunotherapy can create a risk of
T cell aplasia (killing of healthy T cells) as well as fratricide within the CAR-T cell population
[35]. Fratricide is diminished by the phenomenon observed in this report, in which Lym-1-E is
downregulated on Lym-1 CAR-T cells upon epitope-stimulation.
HLA-DR, and plausibly Lym-1-E, are upregulated in activated T cells but are not
expressed in resting T cells [36, 37]. Lym-1-E has also been shown to be expressed by some T
cell lymphoma cell lines (Figure IV.1). The lack of Lym-1-E expression on resting T cells could
minimize the risk of T cell aplasia in the treatment of T cell lymphomas with Lym-1 CAR-T
cells.
This report also suggests that Tu36-E should be investigated as a possible effector for
CAR-T cell therapy. Tu36-E is downregulated in a similar pattern to Lym-1-E upon epitopestimulation, implying that treatment with Tu36 CAR-T cells would minimize the risk of
fratricide in a similar manner. As an epitope of HLA-DR, Tu36-E is also expressed only on
activated T cells, minimizing the risk of T cell aplasia.
HLA-DR is often expressed on APCs such as dendritic cells, macrophages, and B cells.
Therefore, it is necessary to consider the safety of treating T cell lymphoma with CAR-T cells
targeting Lym-1-E or Tu36-E. To prevent CAR-T cells targeting HLA-DR epitopes from killing
other types of immune cells, a dual-targeting CAR construct could be utilized. Dual-targeting
CAR constructs require the recognition of two different antigens to elicit an immune response
against a cell [38]. These CAR-T cells can therefore protect cells only expressing one of the
antigens, increasing the safety of the treatment. There are multiple T cell-specific antigens which
could be used as a target in addition to either Lym-1-E or Tu36-E, including CD4 or CD3 [39].

33
The treatment of T cell lymphomas with CAR-T cells targeting only these T cell-specific
antigens has been explored, however fratricide and safety remain issues in the field. Requiring
engagement with Lym-1-E or Tu36-E in addition to one of these targets could increase the safety
and efficacy of CAR-T immunotherapy treatments for T cell lymphomas [39].
Figure 0.1 Expression of HLA-DR epitopes in various T cell lymphoma cell lines (OCH-Exp-014).

34
Chapter V: Materials and Methods
Cell Culture
T cells
T cells were isolated from Leukopaks received from Gulf Coast Regional Blood Center
using EasySep™ Human T Cell Isolation Kit (StemCell Technologies, Catalog #17951).
T cell medium was made from 43% Click’s Medium (Sigma-Alrich, Catalog #C5572),
43% RPMI-1640 (GenClone Reference #25-506), 10% FBS (Omega Scientific Catalog #FB-01),
2% GlutaMAX (ThermoFisher, Catalog #35050061), 1% non-essential amino acids (Genesee
Scientific Cat #25-536), 1% Penicillin/Streptomycin (Corning, Catalog #30-002-CI), 50ng/mL
IL-7-Fc and 100ng/mL IL-15- Fc (developed in Epstein lab).
T cells were frozen in a mixture of 90% T cell medium and 10% DMSO, placed into a
Mr. Frosty freezing container (ThermoFisher Catalog #5100-0001) at -80ºC, then moved to
liquid nitrogen for storage the following day.
For thawing, cryovials containing T cells were placed briefly in a 37ºC water bath then
added to 5 mL of pre-warmed T cell media. Cells were then centrifuged at 400 rcf and 4ºC for 5
minutes. The supernatant was removed, and cells were resuspended in 1 mL T cell media, then
counted using ThermoFisher Countess 3 Automated Cell Counter. Cells were then diluted to 1 x
106 cells/mL in T cell media and cultured in a 24 well GRex plate (WilsonWolf Catalog
#80192M) in a humidified 37ºC, 5% CO2 incubator.

35
Karpas299 cells
Karpas299 cells were cultured in T75 culture flasks (Laguna Scientific Catalog #4616)
with RPMI-1640 media (GenClone Reference #25-506) with 10% FBS added (Omega Scientific
Catalog #FB-01). Cell media was replaced every 48 hours, and the cell population was split in
half between at approximately 80% confluency.
CAR-T Cell Production
For transduction of the CAR construct into T cells, T cells were first thawed and
expanded in T cell media for three days. In a 24-well suspension plate (Greiner Bio-One Catalog
#662-102), 500 µL of PBS (L) was added to each well being utilized for transduction. 7 µL of 1
µg/µL of RetroNectin® (Takara Bio Catalog # T100B) was then added to each well. The plate
was then left at 4ºC overnight.
The following day, the solution was removed from each well on the plate and 1 mL of
2% filtered BSA was added to each well. After incubation at room temperature for 1 hour, the
BSA was removed, and the wells were washed with 1 mL of PBS (Corning Reference #21-040-
CV).
20 µL of the CAR lentivirus, 10 µL of HEPES buffer, 5 µL of LentiBlast (OZ
Biosciences Catalog #LBPX500), and 215 µL of T cell media were then added to each well. The
plate was centrifuged at 2000g for 1 hour, then 0.5 x 106 T cells from culture were added to each
well. T cell media was added to bring the total volume in each well to 500 µL. The plate was
centrifuged again at 1000rpm for 45 minutes, then placed in a humidified 37ºC, 5% CO2
incubator.

36
The following day, cells were removed, centrifuged at 1000rpm for 6 minutes, and given
new T cell media. The cells were then moved to a 24-well GRex plate (WilsonWolf Catalog
#80192M) and cultured with 25 µL/mL of ImmunoCult™ Human CD3/CD28/CD2 T Cell
Activator (ImmunoCult Catalog #10970). Transduction efficiency was assessed by the
expression of the “261” tag in the CAR construct using an a-261-Dylight650 antibody in flow
cytometry.
Flow Cytometry
Antibodies and Isotype Controls
For flow cytometry, a-261-Dylight650, chLym-1-Dylight488, and LN3-Dylight488
antibodies were developed in the Epstein lab. All antibodies conjugated to a Dylight 488 or
Dylight 650 fluorescent label were conjugated in-house using DyLight™ 488 NHS Ester
(ThermoFisher Catalog #46403) or DyLight™ 650 NHS Ester (ThermoFisher Catalog #62266),
respectively.
Other antibodies utilized in flow cytometry include L243-PerCP (Biolegend Catalog
#307628), Tu36-PE (Invitrogen Catalog #MHLDR04), CD137-APC/Cy7 (Biolegend Catalog
#309830). All isotype controls utilized in flow cytometry are listed in Figure V.1 below
alongside their respective antibodies.

