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Review in translational hematology
Manipulating dendritic cell biology for the active immunotherapy of cancer
David W. O’Neill, Sylvia Adams, and Nina Bhardwaj
Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that have an
unequaled capacity to initiate primary immune responses, including tolerogenic
responses. Because of the importance of
DCs in the induction and control of immu-
nity, an understanding of their biology is
central to the development of potent immunotherapies for cancer, chronic infections, autoimmune disease, and induction of transplantation tolerance. This
review discusses recent advances in DC
research and the application of this knowledge toward new strategies for the clinical manipulation of DCs for cancer immunotherapy. (Blood. 2004;104:2235-2246)
© 2004 by The American Society of Hematology
Introduction
The concept of tumor “immunosurveillance,” whereby the host
immune system is thought to protect against the development of
primary cancers, has been debated for decades and has been
recently resurrected.1 Evidence in support of tumor immunosurveillance includes observations in mice that lymphocytes and molecules essential for immune function, such as interferon-␥ (IFN␥)
and perforin, collaborate to protect against the development of
certain cancers. Additional corroboration has come from identification of numerous human tumor-associated or tumor-specific antigens recognized by T cells and from isolation of tumor antigenspecific T cells from metastatic lesions. Furthermore, infiltration of
certain human cancers by T cells may correlate with dramatically
improved survival.2 The accumulating evidence in favor of tumor
immunosurveillance indicates that immunotherapies or “vaccines”
may prove effective for the treatment of cancer. Indeed, numerous
published reports have shown that vaccination of cancer patients
with killed tumor cells, tumor cell lysates or tumor antigen
proteins, peptides or DNA administered with cytokines or adjuvants can produce immunologic and clinical responses. However,
the immune responses to these vaccines are often weak, and
clinical responses are rarely complete and long lasting.3-5
Dendritic cells (DCs) are bone marrow–derived antigenpresenting cells (APCs) that play a critical role in the induction and
regulation of immune responses. It has been proposed that the
manipulation of DCs as a “natural” vaccine adjuvant may prove to
be a particularly effective way to stimulate antitumor immunity.6,7
This hypothesis has been supported by experiments in mice.
However, published reports of DC-based vaccine trials in humans
have yet to demonstrate improved potency of DC vaccines over
more traditional vaccine preparations.5,8,9 In this review we discuss
the pitfalls of current DC vaccine approaches in the context of
recent advances in DC biology and how improved understanding of
DC biology can be applied to develop more effective immunotherapies for cancer.
From the New York University (NYU) Cancer Institute Tumor Vaccine Center,
NYU School of Medicine, New York, NY.
Submitted December 24, 2003; accepted June 2, 2004. Prepublished online as
Blood First Edition Paper, July 1, 2004; DOI 10.1182/blood-2003-12-4392.
Supported by grants from the National Institutes of Health (CA-84512,
AI-44628), the Cancer Research Institute, the Burroughs Wellcome Fund, and
the Doris Duke Charitable Foundation. N.B. is an Elizabeth Glaser Scientist of
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
DC biology
DC differentiation and subtypes
DCs are a heterogeneous population of cells produced in the bone
marrow in response to growth and differentiation factors fms-like
tyrosine kinase-3 ligand (Flt3L) and granulocyte-macrophage
colony-stimulating factor (GM-CSF). There are 3 generally accepted stages of differentiation for all DC subtypes: DC precursors,
immature DCs, and mature DCs.10 In human blood, immature DCs
and DC precursors are lineage-negative (CD3⫺CD14⫺CD19⫺CD56⫺)
HLA-DR⫹ mononuclear cells6 and are traditionally divided into 2
populations by staining with antibodies to CD11c and CD123
(interleukin 3 receptor ␣ [IL-3R␣]). CD11c⫹CD123lo DCs have a
monocytoid appearance and are called “myeloid DCs” (MDCs),
whereas CD11c⫺CD123hi DCs have morphologic features similar
to plasma cells and are thus called “plasmacytoid DCs” (PDCs).
