Please cite this article in press as: Miller and Sadelain, The Journey from Discoveries in Fundamental Immunology

to Cancer Immunotherapy, Cancer

Cell (2015), http://dx.doi.org/10.1016/j.ccell.2015.03.007

Cancer Cell

Perspective

The Journey from Discoveries in Fundamental

Immunology to Cancer Immunotherapy
Jacques F.A.P. Miller1,* and Michel Sadelain2,*
1The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3050, Australia
2The Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
*Correspondence: miller@wehi.edu.au (J.F.A.P.M.), m-sadelain@ski.mskcc.org (M.S.)
http://dx.doi.org/10.1016/j.ccell.2015.03.007

Recent advances in cancer immunotherapy have directly built on 50 years of fundamental and technological
advances that made checkpoint blockade and T cell engineering possible. In this review, we intend to show
that research, not specifically designed to bring relief or cure to any particular disease, can, when creatively
exploited, lead to spectacular results in the management of cancer. The discovery of thymus immune func-
tion, T cells, and immune surveillance bore the seeds for today’s targeted immune interventions and chimeric
antigen receptors.

Nanos gigantum humeris insidentes. (Like dwarfs sitting notherapy were recognized by the Science magazine as the
on the shoulders of giants.) ‘‘breakthrough of the year’’ in 2013 (Couzin-Frankel, 2013) and
by the US Food and Drug Administration, which approved the
—Bernard de Chartres (1120)
anti-CTLA-4 monoclonal antibody Ipilimumab in 2011 and
granted breakthrough status to the CD19-specific chimeric anti-
If I have seen further than others, it is by standing on the
gen receptors (CARs) utilized at the University of Pennsylvania
shoulders of giants.
and Memorial Sloan Kettering Cancer Center for the treatment
—Isaac Newton (1676) of pediatric and adult acute lymphoblastic leukemia in 2014.
In this review, we aim to expose how basic discoveries in immu-
The immune system is the guardian of our organismal integrity nology led to these promising advances in cancer therapy.
in protecting us from infectious and other foreign invaders, such
as grafts and certain tumors. Immunology and oncology thus Leukemogenesis, the Thymus and Immunological
have a long relationship, in evolutionary terms as well as within Ontogeny
the biomedical sciences. The two fields have intersected again Prior to 1960, the thymus was thought to be a vestigial organ that
and again over the past half-century. We recount here some of had become redundant during evolution and was just a grave-
these events, with a deliberate focus on T lymphocytes—from yard for dying lymphocytes. Even though recirculating small
their discovery to their genetic engineering and use in cancer lymphocytes had been found by Gowans (Gowans et al., 1962)
immunotherapy. Insightful observations into leukemogenesis to be immunocompetent cells able to initiate either cellular or hu-
in mice were at the root of the discovery of thymopoiesis moral immune responses, thymus lymphocytes were deemed
and lymphocyte subsets. Subsequent observations led to the immunoincompetent since they did not recirculate, nor could
concept of immune surveillance, with eventual controversies they transfer immune responses to appropriate recipients.
on the role of immunity in tumor prevention and protection that Furthermore, thymectomy, which had always been performed
were reconciled in the cancer immunoediting hypothesis. As in adult animals, had no untoward effects on immune capacity.
progress was made in our fundamental understanding of antigen In 1959–1961, however, results obtained in a mouse model of
recognition, T cell activation, and T cell costimulation, transla- lymphocytic leukemia induced by the Gross leukemia virus led
tional researchers began to exploit the accumulating knowledge to experiments using neonatally thymectomized mice.
for cancer therapy. Tumor-infiltrating T cells were harnessed for To obtain a high incidence of leukemia in mouse strains that
adoptive cell therapy in a subset of melanoma patients; cancer were not highly prone to develop this malignancy, the virus had
vaccines were developed in an attempt to amplify endogenous to be given at birth, and not later (Gross, 1951). Thymectomy
tumor-specific T cell responses. Two of the most recent, most of virus-inoculated mice at 1 month of age prevented the disease
exciting therapeutic developments rest on the manipulation of (Miller, 1959a), and grafting a neonatal thymus as late as
costimulatory pathways governing T cell function. One, based 6 months after adult thymectomy restored the potential for
on monoclonal antibody technology, enables the release of tu- leukemia development (Miller, 1959b). Clearly, the virus must
mor-infiltrating T cells from inhibition mediated by costimulatory have remained latent, and indeed, it could be recovered from
receptors such as CTLA-4 and PD-1, through either checkpoint the non-leukemic tissues of thymectomized mice not grafted
blockade or depletion of regulatory T cells. The other, based on with thymus tissue (Miller, 1960).
gene transfer technology, enables to repurpose patient’s T cells, It was surmised that to induce a high percentage of leukemia in
targeting them to tumor antigens and augmenting their func- low-leukemic strains of mice, the virus had to be given neonatally
tional properties to overcome barriers erected by tumor cells because it needed to multiply in some cells present only in a
and their microenvironment. These two forms of cancer immu- newborn thymus. To test this hypothesis, the virus was given

Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc. 1

 Please cite this article in press as: Miller and Sadelain, The Journey from Discoveries in Fundamental Immunology to Cancer Immunotherapy, Cancer
Cell (2015), http://dx.doi.org/10.1016/j.ccell.2015.03.007