37
Target Antibody Antibody Isotype Isotype Control
261 tag
(CAR)
a-261-Dylight650
(Epstein lab)
Mouse IgG2A, κ Purified Mouse IgG2a, κ Isotype Ctrl
Antibody
(Biolegend Catalog #401501)
Lym-1-E
(HLA-DR-B)
chLym-1-Dylight488
(Epstein lab)
Human IgG1 ch225-Dylight488 (Epstein lab)
LN3-E
(HLA-DR-B)
LN3-Dylight488
(Epstein lab)
Mouse IgG2b, κ Mouse IgG2b kappa Isotype Control
(eBMG2b), eBioscience™
(Invitrogen Catalog #14-4732-82)
L243-E
(HLA-DR-A)
L243-PerCP
(Biolegend Catalog
#307628)
Mouse IgG2a, κ PerCP Mouse IgG2a, κ Isotype Ctrl
Antibody
(Biolegend Catalog #400256)
Tu36-E
(HLA-DR-B,
M1)
Tu36-PE
(Invitrogen Catalog
#MHLDR01)
Mouse IgG2b Mouse IgG2b Isotype Control, PE
(Invitrogen Catalog #MG2B04)
CD137
(epitopestimulated
cells)
CD137-APC/Cy7
(Biolegend Catalog
#309830)
Mouse IgG1, κ APC/Cyanine7 Mouse IgG1, κ Isotype
Ctrl Antibody (Biolegend Catalog
#400128)
Figure 0.1 Antibodies used in flow cytometry and their respective Isotype Controls.
Flow Cytometry Protocol
For flow cytometry, cells were first counted and diluted to 1x106 cells/mL of media. 100
µL of cells were removed for each flow sample and added into a 12 x 75mm polystyrene tube.
Samples were then washed with 3 mL PBS (Corning Reference #21-040-CV) with 4% FBS
(Omega Scientific Catalog #FB-01) and spun down at 400 rcf and 4ºC for 5 minutes. The
supernatant was then removed. Antibodies or isotype controls were diluted in PBS with 4% FBS
at a 1:10 ratio for greater pipetting accuracy, and 10 µL of diluted antibody was added to each
sample. Samples were placed on a shaker in a room kept at 4ºC, protected from light, for 45
minutes to allow antibody binding. Samples were then washed twice with 3 mL PBS with 4%
FBS, then resuspended in 500 µL PBS with 4% FBS. One drop of Propidium Iodide Ready

38
FlowTM Reagent (Invitrogen Catalog #01-2222-42) was added to each sample to identify dead
cells. Samples were then left at room temperature, protected from light, for 15 minutes prior to
data collection.
UltraComp eBeads™ Compensation Beads (Invitrogen Catalog #01-2222-42) were used
with all antibodies to set compensation between fluorescent channels to avoid channel overlap
during multi-channel flow cytometry experiments.
The Attune NxT Flow Cytometer was used to collect flow cytometric data. Flow
cytometry data was analyzed using FlowJo™ v10.8.1 Software (BD Life Sciences). Dead cells
were identified using the expression of Propidium Iodide Ready FlowTM Reagent and removed
from the data set prior to analysis. Gating for positively staining cells was based upon negative
control samples stained with isotype controls for each respective antibody.
Antibody Interference
Antibody compatibility was assessed between L243 and LN3, chLym-1 and LN3,
chLym-1 and L243, and L243 and Tu36. Karpas299 cells, which show high expression rates of
HLA-DR epitopes, were utilized for these experiments. The flow cytometry protocol described
above was used to test antibody compatibility. Cells were stained in triplicates in six different
ways: 1) Antibody 1 only added, 2) Antibody 2 only added, 3) Antibody 2 added 45 minutes
after Antibody 1, 4) Antibody 1 added 45 minutes after Antibody 2, 5) Antibody 1 and Antibody
2 added at the same time, and 6) Isotype Controls only added. The percentage of cells staining
positively, and the MFI were recorded and compared to determine antibody compatibility. The
experimental design used for each interference experiment is described in Figure V.2.

39
Figure 0.2 Antibody Interference Experimental Design (OCH-Exp-006, -007, -014, -018).
All antibody pairs were found to be compatible except for chLym-1 and LN3, which both
bind epitopes on the beta chain of HLA-DR (Figure V.3). Analysis of antibody interference was
based upon the comparison of the MFI with one antibody added alone and the MFI with both
antibodies added at the same time. Antibodies are all added at the same time during normal flow
cytometry protocol, therefore no significant difference between MFI in these two groups implies
that antibodies will not interfere in flow cytometry. Ultimately, LN3 and L243 antibodies were
used in the same antibody panel for flow cytometry, and chLym-1 and Tu36 antibodies were
used in separate panels.

40
Figure 0.3 MFI Data from Antibody Interference Experiments (OCH-Exp-006, -007, -014, -018),

41
T Cell Activation
T cells and CAR-T cells were activated using ImmunoCult™ Human CD3/CD28/CD2 T
Cell Activator (ImmunoCult Catalog #10970). Cells in culture were diluted to 1x 106 cells /mL
in T cell media, and T Cell Activator was then added at 25 µL/mL.
Surrogate Epitope-Stimulation of CAR-T cells
Epitope-stimulation was simulated in CAR-T cells utilizing the “261” antibody (a-261),
an antibody developed in the Epstein lab against the “261” tag in the extracellular domain of the
CAR construct. a-261 was diluted to 1 ug/mL in PBS (Corning Reference #21-040-CV), and 2
mL (2 ug a-261) of this solution was added to each of three wells in a 6-well non-tissue culture
treated plate (Falcon Reference #351146). The plate was left at 4ºC for 24 hours, then the
solution was removed from each well and 1 mL of 2% filtered BSA was added to each well. The
plate was left at room temperature for 30 minutes, then the BSA was removed. Each well was
washed with 1 mL PBS before the addition of cells.
CAR-T Cell Expansion
CAR-T cells and non-transduced T cells from the same donor were thawed on Day 0
using the protocol described above. Cells were counted and diluted to 0.5 x 106 cells/mL in T cell
media. 0.5 x 106 CAR-T cells and 0.5 x 106 cells non-transduced cells were then added to each of
three wells in a 24-well GRex plate. Flow cytometry was conducted on each well to assess the
expression of the 261 (CAR) tag, Lym-1-E, LN3-E, L243-E, Tu36-E, and CD137. 20 µL of
ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator was then added to each well.

42
On Day 2, 5 mL of T cell media was added to each well. On Day 5, 5 mL of T cell media
was carefully removed from the top of each well and 5 mL of fresh T cell media was added. On
Day 7, cells were counted from each well and flow cytometry was conducted. 2 million cells
from each well were then removed and added to three new wells in the 24-well GRex plate for
the new co-culture. Each well was then diluted to 0.5 x 106 cells/mL to reach a total of 4 mL. 40
µL of ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator was then added to each well.
This process was then repeated until Day 21, with flow cytometry conducted on Days 14
and 21.
Karpas299 cells were used as a positive control for antibody binding.

43
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2. Becar, M.K., A, Physiology, MHC Class I. 2024, Treasure Island, FL: StatPearls
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3. Luckheeram, R.V., et al., CD4(+)T cells: differentiation and functions. Clin Dev
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4. van de Berg, P.J., et al., Cytotoxic human CD4(+) T cells. Curr Opin Immunol, 2008.
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5. Shah, K., et al., T cell receptor (TCR) signaling in health and disease. Signal Transduct
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46
Supplemental Data
Figure 0.1 HLA-DR epitope expression in huLym-1-DAP CAR-T cells on Day 0 of (OCH-Exp-022).
Figure 0.2 HLA-DR epitope expression in non-transduced T cells on Day 0 of (OCH-Exp-022).