Although commonly used, this nomenclature is somewhat misleading. Experiments in mice indicate that both DC populations can be
derived from Flt3-expressing myeloid and lymphoid progenitors.11,12 PDCs and MDCs differ in many ways, including their
tissue distribution, cytokine production, and growth requirements.
PDCs are important in innate antiviral immunity, are found
primarily in blood and lymphoid organs, and are the principal
interferon ␣ (IFN␣)–producing cells in the body. PDCs can activate
antitumor and antiviral antigen responses,13,14 but their potential as
immunotherapeutic adjuvants is largely unexplored because they
are difficult to obtain in large quantities. MDCs, the focus of this
review, are found in many tissues, where they may be classified
into 2 principal subtypes: Langerhans cells (which express the
C-type lectin Langerin, have unique intracellular organelles
called Birbeck granules, and are found in the epidermis and oral,
respiratory, and genital mucosa), and so-called interstitial,
dermal, or submucosal DCs (variously named according to their
anatomic location).15
the Elizabeth Glaser Pediatric AIDS Foundation. D.W.O. is supported in part by
an NYU Cancer Institute Translational Research Program Award. S.A. is
supported in part by an American Society of Clinical Oncology Young
Investigator Award.
Reprints: Nina Bhardwaj, New York University School of Medicine, 550 1st
Ave, MSB 507, New York, NY 10016; e-mail: [email protected].
© 2004 by The American Society of Hematology
2235
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
O’NEILL et al
Antigen uptake, processing, and presentation
DC maturation
DCs capture bacteria, viruses, dead or dying cells, proteins, and
immune complexes through phagocytosis, endocytosis, and pinocytosis. They have an array of cell surface receptors for antigen
uptake, many of which also function in signaling or cell-cell
interactions (Table 1). DCs process captured proteins into peptides
that are loaded onto major histocompatibility complex class I and II
(MHC I and II) molecules, and these peptide-MHC complexes
(pMHC I and II) are transported to the cell surface for recognition
by antigen-specific T cells (pMHC I and pMHC II are recognized
by CD8⫹ and CD4⫹ T cells, respectively).22 Antigens acquired
endogenously (ie, synthesized within the DC cytosol) are typically
processed and loaded onto MHC I, whereas antigens acquired
exogenously (from the extracellular environment) are processed
onto MHC II. Processing of endogenous proteins onto MHC I is
through a cytosolic pathway that involves ubiquitination, degradation by proteasomes, and transport by TAP (transporters for antigen
presentation) into the endoplasmic reticulum. In contrast, exogenously acquired proteins are typically degraded in endosomes/
lysosomes, where the peptides are loaded onto MHC II following
degradation of the MHC II–associated invariant chain (Ii, which
blocks access to the peptide-binding pocket of MHC II).22
An alternative pathway also exists whereby DCs process
exogenous antigens onto MHC I. This pathway, called “crosspresentation,” permits DCs to elicit CD8⫹ as well as CD4⫹ T-cell
responses to exogenous antigens such as apoptotic or necrotic
tumor cells, virus-infected cells, and immune complexes.23-25
Cross-presentation is linked to specific DC antigen uptake receptors (Table 1), which may be targeted in strategies to load
exogenous antigens onto both MHC I and II.24
Lipid and glycolipid antigens expressed on pathogens or self
tissues are presented by DCs to T cells on CD1 molecules
(CD1a-d), which are structurally similar to MHC I but specialized
to bind lipids instead of peptides.26,27 Processing of lipid antigens
onto CD1 molecules is carried out in specialized intracellular
compartments, much like antigen processing onto MHC II. CD1
molecules present lipid antigens to a variety of lymphocytes,
including T cells with substantial T-cell receptor diversity as well as
relatively invariant natural killer T (NKT) cells.
