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Trends Mol Med. Author manuscript; available in PMC 2017 December 01.
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Published in final edited form as:

Trends Mol Med. 2016 December ; 22(12): 1000–1011. doi:10.1016/j.molmed.2016.10.002.

Cell-Intrinsic Barriers of T Cell-Based Immunotherapy

Hazem E. Ghoneim1, Anthony E. Zamora1, Paul G. Thomas1, and Ben A. Youngblood1,*
1Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA

Abstract
Prolonged exposure of CD8+T cells to their cognate antigen can result in exhaustion of effector
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functions enabling the persistence of infected or transformed cells. Recent advances in strategies
to rejuvenate host effector function using Immune Checkpoint Blockade have resulted in
tremendous success towards the treatment of several cancers. However, it is unclear if T-cell
rejuvenation results in long-lived antitumor functions. Emerging evidence suggests that T-cell
exhaustion may also represent a significant impediment in sustaining long-lived antitumor activity
by chimeric antigen receptor T-cells. Here, we discuss current findings regarding transcriptional
regulation during T-cell exhaustion and address the hypothesis that epigenetics may be a potential
barrier to achieving the maximum benefit of T-cell based immunotherapies.

Keywords
Immune checkpoint blockade; chimeric antigen receptor T cells; T cell exhaustion; epigenetics;
histone modification; DNA methylation
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T-Cell Exhaustion
Recent insights into T-cell differentiation have resulted in considerable advances in our
ability to exploit the host immune response to treat chronic infections and cancer. Many of
these new developments have been borne out of studies aimed at gaining a deeper
understanding of the mechanisms involved in a T cell’s ability to acquire stable functional
properties. Upon antigen exposure, naive antigen-specific CD8+ T cells undergo a
differentiation program that promotes their clonal expansion and development into highly
functional cytotoxic effector T cells that can traffic to, and kill pathogen-infected or tumor
cells. Once the antigen is cleared, the majority of effector T cells die with only a subset of
surviving cells differentiating into long-lived memory cells that can confer long-term
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protection against pathogen rechallenge [1]. However, if the source of antigen is not cleared,
the combination of prolonged T-cell receptor (TCR) stimulation and exposure to
inflammatory signals results in progressive repression of effector function, termed T-cell
exhaustion (see Glossary). Originally discovered using the mouse model of chronic

*
Correspondence: [email protected] (B.A. Youngblood).
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 Ghoneim et al. Page 2

lymphocytic choriomeningitis virus (LCMV) infection [2–4], the concept of T-cell

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exhaustion has now emerged as a general mechanism for restricting T-cell effector functions
during infection with chronic viruses and the development of several human cancers [5–9].
Initial investigations into the mechanisms involved in repression of the effector response
focused on changes in gene regulation among functional memory and exhausted T cells.
Genome-wide gene expression profiles of effector and functional memory cells in
comparison with exhausted CD8+ T cells revealed that CD8+ T cells acquire a distinct
transcriptional program during the development of exhaustion. Specifically, it was reported
that functionally exhausted CD8+ T cells maintained an altered metabolism, a defect in
proliferation, and a high level of expression of multiple inhibitory receptors (IRs),
including programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte antigen 4
(CTLA-4), and T cell immunoglobulin mucin receptor 3 (Tim-3) [10–13]. Because
exhausted T cells sustain expression of IRs, several investigators sought to determine their
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role in suppressing T-cell function during the development of exhaustion and to develop
therapeutic strategies limiting IR signaling. It is now believed that T-cell exhaustion can be
at least partially overcome with the administration of monoclonal antibodies (mAbs) against
surface IRs, supporting the idea that effector function can be restored by blocking sustained
inhibitory signals [11, 14–16]. Harnessing the therapeutic potential of host CD8+ T cells to
treat various diseases has also been extended through genetic engineering of human T cells,
either by cloning of endogenous TCR receptors or, by the introduction of a chimeric
antigen receptor (CAR) that recognizes antigen presenting cells. Indeed, recent evidence
suggests that the specificity of a T cell can be redirected to recognize and eliminate tumors,
with dramatic clinical responses seen in patients with metastatic melanoma [17] and B-cell
malignancies [18, 19]. Additionally, such genetic modification strategies appear to be a
promising new immunotherapeutic approach for the treatment of more types of cancer.
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Below we discuss the most recent advancements in T cell-based immunotherapy and the
current limitations and challenges of using Immune checkpoint blockade (ICB) and
adoptive T cell therapy (ACT) approaches.

Immune Checkpoint Blockade Therapy

Many clinical advances in cancer immunotherapy have been based on the finding that
blockade of immune IR signaling on the pool of exhausted T cells can reestablish effector
function and promote antitumor activity. The emergence of ICB therapy as a strategy to treat
cancer was founded on the ability of mAbs to block the interaction between IRs -- such as
CTLA-4 and PD-1-- on exhausted T cells, as well as their corresponding ligands on antigen-
presenting cells [20]. Blocking such inhibitory interactions promotes expansion and recovery
of effector function in exhausted T cells, leading to viral control or tumor regression in
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preclinical models and cancer patients [11, 14, 21]. Indeed, the success of ICB therapy has
been evident for treating a growing list of cancer types, including melanoma, non-small cell
lung carcinoma, Hodgkin’s lymphoma, head and neck cancer, mismatch repair-deficient
tumors, renal cell carcinoma, ovarian, and bladder cancers, highlighting the fact that such
therapeutic strategies are a promising frontier in cancer therapy [20]. The first FDA
approved IR-based therapy, ipilimumab, is a mAb targeting CTLA-4. Its use was shown to
improve the clinical outcome of patients with advanced melanoma, with a survival rate of at