47
Figure 0.3 HLA-DR epitope expression in non-transduced T cells on Day 7 of (OCH-Exp-022).
Figure 0.4 HLA-DR Epitope Expression of Donor 3 and Donor 8 huLym-1-DAP CAR-T cells on Day 7
of (OCH-Exp-025).

48
Figure 0.5 Gating strategy for flow cytometry plots of huLym-1-B-DAP CAR-T cells using isotype
control staining (OCH-Exp-025). Gating for a-261 expression was based off the separation of two distinct
cell populations rather than isotype control staining.
Figure 0.6 Gating strategy for flow cytometry plots of a-CD19-DAP CAR-T cell prep using isotype
control staining (OCH-Exp-025).

49
Figure 0.7 Gating strategy for flow cytometry plots of a-CD19-CD3z CAR-T cell prep using isotype
control staining (OCH-Exp-023).
Figure 0.8 Isotype control gating and a-261 Expression in a-CD19-CD3z CAR-T cell preps (OCH-Exp023).

50
Appendices
Appendix A: OCH-Exp-022 Protocol
Purpose: Test the hypothesis that the preferential expansion seen by Zheng et al (2020) occurs
with huLym-1-DAP CAR T cells from Donor 6. Also test CD137 and HLA-DR epitope
expression.
Materials:
huLym-1-B DAP CAR T cell prep Donor 6, Transduced by OCH, Frozen 2023-
12-13
Mock T cells Donor 6, Made by OCH, Frozen 2023-12-13
T cell medium with cytokines 43% Click's medium, 43% RPMI-1640, 10%
FBS, 2% GlutaMAX, 1% nonessential amino
acids, 1% penicillin/streptomycin)
supplemented with 50 ng/mL IL7-Fc and 100
ng/mL IL15-Fc
ImmunoCult™ Human CD3/CD28/CD2 T
Cell Activator
StemCell Technologies Cat #10970
24-well GRex Plate WilsonWolf Catalog #80192M
RL1 Dylight 650 a-261 CAR tag Made and conjugated by Epstein Lab
RL2 APC/Cy7 4B4-1 CD137 Biolegend Catalog #309830
BL1 Dylight 488 LN3 HLA-DR-B Made and conjugated by Epstein Lab
BL2 PerCP L243 HLA-DR-A Biolegend Catalog #307628
BL1 Dylight 488 chLym-1 HLA-DR-B Made and conjugated by Epstein Lab
YL1 PE Tu36 HLA-DR-B Invitrogen Catalog #MHLDR04
RL1 Dylight 650 a-261 CAR tag Isotype
Control
Purified Mouse IgG2a, κ Isotype Ctrl
Antibody
(Biolegend Catalog #401501)
RL2 APC/Cy7 4B4-1 CD137 Isotype
Control
APC/Cyanine7 Mouse IgG1, κ Isotype Ctrl
Antibody (Biolegend Catalog #400128)
BL1 Dylight 488 LN3 HLA-DR-B
Isotype Control
Mouse IgG2b kappa Isotype Control
(eBMG2b), eBioscience™
(Invitrogen Catalog #14-4732-82), Conjugated
by Epstein Lab
BL2 PerCP L243 HLA-DR-A Isotype
Control
PerCP Mouse IgG2a, κ Isotype Ctrl Antibody
(Biolegend Catalog #400256)
BL1 Dylight 488 chLym-1 HLA-DR-B
Isotype Control
ch225-Dylight488 (Made and conjugated by
Epstein lab)
YL1 PE Tu36 HLA-DR-B Isotype
Control
Mouse IgG2b Isotype Control, PE (Invitrogen
Catalog #MG2B04)
Flow tubes
Flow buffer PBS + 4% FBS

51
Propidium Iodide Ready Flow™ Reagent Invitrogen Cat # R37169, BL2
Compensation beads Invitrogen Cat #01-2222-42 Lot #2339860
RPMI + 10% FBS GenClone Reference #25-506 + Omega
Scientific Catalog #FB-01
T75 Culture Flask Laguna Scientific Catalog #4616
Day 0 (2024-02-12): Thaw huLym-1-B DAP CAR T cell prep and Mock T cells
1. Warm two tubes containing 5 mL of T cell medium with cytokines using 37ºC water
bath.
2. Briefly thaw one 1 mL vial of huLym-1-B DAP CAR T cell prep and one 1 mL vial of
mock T cells in 37ºC water bath.
3. Wipe the outside of the vials and centrifuge tubes with ethanol.
4. Add the entire vial of huLym-1-B DAP CAR T cell prep to the 15 mL centrifuge tube
containing the 5 ml warmed media. Do the same for the Mock T cells in a separate
centrifuge tube.
5. Spin down (400 rcf, 4ºC, 5 mins)
6. Resuspend in 1 mL T cell medium with cytokines.
a. Remove 10 µL from the cell suspension and add it to a 0.5 mL tube.
b. Add 10 µL CellCounter stain to the tube and pipette up and down to mix.
c. Add 10 µL of the mixture to a slide and place in the CellCounter.
7. Count the cells from each vial.
a. huLym-1-DAP CART: 1.0 x 107 live/mL (82%), 2.15 x 106 dead/mL (18%)
b. Mock T: 2.0 x 106 live/mL (47%), 2.28 x 106 dead/mL (53%)
8. Dilute each cell type to 0.5 million live cells/mL in T cell medium with cytokines.
a. huLym-1-DAP CAR-T: Add 19 mL medium.
b. Mock T: Add 3 mL medium.
9. In three wells on a GRex plate, add 1 ml huLym-1-DAP CAR T cell prep and 1 ml Mock
T cells at a 1:1 ratio of living cells.
a. Each well contains 0.5 x 106 live cells of each cell type/well in 2mL.
10. Add the remaining huLym-1-DAP CAR-T cell prep to two new wells on the GRex plate.
a. 8.5 x 106 live (82%), 1.86 x 106 dead (18%)
b. Dilute to 1 million live cells mL = 5.18 mL total per well
11. Add the remaining Mock T cells to a separate well on the GRex plate.
a. 0.5 x 106 live (47%), 0.56 x 106 dead (53%)
b. Dilute to 0.5 million live cells/mL = 2.12 mL total
12. Place G Rex plate into the incubator for expansion.
Day 0 (2024-02-12): Thaw Karpas299 cells
1. Add 5 mL RPMI + 10% FBS to a 15 mL centrifuge tube.
2. Warm the media in the 37ºC water bath.
3. Thaw one cryovial of Karpas299 cells from liquid nitrogen in 37ºC water bath/
4. Wipe the cryovial and centrifuge tube well with ethanol.
5. Add the entire cryovial of cells into the warm media.
6. Spin down (400 rcf, 4ºC, 5 mins)
7. Discard the supernatant and resuspend in 1 mL RPMI + 10% FBS.
8. Count cells.
a. 2.2 x 106 live (78%), 6.28 x 105 dead (22%)

