Harnessing Stem Cell-Like Memory T Cells for Adoptive Cell Transfer Therapy of Cancer

Abstract

Immunotherapies based on the adoptive transfer of naturally occurring or gene-engineered tumor-reactive T cells can result in durable complete responses in patients with metastatic cancers. Increasing findings from mouse studies and clinical trials indicate that intrinsic properties related to the differentiation state of the transferred T cells are crucial to the success of adoptive immunotherapies. There is now evidence that stem cell-like T cells with enhanced capacity for self-renewal and the ability to derive potent effector T cells might be used to improve persistence and long-term anti-tumor immunity. Here, we describe the molecular, metabolic and cellular aspects of T cell differentiation and their relevance to cancer immunotherapy. We also discuss current efforts and new approaches that might potentiate T cell-based immunotherapies through the modulation of T cell fate and differentiation.

Full text

183© Springer International Publishing Switzerland 2015
P.A. Ascierto et al. (eds.), Developments in T Cell Based Cancer
Immunotherapies, Cancer Drug Discovery and Development,
DOI 10.1007/978-3-319-21167-1_8
Chapter 8
Harnessing Stem Cell-Like Memory T Cells
for Adoptive Cell Transfer Therapy of Cancer
Enrico Lugli and Luca Gattinoni
Abstract Immunotherapies based on the adoptive transfer of naturally occurring or
gene-engineered tumor-reactive T cells can result in durable complete responses in
patients with metastatic cancers. Increasing findings from mouse studies and clinical
trials indicate that intrinsic properties related to the differentiation state of the trans-
ferred T cells are crucial to the success of adoptive immunotherapies. There is now
evidence that stem cell-like T cells with enhanced capacity for self-renewal and the
ability to derive potent effector T cells might be used to improve persistence and
long-term anti-tumor immunity. Here, we describe the molecular, metabolic and cel-
lular aspects of T cell differentiation and their relevance to cancer immunotherapy.
We also discuss current efforts and new approaches that might potentiate T cell-
based immunotherapies through the modulation of T cell fate and differentiation.
Keywords Adoptive T cell therapy • T celldifferentiation • T memory stem cells •
Transcription factors • Immune metabolism • Gene therapy • Reprogramming •
Induced pluripotent stem cells • Small molecules • Homeostatic cytokines
Introduction
Adoptive T cell-based therapies have emerged as a potent and highly effective treat-
ment for patients with advanced solid cancer and hematologic malignancies [1–3].
These therapeutic modalities are based on the ex vivo expansion and re-infusion of
autologous or allogeneic tumor-specific T cells to patients. Early efforts to target
malignancy focused on the use of tumor-infiltrating lymphocytes (TIL) [4], bulk
E. Lugli, Ph.D. (*)
Principal Investigator, Laboratory of Translational Immunology, Humanitas Clinical and
Research Center, Via Alessandro Manzoni 113, Rozzano, Milan, Italy
e-mail: enrico.lugli@humanitasresearch.it
L. Gattinoni, M.D. (*)
Experimental transplantation and Immunology Branch, Center for Cancer Research,
National Cancer Institute, 10 Center Drive, Bethesda, MD, USA
e-mail: gattinol@mail.nih.gov

184
lymphocyte populations in vitro-sensitized against tumor antigens [5] and tumor
antigen-specific clones [6–8]. Recent advances in gene transfer technology have
permitted to convey de novo cancer reactivity to T cells through genetic engineering
of tumor-reactive T cell receptors (TCR) [9–11] or chimeric antigen receptors
(CAR), which consist of a single-chain variable fragment of a tumor-specic anti-
body linked to trans-membrane and cytoplasmic domains of T cell signaling mole-
cules [12–17]. Transfer of naturally occurring or genetically engineered
tumor- reactive T cells has resulted in dramatic and possibly curative responses in
some patients (Fig. 8.1). Increasing experimental and clinical evidence indicate that
the differentiation state, the self-renewal capacity and the ability to derive large
numbers of potent effectors critically influence the ability of tumor-reactive T cells
to mediate effective anti-tumor immune responses. Here, we describe the molecular,
Fig. 8.1 Objective clinical regressions in patients with metastatic tumor treated with cell transfer
therapy. (A) Regression of a large fungating scalp mass in a melanoma patient treated with ex vivo
expanded tumor-infiltrating lymphocyte. (B) Regression of multiple liver and lung metastases in a
melanoma patient treated with peripheral blood lymphocytes genetically engineered to express a
NY-ESO-1-specic T cell receptor (TCR). (C) Regression of splenomegaly and multiple adenopa-
thies in a B-cell lymphoma patient treated with peripheral blood lymphocytes genetically engi-
neered to express an anti-CD19 chimeric antigen receptor (CAR). All patients were treated in the
Surgery Branch of the National Cancer Institute, USA
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185
metabolic and cellular aspects of T cell differentiation and the impact they have in
anti-cancer immunotherapy. Finally, we highlight current efforts and promising
strategies that might potentiate adoptive immunotherapies through the modulation
of T cell fate and differentiation.
T Cell Differentiation
Peripheral T lymphocytes are mature cells of the adaptive immune system but,
differently to other committed cells of the body, they display a tremendous hetero-
geneity and high degree of plasticity. Mature T cells are released from the thymus
into the periphery and harbor a given specicity that is encoded at TCR level.
Following cognate antigen (Ag) recognition, naïve T (TN) cells clonally expand into
effectors, the vast majority of which migrate to peripheral tissues and inflamed sites
to remove the infected targets [18]. As a consequence of infection clearance, ~90–
95 % of activated cells dies while a small pool of T cells ultimately develops into
long-lived memory cells capable to persist in the long term in the putative absence
of Ag [18]. Analogous to other tissues in which terminally differentiated cells are
replaced by the progeny of somatic stem cells [19], survival and maintenance of
memory T cells is thought to occur in a stem cell-like fashion, where less differenti-
ated cells give rise to more committed progeny.
The Diversity of T Cell Subsets
While naïve T cells constitute a fairly homogenous population, memory T cells are
highly heterogeneous in terms of phenotypic and functional composition [20].