Maturation is a terminal differentiation process that transforms
DCs from cells specialized for antigen capture into cells specialized
for T-cell stimulation. DC maturation is induced by components of
pathogens or by host molecules associated with inflammation or
tissue injury. These stimuli are often collectively referred to as
“danger signals.”28 Maturation is characterized by reduced phagocytic uptake, the development of cytoplasmic extensions or “veils”
(Figure 1), migration to lymphoid tissues, and enhanced T-cell
activation potential. Mature DCs express a number of characteristic
markers, including CD83, a cell surface molecule involved in
CD4⫹ T-cell development and cell-cell interactions,29,30 and DCLAMP, a DC-specific lysosomal protein. Maturation signals act on
DCs through receptors that trigger intracellular signaling, including
receptors for host-derived inflammatory molecules such as CD40L,
TNF␣, IL-1, and IFN␣ (Table 2). Microbial products and molecules released by damaged host tissues transmit maturation
signals through Toll-like receptors (TLRs), trans-membrane receptors expressed on DCs and other cell types related to Drosophila
Toll protein.40 There are 11 known TLRs thus far, each with
different expression patterns and each recognizing different sets of
molecules. In humans, MDCs express TLRs 1 through 5 and,
depending on the MDC subset, TLR 7 and/or 8. Human PDCs
express TLRs 1, 7, and 9.41-43 Some TLRs act at the cell surface,
whereas others such as TLRs 3, 7, 8, and 9 are found within
endosomes and are presumably activated following capture and
internalization of pathogens or their products.
TLRs signal through the adapter molecule MyD88, which
recruits other signaling molecules in a pathway that activates
NF-␬B and mitogen-activated protein (MAP) kinases, inducing the
transcription of genes encoding inflammatory mediators such as
TNF␣, IL-1, and IL-6.44 Stimulation of some TLRs can trigger
additional, MyD88-independent, signaling pathways.44 In DCs, the
distinct signaling pathways triggered can influence the direction of
the resulting T-cell response.45 TLR agonists, therefore, can be used
to target DC subsets to induce desired T-cell responses.
On maturation, DCs develop an enhanced ability to form pMHC
II46 and pMHC I,22 and some maturation stimuli can also induce
Table 1. DC antigen uptake receptors
Receptor class
C-type lectin-like
Examples
DC-SIGN (CD209)
Ligands
Notes
Viruses (HIV, Dengue, Ebola),
DC-SIGN also binds adhesion molecules (ICAM-2 and -3) that are important
receptors16
Fc receptors
mycobacteria
MMR (MRC1)
Mannosylated molecules
DEC-205 (LY75)
?
BDCA-2 (CLECSF11)
?
Langerin
?
Dectin-1
␤-glucan
Fc␥RI (CD32)
Immune complexes and
Fc␥RII (CD64)
Integrins
in DC trafficking and cell-cell interactions.
—
opsonized cells
␣V␤5
Apoptotic cells
␣M␤2 (CD11b/CD18, CR3)
Opsonized antigens (via
␣X␤2 (CD11c/CD18, CR4)
Opsonized antigens (via iC3b)
CD36
Apoptotic cells
LOX-1 (OLR1)
Hsp-peptide complexes
CD9119
Hsp-peptide complexes
Aquaporins
Fluids
CR3 binds ICAM-1, found on activated lymphocytes and endothelial cells.
iC3b), bacteria
Scavenger receptors17
Heat shock proteins (Hsps) are highly conserved molecules that are
released by dying cells and that can mature DCs. Their normal function is
to chaperone peptides between subcellular compartments.17,18
Other
—
DC-SIGN indicates dendritic cell-specific ICAM-3-grabbing nonintegrin; ICAM-2, intracellular adhesion molecule 2; MMR, macrophage mannose receptor; BDCA, blood
dendritic cell antigen; CR, complement receptor; LOX-1, low density lipoprotein, oxidized, receptor 1; and CLECSF, C-type lectin superfamily. Antigen uptake by way of
DEC-205,20 Fc␥ receptors,135 ␣V␤5 integrin,21 CD36,21 LOX-1,18 and CD9119 have all been associated with cross-presentation.