52
9. Add 20 mL RPMI + 10% FBS to a T75 flask.
10. Add cells to T75 flask and place in the incubator.
Day 0 (2024-02-12): Flow
1. Create antibody mixes:
Mix 1
Isotype
Controls
RL1 Dylight 650 a-261 CAR tag Isotype Control
RL2 APC/Cy7 4B4-1 CD137 Isotype Control
BL2 PerCP L243 HLA-DR-A Isotype Control
BL1 Dylight 488 LN3 HLA-DR-B Isotype Control
3 tubes =
3 µL each isotype
control +
18 µL flow buffer
Mix 2
Isotype
Controls
RL1 Dylight 650 a-261 CAR tag Isotype Control
RL2 APC/Cy7 4B4-1 CD137 Isotype Control
BL1 Dylight 488 chLym-1 HLA-DR-B Isotype
Control
3 tubes =
3 µL each isotype
control +
21 µL flow buffer
Mix 3
Isotype
Controls
RL1 Dylight 650 a-261 CAR tag Isotype Control
RL2 APC/Cy7 4B4-1 CD137 Isotype Control
YL1 PE Tu36 HLA-DR-B Isotype Control
3 tubes =
3 µL each isotype
control +
21 µL flow buffer
Mix 1 RL1 Dylight 650 a-261 CAR tag
RL3 APC/Cy7 4B4-1 CD137
BL2 PerCP L243 HLA-DR-A
BL1 Dylight 488 LN3 HLA-DR-B
5 tubes =
5 µL each antibody +
30 µL flow buffer
Mix 2 RL1 Dylight 650 a-261 CAR tag
RL2 APC/Cy7 4B4-1 CD137
BL1 Dylight 488 chLym-1 HLA-DR-B
5 tubes =
5 µL each antibody +
35 µL flow buffer
Mix 3 RL1 Dylight 650 a-261 CAR tag
RL3 APC/Cy7 4B4-1 CD137
YL1 PE Tu36 HLA-DR-B
5 tubes =
5 µL each antibody +
35 µL flow buffer
1. Add 200 µL (0.1 million cells) to labelled flow tubes:
a. Tubes 1-6: Well 1
b. Tubes 7-9: Well 2
c. Tubes 10-12: Well 3
d. Tubes 13-18: Karpas299 flask
2. To each tube, add 3 mL flow buffer.
3. Spin down (400 rcf, 5 minutes, 4ºC).
4. Remove supernatant.
5. To each tube, add 10 µL of the listed antibody mix:
Tube Contents Antibody Mix
1 Well 1: huLym-1-DAP CAR
T prep + Mock T cells
Mix 1 Isotype Control
2 Well 1: huLym-1-DAP CAR
T prep + Mock T cells
Mix 2 Isotype Control
3 Well 1: huLym-1-DAP CAR
T prep + Mock T cells
Mix 3 Isotype Control

53
4 Well 1: huLym-1-DAP CAR
T prep + Mock T cells
Mix 1
5 Well 1: huLym-1-DAP CAR
T prep + Mock T cells
Mix 2
6 Well 1: huLym-1-DAP CAR
T prep + Mock T cells
Mix 3
7 Well 2: huLym-1-DAP CAR
T prep + Mock T cells
Mix 1
8 Well 2: huLym-1-DAP CAR
T prep + Mock T cells
Mix 2
9 Well 2: huLym-1-DAP CAR
T prep + Mock T cells
Mix 3
10 Well 3: huLym-1-DAP CAR
T prep + Mock T cells
Mix 1
11 Well 3: huLym-1-DAP CAR
T prep + Mock T cells
Mix 2
12 Well 3: huLym-1-DAP CAR
T prep + Mock T cells
Mix 3
13 Karpas299 cells Mix 1 Isotype Controls
14 Karpas299 cells Mix 2 Isotype Controls
15 Karpas299 cells Mix 3 Isotype Controls
16 Karpas299 cells Mix 1
17 Karpas299 cells Mix 2
18 Karpas299 cells Mix 3
6. Place tubes on shaker (with the lights off) in cold room for 45 minutes.
7. Remove tubes from shaker and add 3 mL flow buffer.
8. Spin down (400 rcf, 5 minutes, 4ºC).
9. Remove supernatant.
10. Repeat the wash process one more time.
11. Add 500 µL flow buffer to each tube.
12. To each tube, add 1 drop of Propidium Iodide Ready Flow™ Reagent.
13. Leave tubes at room temperature, protected from light, for 15 minutes.
14. Run flow using Attune NxT flow cytometer and analyze using FlowJo.
Day 0 (2024-02-12): Stimulation of cell preps with Immunocult Human CD3/CD28/CD2 T
cell activator
1. Add 20µl Immunocult to each of the three wells containing 2ml with 0.5M CAR T and
0.5M Mock T cells = 1.0M cells in 2mL. Pipette up and down with large orifice pipette
tip to mix.
2. To the well containing 1.06M Mock T cells alone, add 21.2µL Immunocult.
Day 2 (2024-02-14): Add medium
1. Add 5 mL fresh T cell medium with cytokines to each well containing huLym-1-B DAP
CAR T cell prep combined with Mock T cells in G Rex plate.
2. Do the same to the two wells of huLym-1-DAP CAR T cell prep alone and the well with
Mock T cells alone.

54
Day 5 (2024-02-17): Change medium
1. Remove 5 mL medium from the top of Wells 1, 2, & 3 containing the mixture of Mock
and huLym-1-B DAP CAR T cell preps in G Rex plate.
2. Add 5 mL fresh medium.
3. Do the same to the two wells of huLym-1-DAP CAR T cell prep alone and the well with
Mock T cells alone.
Day 7 (2024-02-19): Flow of cocultured huLym-1-B CAR T and Mock T cells.
1. Count cells in Wells 1, 2 & 3 in the G Rex plate using CellCounter.
a. Well 1: 8.9 x 106 live (90%), 8.86 x 105 dead (10%)
b. Well 2: 7.8 x 106 live (90%), 8.92 x 105 dead (10%)
c. Well 3: 7.56 x 106 live (90%), 8.7 x 105 dead (10%)
2. Repeat Day 0 Flow Protocol.
3. After removing cells for flow cytometry, take 2 million live cells from each well and
move them to three new wells. Discard the remaining cells in the old wells.
a. Well 1: 225 µL = 2 million cells
b. Well 2: 256 µL = 2 million cells
c. Well 3: 265 µL = 2 million cells
4. Count the two wells of huLym-1-DAP CAR T cell prep alone and the well with Mock T
cells alone using CellCounter.
a. huLym-1-DAP CAR T: 9.01 x 106 live (86%), 1.54 x 106 dead (14%)
i. Dilute to 0.5M live cells/mL in T cell medium with cytokines = 18 mL
total
b. Mock T cells: 4.89 x 106 live (95%), 2.52 x 105 dead (5%).
i. Dilute to 0.5M live cells/mL in T cell medium with cytokines = 9.78 mL
total
Day 7 (2024-02-19): Restimulation of huLym-1-B DAP CAR T cell and Mock T cell preps
cultured alone
1. To the new Wells 1, 2, & 3, add T cell medium with cytokines to reach 4 mL medium
total (2M live cells in 4 mL).
2. Add 40 µL Immunocult Human CD3/CD28/CD2 T cell activator to well and gently
pipette up and down with large orifice blue tip to mix.
3. Add 97.8 µL (20 µL/1M live cells) Immunocult to the well containing Mock T cells
alone.
Day 9 (2024-02-21): Add medium
1. Add 5 mL T cell medium with cytokines to each well containing huLym-1-B DAP CAR
T cell prep and Mock T cells in G Rex plate.
2. Do the same to the two wells of huLym-1-DAP CAR T cell prep alone and the well with
Mock T cells alone.
Day 12 (2024-02-24): Change medium
1. Remove 5 mL medium from the top of Wells 1,2,3 containing cocultured Mock T cells
and huLym-1-B DAP CAR T cell prep and Mock T cells in G Rex plate. Add 5 mL fresh
T cell medium with cytokines.
2. Do the same to the two wells of huLym-1-DAP CAR T cell prep alone and the well with
Mock T cells alone.