Seminal studies in the late 1990s segregated T cells into central memory T (TCM) and
effector memory T (TEM) cell subsets on the basis of migratory capacity (secondary
lymphoid vs. peripheral tissues, respectively) and immediacy of effector functions/
killing activity (TEM > TCM) [21]. CD27, a member of the tumor necrosis factor recep-
tor superfamily, and the lymphoid homing molecules C-C chemokine receptor 7
(CCR7) and L-selectin (CD62L) were initially used together with CD45RA, the
long isoform the CD45 protein, to define heterogeneity in the human memory T cell
compartment. Alternatively, CD45RO, the CD45 short isoform, can replace
CD45RA as the expression of the two molecules is generally mutually exclusive on
the cell surface. Single cell analysis of effector functions revealed that CD45RA−CD8+
memory T cells expressing CD27+ secrete both IFN-γ and IL-2 but lack immediate
killing capacity, while CD45RA+CD27− cells produce IFN-γ and TNF but lack IL-2
production and simultaneously display immediate cytotoxic activity ex vivo [22].
A similar cytokine production profile is shared with CD4+ TCM and TEM cells [23].
An additional subset of terminally differentiated cells, named terminal effectors
(TTE) is abundant in the CD8+ but rare in the CD4+ T cell compartment. These cells
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re-express CD45RA [22], lack CCR7 [23] and costimulatory molecules [22] and
bear high levels of the carbohydrate epitope HNK-1, otherwise known as CD57
[24]. TTE cells stain positive for cytolytic molecules and display immediate effector
functions [25] but are defective in proliferative and survival capacities [24].
In vivo analysis of memory T cell dynamics and tropism in nonhuman primates
allowed the identification of the so-called transitional memory or TTM cells that were
proposed to be intermediate between TCM and TEM cells [26]. Specically, these cells
have down-regulated CCR7 and CD62L but retain the expression of the costimula-
tory molecules CD28 and, in humans, CD27 [26, 20]. TTM cells are highly respon-
sive to IL-15 treatment in vivo [26, 27] and migrate to effector sites following
stimulation. The phenotypic combinations generally used in our laboratories to
define human memory T cell subsets are indicated in Fig. 8.2.
Because of the phenotypic differences between human and mouse T cells, murine
memory T cell subsets are classically defined on the basis of CD44 and CD62L.
Fig. 8.2 Heterogeneity and functionality of peripheral T cell subsets in humans. The combinato-
rial expression of multiple markers on the cell surface, as determined by polychromatic flow
cytometry, allows to identify up to six subpopulations in the peripheral blood and tissues of healthy
individuals. With peripheral maturation, T cells progressively lose or acquire specific functional
capacities, as shown at the bottom of the figure. Following antigen recognition, a given T cell is
activated (in red) and undergoes clonal expansion. The extent of proliferation is dependent on the
initial differentiation status. Among peripheral T cell subsets, TSCM cells retain the greatest prolif-
erative capacity in vivo. When infected/tumor cells are removed, some activated T cells escape
clonal deletion and generate long-lived memory T cells that can divide by homeostatic prolifera-
tion and generate more differentiated progeny. In the model proposed here, duration of antigenic
stimulation dictates the stage of differentiation of activated cells returning to quiescence
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Naïve T cells express CD62L but lack CD44, which is upregulated by CD62L+ TCM
cells and CD62L− TEM cells. Additional markers are available to classify memory
T cells in mice, including CCR7, CD27 and a glycoform of the CD43 molecule.
Both CD27+CD43+ and CD27+CD43− memory T cells contain conventional TCM and
TEM cells, while CD27−CD43− T cells are classified as terminal effectors. These
cells express high levels of the Killer cell lectin-like receptor subfamily G member
1 (KLRG-1), and low levels of the Interleukin-7 receptor α (IL-7Rα) and IL-2Rβ
chains, otherwise known as CD127 and CD122, respectively [28].
Sallusto and Lanzavecchia for the rst time proposed a precursor-progeny rela-
tionship between TCM and TEM cells [23] on the basis of the evidence that TCM were
capable to derive TEM cells in vitro, while the opposite was not observed [29, 30].
Several studies in mice, nonhuman primates and humans later demonstrated that TCM
cells serve as early-differentiated progenitors capable of self-renewing and generat-
ing more-differentiated progeny [21, 31, 32]. TCM cells were thus thought to act as a
reservoir of memory T cells, capable to continuously regenerate the memory T cell
compartment in physiology and following injury (e.g., lymphopenia) in a stem
cell-like manner [33, 31, 34].
Identification of T Memory Stem Cells
In 2005, the Emerson’s group reported that a novel memory T cell population,
characterized by a largely naïve-like phenotype but expressing the memory markers
IL-2Rβ and the chemokine C-X-C motif receptor 3 (CXCR3), was responsible of
maintaining graft-versus-host disease upon serial transplantations in mice [35].
Unexpectedly, classically-dened TCM cells failed to do so when adoptively-
transferred as purified fractions. These cells were termed T memory stem cells
(TSCM) as they could differentiate into TCM, TEM and TTE cells while maintaining their
own pool size through self-renewal. TSCM cells can be successfully generated from
naïve precursors by activating the Wnt signaling pathway using either a physiologi-
cal Wnt ligand, Wnt3A, or inhibitors of glycogen synthase kinase-3β (GSK-3β)
[36]. The generated TSCM cells maintained the undifferentiated CD44−CD62L+
naïve-like phenotype but acquired several memory attributes, including the capacity
to rapidly produce effector cytokines, persist in MHC class I-decient hosts and
reconstitute the diversity of T cell subsets upon serial transplantations [36]. Recently,
we have described a TSCM cell population in humans [37]. Similar to mouse cells,
human TSCM cells display a largely naïve-like phenotype together with few memory
markers such as CD95 and IL-2Rβ (Fig. 8.2). Moreover, these cells exhibit enhanced
stem cell-like properties and superior reconstitution capacity in immunodeficient
hosts compared to TCM cells [37]. TSCM are precursors of TCM cells as regards to
peripheral differentiation as revealed by their phenotypic and gene expression
properties. Despite displaying a transcriptional signature characteristic of memory
cells, TSCM cells retain a core of genes expressed by TN cells [37] and share the recir-
culation patterns and distribution of TN cells in vivo. Indeed, they show relative
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abundance in lymphoid tissues compared to the spleen and bone marrow and are
virtually absent from mucosal surfaces [38]. To date, murine TSCM cells have not
been definitively described for pathogen-specific T cells. Conversely, nonhuman
primates bear virus-specific CD8+ TSCM cells following simian immunodeficiency
virus (SIV) infection. SIV-specic TSCM cells are generated early during acute infec-
tion [38], suggesting that a specific gene expression program underlies their differ-
entiation. TSCM cells are subsequently maintained in the long term even under
chronic stimulation by the cognate antigen, possibly because of the selective over-
expression of transcripts regulating self-renewal (LEF1) and mediating protection
from apoptosis (MCL1 and BCL2) [38]. The superior abilities of TSCM cells to self-
renew, resist apoptosis and survive for long periods of time have been corroborated
by a recent study in HIV patients showing that TSCM cells make increasing contribu-
tions to the total viral CD4+ T cell reservoir over time [39]. Finally, the importance
of TSCM cells in the maintenance of the immune homeostasis is suggested by new
findings in nonhuman primates reveling a perturbation of the TSCM cell compartment
during pathogenic but not nonpathogenic SIV [40].