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least 3 years in approximately 20% of patients receiving the drug [20]. The clinical success
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of ipilimumab paved the way for extending ICB strategies to additional IRs (e.g., PD-1) and
their corresponding ligands. Recently, the FDA approved mAbs to block the interaction
between PD-1 and its ligand, programmed death ligand-1 (PD-L1). Anti-PD-1 mAb drugs
(nivolumab and pembrolizumab), and anti-PD-L1 mAb drug (atezolizumab) have
improved objective response rates and decreased toxicities in several types of cancer,
including advanced melanoma, non-small-cell lung carcinoma, and renal cell carcinoma
[22]. Although some patients exhibit partial or complete responses to ICB therapy, many
patients remain unresponsive [23]. Additionally, a recent phase I clinical trial
(NCT01295827) reported that while pembrolizumab was initially efficacious in patients with
advanced melanoma, approximately 30% developed resistance and subsequent tumor relapse
within 2 years [24]. Because the efficacy of ICB therapy is currently limited, it is important
to understand the molecular barriers preventing treatment success.
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Challenges of Immune Checkpoint Blockade Therapy

Despite unprecedented clinical success, a large proportion of patients with cancer fail to
present an objective response following ICB therapy [20]. In addition, some patients initially
responsive to ICB treatment, experience relapses [24]. These failures in ICB therapy
highlight the need to better dissect the potential mechanisms of resistance, and/or the lack of
durable responses in some patients. Among the potential challenges to achieving successful
ICB responses are (1) the absence of host tumor-specific T cells due to negative selection or
physical deletion by cell death of severely exhausted cells; (2) an inability to reactivate the
exhausted T cells that are present within the immunosuppressive tumor microenvironment
(TME); and/or (3) a failure of re-activated cells to expand and sustain their ability to express
effector cytokines and exert tumor-killing activity. These limitations have prompted
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investigators to question which subset of exhausted T cells might be responding to ICB.

Furthermore, several studies are now moving towards addressing whether ICB-responding T
cells acquire a specific gene expression program that leads to the generation of long-lived
memory T cells, or, whether they revert back to an exhausted state.

Building upon the initial insight into the role IRs play in T-cell exhaustion, further
phenotypic and transcriptional characterization of exhausted T cells have resulted in the
discovery of a functional heterogeneity among the pool of exhausted T cells. For instance,
the cumulative coexpression of multiple IRs, including PD-1 and Tim-3, has been found to
be associated with an increase in the degree of T-cell exhaustion, as indicated by the
progressive inability of the more exhausted cells to produce effector cytokines (IL-2, TNFα,
and eventually IFNγ), and the gradual loss of their proliferative capacity (Key Figure, Figure
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1A) [16, 25, 26]. Key experiments defining the subsets of exhausted T cells and their
lineages in a murine model of chronic LCMV infection and in individuals with chronic or
resolved Hepatitis C Virus (HCV) infections have revealed the differential expression of T-
box transcription factors T-bet and Eomesodermin (Eomes) in these cells [27]. These
studies showed that a progenitor subset of exhausted cells, phenotypically characterized as
T-bethigh–Eomeslow, retained the potential for ICB-mediated rejuvenation [27]. By contrast,
terminally differentiated exhausted T cells (defined as expressing low T-bet and high Eomes)
exhibited poor ICB responsiveness (Key Figure, Figure 1) [27]. Thus, severely exhausted T

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cells (terminally differentiated) have been deemed as refractory to ICB-mediated

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rejuvenation (Key Figure, Figure 1B) [28]. In an effort to further define which subset(s) of
exhausted T cells respond to ICB, two recent studies using the mouse model of chronic
LCMV infection reported that the transcription factor Tcf1 (Tcf7 gene) was critical in
maintaining the ICB-responsive subset of exhausted T cells expanding during PD-1
blockade [26, 29]. Another group reported a similar finding but ascribed the proliferative
burst to a less differentiated CD8+ T cell subset [26], presumably deriving more recently
from the thymus. Broadly, each of these studies conclude that a subset of CD8+ T cells, not
fully committed to the exhausted fate, provide the proliferative T-cell burst following ICB
[30]. However, it remains unclear whether such lack of responsiveness to ICB is due to an
inability to rescue the functional response from severely exhausted T cells or whether it is
due to the decreased number of antigen-specific T cells resulting from cell death. These data
raise new questions about both the current success and failure associated with the clinical
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use of ICB, as well as whether new therapeutic approaches can be designed to reactivate and
enlist fully exhausted T cells into fighting tumors.

Gene expression programs associated with the progressive development of T-cell exhaustion
can become reinforced, meaning that multiple exhaustion-associated transcriptional features,
such as upregulated PD-1 expression and poor ICB-responsiveness, are stably maintained
even after antigen withdrawal. Indeed, this has been demonstrated by adoptive transfer of
exhausted T cells into an antigen-free environment, or by showing viral load control ,as in
the case of HIV-1 elite controllers or patients exhibiting successful viral control following
antiretroviral treatment [31–36]. These findings suggest that a stable cell-intrinsic
mechanism is acquired in exhausted T cells (Key Figure, Figure 1). Stabilization of T-cell
exhaustion programs may not only limit the rejuvenation capacity of exhausted T cells
during ICB treatment but also restrict the ability of rejuvenated cells to generate long-lived
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antitumor immunity after tumor clearance, and this may be linked to the observed lack of
protection against tumor relapses in some treated patients. Although several clinical trials
have suggested that the successful objective response following ICB therapy is durable [20,
24, 37, 38], tumor relapses occur in approximately one-third of patients responding to PD-1
blockade, as stated earlier [24]. Such findings highlight the crucial need to understand the
difference between acquiring a certain degree of longevity of tumor control after ICB
therapy and the requirements for generating tumor-specific T-cell memory [39–41]. If indeed
there are heritable “cell division–transmissible” programs repressing the expression of genes
regulating effector function, expansion potential, and metabolic fitness of exhausted T cells,
in addition to sustaining upregulated expression of IRs, it is plausible to hypothesize that
these programs may ultimately terminate the function of the expanded pool of T cells
following ICB treatment. Thus, it is possible that ICB-responsive T cells would then be
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incompetent to generate durable host immunity against the same tumor antigens. This
potential contribution of cell-intrinsic barriers to ICB failures makes it necessary for future
studies to dissect the molecular mechanisms regulating the maintenance of an exhaustion-
associated transcriptional program.

