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 Abeloff’s
Clinical
Oncology
Issue 1 - 2014
The Immune System in
Melanoma Initiation and Progression
R. Todd Reilly, PhD,* James O. Armitage, MD,† and John E. Niederhuber, MD*
Historical Context of Immunotherapy
Although the notion of leveraging host immunity in the
fight against cancer was first conceived over a century
ago, technological advances in the past 20 years have both
broadened our understanding of the functioning of the
immune system and spurred the development of novel
immunologically based treatment options for a range of
cancers. In this review, we focus specifically on summarizing the current understanding of the complex interplay
between the initiation and progression of cutaneous melanoma and the host immune system, highlighting the key
features of tumor growth and immune function that determine the balance between suppression and activation of
tumor-specific immunity.
William Coley, in the late 1800s, is widely recognized
as the first to postulate the potential for host immunity
to serve as a mechanism for cancer treatment when, as a
surgeon at what was then known as Memorial Hospital in
New York City (now Memorial Sloan-Kettering Cancer Center), he observed the spontaneous regression of “sarcomas”
in patients who had a concomitant bacterial infection.1-3
Coley went on to treat several cancer patients through direct injection of the tumor with bacterial extracts (Coley’s
Toxins), noting the (temporary) growth-inhibitory action
of the treatment but also the considerable “risks of inoculation, when successful.”1 Although Paul Erlich in 1909
first proposed that the immune system may play a role in
eliminating nascent tumors,4 the dangers associated with
inoculating patients with bacterial extracts combined with
the inconsistent outcomes reported resulted in a generally unfavorable view of immunotherapy within the clinical community.
Following Paul Erlich’s introduction of the notion of a
relationship between cancer development and the
host immune system, including the concept of immunosurveillance,4 the hypothesis was largely ignored for
almost 50 years until the convergence of a growing
*Inova Translational Medicine Institute, Inova Health System,
Falls Church, Virginia
†University of Nebraska Medical Center, Omaha, Nebraska
Copyright © 2014 Elsevier, Inc.
understanding
of
allograft rejection
and the recognition
of tumor-associated
neoantigens
in
mouse models of
chemically induced
tumors. The identification of unique
tumor specific antigens led to separate
(and subtly different) reassertions by
Thomas and Burnet
that a critical function of the immune
system is to patrol
for and eliminate
nascent tumor cells.5-9 Support for the immunosurveillance hypothesis dimmed again in the 1970s when immunocompromised mouse models were used as a basis to
study spontaneous and chemically induced tumor incidence.10-13 This line of investigation culminated in a series
of studies in which immunocompromised athymic nude
mice were shown to have the same tumor incidence as
immunocompetent animals,14-16 leading many to seriously
question the concept of immunosurveillance. Not appreciated at the time was the fact that athymic mice are not
completely immunocompromised; they possess natural
killer (NK) cells and maintain a small population of T cells
as well.17-19 Over the subsequent 30 years, as our understanding of the diverse range of immune cells has evolved,
so too has the immunosurveillance hypothesis. The current understanding, which is still being refined as further
knowledge is acquired, encompasses not only immunosurveillance but also immunoediting, whereby an equilibrium process exists in which genetic instability within an
incipient tumor results in the outgrowth of individual
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This material was supported by an educational grant from Bristol-Myers Squibb.
1
2
tumor cells expressing novel antigenic determinants (reviewed20-22). These immunoedited tumor cells are then
eliminated by innate immune effector cells (primarily NK
cells) and by adaptive immune effector cells (primarily T
cells). Ultimately, this equilibrium process is thought to
result in either the elimination (or control) of the incipient tumor, or the outgrowth (escape) of tumor cell variants that evade the immunoediting process and progress
to become clinically detectable tumors.20-22
It wasn’t until the late 1980s and 1990s, when the development of transgenic mouse models and other new technologies made it possible to (relatively) quickly and easily
study the behavior of immune cells at the genetic level
as well as at the population level, that investigators were
able to develop strategies to orchestrate perturbations in
the balance between immune activation and suppression. In this review, we discuss a variety of interventions
developed to promote immune-mediated mechanisms
for melanoma treatment, with emphasis on our evolving
understanding of the immunologic pathways and mechanisms underlying those interventions.
Basic Tumor Immunology
Opening
Because opportunities for immune intervention in cancer
therapy have been identified at virtually every stage of the
immune response, reviewing those targets and interventions within the context of a general review of basic processes underlying immune recognition, activation, and effector function is useful. The mammalian immune system
has evolved to encompass the integrated actions of a range
of cells together with both soluble and membrane bound
ligands and their respective receptors to protect the host primarily against pathogenic microbes while remaining generally nonresponsive to host cells and tissues. In humans,
immunity to foreign pathogens generally involves the sequential engagement of both innate and adaptive immune
functions; the former characterized by a more immediate,
less specific, and shorter duration response that gives way
to the more focused and longer lasting effects of the latter. Effector functions of the innate and adaptive immune
responses are very tightly controlled to prevent both collateral and inappropriately targeted damage to normal host
tissues. It is within this dynamic equilibrium between the
opposing influences of immune activation and tolerance that
the opportunities to induce effective antitumor immunity lie.