Cancer Cell

Perspective

at birth but immediately after neonatal thymectomy, based on All of these results were initially regarded with some skepti-
the prediction that thymus grafting performed later would not cism. Vague criticisms abounded, such as the one stating that
restore leukemogenesis. The neonatally thymectomized mice mice must be unusual and that the results obtained would never
fared well until some weeks after weaning, when many became be seen in humans. What needed to be checked, however, was
sick, wasted, and died. This had never been seen by anyone who that because the mice used had been raised in converted horse
had thymectomized adult mice (Miller, 1961a). Post-mortem stables, the added trauma of neonatal thymectomy made them
examination revealed lesions in the liver suggestive of mouse highly susceptible to infections. This criticism was soon quashed
hepatitis virus infection and marked diminution of lymphocytes when it was shown that mice reared in a germfree facility, when
in the blood and in the lymphoid tissues (Miller, 1961b, 1962a). thymectomized at birth did not develop wasting disease and yet
As lymphocytes were known to be involved in graft rejection were unable to reject skin grafts, even those that differed at the
and other immune responses, the neonatally thymectomized H-2 locus (McIntire et al., 1964). In 1968, the nude mouse was
mice were tested for immunocompetence by grafting them discovered (Rygaard, 1973), and immunologists no longer had
with foreign skin before they had begun to show signs of wasting doubts about the immunological function of the thymus.
disease. Remarkably, they failed to reject foreign skin grafts, In children with thymus aplasia, as in di George syndrome,
even when donors and recipients differed at the major histo- thymus transplantation has reversed immunodeficiency (August
compatibility locus, H-2 (Miller, 1961b, 1962a). The neonatally et al., 1970). The problem lies in donor availability and tissue
thymectomized mice also lacked the ability to produce a normal compatibility. However, the recent spectacular success in
antibody response to certain antigens, such as Salmonella typhi creating a functional thymus organ by enforcing Foxn1 expres-
H antigen (Miller, 1962a) and sheep erythrocytes (Miller et al., sion to reprogram mouse embryonic fibroblasts into fetal thymic
1965). epithelium (Bredenkamp et al., 2014) constitutes a first prom-
Thymus grafting restored immune potential, and grafting of a ising step in the provision of appropriate thymus tissue.
foreign thymus induced specific tolerance to skin from the donor
of the thymus graft (Miller, 1962a). This suggested that the The Identification of T and B Cells in Mice
thymus may be the seat where tolerance is learned: ‘‘Antigenic As stated before, Gowans had clearly shown that recirculating
material might make contact with certain cell types differenti- small lymphocytes could respond both by a cellular immune
ating in the thymus and in some way prevent these cells from response (as in skin graft rejection) and by producing antibody
maturing to a stage when they would be capable of reacting (Gowans et al., 1962). He considered that the same cell could
immunologically’’ (Miller, 1962a). This clear prediction of nega- take part in either, depending on the antigenic stimulus. As
tive selection was proven some years later by using the so-called neonatally thymectomized mice were deficient in both cellular
super antigens and transgenic mice, and both positive and and at least some humoral responses, it was urgent to show
negative selection of thymocytes were then worked out (Bevan, that they had markedly reduced numbers of recirculating lym-
1977; Kappler et al., 1987). The major events in thymus cell dif- phocytes, not just blood lymphocytes. This was performed after
ferentiation have been summarized in a recent Timeline review cannulating the thoracic duct of neonatally thymectomized and
(Miller, 2011). ATxBM mice and collecting the lymph over a 24- to 48-hr period
It seemed important to determine whether the thymus exerted (Miller et al., 1967; Miller and Mitchell, 1967). The conclusion was
its influence by seeding cells into the rest of the lymphoid sys- made that most thoracic duct lymphocytes (TDLs) in mice were
tem. Since no cell-surface markers had at that time been found thymus derived, and this was actually proven in subsequent
to identify cells from different locations, use was made of the experiments (Miller and Sprent, 1971).
T6 strain of mice, the cells of which could be identified at meta- But were the same cells involved in antibody production and
phase by the presence of two minute chromosomes. Neonatally skin graft rejection? Two experimental systems suggested that
thymectomized F1 hybrid mice, in which one parent was T6, this might not be the case. Claman (Claman et al., 1966) showed
were grafted with thymus from the other parental strain and that irradiated mice given syngeneic bone marrow cells and
immunized with skin from various donors. An analysis of the syngeneic thymus cells could produce more antibody than
chromosome constitution of the cells in metaphase in the when given either cell source alone. As no antibody markers
spleen showed that 15% to 20% had originated from the thymus were available at the time, the origin of the antibody-forming cells
graft (Miller, 1962a). This suggested that the thymus did produce could not be easily identified. The situation was different for
cells capable of migrating to the periphery and that presumably chickens in Burnet’s laboratory, where impairment of bursa func-
such recent thymus emigrants would have just matured before tion by testosterone injection caused the birds not to produce
leaving or would mature in the peripheral lymphocyte pool to antibody, whereas thymus atrophy in sick birds prevented graft
become fully competent lymphocytes. rejection (Warner et al., 1962). Because mammals do not have
Although adult thymectomy had no effect on immunological a bursa, Burnet (Burnet, 1962) surmised, ‘‘In mammals it is highly
capacity, it could conceivably have caused a problem in mice probable that the thymus also carries out the function of the
whose lymphoid system had been destroyed in some way, as bursa of Fabricius in the chicken, which is to feed into the
for example by total-body irradiation. This proved to be the body the cells whose descendants will produce antibody.’’
case in partially irradiated mice (Miller, 1962c) and in lethally In 1967 and 1968, Miller and Mitchell (Miller and Mitchell, 1967,
irradiated mice protected with bone marrow (Cross et al., 1968, 1969), reconstituted neonatally thymectomized and adult
1964). The use of adult thymectomized, heavily irradiated mice thymectomized, irradiated CBA strain mice with CBA bone
protected with bone marrow (ATxBM mice) proved invaluable marrow and (CBAXC57BL)F1 TDLs and challenged them with
for further elucidation of immune functions. sheep erythrocytes. The ATxBM mice in those experiments