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Does Epigenetic Programming Stabilize T-Cell Exhaustion?

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Transcriptional programming during cellular differentiation is regulated through coordinated

recruitment and/or eviction of transcription factors [42]. The long-term maintenance of
acquired transcriptional states is often mediated by stable covalent changes, known as
“epigenetic modifications,” to DNA and histone proteins [43]. Epigenetic changes
modulate chromatin structure, which regulates the accessibility of transcription factors to
specific DNA sequences. In general, the acquisition of DNA methylation or “repressive”
histone marks at gene-regulatory regions is coupled with transcriptional repression. By
contrast, DNA demethylation or the acquisition of “permissive” histone marks is coupled
with an active or poised state of gene expression (Key Figure, Figure 1B) [44]. Acquired
epigenetic modifications can be faithfully propagated during cell division after the absence
of the initial triggers of epigenetic programming. Such stability of epigenetic programs is
fundamental to establishing and maintaining “transcriptional memory” of the environmental
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stimuli and differentiation cues that a cell transiently experiences [45]. Mounting evidence
indicates that key developmental and functional aspects of effector and memory CD8+ T
cells are coupled to subset-specific epigenetic modifications, including DNA-methylation
programs and histone modifications [46–54] (Box 2).

BOX 2

Epigenetic Regulation of CD8+ T-Cell Development and Differentiation

Growing evidence suggests that dynamic epigenetic reprogramming is coupled to T-cell
development and differentiation. A recent study reported that a stable DNA-methylation
program at the proximal enhancer of the cd4 locus (CD4) is essential to enforce cd4 gene
silencing in CD8+ T cells, whereas CD4+ T cells lack this program, suggesting an
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important role of epigenetic programming in securing cell-identity during T-cell

development [49]. Genome-wide DNA-methylation profiling of naïve and effector virus-
specific CD8+ T cells during acute LCMV infection have revealed that many promoters
and putative regulatory cis-elements of effector genes become demethylated during
effector T-cell differentiation, along with differential methylation programming at naïve/
effector-specific transcription factor binding sites [47]. In another study using a mouse
model of influenza infection, mapping histone modifications in antigen-specific CD8+ T-
cells showed that the loci of effector genes acquired permissive histone marks during
effector and memory T-cell differentiation [52]. Collectively, these data provide evidence
that changes in epigenetic programs are coupled with transcriptional reprogramming
linked to CD8+ T-cell effector function and fate decision.
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The development of T-cell exhaustion may also be regulated by stable epigenetic

programming. It was recently reported that the Pdcd1 locus (PD-1 gene) in antigen-specific
CD8+ T cells undergoes DNA-demethylation programming during the effector stage of the
immune response after viral infection, as in the case of LCMV infection in mice [33]. While
functional memory T cells, generated after acute antigen exposure, were able to remethylate
the regulatory regions in the Pdcd1 locus, the demethylation epigenetic program was
preserved after chronic antigen exposure and coupled to the sustained expression of PD-1 on
exhausted T cells [33, 34]. Even after antigen levels were diminished -- as observed in

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HIV-1 elite controllers or in patients that have successfully controlled HIV-1 infection
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following antiretroviral treatment --the demethylation status of the Pdcd1 locus was
maintained in HIV-1-specific CD8+ T cells. These data suggest that the stability of
exhaustion-associated transcriptional programming is regulated by cell-intrinsic
mechanisms, possibly through fixation of acquired epigenetic programs [34]. In a recent set
of experiments, it was shown that antigen-specific CD8+ T cells isolated from mice during
the effector stage of chronic LCMV infection, and adoptively transferred into an antigen-free
environment, retained a stable exhaustion-associated demethylation program in the promoter
of the Pdcd1locus, even after the T cells were rested from TCR stimulation for 1 month in
vivo. Importantly, these cells were still partially resistant to therapeutic PD-1 blockade
during a recall LCMV response in the infected mice [35]. These findings further support the
idea that exhaustion-associated transcriptional programs are fixed, even after antigen
clearance. However, given that epigenetic modifications may become reinforced, it is
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unclear how these programs are stabilized, and whether they can provide a cell-intrinsic
resistance mechanism against ICB-mediated rejuvenation of T cells (Key Figure, Figure 1).
The answer to these questions would translate into identifying novel epigenetic targets in
exhausted T cells, aiming to extend the dynamic trajectory of the success in cancer
immunotherapy (Box 3).

BOX 3

Combinatorial Immunotherapies and Epigenetic Reprogramming

Approaches
Despite the significant benefit of ICB monotherapy in some patients with cancer, many
patients fail to show successful and/or durable clinical response. Therefore, various
combinations of treatment modalities with ICB are now under clinical investigation for
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several cancer types. Growing evidence from animal tumor models suggest that
combined blockade of multiple IRs (e.g., PD-1, CTLA-4, Tim-3, LAG-3) enhances the
rejuvenation of exhausted antitumor T cells [15, 74, 75]. These promising preclinical
studies have prompted investigating whether clinical combination of immunotherapies
that target non-redundant inhibitory signaling pathways could enhance the efficacy of
ICB and/or successfully treat a larger proportion of patients. Recent clinical trials in
patients with metastatic melanoma showed that treatment with nivolumab plus
ipilimumab resulted in a greater objective response rate and longer progression-free
survival compared to monotherapy [76, 77]. This has now fueled more clinical trials to
test this combinatorial strategy in other types of cancer.