The Role of Innate Immunity
Innate immunity serves as a first line of host defense and,
as such, is composed of epithelial and mucosal barriers
(skin, epithelia, and mucosa), a cadre of soluble antimicrobial factors (complement, cytokines, chemokines),
pattern recognition receptors that allow rapid identification of pathogen-associated molecular patterns
(PAMPs), and a range of effector cells (including dendritic
cells, eosinophils, macrophages, mast cells, monocytes,
neutrophils, and NK cells). The innate immune response is
The Immune System in Melanoma Initiation and Progression
characterized by rapid initiation (minutes to hours), a low
specificity of response relative to the adaptive immune response, and localized inflammation. The first innate effector cells at the site of infection typically are macrophages
and mast cells, which upon activation secrete a range of
cytokines and chemokines that mediate the physiologic
hallmarks of the innate immune response: the dilation
and increased permeability of nearby blood vessels and
resultant accumulation of fluid and blood proteins and the
recruitment of neutrophils, lymphocytes, and monocytes
(precursors of macrophages) into the inflamed site.
Antigen Presentation and AntigenPresenting Cells
Dendritic cells (DCs) are among the innate effector cells
recruited to sites of inflammation. DCs, referred to as professional antigen-presenting cells (APCs), play a critical
role in bridging innate and adaptive immune responses
by acquiring antigens from the site of an infection and
presenting those antigens to effector cells to initiate the
adaptive immune response (Figure 1). An antigen can be
any macromolecule (protein, polysaccharide, or lipid conjugate thereof) that elicits an immune response. DCs that
are recruited to an inflammatory site take up protein antigens, processing and degrading them internally into short
peptides (or determinants), and packaging and presenting
those peptides in association with major histocompatibility complex (MHC) molecules on their surface. If DCs receive the appropriate stimulation through the engagement
of pattern recognition receptors or other “danger signals”
(e.g., proinflammatory cytokines or certain kinds of cellular debris), they undergo a process of maturation that results in the increased expression of cell surface molecules
(MHC Class I [MHC-I], MHC-II, CD80 and CD86 (a.k.a.
B7-1 and B7-2), and other receptors and ligands that play
a role in modulating the adaptive immune response) and
soluble factors (cytokines and chemokines). Activated DCs
also upregulate specific cell surface adhesion molecules
that facilitate their migration to the lymph node, where
they encounter adaptive immune effector cells.
Rudolph Virchow, in 1863, first observed immune infiltrates within tumors, leading him to hypothesize that
inflammation played a role in tumor development.23 It is
now widely accepted that chronic inflammation–resulting
from chronic infections (e.g., Helicobacter pylori/gastritis
or viral hepatitis) or autoimmunity (e.g., inflammatory
bowel disease)–can promote carcinogenesis (reviewed24).
Chronically activated leukocytes continue to secrete
proinflammatory factors, notably tumor necrosis factor
(TNF), interleukin (IL)-1, and IL-8/CXCL-8, which promote tumor growth and development.25-28 Notably, these
factors are also involved in stimulating the secretion of
transforming growth factor-beta (TGF-β), IL-10, and IL-1β
by melanoma (reviewed24,29,30), which play an important
role in influencing the outcome of the adaptive antitumor
immune response.
The Immune System in Melanoma Initiation and Progression
3
B cell) by CD40L (on the T cell). The
fully activated B cell then proliferates
(clonal expansion) and begins to secrete soluble antibody in the form of
GM-CSF
(pentameric) IgM. After about a week,
the plasma cells undergo class switchIL-4,FLT-3L
ing, resulting in the production of solBone marrow
Dendritic cell
uble IgG antibodies, followed by a proprogenitor
progenitor
cess called affinity maturation, which
Ag uptake/Processing
further refines the genetic constructs
Microbial infection
encoding the antibody’s antigen bindNo danger
danger signals
ing site and leads to the production of
signals
Exogenous Endogenous
“Tolerizing DC”
higher-affinity binding of the BCR/
LPS
TNF
antibody to its target antigen. Upon
CpG
CD40L
clearance of the target antigen, a small
Activated DC
proportion of the plasma cells differentiate into memory B cells, a very
long-lived B cell type capable of proModerate MHC II
High MHC II
Chemokines
Chemokines
ducing high-affinity antigen-specific
Adhesion molecules
Adhesion molecules
IgG much more rapidly than in a priCostimulatory molecules
Costimulatory molecules
mary immune response. Antibodies
can mediate phagocytosis of cells expressing their cognate antigen through
Figure 1. Dendritic cells (DCs) can either activate adaptive immunity or
opsinization of the target cell and can
tolerize T cells depending on their state of maturation.