55
Day 14 (2024-02-26): Flow
1. Count cells in Wells 1, 2, & 3:
a. Well 1: 6.22 x 106 live (73%), 2.26 x 106 dead (27%)
b. Well 2: 7.20 x 206 live (79%), 1.87 x 106 dead (21%)
c. Well 3: 6.85 x 106 live (79%), 1.79 x 106 dead (21%)
d. Karpas299: 1.23 x 107 live (93%), 9.56 x 106 dead (7%)
2. Repeat Day 0 Flow Protocol.
3. After removing cells for flow cytometry, take 2 million live cells from each well and
move them to three new wells. Discard the remaining cells in the old wells.
a. Well 1: 321 µL = 2 million live cells
b. Well 2: 278 µL = 2 million live cells
c. Well 2: 292 µL = 2 million live cells
d. Dilute each well to 0.5M live cells/mL in T cell medium with cytokines = 2M live
cells in 4 mL per well.
4. Count the two wells of huLym-1-DAP CAR T cell prep alone and the well with Mock T
cells alone using CellCounter.
a. huLym-1-DAP CAR T cells: 5.24 x 106 live (73%), 1.9 x 106 dead (27%)
i. Dilute to 0.5M live cells/mL in T cell medium with cytokines = 10.4 mL
total
b. Mock T cells: 2.63 x 107 live (80%), 6.52 x 106 dead (20%)
i. Dilute to 0.5M live cells/mL in T cell medium with cytokines = 5.26 mL
total
Day 16 (2024-02-28): Add medium
3. Add 5 mL T cell medium with cytokines to each well containing huLym-1-B DAP CAR
T cell prep in G Rex plate.
4. Do the same to the two wells of huLym-1-DAP CAR T cell prep alone and the well with
Mock T cells alone.
Day 19 (2024-03-02): Change medium
3. Remove 5 mL medium from the top Wells 1, 2 & 3 containing huLym-1-B DAP CAR T
cells cocultured with Mock T cells in G Rex plate.
4. Add 5 mL fresh T cell medium with cytokines.
5. Do the same to the two wells of huLym-1-DAP CAR T cell prep alone and the well with
Mock T cells alone.
Day 21 (2024-03-04): Flow
1. Count cells in Wells 1, 2, & 3:
a. Well 1: 1.88 x 106 live (57%)
b. Well 2: 2.32 x 106 live (70%)
c. Well 3: 2.79 x 106 live (71%)
d. Karpas299 cell count: 21.7 x 106 live (94%)
2. Repeat Day 0 Flow Protocol.
3. Count the two wells of huLym-1-DAP CAR T cell prep alone and the well with Mock T
cells alone using CellCounter.
a. huLym-1-DAP CAR T cells: 2.02 x 106 live (74%)
b. Mock T cells: 11.6 x 106 live (81%)

56
Appendix B: OCH-Exp-023 Protocol
Purpose: Test the hypothesis that culturing a mixture of Donor 3 a-CD19-CD3z CAR-T cells
and naïve T cells in an a-261 coated plate will increase CD137 and decrease Lym-1-E and Tu36-
E in a-CD19 CAR-T cells but not naïve T cells. Results do not support hypothesis.
Materials:
T cell medium with cytokines 43% Click's medium, 43% RPMI-1640, 10%
FBS, 2% GlutaMAX, 1% nonessential amino
acids, 1% penicillin/streptomycin)
supplemented with 50 ng/mL IL7-Fc and 100
ng/mL IL15-Fc
a-CD19-CD3z CAR T Cell Prep Donor 3, Transduced by CHL, Frozen 2021-
06-28, 91% transduced before freezing
Naïve T cells Donor 3, Frozen 2021-06-03
ImmunoCult™ Human CD3/CD28/CD2 T
Cell Activator
StemCell Technologies Cat #10970
Unconjugated a-261 antibody Made by Epstein lab, 1 mg/mL
PBS Corning Reference #21-040-CV
BSA 2%, Filtered through 0.22 uM syringe
RL1 Dylight 650 a-261 CAR tag Made and conjugated by Epstein Lab
RL2 APC/Cy7 4B4-1 CD137 Biolegend Catalog #309830
BL1 Dylight 488 LN3 HLA-DR-B Made and conjugated by Epstein Lab
BL2 PerCP L243 HLA-DR-A Biolegend Catalog #307628
BL1 Dylight 488 chLym-1 HLA-DR-B Made and conjugated by Epstein Lab
YL1 PE Tu36 HLA-DR-B Invitrogen Catalog #MHLDR04
RL1 Dylight 650 a-261 CAR tag Isotype
Control
Purified Mouse IgG2a, κ Isotype Ctrl
Antibody
(Biolegend Catalog #401501)
RL2 APC/Cy7 4B4-1 CD137 Isotype
Control
APC/Cyanine7 Mouse IgG1, κ Isotype Ctrl
Antibody (Biolegend Catalog #400128)
BL1 Dylight 488 LN3 HLA-DR-B
Isotype Control
Mouse IgG2b kappa Isotype Control
(eBMG2b), eBioscience™
(Invitrogen Catalog #14-4732-82), Conjugated
by Epstein Lab
BL2 PerCP L243 HLA-DR-A Isotype
Control
PerCP Mouse IgG2a, κ Isotype Ctrl Antibody
(Biolegend Catalog #400256)
BL1 Dylight 488 chLym-1 HLA-DR-B
Isotype Control
ch225-Dylight488 (Made and conjugated by
Epstein lab)
YL1 PE Tu36 HLA-DR-B Isotype
Control
Mouse IgG2b Isotype Control, PE (Invitrogen
Catalog #MG2B04)
Flow tubes
Flow buffer PBS + 4% FBS
Propidium Iodide Ready Flow™ Reagent Invitrogen Cat # R37169, BL2
Compensation beads Invitrogen Cat #01-2222-42 Lot #2339860