Epigenetic therapy holds a great potential for combinatorial approaches with ICB
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therapy. Although the rationale for epigenetic therapy has been widely founded on
extensive studies of epigenetic abnormalities in tumor cells driving their transformation --
such as epigenetic silencing of tumor-suppressor genes, emerging evidence suggests a
mechanistic immune evasion role of epigenetic reprogramming in tumor cells by
silencing immune-related genes and/or tumor-specific antigens. Studies using murine
models of ovarian cancer showed that production of Th1-type chemokines (e.g., CXCL9,
CXCL10) in tumors is epigenetically repressed and associated with poor T-cell
infiltration in the tumor. Treatment with epigenetic modulators, such as DNA- or EZH2-

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methyltransferase inhibitors, enhanced CXCL9 and CXCL10 chemokine production and

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T-cell trafficking into the tumor and potentiated tumor regression following ICB therapy
[78, 79]. Another study using a melanoma mouse model showed that treatment with a
DNA demethylating agent induced upregulation of a type I IFN-gene signature and
augmented tumor responses to anti-CTLA-4 mAb treatment [73]. Additionally, several
studies suggested that primary sensitization of tumor cells with DNA demethylating
agents would enhance the clinical benefit of ICB by improving tumor-antigen
presentation and/or upregulating PD-L1 expression on tumor cells (reviewed in [72]).
Given the plausible rationale for combining epigenetic and immune therapies, the
concept of epigenetic stabilization of a T-cell exhaustion program -- and its potential
contribution to restraining ICB efficacy -- may establish another platform for identifying
novel epigenetic targets in exhausted T cells. It may also be possible to better identify the
impaired ability of rejuvenated T cells to generate long-lived memory, providing a
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rationale for developing new combinatorial epigenetic modulator strategies to treat

cancer.

The progressive development of T-cell exhaustion described above is largely associated with
corresponding changes in gene expression [26, 30]. Such progressive changes in
transcriptional programming suggest that the switch from ICB-responsive to unresponsive
may be associated with a stable epigenetic state (Key Figure, Figure 1A). Although the
potential regulatory role of epigenetic programming in reinforcing terminal differentiation of
exhausted T cells remains to be fully explored, one explanation for the restricted
rejuvenation capacity of fully exhausted T cells might be that progressively acquired
repressive epigenetic modifications -- through methylation of CpG sites in gene regulatory
regions and/or deposition of repressive histone marks -- reinforce silencing of genes
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involved in effector function, proliferation, and metabolic fitness (Key Figure, Figure 1B).
As such, fully exhausted T cells could be epigenetically restrained and, consequently,
committed to an ICB-refractory state of differentiation (Key Figure, Figure 1B). Although
speculative, such ideas do merit further investigation. For example, characterization of
histone modifications and DNA methylation changes during the development of T-cell
exhaustion and following ICB may be informative in identifying stable exhaustion-
associated programs. These proposed studies and others may help design novel approaches
to manipulate the epigenome of exhausted T cells.

Irrefutably, ICB therapy can enhance antitumor immune responses by circumventing some
of the extrinsic (employed by the tumor) and intrinsic (implicit to T cells) mechanisms that
generally facilitate T-cell exhaustion. However, this treatment modality may be less
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efficacious for patients whose pool of antitumor T cells contains a disproportionate

frequency of terminally differentiated exhausted T cells. Alternatively, it may also be largely
ineffective in patients failing to establish immunological antitumor memory following ICB
therapy, which may be accompanied by higher rates of tumor relapse. In order to broaden
the scope of treatment options for patients who relapse or fail to exhibit an objective
response following ICB therapy, a viable alternative may be to expand the pool of tumor-
specific T cells by genetically engineering human T cells. In the following section, we

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describe how the use of chimeric antigen receptors (CARs) shows promise in overcoming
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some of the shortcomings of ICB therapy.

Chimeric Antigen Receptor T Cells to Overcome Exhaustion

The field of genetically engineering T cells is one of the newest and fastest growing
branches of cancer immunotherapy. Interest in gene-transfer approaches focus on the ability
of these therapies to bypass negative selection and other mechanisms of immune tolerance
resulting in an increased repertoire of tumor-specific T cells [55]. Recent advances in
genetic engineering enable T-cell transduction with CARs so that they can be redirected to
and activated by tumor antigens. CAR T cells combine single-chain variable fragments
(antibody-derived extracellular domain) with T cell–signaling domains (intracellular
domains) to induce T-cell activation upon antigen binding. Although the field of adoptive T-
cell immunotherapy using transduced T cells with antitumor receptors is still in its infancy,
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clinical trials are underway and have shown clinically significant anti-tumor responses in
chronic lymphocytic leukemia, B-cell lymphoma, melanoma, and neuroblastoma [56]. The
benefits of CAR-based therapies stem from four key abilities of CAR T cells: (1) targeting
tumor antigens with specificity, (2) targeting surface antigens in a major histocompatibility
complex (MHC)-unrestricted manner, (3) harnessing the cytolytic potential of cytotoxic T
lymphocytes, and (4) inducing the expansion and persistence of CAR T cells for long-term
responses. Historically, the field of adoptive T cell therapy was pioneered by Steven
Rosenberg and colleagues, who harnessed the tumor killing activity of autologous
lymphocytes from patients’ tumors (referred to as tumor-infiltrating lymphocytes; TILs).
They developed methods for the isolation and culture of TILs that could be reinfused into
patients following preconditioning with lymphodepleting regimens [17]. As has been shown
in clinical trials evaluating the efficacy of adoptive transfer of TILs and the use of mAbs to
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block the inhibitory signals from co-IRs (e.g., ipilumimab for CTLA-4, and nivolumab or
pembrolizumab for PD-1) on T cells, emerging studies suggest that, as a single modality
treatment, CAR T cells are efficacious in many settings, all but for a minor subset of
patients, [A1] This may be partially due to the redundant regulatory mechanisms that are
inherent to all T cells, in addition to our limitations in understanding which T-cell
characteristics are essential and predictive of antitumor activity.