also facilitate target cell lysis either diFrom Pardoll D. Cancer immunology. In: Niederhuber JE, Armitage JO, Doroshow JH, Kastan MB,
Tepper JE, eds. Abeloff’s Clinical Oncology. 5th ed. Philadelphia: Churchill Livingstone; 2014:85.
rectly, through compliment activation,
or indirectly, by activating antibodydependent cell cytotoxicity (ADCC)
Adaptive Immunity
mechanisms of innate effector cells. Whereas tumorIn contrast to the rapid responses achieved in the innate
specific antibodies have been generated (e.g., trastuzumab
immune response, which is believed to be evolutionariand rituximab), such antibodies are predominantly used to
ly older, the adaptive immune response can require 7 to
block proliferative signaling pathways within cancer cells
14 days to become fully activated, is highly specific for
rather than as a platform for immune-mediate tumor rejecdistinct antigens and antigenic epitopes (peptide detertion, although there are some examples of the latter.
minants derived from antigen processing by APCs), and
The cognate receptor on T cells, unlike its B-cell counresults in the formation of long-lasting memory effector
terpart, does not recognize soluble antigen; TCRs recogcells that are able to rapidly reactivate in the event that
nize peptide antigens in the context of MHC I and II molethe specific pathogen (antigen) is encountered again. The
cules. After APCs (DCs) take up and process the antigenic
exquisite specificity of the adaptive immune response is
proteins, the derived peptides are bound by MHC I and
even more impressive when the incredible diversity of anMHC II molecules, which are then transported to the surtigens that can potentially be “recognized” by the adaptive
face of the APC for inspection by T cells. The TCR of CD4+
effector cells is considered. Both the diversity and specificT cells interact with MHC II-peptide complexes, which
ity of antigen recognition are enabled through a series of
are expressed primarily by APCs, while the TCR of CD8+
unique genetic recombination steps during cell differentiaT cells interact with antigenic peptides complexed with
tion and activation that give rise to the antigen receptors
MHC I molecules, which involves nearly all cells includon the surface of B and T cells, the B-cell receptor (BCR)
ing APCs. TCR engagement with MHC bearing its cognate
and T-cell receptor (TCR).
peptide provides signal 1 for T-cell activation. If the APC is
The BCR is a membrane-bound form of the same improperly activated, it will also express CD80/CD86 on its
munoglobulin (IgM) (antibody) that ultimately is secreted
cell surface. Engagement of CD80/CD86 with CD28 on the
by the fully activated B cell (plasma cell) and is capable
T-cell surface provides signal 2, leading to T-cell proliferaof recognizing soluble antigens. Engagement of the BCR
tion (clonal expansion) and differentiation into an effector
to its cognate antigen is the initial step in B-cell activacell (CD4+ helper T cell or CD8+ cytotoxic T cell).
tion (referred to as signal 1). The BCR-antigen complex is
Activated CD8+ T cells undergo rapid, IL-2–dependent
then internalized and degraded, and antigenic epitopes are
proliferation and upregulation of surface receptors, such
presented on MHC II on the B-cell surface for inspection
as the cytokine receptor CXCR-3, to facilitate trafficking
by (activated) CD4+ T cells, which provide signal 2 to the
to the peripheral tissues and secretion of proinflammatory and antiviral cytokines such as TNF-α and interferon
B cell through engagement of the CD40 coreceptor (on the
4
(IFN)-γ. When activated CD8+ T cells encounter cognate
peptide in the context of MHC I expressed on the surface
of somatic cells, they release perforin and granzyme from
lytic granules. Perforin polymerizes within the cell membrane of the target cell, forming a pore through which granzyme enters. The granzymes, which are serine proteases,
then trigger apoptosis in the target cell. Some CD8+ T cells
also express Fas, which can activate Fas-L on target cells to
trigger apoptosis; however, this pathway is primarily used
to terminate lymphocytes upon elimination of a pathogen.
In addition to Fas-mediated elimination of lymphocytes,
some CD8+ T cells also secrete IL-10 in the effector phase
as a means to attenuate cytolytic effector function, thereby minimizing damage to uninfected bystander cells.
Effector cell function by CD4+ helper T cells, as their
name implies, involves providing secondary signals to other
innate and adaptive effector cells; however, the phenotype
of activated CD4+ T cells can vary depending on a number
of factors, including the subtype of DC encountered by the
naïve CD4+ T cell, the levels of cognate antigen-MHC encountered at activation, and the cytokine milieu present at
activation. Th1 CD4+ helper T cells tend to secrete IFN-γ
and IL-2, and are primarily involved in facilitating innate
and adaptive immune responses to intracellular pathogens
as well as some autoimmune responses. Th2 CD4+ helper
T cells secrete a more diverse array of cytokines (including
IL-4, IL-5, IL-9, and IL-10) and are involved primarily in facilitating innate and adaptive immunity to extracellular parasites as well as allergy and asthma. The Th17 phenotype
is associated with the secretion of IL-17, IL-21, and IL-22
and is involved with facilitating immunity to extracellular
bacteria and fungi as well as some autoimmune responses.