Environmental and Cell Intrinsic Cues Regulating
T Cell Differentiation
Transcriptional Control of T Cell Differentiation
Despite the human memory T cell compartment has been extensively characterized
in the past years at the phenotypic and functional level, the molecular determinants
leading to the formation of long-lived memory T cells have been mostly defined
using mouse models. The study of the transcriptional regulators of memory T cell
differentiation exploded after the identification of a population of cells capable to
survive the effector phase of the immune response and enter into the memory pool
and it has been extensively reviewed elsewhere [41, 42]. At least two independent
reports identified heterogeneity in the effector T cell pool at the peak of the immune
response (i.e., at day 7 post infection in mouse models) on the basis of IL-7Rα and
KLRG-1 [43] or CD62L [44] expression. Adoptive transfer of discrete populations
indicated that IL-7Rα+ KLRG-1− cells are activated T cells capable to persist and
further differentiate into long-lived memory T cells [43]. Conversely,
IL-7Rα−KLRG-1+ T cells are potent short-lived effectors, able to migrate to inflamed
sites and remove infected targets. Gene expression proling of these two T cell
populations helped revealing the role of specific transcription factors and molecular
regulators in driving memory vs. effector differentiation [45, 46].
Two T-box transcription factors, T-bet (encoded by Tbx21) and Eomesodermin
(Eomes, encoded by Eomes) play a pivotal role in this regard. Their expression
seems to be reciprocal with progressive memory differentiation in the mouse, as
Eomes is highly expressed in TCM but not in TEM, while the opposite pattern is
observed for T-bet. Conversely, EOMES mRNA is low in human naïve T cells and
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is progressively upregulated in more differentiated memory subsets [37], highlighting
potentially different mechanisms at the basis of memory T cell differentiation in the
two species. Both proteins are involved in effector differentiation following antigen
encounter by naïve T cells, as they are required for the optimal acquisition of killing
activities [47]. Perforin, granzyme B and IFN-γ as well as CXCR3, which directs
effector cells towards inflamed sites, are partially under the control of these tran-
scription factors [48, 49]. Tbx21−/− mice fail to develop short-lived effectors in
response to antigen stimulation but are enriched in memory precursors [46].
Conversely, T cells lacking Eomes generate high levels of memory precursors,
which, however, fail to differentiate into long-lived memory T cells [50]. This is, at
least in part, due to their inability to respond to IL-15 because of the lack of IL-2Rβ
chain on the surface of Eomes−/− and Tbx21−/− T cells [47]. IL-15-dependent survival
and homeostatic proliferation of memory T cells and their localization to the bone
marrow niche is consequently abrogated [50].
Similarly to Tbet, the transcription factor Blimp-1 encoded by the Prdm1 gene
regulates effector differentiation from naïve precursors, and ensures effector recall
responses from mature memory T cells [51, 52]. Blimp-1-deficient T cells are
highly enriched in memory precursors following infection with different viruses,
and more rapidly develop into IL-2-secreting TCM cells than wild-type mice [51].
Consistent with their defective effector differentiation and accelerated memory
formation, Ag-specific Prdm1−/− T cells displayed decreased levels of T-bet and
increased levels of Eomes [52]. Blimp-1 limits the ability of short-lived effectors to
enter in the memory pool by repressing the inhibitor of DNA binding 3 (Id3), an
important regulator of genome stability [53]. Accordingly, Id3high T cells at the peak
of the immune response identified CD8+ T cells capable to develop into long-lived
memory T cells [54]. In turn, Bach2, originally identified in B cells as a regulator of
class switch recombination and somatic hypermutation of immunoglobulin genes,
represses Blimp-1 expression in both B cells and CD8+ T cells, induces the expres-
sion of Id3 and enhances the formation of memory T cells by increasing the fre-
quency of CD62L+KLRG-1− T cells [55]. In CD4+ cells, the T cell-specific
transcription factor Menin binds to the Bach2 locus and ensures Bach2 expression
through histone acetylation [56]. Menin overexpression inhibits senescence in CD4+
T cells and restricts the acquisition of the senescence-associated secretory pheno-
type, characterized by the overexpression of pro-inammatory cytokines and matrix
remodeling factors [56]. The role of the Menin-Bach2 axis in the function of mem-
ory T cells during a recall response is, at present, still to be defined.
Members of Forkhead box O (FoxO) protein family are also emerging as key
regulators of memory T cell differentiation and homeostasis. In the unphosphory-
lated form, Foxo proteins are found in the nucleus where they regulate gene expres-
sion by binding regulatory DNA motifs [57]. FoxO proteins are mostly involved in
the regulation of genes involved in cell cycle and apoptosis, and can either induce or
block these specic functions. Upon phosphorylation, the FoxO proteins are retained
in the cytoplasm and are subsequently inactivated through proteasomal- mediated
degradation [57]. Growth factor, hormone and cytokine stimulations activate the AKT
pathway, which in turn mediate FoxO proteins inactivation. The best characterized
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members of the FoxO family, as regards to memory T cell biology, are FoxO3a and
Foxo1. The former was initially described as a regulator of human CD4+ T cell
survival. TCM cells harbor slightly lower levels of the native protein compared to TEM
cells but show a two- to five-fold higher levels of the phosphorylated form on mul-
tiple residues ex vivo, thus resulting in increased in vitro resistance to spontaneous
and Fas ligand (FasL also known as CD95L)-induced apoptosis [58]. TCM cells also
show reduced levels of the pro-apoptotic protein Bcl2-Like 11 (also known as Bim),
which is as direct FoxO3a transcriptional target [58]. Accordingly, FoxO3a-deficient
mice show reduced levels of Bim and Bcl2 binding component 3 (also known as
PUMA) pro-apoptotic proteins, generate higher numbers of memory precursors fol-
lowing cognate antigen activation and preferentially persist in the long-term [59].