2 Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc.

 Please cite this article in press as: Miller and Sadelain, The Journey from Discoveries in Fundamental Immunology to Cancer Immunotherapy, Cancer
Cell (2015), http://dx.doi.org/10.1016/j.ccell.2015.03.007

Cancer Cell

Perspective

Dr Miller’s experiments I would have assumed that a single

variety of small lymphocyte was involved in each of our experi-
ments. If we have two cell lines that are collaborating, then
we have specificity residing in two cell lines, one thymus-derived
and the other marrow-derived. The problem is to bring these two
specific cell lines together. Does this necessity for the two cells
to find one another raise problems? It seems an inefficient mech-
anism if it rests only on chance contacts.’’ The then Professor of
Immunology at the National University in Canberra, Australia,
offered a less diplomatic critique and simply likened B and
T cells to the first and last letter of the word ‘‘bullshit.’’
The above somewhat simple experiments, performed without
the use of modern technologies such as gene targeting, cluster
of differentiation (CD) antigens, monoclonal antibody, and flow
cytometry, changed the course of immunology. Thus, the exis-
tence of T and B cells required a reinvestigation of numerous
Figure 1. The Origins of T Cells and B Cells
Percent reduction of antibody-forming cells per spleen of adult thymectomized
immunological phenomena in terms of the role played by the
and heavily irradiated CBA protected with CBA bone marrow (ATxXBM), given two distinct cell types, including the carrier effect, immunological
(CBAXC57BL)F1 thoracic duct lymphocytes, and challenged with sheep tolerance, immunological memory, immunodeficiency, autoim-
erythrocytes. To the incubation mixture was added normal mouse serum munity, and genetically determined immune responsiveness.
(NMS; slightly toxic), C57BL anti-CBA serum (aCBA), or CBA anti-C57BL
serum (aC57BL) as shown. The number of mice providing spleens in each An avalanche of work soon followed as investigations were
group was three to six. Adapted from Mitchell and Miller (1968). These ex- focused on defining and investigating further lymphocyte sub-
periments demonstrated the existence of thymus-derived cells (later known sets and their function.
as T cells) that were not antibody formers but were essential to allow cells
derived from the bone marrow (later known as B cells) to produce antibody
It was clear that the antigen recognition receptor on B lympho-
to certain antigens (later known as thymus-dependent antigens). cytes was immunoglobulin (Ig), but controversy raged concern-
ing the nature of the T cell receptor (TCR) for antigen. Some
argued strongly in favor of the existence of a different type of
were injected with immunocompetent TDLs, and antibody-form- Ig molecule (IgT) (Marchalonis et al., 1972). Others, however,
ing cells were produced that lysed sheep erythrocytes. This was showed that Ig on T cells had originated from B cells and bound
shown by plaques when sheep red cells and spleen cells were to the T cell Fc receptor (Hudson et al., 1974) and that no RNA
layered onto agar plates. Now the time was appropriate to deter- molecules bearing Ig VH nucleotide sequences could be de-
mine whether the TDLs gave rise to antibody-forming cells. It tected in T cells (Kemp et al., 1982). The discovery of monoclonal
was done by simply adding to the plates antiserum against antibodies (Köhler and Milstein, 1975) was finally instrumental in
CBA strain antigens made by immunizing C57BL mice with elucidating the TCR structure, and its coding sequences were
CBA tissues or antiserum against C57BL made in CBA mice. soon cloned independently in mouse and man (Hedrick et al.,
The latter would be expected to kill any antibody-forming cells 1984; Yoshikai et al., 1984). The TCR chains (a and b, or in
if they were derived from the (CBAXC57BL)F1 TDL and hence some cases g and d) are highly homologous to Ig and are
from the thymus-derived cells (Figure 1). The results were spec- associated on the T cell membrane with CD3, a complex of three
tacular, showing beyond any doubt that the antibody-forming polypeptides, g, d,and ε, and with a disulphide-linked z-z homo-
cells did not originate from the TDLs, but rather from the bone dimer or z-h heterodimer (Gold et al., 1986). These complexes
marrow (Mitchell and Miller, 1968). It proved that lymphocytes not only regulate the assembly and expression of the TCR, but
could be subdivided into two major groups: thymus-derived are also responsible for trans-membrane transduction of signals
cells (later known as T cells) were not antibody formers but after antigen occupation (Oettgen et al., 1985). The demonstra-
were essential to allow cells derived from the bone marrow (later tion that the TCR was sufficient to direct antigen specificity in
known as B cells) to produce antibody in response to certain transgenic mice (Dembic  et al., 1986) would set the stage for
antigens (later known as thymus-dependent antigens). They TCR-based T cell engineering two decades later.
were thus helper cells collaborating with other cell types, derived Since T and B cells utilize different molecules as antigen-spe-
from the bone marrow, to enable these to produce antibody. The cific receptors, do they perceive antigen in different ways? This is
murine equivalent of the bursa was thus the bone marrow. indeed the case: whereas B cells can bind soluble antigen,
The existence of two major lymphocyte subsets was first T cells generally recognize antigen only if displayed on the sur-
regarded with some skepticism. At a meeting in Brooke Lodge faces of antigen-presenting cells (APCs), such as dendritic cells
(Augusta, Michigan) held in 1968, Good was ‘‘concerned at (Steinman and Cohn, 1973) or virus-infected cells. Cytotoxic
separating thymus-derived from marrow-derived cells’’ since T cells isolated from mice recovering from a virus infection
the former ‘‘are in fact marrow derived-cells.’’ He also claimed were tested for their capacity to kill virus-infected target cells
to ‘‘have evidence that in the rabbit it (the bursa equivalent) re- in vitro. Killing was observed but only if the target cells had the
sides in the ilial lymphoid tissue and in the lymphoid tissue of same major histocompatibility complex (MHC) haplotype as
the appendix’’ (Good, 1969). At the same meeting, Gowans the mice from which the T cells were obtained (Zinkernagel
(Gowans, 1969), who had proven that recirculating small lym- and Doherty, 1974). This suggested that there might be an asso-
phocytes were immunocompetent, stated, ‘‘Had it not been for ciation between a virus product and MHC molecules at the cell

Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc. 3

 Please cite this article in press as: Miller and Sadelain, The Journey from Discoveries in Fundamental Immunology to Cancer Immunotherapy, Cancer
Cell (2015), http://dx.doi.org/10.1016/j.ccell.2015.03.007

Cancer Cell

Perspective

surface and that the specificity of the TCR was directed to both of antimicrobial bodies but is determined purely by cellular fac-
MHC molecules and virus-encoded products. The phenomenon tors. These may be weakened in older age groups where cancer
became known as MHC restriction and the MHC molecules is more prevalent.’’ Burnet echoed this view in his paper on tu-
involved as restriction elements. T cells in fact recognize mor immunosurveillance (Burnet, 1971).
relatively short peptide fragments (Townsend et al., 1986) that A large amount of work has been done by many to determine
become wedged in the jaws of the MHC molecules. How the importance of immunocompetent lymphocytes in guarding
T and B cells perceive antigen is highly relevant for tumor against the emergence of neoplastic clones of cells. There is
immunity and the escape of immunogenic tumor cells from any no doubt that a deficiency of such cells is associated with an
host immune response. Loss of cell-surface antigen or MHC increased incidence of those neoplasms that happen to be
molecules from genetically unstable tumor variants would of strongly immunogenic and to be produced by strong carcino-
course prejudice or prevent the response of any specific B and gens as cited above (Miller et al., 1963). However, extensive
T cells. CARs would later capitalize on the respective advan- studies performed using athymic nude mice failed to provide
tages of both targeting modalities—and Ig VH genes would any evidence of an increased susceptibility to the development
make their way into T cells, after all (see below). of spontaneous tumors (Rygaard and Povlsen, 1976). On the
Further T cell subsets and their function were soon identified: other hand, RAG-deficient mice (lacking T, B, and NKT cells)
CD4 and CD8 T cells (Kisielow et al., 1975; Cantor and Boyse, do spontaneously develop gastrointestinal epithelial malig-
1975), natural killer T (NKT) cells (Makino et al., 1995; Godfrey nancies by the age of 18 months (Shankaran et al., 2001);
et al., 2004), and CD4+CD25+FoxP3+ regulatory T (Treg) cells immunocompromised pathogen-free severe combined immu-
(Sakaguchi et al., 1985). Self-tolerance, as mentioned above, nodeficiency (SCID) mice have a high incidence of spontaneous
is mostly achieved intra-thymically by negative selection of thymic lymphomas, and about 2% of retired breeders developed
self-reactive thymocytes. Since, however, some self-reactive a variety of non-thymic tumors (Huang et al., 2011).
T cells (e.g., those with low affinity to self-antigens) escape to It is well established that immunosuppressed humans, e.g.,
the periphery, a peripheral tolerance mechanism exists, enabling following therapy for transplantation, have an increased inci-
Treg cells to prevent autoimmunity (reviewed in Josefowicz et al., dence of tumors such as skin or cervical carcinomas and lym-
2012). Treg cells are either produced in the thymus during T cell phomas (Penn and Starzl, 1973). A key question is whether this
development (natural Treg [nTreg] cells) or induced from naive is the result of failed immunosurveillance or of increased suscep-
CD4+ T cells in the periphery (so-called induced Treg [iTreg] tibility to infection by potentially oncogenic viruses (human papil-
cells) and are marked by expression of the transcription factor lomavirus, hepatitis C virus, human immunodeficiency virus, and
Foxp3 (Sakaguchi and Powrie, 2007). These cells soak up inter- Epstein-Barr virus [EBV]).
leukin-2 (IL-2) (Smith, 1989) through their high-affinity trimeric Many of the antigens expressed by spontaneous tumors are
IL-2 receptor, thus depriving other T cells of this growth factor, expected to be self-antigens, and hence thymus-derived lym-
but the exact mechanism by which, when recruited into the phocytes responding to these antigens would have been deleted
tumor environment, they prevent the activity of other immune in the thymus. Low-affinity cells specific for these self-antigens
cells capable of destroying the tumor is not clear. It may involve may, however, have escaped deletion and migrated to the pe-
the production of inhibitory cytokines such as transforming riphery, where they may be held in check by the growing tumors
growth factor (TGFb) (Powrie et al., 1996), IL-10 (Asseman as described below. Some spontaneous tumors have a high fre-
et al., 1999), and IL-35 (Collison et al., 2007). quency of mutations that may therefore generate de novo anti-
It is not the aim of this review to describe in further detail each gens (e.g., BCR-Abl, mutated forms of p53) that will not have
of the key advances that have led to our current knowledge of been present in the thymus and may thus appear foreign to the
the function of T and B cells, since this has been extensively immune system (Coulie et al., 2014; Gubin et al., 2014). In these
covered recently in a Timeline review (Miller, 2011). It is note- cases, therefore, both T cells and antibodies directed to tumor
worthy that several genes that govern T cell development or antigens should be able to eliminate the tumor. It is, however,
function, e.g., those encoding the IL receptor common g chain, not clear how likely it is that a point mutation in a single oncogene
the Wiskott-Aldrich syndrome protein, and others, are now being would give rise to an antigenic peptide. In some instances,
developed as genetic therapies for the treatment of severe pathogen-elicited thymus-derived cells that cross-react with
immunodeficiencies (Fischer et al., 2013) neoantigens acquired through somatic mutation may contribute
to anti-tumor responses (Snyder et al., 2014).
From Immune Surveillance to Cancer Vaccines The bulk of the observations on immune surveillance have in
and Adoptive T Cell Therapy fact shown that T cells and antibodies could recognize and elim-
The finding that neonatally thymectomized mice were more sus- inate tumors (Boon et al., 1994). How tumors spontaneously
ceptible than normal mice to the cancer-producing activities of generate tumor immunity has led to the cancer immunoediting
strong carcinogens (e.g., 3,4-benzopyrene) (Miller et al., 1963) hypothesis (Schreiber et al., 2011). This consists of three
and polyoma virus (Miller et al., 1964), (Miller, 1967) provided sequential phases: elimination, equilibrium, and escape. In the
some experimental support for Paul Ehrlich’s concept of immu- first phase, tumors developing before being clinically apparent
nological surveillance (Ehrlich and Himmelweit, 1957): ‘‘I am are destroyed and eliminated by both innate and adaptive im-
convinced that during development and growth malignant cells mune mechanisms (e.g., Shankaran et al., 2001). If variants arise
arise extremely frequently, but in the majority of people they and fail to be eliminated, cells of the adaptive immune system
remain latent due to the protective action of the host. I am also may restrain their growth during the lifetime of their host and
convinced that this natural immunity is not due to the presence a state of equilibrium is reached. Constant immune selective