Finally, a subset of CD4+ regulatory T cells (Treg) can arise
in the thymus or can be induced peripherally under certain
conditions (e.g., stimulation in the presence of TGF-β and
the absence of proinflammatory cytokines). Treg cells, identified in the mid 1990s by Sakaguchi and colleagues,31 play
a vital role in limiting T-cell proliferation and attenuating
effector function32,33 (reviewed34).
Because of the specificity and potent effector function of
the adaptive immune response, we have evolved mechanisms of immunologic tolerance to protect from the inappropriate activation of immunity against self-antigens
(i.e., autoimmunity). Immunologic tolerance arises as the
sum of two distinct processes, central tolerance and peripheral tolerance. Central tolerance involves the elimination of potentially self-reactive T cells in the thymus during the early phases of their development. Because central
tolerance cannot reliably eliminate all potentially selfreactive T cells, we have also evolved processes termed
peripheral tolerance. For example, T cells that receive signal
1, cognate antigen in the context of MHC, in the absence
of signal 2, appropriate costimulation, undergo a process
termed anergy, in which the T cells become refractory to
activation or effector function on subsequent encounters
with cognate antigen. Similarly, T cells that repeatedly
receive signal 1, even with appropriate costimulation, can
The Immune System in Melanoma Initiation and Progression
undergo clonal exhaustion, in which further responses to
antigenic stimulation are highly attenuated.
Immune Modulation
Although mechanisms of central and peripheral tolerance play critical roles in shaping the immune repertoire,
in many ways the more important processes of immune
regulation–in particular, where clinical interventions for tumor immunotherapy and autoimmunity are concerned–lie
in the pathways activated by the costimulatory and coinhibitory receptors that serve to modify the response to signal
1 plus signal 2 for T-cell activation. In fact, because APCs
and T cells express on the surfaces not only CD28-B7 but
an array of costimulatory and coinhibitory ligand/receptor
pairs, signal 2 is viewed more accurately as the net sum of
the activating and attenuating signaling cascades that are
initiated in conjunction with TCR engagement with cognate peptide-MHC complexes. The requirement for activating signals beyond TCR engagement was first recognized
in the 1980s and 1990s when the CD28/B7-1 costimulatory
receptor/ligand pair were first cloned (with B7-2 discovered
shortly thereafter) and their role in stimulating IL-2 secretion by Th1 CD4+ Helper T cells was identified.35 During
the same time period, another receptor expressed on the
T-cell surface was discovered that had high sequence homology with CD28. That receptor, cytotoxic T-lymphocyteassociated protein 4 (CTLA-4), was subsequently shown to
bind to B7-1 with much greater affinity than CD2836. An inhibitory role for CTLA-4 was first postulated separately by
Bluestone37 and Allison,38 but that role was not confirmed
definitively until the mid-1990s when CTLA-4 knockout
mice were shown to develop a generalized lymphoproliferation with massive lymphatic infiltration of all organs.39,40
In fact, this time from the mid 1980s through the late 1990s
represents a period of rapid progress in identifying and
characterizing the myriad of accessory receptors that are
either constitutively expressed or up/downregulated on
T cells on TCR ligation and that play a role in determining
the outcome of that encounter with antigen.
Costimulatory receptors such as CD27, CD134 (OX40),
CD137 (4-1BB), glucocorticoid-induced tumor necrosis factor receptor (GITR), herpes virus entry mediator (HVEM),
and inducible costimulator (ICOS) enhance T-cell proliferation, cytokine secretion, and survival. Their ligands (CD70,
CD1334-L, CD137-L, GITRL, LIGHT, and ICOS-L, respectively) generally are expressed by APCs (B cells, DCs, macrophages). Each of these costimulatory molecules has been
targeted in animal models and has shown promise for the
augmentation of antitumor immunity and, in some cases,
has progressed to clinical evaluation (reviewed41).
Although somewhat smaller in number, the coinhibitory
receptors have come to play a much larger role than costimulators in preclinical and clinical investigations of immune modulation. Unlike CD28, CTLA-4 is not expressed
constitutively on the T-cell surface but is upregulated and
expressed on the surface on TCR ligation.37,42 Because of
its higher affinity for B7-1 and B7-2, CTLA-4 effectively
5
The Immune System in Melanoma Initiation and Progression
outcompetes CD28 for ligand binding and acts to block Tcell activation and proliferation36 (reviewed41,43). Preclinical studies of anti-CTLA-4 blocking monoclonal antibodies
(mAbs) demonstrate that CTLA-4 blockade can enhance
CD8+ T-cell responses44 and mediate the rejection of established tumors in mice.45,46
In addition to CTLA-4, programmed cell death protein-1
(PD-1)47 has also emerged as an important target for the
modulation of antitumor immunity (Figure 2). PD-1 is
found on the surface of activated T cells, as well as on B
cells, DCs, NK T cells, and monocytes. PD-1 ligation initiates a series of events that result in the attenuation of signals stemming from TCR ligation. Unlike the massive and
generalized lymphoproliferation seen in CTLA-4 knockout
mice, PD-1 knockout mice show milder and antigen-restricted autoimmunity that varies in a strain-dependent
manner.48,49 Expression of the ligands for PD-1, PD-L1 (B7H1) and PD-L2 (B7-DC) is not restricted to lymphocytes.