Chimeric Antigen Receptor Limitations and Strategies for Improvement

Current clinical protocols use polyclonal T-cell populations as a starting point for the
transduction of CAR-modified T cells, but few studies have addressed whether an optimal T-
cell subtype and/or manufacturing system should be followed when generating CAR T cells.
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Recent evidence from clinical reports suggests that the starting cell population, method of
expansion, tumor type being treated, and design/engineering approach can all greatly affect
the persistence and function of CAR T cells [19, 57]. CAR T-cell persistence most likely
plays a major role in determining the efficacy of the anti-tumor response, but due to the lack
of consensus on the design of clinical trials among U.S. research institutions, it is difficult to
pinpoint which of these factors will contribute to long-term memory against tumor-specific
antigens. CAR T cells designed with the 4-1BB–costimulatory domain (as opposed to
CD28 signaling components) have been shown to display superior antitumor activity,

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characterized by reduced tumor burden, decreased exhaustion during ex vivo expansion, and
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increased persistence in vivo in xenograft mouse models [58, 59]. Furthermore, in a recent
clinical trial utilizing CD19 CAR T cells containing the 4-1BB signaling domain to treat
patients with refractory CD19+ leukemia and lymphoma, prolonged persistence of CAR T
cells was observed (out to 2 years); this exceeded the persistence observed in a similar study
utilizing CD19+ CAR T cells containing the CD28 signaling domain [60, 61]. However, the
differentiation status of transduced T cells and the conditioning regimen a patient undergoes
prior to reintroducing the engineered T cells (e.g., addition or exclusion of alkylating agents,
fludarabine, total body irradiation) can also profoundly affect the persistence and cytotoxic
potential of CAR T cells [62] (Figure 2). In fact, several studies have shown that
lymphodepletion, either by total-body irradiation or cytotoxic drug administration, can
create a niche for transferred T-cell expansion and induce homeostatic proliferation due to
the accumulation of homeostatic cytokines (e.g., IL-7 and IL-15) [56, 63–65] . Moreover, in
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vitro culture conditions that stimulate (via anti-CD3/CD28 or stimulator cells) and expand
(via cytokines, such as IL-2) T-cell populations, can alter the differentiation status and
effector function of such engineered T cells (Figure 2). Although it is unclear whether
specific frequencies of infused CAR T cell subsets (e.g., naïve, TN; stem cell memory,
TSCM; effector, TEFF; central memory, TCM; or effector memory TEM) have greater clinical
efficacy in the context of adoptive T cell therapy, the frequency of CAR-modified T cells
with a TSCM-like phenotype positively correlates with their in vivo expansion in patients
with B-cell malignancies [66]. Thus, since the differentiation status and effector function of
such engineered T cells markedly affect antitumor efficacy, engraftment, and T-cell
expansion, efforts are increasing to preserve a more primitive (i.e., less differentiated) T-cell
pool and increase the benefits afforded by adoptive T cell therapy (Figure 2) [67, 68].

In addition to cell-intrinsic factors, several extrinsic factors limit the clinical efficacy
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afforded by CAR T cells. For instance, some extrinsic challenges are attributed on the one
hand to suppressive components of the TME, such as inhibitory cytokines (e.g., TGF-β and
IL-10), inhibitory cell types (e.g., regulatory T cells (Tregs) and myeloid-derived
suppressor cells (MDSCs)), as well as inhibitory surface proteins (e.g., PD-L1 and PD-L2
signaling pathways [69, 70]. On the other hand, other challenges are inherent to the nature
and tissue-specificity of different solid tumors, which can greatly limit the quantity and
quality of tumor-specific antigens being targeted, due to an increased likelihood of off-target
toxicities in these types of tumors [69, 70]. One of the greatest obstacles with CAR T cell
therapy to be overcome is the identification of suitable targets. This is problematic because
of the lack of technological resources to identify novel tumor-specific antigens, as well as
the fact that several candidate antigens are also expressed in vital organs. Importantly,
several approaches to overcome these limitations are actively being explored by various
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research institutions. Indeed, attempts are being made to modulate the epigenetic profile of
exhausted T cells exposed to chronic levels of antigen (Key Figure, Figure 1). For instance, a
recent study reported that exhausted CD8+ T cells isolated from chronic LCMV-infected
mice presented reduced levels of the permissive diacetylated histone H3 epigenetic mark
compared to effector or memory CD8+ T cells [71]. In vitro treatment of exhausted T cells
with an HDAC inhibitor restored diacetylated histone H3 levels, which were associated with
improved CD8+ T cell potential to express effector cytokines upon antigen-stimulation.

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Interestingly, treated T cells could develop into functional memory cells upon adoptive
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transfer into an antigen-free environment [71]. Of note, studies are currently ongoing to
modulate the intracellular signaling domains within a same CAR; these involve CAR T cells
harboring third generation CARs that combine CD3ζ, CD28, and 4-1BB signaling domains,
yielding promising results. Thus, it will be interesting to follow how these exciting
investigations unfold.