Differently, FoxO1 was found to promote memory T cell persistence through the
induction of genes promoting cell survival (BCL2, Il7r, Tcf7) and maintenance of
the TCM cell status (Sell, encoding for CD62L, and Ccr7) [60]. ChIP sequencing
analysis demonstrated that Tcf7 and Ccr7 are direct transcriptional targets of FoxO1
[60]. Deletion of FoxO1 from activated CD8+T cells did not affect effector differen-
tiation but abrogated the formation of memory T cells [60, 61]. Moreover, the few
T cells transitioning to the memory phase showed virtually no capacity to respond
to recall antigenic stimulation.
Therefore, two members of the same protein family that are inactivated down-
stream of the Akt pathway by similar mechanisms regulate memory T cell differen-
tiation by promoting the transcription of genes with opposite functions. It is possible
to speculate that FoxO1 and FoxO3a are repressed by different concentrations of Akt
stimulators or with different kinetics, thus deciding whether a T cell transits to the
memory phase or dies during the effector phase. In addition, different concentrations
of the two proteins at the single cell level might increase the heterogeneity of the
memory precursors population even further.
Metabolic Regulation of T Cell Differentiation
A single naïve T cell is able to generate thousands of daughter cells by dividing
every 4–6 h in response to antigen recognition [62]. It is becoming increasingly
evident that such a fast dynamics requires profound changes not only at the tran-
scriptional level but also at the metabolic level [63]. In the absence of antigen, naïve
T cells are quiescent, are characterized by a very high nuclear:cytoplasmic ratio
(indicative of little protein synthesis), divide only rarely in response to homeostatic
proliferation, mainly mediated by IL-7, and have low energetic demand [63].
Memory T cells are also quiescent in the absence of antigenic stimulation but dis-
play greater mitochondrial mass, which provides a bioenergetic advantage for sup-
porting rapid recall responses after antigen re-exposure [64, 65]. Quiescent naïve
and memory T cells rely almost completely on energy derived from mitochondrial
oxidative phosphorylation (OXPHOS) and fatty acid β-oxidation (FAO), however
following antigen-induced activation they switch to a glycolytic metabolism even in
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oxygen-replete microenvironments [63, 66]. It remains unclear why T cells adopt a
less efficient pathway for ATP generation under conditions of high-energy demand.
For years, it has been proposed that this phenomenon, also known as the Warburg
effect, was necessary to generate precursors of deoxyribonucleotides that are subse-
quently used for DNA replication [67]. However, recent findings indicate that gly-
colysis is critical for effector differentiation as it is required for the post-transcriptional
regulation of specific effector function such as IFN-γ production [68]. Moreover,
T cells activated in limiting concentrations of glucose failed to upregulate killing
molecules, such as perforin and granzymes [69].
These observations clearly indicate that distinct T cell subsets exhibit unique
metabolic programs. Whether these metabolic characteristics reflect functional
changes orchestrated by diverse transcriptional programs or rather instructively dic-
tate T cell fate decisions has just begun to be addressed. The first evidence that
memory T cell formation is regulated at the metabolic level came from the analysis
of mice lacking tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6)
[70]. Despite effector differentiation was maintained in Traf6−/− mice, the generation
of memory T cells was abrogated almost completely. Gene expression studies
revealed that Traf6−/− T cells were incapable to induce transcripts involved in FAO,
and displayed a reduced 5′ adenosine monophosphate-activated protein kinase
(AMPK), a master regulator of fatty acid metabolism [70]. Accordingly, the overex-
pression of carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme of
FAO, was sufficient to augment CD8+ T cell memory and hence potentiate recall
immune responses [71]. These findings clearly demonstrate that FAO can influence
the establishment of immunological memory, indicating that changes in metabolism
play a direct role in regulating T cell differentiation. Recently, we have demon-
strated that also glycolysis can directly influence the generation of memory and
effector T cells [72]. Activated T cells displaying high glycolytic activity tended to
be short-lived, while cells with low glycolytic metabolism established memory.
Moreover, enforcing glycolysis by overexpression of the glycolytic enzyme Pgam1
severely impaired the ability of CD8+ T cells to persist in the long term whereas
inhibition of glycolytic ux using 2-deoxyglucose (2-DG) increased CD8+ T cell
memory formation by preserving the expression of Tcf7, Lef1 and Bcl6 and repress-
ing the upregulation of Prdm1 and effector-associated genes.
AKT, also known as protein kinase B, has been shown to integrate cell growth
signals with glycolytic metabolism in a variety of cellular systems. Increased AKT
activity in effector T cells leads to the activation of the mammalian target of rapamycin
(mTOR) which favors cell growth, protein synthesis and proliferation [73]. In mam-
malian cells, mTOR is found in two different multiprotein complexes, mTORC1
and mTORC2, that are activated by a plethora of extrinsic and intrinsic signals in
T cells, including antigen, cytokines, glucose and amino acids, among others. For
more detailed information on mTORC1 and mTORC2, excellent reviews were
recently published [74, 75]. The increased function of mTOR has been linked to a
number of molecular events involved in effector differentiation. TCR-dependent
immune activation leading to the down-regulation of CCR7 and CD62L depends on
increased PI3K-mTOR function, which subsequently controls migratory capacity
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in vitro and in vivo [166]. Furthermore, excessive mTOR stimulation drives T cells
towards a terminally differentiated effector state whereas mTOR inhibition by the
immunosuppressive drug rapamycin resulted in increased numbers of memory
T cells [76]. These results appear counterintuitive as rapamycin is widely used in
solid organ and hematopoietic stem cell transplantations to inhibit allogeneic T cell
immune responses [73]. Despite the exact mechanisms at the basis of mTOR inhibi-
tion by rapamycin and the outcome of the T cell response are still to be defined, it is
possible to hypothesize that the dose, the timing and the duration of administration
are important variables in this regard.