Importantly, PD-1 ligands are upregulated in hematopoietic, epithelial, and endothelial cells in response to inflammatory cytokines (e.g., type I and type II interferons).50-52
PD-L1 expression has also been shown in a variety of tumor types,53-56 including melanoma.57 Although less fully
characterized, PD-L2 expression is also upregulated on a
variety of normal and malignant cells in response to certain proinflammatory signals. Because of the timing and
localization of PD-1 expression and its ligands, PD-1 is hypothesized to help attenuate peripheral T-cell responses
during the latter stages of pathogen clearance during infections and to prevent autoimmunity, but also to limit Tcell responses to persistent antigens.58-61 Blocking PD-1 signaling on T cells in animal models can restore CD8+ T-cell
responses in models of chronic infection 61 and mediate
tumor rejection.62,63 Conversely, the engineered expression of PD-L1 by tumor cell lines was shown to limit effective CD8+ T-cell–mediated tumor rejection.53
Other coinhibitory receptor/ligand interactions are also
potential targets for immunomodulation. These include
the B7-H4 ligand (B7S1, B7x), which is found on activated
B and T cells as well as monocytes.64,65 Although a T-cell–
expressed receptor for B7-H4 has not yet been definitively
identified, the blockade of B7-H4 using mAbs has been
shown to enhance T-cell activation and effector function
in vitro and in vivo.64-66 B7-H4 expression has been demonstrated in a range of cancers.66-69 In addition, expression of
B7-H4 by tumor-associated macrophages (TAMs) has been
shown to play a role in suppressing antitumor immunity.70 Expression of lymphocyte activation gene-3 (LAG-3)
is upregulated on T cells after activation and competes
with CD4 for binding to MHC II, delivering a signal that
inhibits T-cell proliferation and cytokine secretion.71,72 In
addition to its expression on activated CD4+ T cells, LAG-3
expression is also seen in some Treg cells. Thus, blockade
of LAG-3 signaling is believed to promote antitumor effector function both by restoring T-cell activation and inhibiting Treg-mediated suppression.73,74
The Unique Relationship Between Melanoma
and Host Immunity
Although various mechanisms of immune activation
against cancer have been targeted for virtually all tumor
types, melanoma historically has drawn the most attention for the development of immunologically based therapeutic regimens. The relative breadth of melanoma-specific
immunotherapies has its
roots in three primary
B7.1/2
CD28
B7.1/2
CD28
observations.
T cell still in
+
+
First is the observation
secondary
APC
APC
lymphoid tissue
that a significant proportion
of individual melanoma tuSignal 1
Signal 1
mors undergo spontaneous
–
regression, and there is evCTLA-4
idence to suggest that reActivation of naïve
Antigen experienced
or resting T cells
T cell
gression is associated with
an immunologic tumor infiltrate.75-77 Whether a simiB7.1/2
CD28
lar rate of (presumably)
Tissue
+
Traffic to
or
immune-mediated tumor
DC
periphery
tumor
rejection occurs in other
Signal 1
Signal 1
tumor types is currently
unknown; however, the lo–
calization and pigmentaPD-L1
PD-1
tion of melanomas make
Activation of naïve
Antigen experienced
these spontaneous regresor resting T cells
T cell
Inflammation
sions more apparent.
Figure 2. CTLA4 and PD1 checkpoints act to regulate different elements of the T-cell
Second, the observation
response.
of spontaneous melanoFrom Pardoll D. Cancer immunology. In: Niederhuber JE, Armitage JO, Doroshow JH, Kastan MB, Tepper JE,
ma regression is often aseds. Abeloff’s Clinical Oncology. 5th ed. Philadelphia: Churchill Livingstone; 2014:88.
sociated with vitiligo, an
6
autoimmune-mediated depigmentation of the skin. Vitiligo is often accompanied by the presence of autoantibodies
against self-antigens expressed by both melanocytes and
melanoma cells.78,79
In addition, tumor-associated antigens (TAAs) from
melanoma were among the first tumor-specific antigens
to be identified. In melanoma, TAAs tend to fall into three
main categories: lineage specific or differentiation antigens (those expressed by both normal and malignant melanocytes), cancer-testis antigens (those expressed during
tissue development, but absent from adult tissues–except
the testis and placenta), and overexpressed/mutated proteins (those expressed at higher levels or mutated in tumor
cells relative to normal cells). Melanocyte lineage-specific
TAAs (reviewed80-82) include gp75, gp100, Melan A/MART1, tyrosinase, and TRP-1. Cancer-testis antigens relevant
to melanoma include the BAGE, GAGE, and MAGE family
proteins as well as NY-ESO-1. Other important melanoma
TAAs include mutated forms of β-catenin and CDK4.