Concluding Remarks
There are various instances of ICB success to treat different forms of cancer. In addition,
several promising preclinical results and emerging models of immunotherapeutic approaches
such as CAR therapy have been reported. Consequently, there is a sound rationale for
combining treatment modalities to induce broader antitumor responses. Although the
number of potential immunotherapeutic combinations is almost endless, there is growing
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evidence of the critical role of epigenetic programming in cell transformation and

malignancy; this area has attracted special attention in the design of strategies that combine
epigenetic modulators and ICB therapy [72]. The exact mechanisms by which epigenetic
sensitization improves immunotherapy are still unknown, but several hypotheses are being
tested, including that epigenetic therapy (1) increases the expression of tumor-specific
antigens (such as cancer testis antigens), especially important in cancers that alter antigen
processing to evade recognition by the immune system; (2) inhibits the suppressive function
of the TME, by altering epigenetic programs involved in immune evasion, either in the
tumor (Box 3), or in immunosuppressive cell types (e.g., Tregs and MDSCs); and/or (3)
positively modulates directly and/or indirectly the development and effector function of
cytotoxic immune cells, ultimately increasing the killing of transformed cells. Interestingly,
treatment with DNA demethylating agents was recently shown to enhance the response to
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anti-CTLA-4 treatment in a melanoma mouse model; this was achieved by inducing re-
expression of endogenous retrovirus dsRNA that triggers type I interferon immune responses
in cancer cells [73].

While highly compelling, the specific role of exhaustion-associated epigenetic programs on

the TILs’ responsiveness to ICB, and their ability to generate antitumor memory, remains
largely unexplored. A better understanding of the fundamental mechanisms involved in the
development of T-cell exhaustion is urgently needed to enhance the therapeutic efficacy of
ICB and combination therapies to treat malignancies. This information will also very likely
contribute to the optimization of existing and newly emerging CAR T cell strategies.
Undoubtedly, the recent success of T cell-based immunotherapies is the result of a brilliant
marriage between clinical and basic research. While extremely exciting, it is likely that we
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have just scratched the surface of the potential that T-cell immunotherapy has to offer (see
Outstanding Questions and Box 1). It will be of great interest to see how resolution of the
questions outlined above furthers the development of this exploding field.

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 Ghoneim et al. Page 11

BOX 1
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The Clinician’s Corner

• Exhaustion of persistently antigen-stimulated CD8+ T cells, as in the
case of cancer or chronic infections, is associated with a unique
transcriptional program characterized by upregulated expression of
surface IRs (e.g., PD-1, CTLA-4) and impaired effector functions.

• Rejuvenation of exhausted anti-tumor T cells by blocking their surface

IRs (immune checkpoint blockade; ICB) has revolutionized the field of
cancer immunotherapy with unprecedented success in treating multiple
types of cancer.

• Many patients with cancer fail to show clinical benefit following ICB
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and some treated patients experience tumor relapses, raising questions

on the molecular mechanisms regulating T cell responsiveness to ICB
therapy.

• Growing evidence indicates that partially exhausted T cells are the

main responders to ICB, whereas severely exhausted cells exhibit poor
ICB-responsiveness and stably retain many of the exhaustion-
associated transcriptional features even after antigen clearance,
suggesting cell-intrinsic barriers in exhausted T cells to ICB therapy.

• Epigenetic stabilization of exhaustion-associated transcriptional

programs in CD8+ T cells may remain as a potential cell-intrinsic
barrier that restrains their ICB-responsiveness and possibly enforces
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reversion of rejuvenated T cells into the exhausted state. Identifying

these epigenetic programs will facilitate development of novel
therapeutic strategies to manipulate these epigenetic targets. Preclinical
and ongoing clinical studies show promising synergistic potential of
standard epigenetic therapies in combination with T cell-based
immunotherapy.

Outstanding Questions
• What cell-intrinsic mechanisms reinforce the stability of transcriptional
repression during the development of T cell exhaustion?

• Are specific epigenetic programs progressively acquired during the

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terminal differentiation of exhausted T cells?

• Does this exhaustion-associated epigenetic programming restrict the

therapeutic efficacy of immune checkpoint blockade?

• How are combination therapies independently and mutually influenced

by epigenetic changes in a cell? Conversely, how do combination

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 Ghoneim et al. Page 12

therapies independently and mutually influence epigenetic changes in a

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cell? How might these changes affect treatment outcomes?

• Does the fixation of exhaustion-associated epigenetic programs restrain

the ability of rejuvenated T cells to generate long-lived immunity after
ICB therapy?

• Can developmental reprogramming of terminally differentiated

exhausted T cells restore their responsiveness following ICB treatment?

• Which is the best human T cell subset for adoptive T cell therapy,
taking into account balanced attributes for optimum activation, in vitro
expansion, killing capacity, and in vivo persistence?

• Can epigenetic reprogramming of CAR T cells during in vitro

expansion modulate their long-term potential for in vivo persistence
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and killing activity?

Acknowledgments
We thank Dr. Nisha Badders for scientific editing (St. Jude Children’s Research Hospital). This work was supported
by the National Institutes of Health grant 1R01AI114442 (to B.Y.), R01 AI107625 (to P.T.) and ALSAC (to B.Y.
and P.T.).

Glossary
T-cell exhaustion
Dysfunctional state acquired by T cells experiencing persistent TCR stimulation
characterized by upregulated expression of immune inhibitory receptors (e.g., PD-1,
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CTLA-4, Tim-3), impaired effector function, poor proliferation, and metabolic defects.