4 Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc.

 Please cite this article in press as: Miller and Sadelain, The Journey from Discoveries in Fundamental Immunology to Cancer Immunotherapy, Cancer
Cell (2015), http://dx.doi.org/10.1016/j.ccell.2015.03.007

Cancer Cell

Perspective

pressure of genetically unstable tumors may, however, lead to ligands CD80 (B7-1) and CD86 (B7-2) on the APC (Lafferty and
variants that can no longer be recognized by effector T cells Cunningham, 1975; Harding et al., 1992). Additional costimula-
because they have downregulated antigen or MHC, secreted tory receptors support T cell expansion and the maintenance
immunosuppressive cytokines such as TGFb or IL-10, or of T cell memory (Croft, 2003). Both activating and costimulatory
recruited Treg cells in their environment. They then enter the molecules would eventually be turned into powerful anti-cancer
escape phase and become clinically apparent. agents, via very different therapeutic strategies, one based
The administration of a cancer vaccine, such as GVAX (Dranoff on monoclonal antibody technology and the other on genetic
et al., 1993), has been used in the management of non- or weakly engineering.
immunogenic cancers. GVAX expresses tumor antigens in the
context of two important adjuvant activators, granulocyte- Reversing Tumor-Protective Immune Inhibition
macrophage colony-stimulating factor (GM-CSF) and pathogen Under normal circumstances, T cell responses are regulated
associated molecular patterns (PAMPs), that promote the acti- by inhibitory checkpoints, mediated in part by CTLA-4 (CD152),
vation of T cells with low affinity for self-antigens expressed by PD-1 (CD279), and BTLA (CD272), to prevent unrestrained multi-
tumors. This vaccine, together with the DNA-damage-inducing plication, collateral damage by cytotoxic effector molecules, and
chemotherapeutic drug cyclophosphamide that preferentially even autoimmunity.
targets Treg cells in a mixed cell population (Ghiringhelli et al., CTLA-4 competes with CD28 for the ligands CD80 and CD86
2007), with further assistance from the dual cocktail of anti- and acts as a signal dampener during the early stages of activa-
CTLA-4 plus anti-PD-1 (or anti-PD-L1) antibodies, discussed tion of naive and memory T cells, in fact 24–48 hr after antigen
below, may be successful in the treatment of certain tumors— presentation (Krummel and Allison, 2011). It has been shown
if potent, truly tumor-specific T cell responses are indeed eli- to act as an effector molecule that inhibits CD28 costimulation
cited. This goal may be harder to reach in tumors with a low by the cell-extrinsic depletion of its ligands (Qureshi et al.,
rate of mutations (Alexandrov et al., 2013). 2011). CTLA-4 is expressed on T cells, and its main physiological
The focus on T cell responses inspired yet another approach in function is to down-modulate CD4 T helper cell activity and
cancer immunotherapy, pioneered by Rosenberg and col- increase CD4+CD25+FoxP3+ Treg-cell-mediated immunosup-
leagues, focusing directly on the patient’s own tumor-infiltrating pression (reviewed by Pardoll, 2012). A deficiency or mutation
lymphocytes (TILs) when those exist and are accessible. In TIL in CTLA-4 in humans has contributed to the development of
therapy, T cells retrieved from surgical melanoma specimens autoimmune diseases including autoimmune hypothyroidism,
are cultured under activating conditions and reinfused with IL-2 type I diabetes, systemic lupus erythematosus, and celiac dis-
(Rosenberg et al., 1986) after host preconditioning (Gattinoni ease (reviewed by Watanabe and Nakajima, 2012). Recently, a