57
2024-03-20: Thaw Cells
13. Warm two tubes containing 5 mL of T cell medium with cytokines using 37ºC water
bath.
14. Briefly thaw one 1 mL vial of a-CD19 CAR T cell prep and one 1 mL vial of T cells in
37ºC water bath.
15. Wipe the outside of the vials and centrifuge tubes with ethanol.
16. Add the entire vial of a-CD19 CAR T cell prep to the 15 mL centrifuge tube containing
the 5 ml warmed media. Do the same for the T cells in a separate centrifuge tube.
17. Spin down (400 rcf, 4ºC, 5 mins)
18. Resuspend in 1 mL T cell medium with cytokines.
19. Count the cells from each tube.
19.6 Remove 10 uL from each well and add to sterile 0.5 mL Eppendorf tubes.
19.7 Combine each sample with 10 uL DAPI stain.
19.8 Add 10 uL to a slide and count using Cell Counter.
19.9 Naïve T cells: 1.69 x 106 live (84%)
19.10 a-CD19-CD3z CAR T prep: 3.48 x 106 live (94%)
20. Dilute both cell preps separately to 0.5 million/mL in T cell medium with cytokines. Add
cells to two separate wells in a GRex plate.
21. Add 25 uL/mL of Immunocult T Cell Activator to each well.
22. Place GRex plate into incubator for expansion.
2024-03-22: Change cell media
1. Remove 5 mL media from each well and add 5 mL fresh T cell media in.
2024-03-25: Change cell media & Count Cells
1. Spin down each well of cells and resuspend in 1 mL T cell medium with cytokines.
2. Count naïve T cells and a-CD19-CD3z CAR T prep.
1. Remove 10 uL from each well and add to sterile 0.5 mL Eppendorf tubes.
2. Combine each sample with 10 uL CellCounter stain.
3. Add 10 uL to a slide and count using Cell Counter.
4. Donor 3 T cells: 8.68 x 106 live (84%)
5. a-CD19-CD3z CAR-T: 4.74 x 106 live (78%)
3. Add each cell type to separate wells on GRex plate.
4. Dilute each cell type to 1 million cells/mL in T cell medium with cytokines.
2024-03-25: Coat plate with a-261
1. Obtain a new 6-well untreated plate.
2. Label the wells 1-6.
3. Add 6 uL of 1 mg/mL a-261 to 6 mL of PBS in a 15 mL centrifuge tube (final = 1ug/ml).
4. Pipette solution up and down to mix.
5. To wells 1-3, add 2 mL (2ug) of the solution.
6. Place the plates in the refrigerator overnight.
Day 0 (2024-03-26): Start Co-Culture
1. a-261 Coating:
a. Remove the plate with a-261-coated wells from the refrigerator.
b. Remove the solution from wells 1-3.
c. Add 1 mL BSA to each well and let sit for 30 minutes.
d. Remove BSA from each well.

58
2. Count cells in the two preps.
a. Remove 10 uL from each well and add to sterile 0.5 mL Eppendorf tubes.
b. Combine each sample with 10 uL DAPI stain.
c. Add 10 uL to a slide and count using Cell Counter.
d. Donor 3 T cells: 1.07 x 107 live (79%)
e. a-CD19-CD3z CAR-T: 5.44 x 106 live (84%)
3. Dilute cells to 0.5 million live cells/mL with T cell media with cytokines.
4. Combine 8.1 mL (4.05 million cells) of each cell type (Total = 8.1 million cells) in a 15
mL centrifuge tube.
5. In Wells 2-6 of the plate, add 2.6 mL of the cell mixture (0.65 million of each cell type).
(Total = 1.3 million cells. Remove 0.6 ml (0.3 million cells for Day 2 flow, leaves 1
million in each well.)
6. In Well 1, add 3.2 mL of the cell mixture. (Total = 1.6 million cells. Remove 1.2 ml (0.6
million cells) for Day 2 flow (some cells used for isotype controls) leaves 1 million in coculture).
Day 0 (2024-03-26): Flow
1. Create antibody mixes:
Mix 1
Isotype
Controls
RL1 Dylight 650 a-261 CAR tag Isotype
Control
RL2 APC/Cy7 4B4-1 CD137 Isotype Control
BL2 PerCP L243 HLA-DR-A Isotype Control
BL1 Dylight 488 LN3 HLA-DR-B Isotype
Control
1 tube =
1 uL each isotype control +
9 uL flow buffer
Mix 2
Isotype
Controls
RL1 Dylight 650 a-261 CAR tag Isotype
Control
RL2 APC/Cy7 4B4-1 CD137 Isotype Control
BL1 Dylight 488 chLym-1 HLA-DR-B Isotype
Control
1 tube =
1 uL each isotype control +
9 uL flow buffer
Mix 3
Isotype
Controls
RL1 Dylight 650 a-261 CAR tag Isotype
Control
RL2 APC/Cy7 4B4-1 CD137 Isotype Control
YL1 PE Tu36 HLA-DR-B Isotype Control
1 tube =
1 uL each isotype control +
9 uL flow buffer
Mix 1 RL1 Dylight 650 a-261 CAR tag
RL3 APC/Cy7 4B4-1 CD137
BL1 Dylight 488 LN3 HLA-DR-B
BL2 PerCP L243 HLA-DR-A
6 tubes =
6 uL each antibody +
36 uL flow buffer
Mix 2 RL1 Dylight 650 a-261 CAR tag
RL2 APC/Cy7 4B4-1 CD137
BL1 Dylight 488 chLym-1 HLA-DR-B
6 tubes =
6 uL each antibody +
36 uL flow buffer
Mix 3 RL1 Dylight 650 a-261 CAR tag
RL3 APC/Cy7 4B4-1 CD137
YL1 PE Tu36 HLA-DR-B
6 tubes =
6 uL each antibody +
36 uL flow buffer
2. Add 200 uL (0.1 million cells) to labelled flow tubes:
a. Tubes 1-6: Well 1

59
b. Tubes 7-9: Well 2
c. Tubes 10-12: Well 3
d. Tubes 13-15: Well 4
e. Tubes 16-18: Well 5
f. Tubes 19-21: Well 6
3. To each tube, add 3 mL flow buffer.
4. Spin down (400 rcf, 5 minutes, 4ºC).
5. Remove supernatant.
6. To each tube, add 10 uL of the listed antibody mix:
Tube Contents Antibody Mix
1 Well 1 (a-261 coating) Mix 1 Isotype Control
2 Well 1 (a-261 coating) Mix 2 Isotype Control
3 Well 1 (a-261 coating) Mix 3 Isotype Control
4 Well 1 (a-261 coating) Mix 1
5 Well 1 (a-261 coating) Mix 2
6 Well 1 (a-261 coating) Mix 3
7 Well 2 (a-261 coating) Mix 1
8 Well 2 (a-261 coating) Mix 2
9 Well 2 (a-261 coating) Mix 3
10 Well 3 (a-261 coating) Mix 1
11 Well 3 (a-261 coating) Mix 2
12 Well 3 (a-261 coating) Mix 3
13 Well 4 Mix 1
14 Well 4 Mix 2
15 Well 4 Mix 3
16 Well 5 Mix 1
17 Well 5 Mix 2
18 Well 5 Mix 3
19 Well 6 Mix 1
20 Well 6 Mix 2
21 Well 6 Mix 3
7. Place tubes on shaker (with the lights off) in cold room for 45 minutes.
8. Remove tubes from shaker and add 3 mL flow buffer.
9. Spin down (400 rcf, 5 minutes, 4ºC).
10. Remove supernatant.
11. Repeat the wash process one more time.
12. Add 500 uL flow buffer to each tube.
13. To each tube, add 1 drop of Propidium Iodide Ready Flow™ Reagent.
14. Leave tubes at room temperature, protected from light, for 15 minutes.
15. Run flow using Attune NxT flow cytometer and analyze using FlowJo.
Day 1 (2024-03-27): Flow
1. Pipette cells in each well up and down well to mix.
2. Count cells in each well.
a. Remove 10 uL from each well and add to sterile 0.5 mL Eppendorf tubes.