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
MANIPULATING DC BIOLOGY FOR IMMUNOTHERAPY
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Figure 1. DC morphology. (A) Immature monocyte-derived DC.
(B) Monocyte-derived DC matured with IL-1␤, IL-6, tumor necrosis
factor ␣ (TNF␣), and prostaglandin E2 (PGE2). Dif-Quick–stained
cytocentrifuge preparations are shown. Viewed at 1000⫻ magnification using an Olympus BX51 microscope with an Olympus U Plan
Fluorite 100⫻/1.30 NA oil immersion objective. Images were captured
using an Optronics MicroFire digital camera and MicroFire image
acquisition software and processed with Adobe Photoshop.
cross-presentation.47-49 Maturation also results in increased expression of adhesion and costimulatory molecules involved in the
formation of the immunologic synapse (Figure 2) and induces DCs
to secrete cytokines that are critical in determining the nature of the
ensuing immune response (Figure 3). Another important effect of
maturation is the induced secretion of chemokines that recruit
monocytes, DCs, and specific subsets of T cells into the local
environment (Table 3). Finally, maturation imparts on peripheral
DCs the ability to migrate from the tissues to T-cell zones of lymph
nodes. This is mediated, at least in part, through differential
regulation of DC chemokine receptors such as CCR1, CCR5, and
CCR7 (Table 3).
DC interactions with lymphocytes
DCs initiate or “prime” T-cell responses in secondary lymphoid
organs such as lymph nodes, spleen, or mucosal lymphoid tissues.62-64 Effective priming of naive T cells is manifested by their
Table 2. DC maturation stimuli
Classes
TNF family molecules
TLR agonists
Examples
Receptor on DC
TNF␣ (TNF)
TNFR (TNFRSF1A)
CD40L (CD154, TNFSF5)
CD40 (TNFSFR5)
FasL (TNFSF6)
Fas (TNFRSF6)
TRANCE (TNFSF11)
RANK (TNFRSF11A)
Notes
TNF family molecules are found on a variety
of activated immune cells.
LIGHT (TNFSF14)
HVEM (TNFRSF14)
Bacterial lipopeptides
TLR1
Pathogen-associated peptidoglycans, lipoproteins, glycolipids,
TLR2
breakdown products of host extracellular
dsRNA, polyl:C
TLR3
antimicrobial peptides. Imiquimod and
LPS, Hsp60, Hsp70, oligosaccharides of hyaluronan, ␤-defensins
TLR4
R848 are synthetic antiviral compounds
Flagellin
TLR5
(imidazoquinolines).
Imiquimod
TLR7
R-848, ssRNA31,32
TLR7, TLR8
Oligosaccharides of hyaluronan are
matrix. ␤-defensins are host-derived
Hsp70
CpG DNA, HSV DNA33
TLR9
IL-1␤
IL-1R
IL-6
IL-6R
Growth factors
TSLP
IL-7R␣/TSLPR heterodimer
Interferons
IFN␣35
IFNAR1
Adhesion molecules
Agonistic antibody
CEACAM-1 (CD66a)
—
Costimulatory
Agonistic antibody
B7-DC36
—
Immune complexes
Opsonized antigens
Fc receptors
Microbes
Viruses (Influenza, HIV)
Cytokines
—
Expressed by inflamed epithelial cells and
stimulates Th2 responses.34
—
molecules
Triggers signaling via Syk kinase37
—
—
—
—
Bacteria
Activated
lymphocytes
CD4⫹ and CD8⫹ T cells
NK cells
NKT cells
V␦1⫹ ␥:␦ T cells
Other
Uric acid38
?
Necrotic cells
Agonistic antibody
Uric acid is released from dying cells.