In summary, T cell fate is tightly regulated at the transcriptional, signaling and
metabolic levels. What is clear is that these different aspects are not independent but
are closely interconnected. Most importantly, they can be modulated by using small
molecules that are approved for clinical use [77]. The implications of memory T cell
differentiation in the regulation of anti-tumor immunity at the preclinical and clinical
levels are described below.
T Cell Differentiation and Adoptive Immunotherapy Efficacy
Lessons from Mouse Models
It had long been unclear whether the differentiation state represented a crucial deter-
minant of the ability of tumor-reactive T cells to mediate anti-tumor responses upon
adoptive transfer. Because of the vast heterogeneity of T cell preparations employed
in adoptive immunotherapy studies it was impossible to precisely separate the thera-
peutic contribution of dened T cell subsets from the impact of distinct TCRs before
the advent of TCR transgenic mice. Since the nal goal of adoptive immunotherapy
is to generate T cells capable of patrolling the body in search of cancer cells to
destroy, it was initially assumed that TTE and TEM cells were the ideal T cells to
transfer, as they possess a propensity to migrate into peripheral tissues and display
immediate cytotoxic functions upon antigen encounter. Accordingly, the potency of
T cell products was exclusively determined by assessing the ability of tumor-
reactive T cells to release IFN-γ and kill tumor cells upon in vitro co-culture [78].
It was somehow surprising to realize that these two subsets, on the contrary, were
poorly capable of destroying tumors upon adoptive transfer compared to less dif-
ferentiated T cells. First evidence came from two sets of experiments conducted in
the pmel-1 model of adoptive immunotherapy that employs gp100-specific CD8+
T cells derived from the TCR transgenic mouse pmel-1 to target B16 melanoma [79].
Terminally differentiated KLRG-1+ TTE cells generated from reiterative stimulations
of pmel-1 cells with cognate antigen and IL-2 were found to be 100-fold less effec-
tive in vivo on a per-cell basis than T cells at an early stage of differentiation [80].
Parallel experiments evaluating the antitumor efficacy of tumor-specific CD8+
memory subsets revealed that less differentiated TCM cells were capable of inducing
durable complete responses while mice receiving TEM cells ultimately succumbed to
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unrestrained tumor growth [81]. The inadequacy of CD62L− T cell subsets to mediate
profound immune responses following adoptive transfer has been also documented
by several other groups in diverse settings including models of tumor treatment
[82–84], viral protection [85, 86] and allogeneic hematopoietic stem cell transplan-
tation [87, 88].
The paradoxical inability of TTE and TEM at triggering tumor regression upon
adoptive transfer finds its roots in several biological hurdles that are integral com-
ponents of the effector differentiation program. For instance, the inefficient traffick-
ing to peripheral lymphoid tissues due to the loss of CD62L and CCR7 expression
can disrupt the intimate interactions with dendritic cells that are fundamental for the
induction of productive T cell responses [80]. Indeed, anti-tumor responses were
virtually abrogated in hosts devoid of secondary lymph nodes and with a disrupted
splenic structure [81]. Moreover, Sell−/− tumor-specific CD8+ T cells were impaired
in their ability to inhibit tumor growth compared with wild-type T cells [80, 81].
A profound reshaping of the co-stimulatory and inhibitory receptor landscape also
accompanies the differentiation process. Down-regulation of CD28 and CD27
expression in TTE and TEM cells can limit co-stimulatory signals resulting in
decreased cell proliferation and long-term survival [89–92]. This dysfunctionality
can also be aggravated by the concomitant overexpression of KLRG-1 and multiple
inhibitory receptors such as PD-1, LAG-3 and 2B4, which have detrimental effects
on cell growth and function [93, 94]. TTE and TEM cells might also not receive
sufficient pro-survival and activating signals from common γ chain (γC) cytokines.
As T cells progressively differentiate into TTE cells, they lose the ability to utilize
IL-2 in autocrine fashion [95, 80] and to respond to IL-7 cues due to the down-
regulation of IL-7rα expression [43, 44]. Finally, gradual telomere erosion [96, 23,
97] and up-regulation of pro-apoptotic molecules, including BID (B-cell lymphoma
2 (BCL-2)-homology domain 3 (BH3)-interacting-domain death agonist) and BAD
(BCL-2-antagonist of cell death) [80, 81] might ultimately result in TTE replicative
senescence and cell death. Altogether, these phenotypic and functional changes
characterizing the differentiation program severely impair the ability of TTE and TEM
cells to engraft, expand and persist long-term following adoptive transfer into tumor-
bearing hosts [80, 81].
It is now clear that the proliferative potential and survival capacity are key attri-
butes to seek in tumor-reactive T cells for adoptive transfer. Among T cell memory
subsets, TSCM cells possess a robust proliferative capacity and a superior ability to
persist in the long-term, which make them a desirable cell population to employ in
adoptive immunotherapy [98]. When tested in the pmel-1 model, minuscule numbers
of TSCM cells mediated dramatic tumor regression of large established B16 melanoma
[36]. Paralleling their engraftment and proliferative potentials, the ability of memory
T cells to mediate tumor regression progressively decreased from TSCM cells to TCM
cells and TEM cells [36]. These findings were corroborated in subsequent experiments
using human T cell memory subsets genetically engineered to express an anti-meso-
thelin CAR to treat human mesothelioma xenografts in immunodecient mice [37].
In conclusion, findings made in mice have established an inverse relationship between
T cell differentiation status and the relative capacities of transferred T cells to engraft,
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proliferate, and mediate antitumor immunity. These data strongly support the use of
the less differentiated CD62L+ subsets and particularly TSCM cells over the CD62L−
TEM and TTE cells for adoptive immunotherapies.