et al., 2006). This approach is, however, not applicable to all heterozygous nonsense mutation in exon 1 of CTLA4 has been
melanoma patients nor many other cancers. In EBV-associated identified in one of the commoner subgroups of common
lymphoproliferative disease, for which EBV-transformed B cells variable immunodeficiency syndromes (CVIDs) characterized
uniquely serve as effective APCs for tumor antigens of viral by hypogammaglobulinemia, recurrent infections, and multiple
origin, tumor-specific T cell lines have been successfully gener- autoimmune manifestations (Schubert et al., 2014).
ated from peripheral blood of healthy donors and used to treat PD-1 is expressed following T cell activation and during
immunocompromised recipients (Papadopoulos et al., 1994; chronic antigen-induced T cell stimulation, and it limits the activ-
Heslop et al., 1996). Artificial APC systems substituting for ity of T cells that have already been activated (Keir et al., 2008).
EBV-transformed B cells or dendritic cells (Kim et al., 2004) Unlike CTLA-4, PD-1 is expressed not only on all T cells subsets
have since been developed to facilitate the selection and/or but also on activated B cells and NK cells. It exerts its main func-
expansion of therapeutic T cells. tion during the inflammatory response associated with infection.
Chronic antigen exposure, as occurs in certain viral infections,
From T Cell Activation Biology to Checkpoint Blockade can induce exhaustion of cognate antigen-specific activated
and T Cell Engineering T cells. Furthermore, signals from inflammatory tissues induce
Most spontaneous tumors express self-antigens and present the expression of the PD-1 ligands, PD-L1 (B7-H1) or PD-L2
peptides derived from them embedded in MHC proteins on their (B7-DC), that downregulate T cell activity, thereby limiting collat-
cell surface. T cells with high-affinity TCRs for these would have eral damage and even autoimmunity. As with CTLA-4, mutations
been deleted by negative selection in the thymus. In melanomas, in PD-1 have also been associated with autoimmunity in humans
which are known to undergo a high mutation rate (Berger et al., (Watanabe and Nakajima, 2012).
2012), some mutated peptides are displayed as altered self- Theoretically, therefore, antagonists directed to these immune
antigens on the cell surface, where they can be recognized by inhibitory checkpoints should enable T cells to proliferate and
cytotoxic CD8 T cells. So why are these immunogenic tumors respond more vigorously and would be beneficial in controlling
not spontaneously killed by activated CD8 cells? Extensive virus infections and suppressing tumor growth (Figure 2). Ago-
studies of the molecules and processes involved in T cell acti- nists of CTLA-4 or PD-1, on the other hand, may prevent unde-
vation have shed light on this conundrum and yielded levers to sirable immune responses as occur in autoimmunity, allergy,
enable or restore T cell function. and transplant rejection (Okazaki and Honjo, 2007). In many
T cell activation initially requires two signals, one from the cases, cancer cells express the ligand PD-L1 on their surface,
recognition of antigen-derived peptides displayed in the context which may restrict tumor-reactive T cell responses by engaging
of MHC molecules on the surface of APCs and one from the the inhibitory receptor PD-1. Furthermore, PD-L1 on tumor cells
interaction of the T cell costimulatory molecule CD28 with its and the surrounding stromal cells is upregulated by g-interferon

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Cell (2015), http://dx.doi.org/10.1016/j.ccell.2015.03.007