Murine lymphocytic choriomeningitis virus (LCMV) infections

Two strains of LCMV—Armstrong and Clone 13—differing in two amino acids only, have
been widely used to study host immune responses during acute vs. chronic virus infection.
In mice, the Armstrong strain is cleared within 8–10 days post-infection, whereas the Clone
13 strain causes a more disseminated chronic infection with higher viral titers that persist for
a prolonged period of time.

Inhibitory receptors (IRs)

Various immune checkpoints are inducibly expressed on the surface of activated T
lymphocytes to tightly control T cell function, not only during exhaustion, but also during
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acute T cell activation, differentiation, and autoimmune responses. Immune IRs, such as
CTLA-4 and PD-1, negatively regulate T cell function using different regulatory
mechanisms, including competition against co-stimulatory receptors for the binding of a
common ligand; interfering with activating signals downstream of the TCR; or upregulating
inhibitory genes involved in T cell suppression.

Immune checkpoint blockade (ICB)

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 Ghoneim et al. Page 13

Therapeutic strategy to block inhibitory signaling from immune IRs and their ligands.
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Several mAbs blocking CTLA-4 or PD-1—PD-L1 inhibitory interactions (generally on T

cells) have recently gained FDA approval for the treatment of various malignancies, and
remain some of the most promising anti-oncogenic treatment strategies.

Chimeric antigen receptors (CARs)

Genetically engineered receptors designed to combine both antigen-specificity and T cell-
activating properties in a single molecule. They can be expressed on human T cells to target
specific tumor antigens.

Ipilimumab
Fully human mAb that blocks the surface inhibitory receptor CTLA-4 (Yervoy, Bristol-
Myers Squibb Company). It was approved by the FDA in March 2011 to treat metastatic
melanoma, and the approval was extended in 2015 to treat surgically resectable melanoma.
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Nivolumab
Fully human mAb that blocks the surface inhibitory receptor PD-1 (Opdivo, Bristol-Myers
Squibb Company). It was approved by the FDA in December 2014 to treat metastatic
melanoma. Growing clinical evidence of its efficacy in several types cancer resulted in
extending the FDA approval of nivolumab to also treat stage IV non-small cell lung cancer
(NSCLC; squamous and adenocarcinoma), renal cell carcinoma, Hodgkin’s lymphoma in
2015 and 2016. A combination of nivolumab with ipilimumab has been approved to treat
unresectable or metastatic melanoma.

Pembrolizumab
Humanized mAb that blocks the PD-1 receptor (Keytruda, Merck Sharp and Dohme Corp.).
It was approved by the FDA in September 2014 to treat metastatic melanoma, then extended
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to treat stage IV NSCLC and recurrent or metastatic head and neck squamous cell carcinoma
in 2015 and 2016.

Atezolizumab
Humanized mAb that block the PD-L1 ligand (Tecentriq, Genentech Inc.). It is the first anti-
PD-L1 product to be approved by the FDA in 2016 and has been indicated to treat the most
common type of bladder cancer, called urothelial carcinoma.

Antigen-presenting cells
Heterogeneous populations of immune cells that can display antigen-derived epitopes over
their surface after being complexed with the major histocompatibility complex proteins
(MHC) class I or II. These epitopes can be recognized by T cells using their antigen-specific
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T-cell receptor (TCR). Intracellular pathogen-infected or transformed cells can also present
pathogen- or tumor-derived epitopes complexed with MHC class I proteins to be recognized
by CD8+ T cells.

T-bet and Eomesodermin

T-bet (T-box expressed in T cells) and Eomes (eomesodermin): T-box transcription factors
that play regulatory roles in the development and functions of immune cells including T
lymphocytes and NK cells. T-bet was originally identified as a master lineage-defining

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 Ghoneim et al. Page 14

transcription factor that promotes the differentiation of mouse Th1 CD4+ T cells while
inhibiting Th2 and Th17 CD4+ T cell differentiation. Additionally, upregulated expression of
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T-bet and Eomes is critical for regulating effector and effector memory CD8+ T cell
differentiation. Differential expression of T-bet and Eomes in exhausted CD8+ T cells
defined the functional heterogeneity in exhausted T cell populations.

HIV-elite controllers
A rare subset of patients with human immunodeficiency virus (HIV) infection who are able
to control the viral burden, maintaining undetectable viremia without any treatment (in
conventional assays).

Epigenetic modifications
Covalent heritable modifications that are acquired on histone tail proteins or DNA without
changing DNA sequences. They regulate gene expression programs either through repressive
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epigenetic changes that induce gene silencing, or permissive changes that promote active or
poised states of gene expression. Because such changes are cell division-transmissible, they
play critical regulatory roles in cell differentiation and cell identity maintenance.

Tumor microenvironment (TME)

Tissue environment that surrounds and feeds tumor cells and includes immune cells, other
non-transformed cells, secreted molecules, extracellular matrix, and blood vessels. Tumor
cells can dynamically change their microenvironment and TME can affect the growth and
spread of a tumor.

Tumor-infiltrating lymphocytes (TILs)

Lymphocytes that are recruited to, and infiltrate the TME to control tumor growth. These
cells have been exploited for adoptive T cell therapy by isolating them from patients’
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tumors, and expanding them ex vivo before being reinfused back into patients.

Regulatory T cells (Tregs)

Subset of CD4+ T cells that suppress the effector function of other T cell subsets. One role
consists of conferring protection against autoimmune responses by maintaining tolerance to
self-antigens. They also control the development of immunopathologies in the host by
modulating immune responses to non-self-antigens.