Insights from the Clinic
The question of which T cell subset is more effective for adoptive immunotherapy
becomes murkier when considering the available clinical data. Clinical trials employ-
ing well-defined tumor-reactive T cell subsets are still lacking due to the technical
complexity associated with the isolation procedures, however some conclusions can
be drawn from key observations and retrospective analyses. Consistent to what was
observed in mouse studies [80, 84], tumor-specific CD8+ T cell clones that were
generated and expanded ex vivo through multiple stimulations in the presence of
IL-2, a cytokine that promotes terminal differentiation [99], failed to persist after
infusion and did not meditate clinically meaningful tumor regressions [6–8].
Conversely, T cell persistence has been highly correlated with tumor responses
across multiple clinical trials [100–102, 15] and has been linked to intrinsic T cell
properties that are reflective of their differentiation state and replicative history. Early
studies revealed that a short duration of tumor infiltrating lymphocyte (TIL) culture
or a relatively rapid doubling time were associated with clinical responses [103,
104]. Additional parameters such as the length of telomeres [105, 100], the expres-
sion of CD27 [106, 100] and CD28 [105] and the frequency of TCM cells in the infu-
sion product [107] have also been correlated with tumor responses in patients with
cancer. However, recent TIL analyses from a limited cohort of patients failed to
observe a correlation with telomere length and tumor responses [108]. Furthermore,
in this set of patients, objective responses were associated with the infusion of
CD45RA−CD62L−CD27− TTE [108]. These discrepancies might be related to the
prevalence of tumor-specific T cells within a given T cell subset. For instance, TTE
might have been relatively enriched for tumor-reactive T cells or highly avid TCR
clonotypes. Taken together, the majority of data in humans is consistent with the
notion that less-differentiated T cells confer superior antitumor efficacy relative to
TEM and TTE cells.
Potentiating Adoptive T Cell Therapies by Modulating T Cell
Differentiation
Restraining T Cell Differentiation
Current approaches employed to generate T cells for adoptive transfer often rely on
variations of a protocol established more than 20 years ago [109, 110], before the
implication of T cell differentiation on in vivo anti-tumor efficacy was completely
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appreciated [111]. This strategy consist of potent activating stimuli, such as anti- CD3
antibodies, high concentrations of IL-2 and allogeneic feeders, which result in the
generation of large numbers of tumor-reactive T cells but inevitably drive cells
towards terminal differentiation and senescence.
To limit the negative influence of ex vivo expansion on T cell differentiation, new
methods have been investigated (Fig. 8.3A). Early studies focused on the use of γC
cytokines alternative to IL-2, such as IL-15 and IL-21. IL-15 was found to sustain T
cell expansion without the robust pro-differentiating activity that is typical of IL-2.
Contrary to IL-2, which generates TTE and TEM cells, stimulation of T cells in the
presence of IL-15 promoted the formation of T cells with the phenotypic, functional
and metabolic qualities found in naturally occurring TCM cells [81, 80, 71, 112].
IL-15-generated tumor-reactive T cells exhibited enhanced anti-tumor responses
Fig. 8.3 Programming and reprogramming T cell fates for therapeutic use. (A) Restraining T cell
differentiation. Differentiation of activated naive T (TN) cells can be withheld by small molecules
targeting key metabolic and developmental pathways or γC cytokines alternative to interleukin-2
(IL-2), such as IL-21. (B) Two step reprogramming of terminally differentiated effector T (TTE)
cells through an induced pluripotent stem (iPS) cell intermediate or a possible stimulus-triggered
acquisition of pluripotency (STAP) cell intermediate. TTE cells are reprogrammed into iPS cells by
enforced expression of OCT4, Kruppel-like factor 4 (KLF4) and sex determining region Y (SRY)
BOX 2 (SOX2) with or without MYC or by ectopic expression of the microRNA (miRNA) cluster
302–367. TTE cells might also be converted into STAP cells by exposure to strong external stimuli
such as a transient low-pH stressor. iPS and STAP cells can be then re-differentiated into TN cells
through the induction of NOTCH signaling. (C) Direct reprogramming of TTE into TN or memory
stem (TSCM) cells by enforced expression of TN or TSCM-associated transcription factors or miRNAs.
TCM, central memory; TEM, effector memory; GSK-3β, glycogen synthase 3β
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compared to those grown in the presence of IL-2 [113]. Additionally, human T cells
activated in the presence of low doses of IL-15 and IL-7 generated TSCM-like cells
capable of expanding and mediating GVHD on serial transplantation [114]. More
recently, several investigators have evaluated the activity of IL-21 on the expansion
and differentiation of tumor-specific CD8+ T cells. In both mouse and human studies,
IL-21 profoundly repressed T cell differentiation as manifested by the generation
of T cells lacking the expression of conventional memory cell markers [115, 116].
T cells activated in IL-21 maintained a naive-like phenotype and the ability to
secrete high amounts of IL-2 [116, 115, 117]. Although a comprehensive pheno-
typic characterization of these cells was not done in these studies, it is likely that
IL-21-generated cells might comprise TSCM-like cells.
In the past decade there has been an increasing understanding of the signaling
pathways and transcriptional circuitry that regulate memory and effector T cell differ-
entiation [42, 41]. Many of these pathways can now be targeted by small molecules,
which are already approved, or under clinical evaluation for other indications. This
raises the exciting possibility of repurposing these drugs to modulate T cell differ-
entiation to potentiate T cell therapeutic fitness [77]. As discussed above, mTOR
has emerged as a key modulator of CD8+ T cell fate commitment and its modulation
by rapamycin as well as by temsirolimus, a rapamycin analogue that is approved for
treating advanced renal cell carcinoma, were shown to enhance the formation of
CD8+ memory T cells and augment their anti-tumor efficacy [118, 119].
Analogous results can also be obtained by targeting molecules upstream from
mTOR such as AMPK and AKT. Metformin, an AMPK agonist used for the
treatment of type 2 diabetes [120], has been shown to enhance T cell survival, mem-
ory responses and anti-tumor treatment [70]. Pharmacological blockade of AKT
limited the acquisition of effector molecules and function while preserving a TCM-
like phenotype and migratory capacity [69]. Although the AKT inhibitor employed
in this study is not available for use in humans, several other AKT inhibitors are
currently under clinical evaluation for the treatment of solid tumors and hemato-
logic malignancies [77]. Additionally, saracatinib, an Src family inhibitor undergo-
ing clinical investigation for the treatment of cancer and Alzheimer's disease, was
found to enhance the generation of TCM cells in responses to vaccination and confer
superior protection against tumor challenge through an unresolved signaling path-
way regulating the AKT–mTOR pathway [121].