Cancer Cell

Perspective

stricted proliferation, thereby enhancing their capacity to kill

their tumor targets. The blockade of CTLA-4 using monoclonal
antibodies, as first shown by Allison (Leach et al., 1996), restored
tumor immunity in mice bearing immunogenic tumors. There are
two anti-CTLA-4 antibodies in use against metastatic melanoma:
ipilimumab and tremelimumab. There are eight anti-PD-1/PD-L1
antibodies in clinical trials: nivolumab, pembrolizumab /MK-
3475, pidilizumab, and AMP-224, targeting PD-1; and BMS-
935559, MEDI4736, MPDL3280A, and MSB0010718C, targeting
PD-L1. In addition, inhibitors against other immune checkpoint
regulators expressed on the cell surface, such as TIM3, LAG3,
and VISTA, and agonist antibodies acting on T cell costimulatory
receptors, such as OX40 and CD137, are being developed
for cancer immunotherapy (European Society for Medical
Oncology, 2014). The dual cocktail of ant-CTLA-4 and anti-PD-
1 has been most effective in treating some melanoma patients
(Curran et al., 2010).
Monoclonal antibodies targeting CCR4, a chemokine receptor
Figure 2. Antibody-Mediated Targeting of Negative Regulators of that is highly expressed on Treg cells, and PI3 kinase inhibitors
T Cell Responses that preferentially block Treg cell activity, may soon complement
Anti-CTLA4 monoclonal antibodies can alleviate CTLA4-mediated abrogation the T cell activation checkpoint blockade modulators (e.g., anti-
of CD28 signaling in T cells and deplete intra-tumoral regulatory T cells
(Treg cells), which act exogenously to suppress anti-tumor effector T cells. bodies against CTLA4 or PDL1) in immunotherapy of cancer
PD-1/PD-L1 blockade interrupts the PD-L1 tumor adaptive response, which (Sugiyama et al., 2013; Abu-Eid et al., 2014).
may be initiated by T-cell-derived g-interferon secretion, inducing PD-L1
expression by the tumor cell, and the induction of PD-1 expression by the
activated T cells.
The Promise of T Cell Engineering
The advent of gene transfer technologies, in particular those
enabling the transduction of human T lymphocytes using gibbon
secreted by the very activated T cells that have infiltrated the ape leukemia virus envelope-pseudotyped g-retroviral vectors
tumor environment (Iwai et al., 2002). Treg cells, which specif- (Mavilio et al., 1994; Bunnell et al., 1995; Gallardo et al., 1997),
ically suppress activated T cells during a normal immune created new opportunities for immune intervention based on
response to limit collateral damage and autoimmunity, are T cell engineering (Sadelain et al., 2003). Patients’ T cells, easily
recruited to the tumor environment and act as another dampener accessible in peripheral blood, can be genetically instructed to
that limits the activity of tumor-infiltrating cytotoxic T cells (Cu- target tumors by transduction of receptors for antigen, utilizing
riel, 2007). Treg cells express high levels of immune checkpoint either the physiological TCR (Ho et al., 2003; Stone and Kranz,
receptors, including CTLA-4 and PD-1, wherein these inhibitors 2013) or synthetic receptors now known as CARs (Sadelain
of effector T cells paradoxically enhance Treg cell activity. These et al., 2009). Both approaches have shown clinical successes,
receptors may thus serve as targets for antibody-mediated Treg particularly in melanoma (Robbins et al., 2011), targeting NY-
cell depletion (Simpson et al., 2013) (Figure 2). A deficiency of ESO1 (Chen et al., 1997), and in acute lymphoblastic leukemia,
CTLA-4 in Treg cells in mice results in the spontaneous develop- (Brentjens et al., 2013; Grupp et al., 2013; Davila et al., 2014;
ment of systemic lymphoproliferation, fatal T-cell-mediated Lee et al., 2015; Maude et al., 2014), targeting CD19 (Brentjens
autoimmune disease, and hyperproduction of IgE, and the loss et al., 2003).
of CTLA-4 also potentiates tumor immunity (Wing et al., 2008). CARs are artificial, composite receptors for antigen that
Furthermore, the immune-cell-expressed ligand semaphorin- integrate principles of B cell and T cell antigen recognition
4a (Sema4a) and the Treg-cell-expressed receptor neuropilin-1 (Figure 3A). They are particularly attractive in that they elude hu-
(Nrp1) interact to potentiate Treg cell function and survival. man leucocyte antigen (HLA) restriction and are thus applicable
Nrp1 is required by Treg cells to limit anti-tumor immune re- to all patients irrespective of their HLA haplotypes, unlike TCRs.
sponses, suggesting that the Nrp1-Sema4a pathway may be a CARs may also overcome HLA downregulation by tumors, which
potential target to limit Treg-cell-mediated tumor-induced toler- deprives T cells of a ligand for their endogenous TCR (Seliger,
ance (Delgoffe et al., 2013). 2008). The critical function of CARs is, however, not to merely
Inhibitory myeloid cells also accumulate in certain tumor types target the T cells to a tumor antigen, but to enhance T cell func-
and may negatively impact on anti-tumor T cell responses tion. Thus, effective CARs further integrate principles of T cell
(reviewed in Ostrand-Rosenberg, 2010). costimulation (Figures 3B and 3C) and provide a broad spectrum
Monoclonal antibody technology (Köhler and Milstein, 1975) of functional enhancements acquired by directly soliciting
has enabled a range of immunotherapies targeting cell-surface selected costimulatory pathways (Sadelain et al., 2013; Jensen
molecules. What has recently been achieved with clinical suc- and Riddell, 2015).
cess in the case of some tumors, in particular malignant meta- The CARs that have recently shown impressive clinical out-
static melanoma, is the use of blocking monoclonal antibodies comes in patients with B cell malignancies are second-genera-
directed against CTLA-4 and PD-1 or PD-L1 (Figure 2). These tion CARs, so named to highlight their fundamental difference
allow the specific tumor infiltrating T cells to undergo unre- with the CD3z chain fusion receptors that preceded them

6 Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc.