Myeloid-derived suppressor cells (MDSCs)

Heterogeneous myeloid cell populations found in tumor, infection, or inflammatory sites;
they are able to suppress T cell functions.

Epigenetic modulators
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Chemical compounds that can modulate disease-related epigenetic abnormalities by

targeting regulators of epigenetic processes including epigenetic writers (e.g., DNA
methyltransferases—DNMTs, histone methyltransferases—HMTs, histone
acetyltransferases—HATs), epigenetic readers (e.g., bromodomain-containing proteins), and
epigenetic erasers (e.g., histone deacetylases—HDACs, lysine demethylases—KDMs). For
example, 5-azacytidine and 5-aza-2′-deoxycytidine are FDA-approved DNA demethylating
agents (inhibitors of DNMTs) to treat some hematologic cancers, such as myelodysplastic

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 Ghoneim et al. Page 15

syndrome. HDAC inhibitors are also approved for the treatment of cutaneous and peripheral
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T-cell lymphoma.

Adoptive T cell therapy

Therapeutic approach that depends on isolating autologous TILs from a patient’s tumor and
expanding them ex vivo to achieve greater numbers of TIls. Expanded TILs are reinfused
back into the patient after preconditioning with lymphodepleting regimens.

Negative selection
the process by which lymphocytes bearing receptors that react too strongly with self-
antigens are eliminated from the repertoire in order to maintain self-tolerance.

4-1BB-co-stimulatory domain
The portion of the CAR’s cytoplasmic tail that includes the addition of the CD137 (also
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known as 4-1BB) costimulatory protein receptor’s signaling domain in order to enhance cell
signaling and antitumor potency.

Homeostatic proliferation
Proliferative response of lymphocytes induced by a lymphopenic environment to maintain a
constant level of lymphocytes.

Allogeneic feeder cell lines

Cell lines (stimulators) that are used to stimulate the activation and expansion of responder T
cells due to their expression of foreign histocompatibility antigens (e.g., MHC class I or
MHC class II molecules).

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Trends
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• Immune checkpoint blockade-mediated expansion of partially

exhausted CD8+ T cell has resulted in unprecedented clinical responses
in multiple types of cancer.

• Multiple hallmarks of transcriptional programming during the

development of T cell exhaustion are stably inherited, even after the
level of specific antigen has diminished.

• Stability of a permissive transcriptional program at the PD-1 promoter

in exhausted CD8+ T cells is coupled to stable epigenetic
modifications.

• Exhausted T cells remain partially refractory to immune checkpoint

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blockade therapy, even after resting in an antigen-free environment.

• Current CAR T cell therapeutic approaches remain vulnerable to T cell

exhaustion
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 Ghoneim et al. Page 21
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Key Figure, Figure 1. Epigenetic Barriers to Immune Checkpoint Blockade-Mediated

Rejuvenation of T Cells
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(A) The schematic depicts the repression in developmental plasticity acquired by antigen-
specific T cells during clonal expansion in response to acute or chronic antigen exposure.
Upon acute antigen exposure, naive CD8+ T cells clonally expand and differentiate into
effector cells. During chronic high-level antigen exposure, persistent T cell receptor (TCR)
stimulation can cause T cell exhaustion. Effector function is progressively repressed during
the development of T cell exhaustion, thereby leading to a heterogeneous population of T
cells at various levels of exhaustion. Partially exhausted T cells are phenotypically defined
by sustained expression of a minimal number of immune inhibitory receptors (IRs) and
characterized by differential expression of T-bet and Eomes (T-bethigh Eomeslow T cells),
whereas fully exhausted T cells are marked by co-expression of multiple IRs. The immune
checkpoint blockade (ICB) obstructs inhibitory signals from immune IRs, resulting in
rejuvenation of partially exhausted T cells. (B) The schematic depicts the effect of
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progressive epigenetic programming that reinforces silencing of genes involved in effector

function, proliferation, and metabolic fitness of exhausted T cells. Partially exhausted T cells
may retain a degree of epigenetic plasticity at exhaustion-specific silenced genes manifested
by partially methylated DNA and deposition of fewer repressive, more permissive histone
marks. Terminally differentiated exhausted T cells may be more epigenetically restrained
through fully methylated DNA, the deposition of more repressive histone marks, and the loss
of permissive histone marks. (C) The diagram models the potential reprogramming of

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exhaustion-associated epigenetic programs that could be done to remove cell-intrinsic

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barriers to achieving a better, possibly durable rejuvenation response of fully exhausted T

cells following ICB therapy.
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Figure 2. Genetically-Engineered T cell Immunotherapeutic Approaches

The schematic depicts the multiuse process of generating genetically engineered human T
cells targeted against specific tumor antigens.
(Step 1) Autologous T cells can be isolated from a patient’s peripheral blood mononuclear
cells or an excised tumor.
(Step 2) T cell pools or specific subsets can be stimulated with (1) activating CD3 antibody
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(OKT3), (2) anti-CD3/CD28–coated beads, or (3) allogeneic feeder cell lines.

(Step 3) Specific T cell subsets can be sorted or enriched according to their differentiation
status to exploit proliferative and effector functions.
(Step 4) Engineered T cells can be generated by cloning either T cell receptors (TCRs) or
chimeric antigen receptors (CARs) and transducing a patient’s T cells with retro- or lenti-
viruses, thus redirecting recognition toward tumor-associated antigens.
(Step 5) Engineered T cells can then be expanded in vitro in the presence of conditioning
cytokines (e.g., IL-2 or IL-15) to increase the frequency of tumor-specific T cells generated.

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(Step 6) Engineered T cells specific for an antigen (target) of interest are selected.
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(Step 7) Engineered T cells can then be reinfused into the patient (usually post-
lymphodepletion).
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