60
b. Combine each sample with 10 uL DAPI stain.
c. Add 10 uL to a slide and count using Cell Counter.
3. Add 0.1 million cells to labelled flow tubes:
a. Tubes 1-6: Well 1
b. Tubes 7-9: Well 2
c. Tubes 10-12: Well 3
d. Tubes 13-15: Well 4
e. Tubes 16-18: Well 5
f. Tubes 19-21: Well 6
4. Repeat Day 2 Flow Protocol starting with Step 3.
Day 2 (2024-03-28): Flow
1. Repeat Day 1 Flow protocol.

61
Appendix C: OCH-Exp-025 Protocol
Purpose: Test the hypothesis that the preferential expansion seen in huLym-1-DAP CAR-T cells
made with T cells from Donor 6 is reproducible in huLym-1-DAP CAR-T cells made with T
cells from Donors 3 and 8. Test the hypothesis that a-CD19-DAP CAR-T cells made from T
cells from Donor 6 do not show the same preferential expansion.
Materials:
huLym-1-B DAP CAR T cell prep Donor 3, Transduced by OCH 2024-03-18
huLym-1-B DAP CAR T cell prep Donor 8, Transduced by OCH 2024-03-18
a-CD19-DAP CAR T cell prep Donor 6, Transduced by OCH 2024-03-18
T cell medium with cytokines 43% Click's medium, 43% RPMI-1640, 10%
FBS, 2% GlutaMAX, 1% nonessential amino
acids, 1% penicillin/streptomycin)
supplemented with 50 ng/mL IL7-Fc and 100
ng/mL IL15-Fc
ImmunoCult™ Human CD3/CD28/CD2 T
Cell Activator
StemCell Technologies Cat #10970
24-well GRex Plate WilsonWolf Catalog #80192M
RL1 Dylight 650 a-261 CAR tag Made and conjugated by Epstein Lab
RL2 APC/Cy7 4B4-1 CD137 Biolegend Catalog #309830
BL1 Dylight 488 LN3 HLA-DR-B Made and conjugated by Epstein Lab
BL2 PerCP L243 HLA-DR-A Biolegend Catalog #307628
BL1 Dylight 488 chLym-1 HLA-DR-B Made and conjugated by Epstein Lab
YL1 PE Tu36 HLA-DR-B Invitrogen Catalog #MHLDR04
RL1 Dylight 650 a-261 CAR tag Isotype
Control
Purified Mouse IgG2a, κ Isotype Ctrl
Antibody
(Biolegend Catalog #401501)
RL2 APC/Cy7 4B4-1 CD137 Isotype
Control
APC/Cyanine7 Mouse IgG1, κ Isotype Ctrl
Antibody (Biolegend Catalog #400128)
BL1 Dylight 488 LN3 HLA-DR-B
Isotype Control
Mouse IgG2b kappa Isotype Control
(eBMG2b), eBioscience™
(Invitrogen Catalog #14-4732-82), Conjugated
by Epstein Lab
BL2 PerCP L243 HLA-DR-A Isotype
Control
PerCP Mouse IgG2a, κ Isotype Ctrl Antibody
(Biolegend Catalog #400256)
BL1 Dylight 488 chLym-1 HLA-DR-B
Isotype Control
ch225-Dylight488 (Made and conjugated by
Epstein lab)
YL1 PE Tu36 HLA-DR-B Isotype
Control
Mouse IgG2b Isotype Control, PE (Invitrogen
Catalog #MG2B04)
Flow tubes
Flow buffer PBS + 4% FBS
Propidium Iodide Ready Flow™ Reagent Invitrogen Cat # R37169, BL2
Compensation beads Invitrogen Cat #01-2222-42 Lot #2339860

62
Day 0 (2024-03-20): Flow
23. Count cells preps and Mock T cells from each transduction.
a. huLym-1-DAP CAR-T cell prep (Donor 3): 7.74 x 105 live (81%)
b. huLym-1-DAP CAR-T cell prep (Donor 8): 3.93 x 105 live (66%)
c. a-CD19-DAP CAR-T cell prep (Donor 6): 6.04 x 105 live (82%)
24. Dilute each cell type to 0.5M live cells/mL in T cell medium with cytokines.
25. Add each cell prep to a separate well on GRex plate.
26. Obtain 6 clean flow tubes and add cells to each tube:
a. Tube 1: 200 µL (0.1 x 106 live cells) from huLym-1-DAP CAR-T cell prep (Donor 3)
b. Tube 2: 200 µL (0.1 x 106 live cells) from huLym-1-DAP CAR-T cell prep (Donor 3)
c. Tube 3: 200 µL (0.1 x 106 live cells) from huLym-1-DAP CAR-T cell prep (Donor 8)
d. Tube 4: 200 µL (0.1 x 106 live cells) from huLym-1-DAP CAR-T cell prep (Donor 8)
e. Tube 5: 200 µL (0.1 x 106 live cells) from a-CD19-DAP CAR-T cell prep (Donor 6)
f. Tube 6: 200 µL (0.1 x 106 live cells) from a-CD19-DAP CAR-T cell prep (Donor 6)
27. To each tube, add 3 mL flow buffer.
28. Spin down (400 rcf, 5 minutes, 4ºC).
29. Remove supernatant.
30. Dilute antibodies in flow buffer:
a. 3 µL RL1 Dylight 650 a-261 CAR tag Isotype Control + 27 µL flow buffer
b. 3 µL RL1 Dylight 650 a-261 CAR tag Antibody + 27 µL flow buffer
31. To tubes 1, 3, & 5, add 10 µL diluted RL1 Dylight 650 a-261 CAR tag Isotype
Control.
32. To tubes 2, 4 & 6, add 10 µL diluted RL1 Dylight 650 a-261 CAR tag Antibody.
33. Place tubes on shaker (with the lights off) in cold room for 45 minutes.
34. Remove tubes from shaker and add 3 mL flow buffer.
35. Spin down (400 rcf, 5 minutes, 4ºC).
36. Remove supernatant.
37. Repeat the wash process one more time.
38. Add 500 µL flow buffer to each tube.
39. To each tube, add 1 drop of Propidium Iodide Ready Flow™ Reagent.
40. Leave tubes at room temperature, protected from light, for 15 minutes.
41. Run flow using Attune NxT flow cytometer and analyze using FlowJo.
Day 0 (2024-03-20): Stimulation of cell preps with Immunocult Human CD3/CD28/CD2 T
cell activator
3. To each of the three wells containing the cell preps, add 20 µL/1M cells Immunocult T
cell Activator.
Day 2 (2024-03-22): Add medium
3. Add 5 mL fresh T cell medium with cytokines to each cell prep.
Day 5 (2024-03-25): Change medium
4. Remove 5 mL medium from the top of each well containing cell preps.
5. Add 5 mL fresh medium.
Day 7 (2024-03-27): Flow
2. Count cells in each cell prep.
a. huLym-1-DAP CAR-T cell prep (Donor 3): 1.62 x 106 live (87%)
b. huLym-1-DAP CAR-T cell prep (Donor 8): 1.75 x 106 live (83%)