Another important pathway that has recently been implicated in the regulation of
T cell differentiation and memory formation is the WNT–β-catenin signaling path-
way [122, 123]. GSK3-β inhibitors that are under clinical evaluation for Alzheimer's
disease and other neurodegenerative diseases [124] could be use to induce down-
stream signals of the WNT–β-catenin pathway to generate TSCM-like cells capable of
triggering potent anti-tumor immune responses [37, 36].
More recently, direct targeting of metabolic rate-limiting enzymes has been
shown to be an effective strategy to restrain differentiation and enhance T cell mem-
ory and anti-tumor function [71, 72]. Inhibition of glycolytic ux by 2-DG, a hexo-
kinase inhibitor currently under evaluation in clinical trials because of its direct
negative impact on glycolytic tumor cells [125], limited T cell differentiation,
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resulting in the generation of T cells with improved anti-tumor efficacy [72]. Taken
together, these studies underscore the ever-increasing number of reagents available
that can be immediately integrated in the next generation protocols for the production
of tumor-specific T cells for adoptive immunotherapy.
Reprogramming Terminally Differentiated T Cells
Restraining T cell differentiation during ex vivo expansion can be an effective strategy
for therapies based on the adoptive transfer of T cells genetically engineered with a
tumor-reactive TCR or CAR as a large fraction of peripheral blood lymphocytes
comprises TN and TCM cells [37, 126]. However, this approach become less relevant
for therapies relying on naturally occurring tumor-specific T cells, which are often
found in a state of terminal differentiation and exhaustion due to chronic antigen
stimulation in the tumor-bearing host [127–130]. Recent advances of regenerative
medicine demonstrating successful reprogramming of mature cell lineages into
induced pluripotent stem (iPS) cells have opened the exciting possibility of rejuve-
nating exhausted and senescent T cells [131]. Since Yamanaka’s seminal discovery,
mature T lymphocytes have been reprogrammed into iPS cells by enforcing expres-
sion of the transcription factors OCT4, SOX2, KLF4 and MYC [132–134].
Importantly, T cell-derived iPS cells retain the rearranged variable (V), diversity (D)
and joining regions (J) of the TCR chains, indicating that iPS cells generated from
tumor-specific T cells could maintain their anti-tumor reactivity. Increasing under-
standing of the signaling required for T cell development during thymopoiesis has
led to the development of ex vivo protocols that support the generation of T cells
from stem cell precursors including iPS cells, providing a feasible methodology for
re-differentiating T cell-derived iPS cells [135–139]. Recently, Vizcardo et al. [140]
and Nishimura et al. [141] have put this two-step reprogramming concept into prac-
tice (Fig. 8.3B). These groups obtained iPS cells from T cell clones specic for the
melanoma-associated antigen MART-1 or the HIV-1 protein Nef, respectively, and
re-differentiated them into mature T cells by coculture with OP9 feeder cells over-
expressing the Notch ligand Delta like-1 (DLL1). Although reprogrammed T cells
maintained the original TCR rearrangement and the ability to mediate specic
effector functions, it remains unclear whether these cells could be truly considered
rejuvenated. These cells exhibited elongated telomeres, indicating an increased pro-
liferative potential, however they displayed phenotypic traits of TEM cells rather than
naïve or early memory subsets. A recent paper indicated that reprogrammed T cells
from iPS cells acquired the phenotype and functional characteristics of innate-like
γδ T cells [142]. When redirected with a CD19-specic CAR, they were able to
mediate potent tumor regression in a human lymphoma xenograft tumor model.
Although feasible, the two-step reprogramming approach is currently inefficient
both in terms of the frequency of cells successfully reprogrammed and the duration
necessary to achieve full reprogramming. Recently, Obokata and colleagues have
reported that pluripotency could be induced faster and more efficiently by exposing
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198
lymphocytes to strong external stimuli such as a transient low-pH stressor [143].
Since then, misconduct proceedings have surfaced calling into question the validity
of the results [144]. If confirmed and reproduced in adult human cells, however,
Obokata’s findings could make the two-step reprogramming method more practical
(Fig. 8.3B).
An alternative approach to overcome the inefficiencies of two-step reprogram-
ming might be the direct reprogram of terminally differentiated T cells into TN and
TSCM cells. An increasing number of reports have revealed that direct reprogram-
ming can be used to differentiate diverse mature cell types into alternative differen-
tiated lineages such as neurons [145, 146], hepatocytes [147], cardiomyocytes
[148], blood progenitors [149], and pancreatic β cells [150, 151] by ectopic expres-
sion of cell-specific transcription factors. Adapting this strategy, enforced expres-
sion of transcription factors or miRNAs essential to TN and TSCM identity might
result in the intra-lineage reprogramming of TTE cells into less-differentiated T cells
(Fig. 8.3C).
Current Clinical Efforts and Future Directions
The realization that T cell differentiation and in vivo anti-tumor effectiveness are
inversely correlated has recently prompted a series of new clinical trials designed to
test the efficacy of less-differentiated T cell populations (Table 8.1). Because pro-
longed TIL cultures can drive cells towards terminal differentiation, changes were
made to standard TIL protocols to shorten the period of ex vivo expansion [152–
154]. These minimally cultured TILs were called young TILs, as they possessed
characteristic of less-differentiated T cells including longer telomeres and higher
expression of CD27 and CD28 compared to TIL cultured with a conventional pro-
tocol. Early trials using young TIL preparations have demonstrated anti-tumor effi-
cacies comparable to standard TILs with objective responses of 28–58 % [155, 156,
153, 157, 104]. It is unclear the reason why young TILs did not induce increased
anti-tumor responses compared to the historical experience using standard TILs.
However, it is becoming evident that “young TILs” is a misnomer as the rapid
expansion protocol employed to expand TILs prior to infusion virtually nullify the
benefit of initial short TIL cultures resulting in low frequencies of less- differentiated
T cells [152]. Furthermore, in a recent young TIL trial characterized by an unusu-
ally low response rate, infused TILs, rather than being minimally cultured, were
grown for period of time comparable to standard TILs [156].