Although our understanding of the factors mediating spontaneous regression of melanoma is still very limited, including what is effectively only the assumption of a role for the
immune system in mediating that regression, the relative
wealth of melanoma-specific TAA and the availability of effective animal models of melanoma historically have favored
the development of melanoma-specific immunotherapies.
The Complex Balance Between Immune
Activation and Suppression in Melanoma
Nonspecific T-Cell Activation
The advances that promoted the re-emergence of
immunosurveillance-immunoediting along with the studies identifying melanoma TAAs and the characterization
of melanoma-specific immune responses set the stage
for the development of immunotherapies designed to activate pre-existing melanoma-specific immunity. Among
the first such immunotherapies was the use of IFN-α and
IL-2 as adjuvant therapy for melanoma. INF-α is known
to activate NK cell-mediated cytotoxicity and to enhance
antigen presentation to T cells. Similarly, IL-2 promotes
T-cell proliferation and survival and mediates T-cell
differentiation into effector cells. Both are presumed to
activate innate effector responses to tumor cells as well
as the generation of tumor-specific adaptive immunity.
Clinical trials of adjuvant therapy with IFN-α and IL-2 in
the 1980s and 1990s showed sufficient improvements in
relapse-free survival (IFN-α) and overall survival and durable regression (IL-2) to merit FDA approval. Given the
role of each of these cytokines in the nonspecific activation of immunity, it is not surprising, however, that both
cytokines have significant associated toxicities related to
the activation of immunity to auto-antigens (in addition
to melanoma-specific antigens) and nonspecific inflammation leading to capillary leak syndrome and, thus,
require careful monitoring and compensatory actions in
the clinical therapeutic setting (reviewed83).
The Immune System in Melanoma Initiation and Progression
Modulating the summative coreceptor signaling (signal
2) during T-cell activation also provides an opportunity
to expand endogenous melanoma-specific T cells. As the
first and best characterized T-cell coinhibitory receptor,
CTLA-4 has been an important target for immunotherapeutic intervention. In the late 1990s, CTLA-4 blocking
was shown to mediate the spontaneous rejection of immunogenic tumors in mice45 as well as the enhancement
of vaccine-induced antitumor immunity in less immunogenic models84-86 without the generalized lymphoproliferation seen in CTLA-4 knockout mice. Clinical testing of a
humanized CTLA-4-blocking mAb was initiated in 2008,
and FDA approval was eventually granted in 2011.87-90 Like
IL-2 therapy, treatment with anti-CTLA-4 provides some
clinical benefit; however, it also elicits the induction of autoinflammation (colitis and hypophysitis)89,91-93 that must
be carefully managed.
Active (Antigen-Specific) Vaccination and
Adoptive Transfer
Following the advent of systemic cytokine immunotherapies for melanoma, a variety of approaches aimed at
activating adaptive immunity (primarily focused on the
development of CD8+ cytotoxic T cells) targeting specific
melanoma TAAs or multiple TAAs have developed. Given that melanoma antigens were among the first human
TAAs identified in the early 1990s,80,81,94 it is not surprising that these became the focal point for initial vaccination efforts. Further characterization of TAAs led to the
discovery of individual epitopes known to bind to human
MHC I and MHC II molecules (human leukocyte antigens
[HLAs]). Using peripheral T cells from melanoma patients, peptides derived from Melan A/Mart-1 and gp100
were shown to activate T-cell–specific immune responses
both in vitro and in vivo95-97; however, there was no direct
correlation between ex vivo T-cell activation and objective clinical response.98,99 In many of these early clinical
studies, putative immunogenic peptides were delivered in
the absence of adjuvant (which provides the immunologic
“danger” signals necessary to evoke signal 2 during T-cell
activation), potentially leading to tolerization of endogenous melanoma-specific T-cell populations rather than
activation. Overall, vaccination with melanoma-specific
peptides has been proven safe but has not shown significant clinical benefit. Using current technologies, immunogenic peptides for clinical use can be produced relatively
inexpensively; however, the use of individual peptides
(alone or even in combination) restricts the potential pool
of activated melanoma-specific T cells. Moreover, peptide
vaccines are, by definition, restricted to individual HLAtypes, thereby limiting their application to only those
patients who express the relevant HLA molecule(s).