 Please cite this article in press as: Miller and Sadelain, The Journey from Discoveries in Fundamental Immunology to Cancer Immunotherapy, Cancer
Cell (2015), http://dx.doi.org/10.1016/j.ccell.2015.03.007

Cancer Cell

Perspective
Figure 3. Principles of T Cell Engineering
and CAR Design
(A) Integration of B cell and T cell antigen recog-
nition principles in the design of CARs. The heavy
and light chain chains, which are components
of the B cell receptor and Igs, are fused to the
T-cell-activating z chain of the TCR-associated
CD3 complex to generate non-MHC restricted,
activating receptors capable of redirecting T cell
antigen recognition and cytotoxicity.
(B and C) Integration of T cell activation and
costimulation principles in dual signaling CARs
designed to enhance T cell function and persis-
tence in addition to retargeting T cell specificity.
In (B), the physiological abTCR associated with
the CD3 signaling complex is flanked by the CD28
costimulatory receptor. (C) shows a prototypic
second-generation CAR, which comprises three
canonical components: an scFv for antigen
recognition, the cytoplasmic domain of the CD3z
chain for T cell activation, and a costimulatory
domain to enhance T cell function and persistence
(here CD28, as described in Maher et al. [2002],
and currently utilized in ALL patients as reported in
Brentjens et al. [2013] and Davila et al. [2014]).
Unlike the abTCR/CD3 complex, which comprises
g, d, ε, and z signaling chains and is modulated
by a multitude of costimulatory receptors, CARs
possess in a single molecule the ability to trigger
and modulate antigen-specific T cell functions.

CARs to be unable to command T cell

expansion upon repeated exposure to an-
tigen (Gong et al., 1999), consistent with
the two-signal model of T cell activation
(Lafferty and Cunningham, 1975) and
transgenic mouse studies of first-genera-
tion CARs (Brocker, 2000). These findings
prompted us to combine chimeric costi-
mulatory receptors that we had previously
studied in human primary T cells (Krause
et al., 1998) with T-cell-activating CARs,
thus creating dual-signaling receptors
(Figure 3C) that we found were able to
instruct human peripheral blood T cells
to expand and retain function upon
repeated exposure to antigen (Maher
(Sadelain et al., 2009). These molecules evolved in three critical et al., 2002). These and other contemporary studies (Finney
stages. When the z chain of the CD3 complex was independently et al., 1998; Hombach et al., 2001; Imai et al., 2004) paved the
cloned by the Weiss, Seed, and Klausner groups, the newly way for engineering persisting functional T cells, which we later
discovered T-cell-specific chain, which lacks an extracellular named ‘‘living drugs.’’ Second-generation CARs are a new class
domain (Figure 3B), was fused to CD8, CD4, or CD25 (Irving of drugs with exciting potential for cancer immunotherapy (Sade-
and Weiss, 1991; Letourneur and Klausner, 1991; Romeo and lain et al., 2013; Jensen and Riddell, 2015). Recent clinical results
Seed, 1991) to enable antibody-mediated cross-linking of the z obtained with CD19 CARs are reviewed in Ramos et al. (2014) and
chain fusion receptor. These studies, conducted in leukemic Kochenderfer and Rosenberg (2013). CAR therapy promises to
T cells, revealed the activating function of the z chain. The addi- produce more durable cancer regression in other hematological
tion of an scFv derived from an Ig, first reported by Eshhar and malignancies and solid tumors, including those that do not pre-
colleagues (Eshhar et al., 1993; Brocker et al., 1993), diversified sent altered self-antigens.
the binding capacity of these fusion receptors and redirected CARs may be combined with costimulatory ligands (Stephan
the specificity of transfected cytotoxic T cell hybridomas (Eshhar et al., 2007), chimeric costimulatory receptors (Krause et al.,
et al., 1996). Having established methods for the transduction of 1998; Duong et al., 2011; Wilkie et al., 2012; Prosser et al.,
human peripheral blood T cells (Gallardo et al., 1997), we could 2012; Kloss et al., 2013), or cytokines such as IL-15 (Hoyos
for the first time test the functional features of z-chain-based et al., 2010; Markley and Sadelain, 2010) or IL-12 (Chinnasamy
CARs in clinically relevant cell types. We found z-chain-based et al., 2012; Chmielewski and Abken, 2012; Pegram et al.,

Cancer Cell 27, April 13, 2015 ª2015 Elsevier Inc. 7

 Please cite this article in press as: Miller and Sadelain, The Journey from Discoveries in Fundamental Immunology to Cancer Immunotherapy, Cancer
Cell (2015), http://dx.doi.org/10.1016/j.ccell.2015.03.007

Cancer Cell

Perspective

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ACKNOWLEDGMENTS
Chinnasamy, D., Yu, Z., Kerkar, S.P., Zhang, L., Morgan, R.A., Restifo, N.P.,
We thank Drs. Tony Basten, Marc Feldmann, Dale Godfrey, Robyn Slattery, and Rosenberg, S.A. (2012). Local delivery of interleukin-12 using T cells
and Andreas Strasser for reading the manuscript and for helpful suggestions. targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice.
Clin. Cancer Res. 18, 1672–1683.

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