Alternative γC cytokines have been used in recent clinical protocols for the gen-
eration of tumor-reactive T cells for adoptive immunotherapy. Autologous MART1-
specific CD8+ T cells were generated in vitro using artificial antigen presenting cells
in the presence of a combination of IL-2 and IL-15 [158]. This approach resulted in
the generation of a mixture of TCM and TEM memory cells capable of engrafting and
persist in patients for prolonged periods in the absence of previous lymphodepletion
conditioning or cytokine support. Notably, these cells trafficked to the tumor and
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Table 8.1 Adoptive immunotherapy trials designed to administer less differentiated T cells
Target Cancer Cell product ID Center Status
Undened Melanoma Young tumor infiltrating lymphocytes NCT00513604 NCI Completed (Dudley
et al. [156])
Undened Melanoma Young tumor infiltrating lymphocytes NCT00287131 Sheba Medical
Center
Completed (Besser
et al. [104])
Undened GI cancer Young tumor infiltrating lymphocytes NCT01174121 NCI Recruiting
E6; E7 HPV- associated
cancers
Young tumor infiltrating lymphocytes NCT01585428 NCI Recruiting
MART-1 Melanoma IL-15/IL-2 modulated, MART-1 specic CD8+ T cells NCT00512889 Dana-Farber
Cancer Institute
Completed (Butler
et al. [158])
WT-1 Melanoma IL-21 modulated, WT-1 specific CD8+ T cell clones NCT00052520 Fred Hutchinson
Cancer Research
Completed (Chapuis
et al. [159])
MART-1 Melanoma IL-21 modulated, MART-1 specic CD8+ T cell clones NCT01106235 Fred Hutchinson
Cancer Research
Completed
gp100 Melanoma gp100-specific CD8+ T cell clones derived from high IL-2:
IFNG index precursors
NCT00665470 NCI Completed (Wang
et al. [162])
CD19 CD19+ B cell
malignancies
CD19-CAR specic CD8+ T-cells derived from virus-specific
TCM
NCT01475058 Fred Hutchinson
Cancer Research
Recruiting
CD19 B-Lineage NHL CD19-CAR CD8+ T cells derived from TCM enriched cells NCT01318317 City of Hope
Medical Center
Active, non- recruiting
CD19 B-Lineage NHL CD19-CAR T cells derived from TCM enriched cells NCT01815749 City of Hope
Medical Center
Recruiting
NY-ESO-1 Melanoma NY-ESO-1 TCR T cells derived from CD62L+ cells NCT02062359 NCI Not yet recruiting
GI gastrointestinal, HPV human papilloma virus, IL interleukin, IFNG interferon-γ, NHLnon- Hodgkin’s lymphoma, TCM central memory T cells, NCI National
Cancer Institute
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mediated biological and clinical responses. IL-21 has also been used to limit terminal
differentiation of WT1-specific donor-derived CD8+ T cell clones [159]. WT1-
specific clonal populations generated with exposure to IL-21 displayed higher
CD27, CD28, or IL-7Rα compared to clones generated in the absence of this cyto-
kine. Consistent with previous studies [6–8], clones generated without IL-21 failed
to persist longer than two weeks in vivo. Remarkably, WT-1-specic clones gener-
ated in the presence of IL-21 survived long-term after infusion, establishing immu-
nological memory. Most importantly, IL-21 generated clones exhibited direct
evidence of anti-leukemic activity.
Another promising strategy currently under clinical evaluation is the use of cell
products derived from the expansion of TCM cells. These studies stem from preclini-
cal evidence in mice and nonhuman primates indicating that progenies of isolated
TCM cells have enhanced capacity to persist and form long-lived memory cells fol-
lowing adoptive transfer compared to TEM-derived cells [160, 161]. Since reagents
necessary to isolate TCM in a high-throughput manner were not initially available,
Wang and colleagues took advantage of the well-known ability of TCM cells to pro-
duce greater amount of IL-2 than their TEM counterparts to develop a PCR-based
assay for early detection of TCM clones [162]. This strategy enabled the isolation,
expansion, and transfer of rare human melanoma-specific CD8+ TCM cells. TCM-
derived T cell clones engrafted and persisted at high frequencies in 4 out of 5
patients, 1 month after the transfer, and were associated with some minor and mixed
tumor regression [162]. More recently, the development of GMP-compliant beads
for TCM cell isolation [163] has led to the initiation of a series of trials employing
CD19-CAR engineered CD8+ T cells derived from TCM cells for the treatment of B
cell malignancies (Table 8.1). Recently, two studies have suggested that T cells
derived from naïve rather than TCM cells might allow superior efficacy upon adop-
tive transfer [164, 126]. TN-derived cells exhibited greater proliferative potential
and mediated enhanced anti-tumor function compared to TCM cells in a murine
tumor model [164]. Moreover, in human studies, TN-derived cells displayed higher
expression of CD27 and longer telomeres compared to cells derived from conven-
tional memory subsets, indicating that the TN-derived progeny possess traits that
correlate with tumor responses in clinical trials [126]. Thus, a new study employing
tumor-specific T cells derived from CD62L+ precursors, which comprise naïve,
TSCM and TCM cell has recently been planned (Table 8.1).
In summary, several new studies investigating the safety and efficacy of less-
differentiated cells have been initiated or planned. Preliminary results are starting to
reveal significant improvements in terms of T cell persistence, which hopefully, will
translate into increased tumor response rates. New clinical-grade protocols for TSCM
cell generation are also under development, paving the way for a rapid translation of
TSCM into future clinical trials [165, 114].
Acknowledgments This work was supported by the Intramural Research Programs of the US
National Institutes of Health, National Cancer Institute and by grants from the Associazione
Italiana per la Ricerca sul Cancro (MFAG10607), Fondazione Cariplo (2012/0683), Italian
Ministry of Health (Bando Giovani Ricercatori GR-2011-02347324) and the European Community
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(Marie Curie Career Integration Grant 322093) to E.L. The authors would like to thank Steven
Rosenberg for providing images of the patients treated in the Surgery Branch, and Yun Ji and
Alessandra Roberto for critical discussion. E.L. is an International Society for the Advancement of
Cytometry (ISAC) scholar.
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8 Harnessing Stem Cell-Like Memory T Cells for Adoptive Cell Transfer Therapy…