To provide more potent immunostimulation with peptide vaccines, many have turned to pulsing peptide antigens directly onto ex vivo activated DCs. Initial studies
using animal models in the early 1990s demonstrated
that DCs derived from peripheral blood could be used to
7
The Immune System in Melanoma Initiation and Progression
elicit peptide-specific T-cell responses.100,101 The identification of culture methods leading to the generation of significant quantities of activated DCs from peripheral blood
monocytes in the mid 1990s further advanced the field,102
ushering in clinical trials of DC-based immunotherapies
for melanoma103-106 and a range of other cancer types and
leading to FDA approval of sipuleucel-T (an autologous DC
vaccine preparation) for prostate cancer in 2010.107 With
the advent of more careful characterization of DC subtypes, investigators currently are characterizing the differences in T-cell responses elicited by plasmacytoid DCs and
myeloid DCs compared with monocyte-derived DCs and
their implications for melanoma immunotherapy.108,109
To overcome the limited scope of antigenic targets associated with peptide-based vaccines, various vaccine platforms
have been developed to target whole-protein TAAs and multicomponent TAAs, allowing for endogenous processing
and presentation of potentially multiple antigenic epitopes
on endogenous HLA alleles. DNA-based vaccines can encode one or more TAAs and also contain CpG motifs within
the DNA vaccine that activate a specific pattern recognition
receptors on innate effector cells (toll-like receptor 9), providing a strong adjuvant effect.110 Preclinical studies of DNA
vaccines targeting a range of melanoma antigens in the
B16 mouse model have shown protective effects.111-113 DNAbased vaccines generally are delivered intradermally or intramuscularly and are thought to result in transcription and
translation of the encoded antigen directly by APCs (DCs,
in particular) and/or indirectly through the transfection of
bystander cells in the area of vaccination.114 DNA vaccines
have shown safety and efficacy for the prevention of certain
infectious diseases; however, clinical trials of a melanomaspecific DNA vaccine were discontinued when the drug did
not meet expectations.115-121 Other vector-based methods to
evoke melanoma-specific immunity include the use of recombinant viruses122 and bacteria.123-125 The use of recombinant pathogens to deliver TAAs provides the advantage of
introducing one or more antigenic melanoma proteins in
the context of PAMPs, providing a potent adjuvant effect for
the generation of melanoma-specific immunity; however,
these approaches also induce highly potent neutralizing antibodies to the vector (virus or bacteria), which can diminish the effectiveness of subsequent vaccinations. The use
of whole-cell vaccination (reviewed126-128), which includes
autologous vaccine preparations derived from patient
tumor samples as well as allogeneic vaccines comprised of
well-defined cell lines, similarly provide the opportunity
to elicit immunity across a range of potential melanoma
antigens. In particular, whole-cell vaccines can include a
range of known and as yet undefined melanoma antigens.
As with peptide-based vaccines, vector-based vaccines have
shown promise in preclinical models and are able to elicit
melanoma-specific T-cell responses129,130 but have shown
only limited efficacy in clinical application.131,132
To bypass altogether the difficulties inherent in eliciting potent antitumor immunity in vivo, many have turned
to the ex vivo activation and expansion of tumor-specific
CD8+ cytotoxic T cells. Melanoma in particular has
been associated historically with the presence of tumorinfiltrating lymphocytes (TILs). Adoptive T-cell therapy
(ACT) using expanded, patient-derived TIL preparations
was first developed by the Rosenberg lab in the 1980s.133
This approach has evolved to include not only the isolation of tumor-specific T cells from TILs (and peripheral
blood), their ex vivo expansion and readministration to
the patient, but also two methods through which patient
lymphocytes are modified for more potent antitumor
effect. One approach addresses the relatively low proportion of tumor-specific T cells in patient-derived samples
by modifying peripheral blood lymphocytes (PBLs) to express a melanoma-specific TCR. The recombinant TCRexpressing T cells are then adoptively transferred back to
the patient. The use of chimeric antigen receptors (CARs)
takes this approach a step further, bypassing the need for
TCR-peptide/MHC engagement by modifying T cells to express an engineered receptor composed of a single-chain
antibody against a melanoma-specific antigen fused with
an intracellular signaling domain (e.g., CD3 or Fc receptor) to elicit T-cell–mediated cytotoxicity when the singlechain antibody engages its cognate antigen on tumor cells.
Of the three approaches, ACT has been studied far more
extensively and has shown promising results,134,135 whereas TCRs and CARs have been less well studied to date.136-139
Overcoming of Treg-Mediated Suppression
The challenges associated with activating melanomaspecific T cells (those that have survived central tolerance
mechanisms) are many; however, the challenges do not
end with activation. Whether activated spontaneously,
through active vaccination (by various means), or through
ex vivo manipulation, tumor-specific CD8+ cytotoxic T
cells must persist, proliferate, and carry out effector functions in vivo to exert a productive antitumor effect. Animal studies using the B16 melanoma model have shown
that CD4+ CD25+ Treg cells actively suppress the effector
function of tumor-specific CD8+ T cells in tumor-bearing
mice.140 Similar studies in separate mouse melanoma
models showed that depletion of Treg was critical for successful tumor eradication after adoptive transfer of tumorspecific CD8+ T cells and IL-2.141,142 Simply depleting all
CD4+ CD25+ T cells is problematic, however, because
CD25+ is also expressed on effector T-cell populations.