63
c. a-CD19-DAP CAR-T cell prep (Donor 6): 3.08 x 106 live (87%)
3. Dilute each cell prep to 0.5M cells/mL in T cell medium with cytokines.
4. Create antibody mixes:
Mix 1
Isotype
Controls
RL1 Dylight 650 a-261 CAR tag Isotype
Control
RL2 APC/Cy7 4B4-1 CD137 Isotype Control
BL2 PerCP L243 HLA-DR-A Isotype Control
BL1 Dylight 488 LN3 HLA-DR-B Isotype
Control
3 tubes =
3 µL each isotype control +
18 µL flow buffer
Mix 2
Isotype
Controls
RL1 Dylight 650 a-261 CAR tag Isotype
Control
RL2 APC/Cy7 4B4-1 CD137 Isotype Control
BL1 Dylight 488 chLym-1 HLA-DR-B
Isotype Control
3 tubes =
3 µL each isotype control +
21 µL flow buffer
Mix 3
Isotype
Controls
RL1 Dylight 650 a-261 CAR tag Isotype
Control
RL2 APC/Cy7 4B4-1 CD137 Isotype Control
YL1 PE Tu36 HLA-DR-B Isotype Control
3 tubes =
3 µL each isotype control +
21 µL flow buffer
Mix 1 RL1 Dylight 650 a-261 CAR tag
RL3 APC/Cy7 4B4-1 CD137
BL2 PerCP L243 HLA-DR-A
BL1 Dylight 488 LN3 HLA-DR-B
5 tubes =
5 µL each antibody +
30 µL flow buffer
Mix 2 RL1 Dylight 650 a-261 CAR tag
RL2 APC/Cy7 4B4-1 CD137
BL1 Dylight 488 chLym-1 HLA-DR-B
5 tubes =
5 µL each antibody +
35 µL flow buffer
Mix 3 RL1 Dylight 650 a-261 CAR tag
RL3 APC/Cy7 4B4-1 CD137
YL1 PE Tu36 HLA-DR-B
5 tubes =
5 µL each antibody +
35 µL flow buffer
5. Add 200 µL (0.1 million cells) to labelled flow tubes:
a. Tubes 1-6: huLym-1-DAP CAR-T cell prep (Donor 3)
b. Tubes 7-12: huLym-1-DAP CAR-T cell prep (Donor 8)
c. Tubes 13-18: a-CD19-DAP CAR-T cell prep (Donor 6)
6. To each tube, add 3 mL flow buffer.
7. Spin down (400 rcf, 5 minutes, 4ºC).
8. Remove supernatant.
9. To each tube, add 10 µL of the listed antibody mix:
Tube Contents Antibody Mix
1 huLym-1-DAP CAR-T cell
prep (Donor 3)
Mix 1 Isotype Control
2 huLym-1-DAP CAR-T cell
prep (Donor 3)
Mix 2 Isotype Control
3 huLym-1-DAP CAR-T cell
prep (Donor 3)
Mix 3 Isotype Control

64
4 huLym-1-DAP CAR-T cell
prep (Donor 3)
Mix 1
5 huLym-1-DAP CAR-T cell
prep (Donor 3)
Mix 2
6 huLym-1-DAP CAR-T cell
prep (Donor 3)
Mix 3
7 huLym-1-DAP CAR-T cell
prep (Donor 8)
Mix 1 Isotype Control
8 huLym-1-DAP CAR-T cell
prep (Donor 8)
Mix 2 Isotype Control
9 huLym-1-DAP CAR-T cell
prep (Donor 8)
Mix 3 Isotype Control
10 huLym-1-DAP CAR-T cell
prep (Donor 8)
Mix 1
11 huLym-1-DAP CAR-T cell
prep (Donor 8)
Mix 2
12 huLym-1-DAP CAR-T cell
prep (Donor 8)
Mix 3
13 a-CD19-DAP CAR-T cell
prep (Donor 6)
Mix 1 Isotype Controls
14 a-CD19-DAP CAR-T cell
prep (Donor 6)
Mix 2 Isotype Controls
15 a-CD19-DAP CAR-T cell
prep (Donor 6)
Mix 3 Isotype Controls
16 a-CD19-DAP CAR-T cell
prep (Donor 6)
Mix 1
17 a-CD19-DAP CAR-T cell
prep (Donor 6)
Mix 2
18 a-CD19-DAP CAR-T cell
prep (Donor 6)
Mix 3
10. Place tubes on shaker (with the lights off) in cold room for 45 minutes.
11. Remove tubes from shaker and add 3 mL flow buffer.
12. Spin down (400 rcf, 5 minutes, 4ºC).
13. Remove supernatant.
14. Repeat the wash process one more time.
15. Add 500 µL flow buffer to each tube.
16. To each tube, add 1 drop of Propidium Iodide Ready Flow™ Reagent.
17. Leave tubes at room temperature, protected from light, for 15 minutes.
18. Run flow using Attune NxT flow cytometer and analyze using FlowJo.

Abstract (if available)

Abstract HLA-DR is a well-recognized marker of activated T cells. However, the mechanism and functional relevance behind HLA-DR upregulation in T cells are not well understood.

HLA-DR is a Class II MHC molecule expressed on the surface of antigen-presenting cells (APCs). HLA-DR presents peptides derived from exogenous antigens to CD4+ T cells, allowing the CD4+ T cells to regulate immune responses against the presented antigen and/or kill the cell presenting the peptide.

The Lym-1 antibody recognizes an epitope (Lym-1-E) on the beta subunit of HLA-DR. It had been previously discovered that epitope-stimulation of CAR-T cells utilizing either DAP10/12 or CD3z signaling domains downregulated the expression of Lym-1-E. This thesis further characterizes changes in the HLA-DR structure caused by epitope-stimulation and discusses its possible functional relevance.

Epitope-stimulation of a T cell was found to also lead to the downregulation of Tu36-E, an epitope expressed on the beta subunit of “M1” HLA-DR but not “M2” HLA-DR. M1 HLA- DR is a conformation considered more likely to present peptides to T cells than the M2 conformation. The downregulation of Tu36-E on epitope-stimulated T cells therefore prompts the hypothesis that epitope-stimulation leads to a conformational change in HLA-DR from M1 to M2, making the molecule less likely to present peptides and protecting the T cell from killing by a CD4 cytotoxic lymphocyte.

These results prompt further investigation into the regulation of HLA-DR function in T cells, as well as the possibility of treating T cell lymphomas that express M1 HLA-DR with CAR-T cells targeting its epitopes.

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Asset Metadata

Creator Hart, Olivia (author)

Core Title T cell regulation of HLA-DR

School Keck School of Medicine

Degree Master of Science

Degree Program Medical Physiology

Degree Conferral Date 2024-05

Publication Date 05/22/2024

Defense Date 04/02/2024

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

Tag cancer immunotherapy,CAR-T cell immunotherapy,CAR-T cells,HLA-DR,immunology,immunotherapy,Lym-1,Lym-1 CAR-T cells,MHC class II,OAI-PMH Harvest,T cell activation,T cell lymphoma,T cells

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Kaslow, Harvey (committee chair), Bonnin, Alexandre (committee member), Epstein, Alan (committee member)

Creator Email ohart@usc.edu,oliviahart99@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113953808

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Legacy Identifier etd-HartOlivia-13002

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