Moreover, Treg populations in humans appear to uniformly express CD25 or Foxp3, another surface receptor
associated with the Treg phenotype.143 Early studies of Treg
populations isolated from melanoma patients indicated
antigen-specific suppression associated with CD4+ CD25+
T cells expressing IL-10.144 Treg cells expressing the immunosuppressive cytokines IL-10 and TGF-β have been
isolated from patients with metastatic melanoma with various surface phenotypes (CD4+ CD25+, CD4+ CD25+, and
CD4+ CD25hi Foxp3+).145-147 There currently are no clear
mechanisms to specifically deplete Treg populations in humans, although more generalized approaches to achieve
8
nonmyeloablative lymphodepletion have been used.148,149
In addition, further characterization of putative Treg surface markers such at LAG-3 and GITR may lead to the
development of Treg-depleting therapeutics.41
Immune suppression within the tumor microenvironment is also mediated through the action of myeloidderived suppressor cells (MDSCs), which are believed to
be derived in conditions of chronic inflammation.150,151
MDSCs can suppress immune effector function (both innate and adaptive) by depleting arginine and tryptophan
from the local environment152 or through the production of
immunosuppressive cytokines such as IL-10 and TGF-β.153
Because the presence of MDSCs within some tumors is
correlated with a poor prognosis,154,155 countering their
suppressive effects is an area of intense effort.156-158
Modulation of the Effector Response
In addition to the indirect immunosuppressive mechanisms at work in the tumor microenvironment described
previously, tumor cells can directly suppress T-cell effector function through their surface expression of PD-L1.
Although autoimmunity in PD-1 knockout mice was mild
compared with that seen in CTLA-4 knockout mice,48,49
subsequent studies of PD-L1/PD-L2 knockout mice predisposed to autoimmunity showed rapid, organ-specific
autoimmunity,159 suggesting a peripheral role for PD-1 in
limiting T-cell effector function. Consistent with this hypothesis, preventing PD-1 mediated signaling was shown
to enhance vaccine response and even reverse aspects of
clonal exhaustion in T cells.61 A role for PD-1 blockade in
tumor immunotherapy was first demonstrated in 2002,
when PD-L1 expression was shown in melanoma as well as
several other human cancers and its expression on tumor
cells was shown to suppress tumor-specific T-cell responses in mice.53,160 Clinical trials of several anti-PD-1 biologics
have been initiated, and the approach has shown promise
(reviewed60). There are currently four PD-1-targeted therapeutics under clinical evaluation; three of these are mAbs,
and one is a fusion protein composed of the ligand PD-L2
on a human IgG1 backbone. In addition to evaluating the
safety and efficacy of anti-PD-1 therapeutics in the clinical management of melanoma, these agents also are being
evaluated for the treatment of other solid and hematologic
malignancies as well as for the reversal of T-cell exhaustion associated with chronic infection. By comparison with
anti-CTLA-4, which is thought to nonspecifically promote
T-cell activation/proliferation and thereby predispose the
patient to autoinflammation, anti-PD-1 has not shown the
same severity of autoinflammation (pneumonitis).60
Conclusion
Beginning in the late 1980s, technological advances in gene
manipulation significantly accelerated our understanding
of the basic immunologic principals underlying the generation of immune response, particularly with respect to
T-cell activation and effector function. This paved the way
for the first truly productive forays into the generation of
The Immune System in Melanoma Initiation and Progression
immunotherapies for cancer, with particular emphasis
on melanoma, in which broad promotion of endogenous
antitumor immune responses was sought. The subsequent discovery of a range of T-cell costimulatory and
coinhibitory receptors in the early 1990s sparked the development of the next generation of improved approaches
or melanoma immunotherapy and also directed our
attention not only to the complexities of T-cell activation, but also the myriad of ways in which peripheral
tolerance mechanisms–particularly within the tumor
microenvironment–serve to further limit antitumor
effector function. These advances sought to elicit more
specific antitumor immunity through vaccination and
also to promote T-cell expansion through the elimination of coinhibitory signaling. As we move towards the
third generation of immunotherapeutic development, we
must now expand our perspective once again, taking into
account our evolving understanding of T-cell activation
to more carefully orchestrate and integrate the multiple
immunomodulatory signaling pathways to achieve a more
effective and durable antitumor immune response. Moreover, we must consider and account for the various peripheral tolerance mechanisms occurring within the tumor
microenvironment to promote better effector function.
Lastly, we should consider and evaluate the integration of
nonimmune-based therapeutics, the mechanisms of which
are not antagonistic.
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