Stimulation of TREM2 on DCs triggers
TREM2
signaling via DAP12.39
TNF indicates tumor necrosis factor; TNFRSF, TNF receptor superfamily; CD40L, CD40 ligand; TNFSF, TNF ligand superfamily; TRANCE, TNF-related activation-induced
cytokine; RANK, receptor activator of NF-␬B (nuclear factor-␬B); LIGHT, homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for
HVEM, a receptor expressed by T lymphocytes; HVEM, Herpes virus entry mediator; TLR, Toll-like receptor; Hsp, heat shock protein; dsRNA, double-stranded viral RNA; LPS,
bacterial lipopolysaccharide; ssRNA, single-stranded RNA; CpG DNA, bacterial unmethylated CpG motif DNA; HSV, Herpes simplex virus; TSLP, thymic stromal
lymphopoietin; Th2, T helper cell type 2; IFN, interferon; CEACAM-1, CEA (carcinoembryonic antigen)—related cell adhesion molecule 1; TREM, triggering receptor expressed
on myeloid cells; DAP12, DNAX activation protein 12.
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O’NEILL et al
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
including OX40 and 4-1BB, which may be critical for both
initiating and sustaining long-lived T-cell immunity (Figure 2).71-75
DCs also interact directly with B cells and lymphocytes of the
innate immune system. Activated MDCs can directly induce B-cell
proliferation, immunoglobulin isotype switching, and plasma cell
differentiation through the production of the B-cell activation and
survival molecules BAFF (B-cell–activating factor belonging to
the TNF family) and APRIL (a proliferation-inducing ligand),76-78
and activated PDCs can induce the differentiation of CD40activated B cells into plasma cells through the secretion of IFN␣/␤
and IL-6.79 DCs can also activate and induce the expansion of
resting NK cells by mechanisms that are just beginning to be
understood. Requirements for direct cell contact or soluble factors
have both been described.80 Activated NK cells can kill immature,
but not mature, DCs and can stimulate DCs to induce protective
CD8⫹ T-cell responses.80,81 Finally, DCs presenting the synthetic
glycolipid ␣-galactosylceramide (␣-GalCer) on CD1d can activate
NKT cells to produce IFN␥ and promote resistance to tumors.82
Activated NKT cells can in turn rapidly induce the full maturation
Figure 2. Molecules involved in the immunologic synapse between DCs and T
cells. Molecules expressed on DCs are listed on the left, with their corresponding
T-cell ligands listed on the right. In the right-hand column, stimulatory interactions are
indicated in black text and inhibitory interactions are indicated in red. Signaling from T
cells to DCs also occurs but is not shown here. Initial DC–T-cell interactions are
mediated by adhesion molecules and semaphorins such as neuropilin-1.50 Following
engagement of the T-cell receptor by pMHC complexes (signal 1) and engagement of
CD28 by B7-1 and B7-2 (signal 2), additional molecules are up-regulated on both cell
types that determine the nature of the ensuing T-cell response. Up-regulated
molecules include semaphorins such as SEM4-A and members of the B7, CD28,
TNF, and TNFR families of costimulatory molecules. Bidirectional signaling between
these molecules results in either further T-cell activation or in attenuation of the T-cell
response, depending on the molecules involved. Both B7-H1 and B7-DC interact with
PD-1 to inhibit activated T cells, but B7-DC can also work synergistically with B7-1
and B7-2 to enhance T-cell activation through an unknown receptor.51,52 B7x
transmits an inhibitory signal by way of BTLA (B and T lymphocyte attenuator),53 and
B7-H3 can also transmit an inhibitory signal but through an unknown receptor.54
Thromboxane A2 (TXA2) secreted by the DCs also attenuates the DC–T-cell
interaction by way of the thromboxane receptor (TP) on the T cell.55 Inhibitory
molecules are thought to prevent excessive inflammation and autoimmunity. Alternative names for B7 family members are CD80 (B7-1), CD86 (B7-2), PD-L1 (B7-H1),
PD-L2 (B7-DC), B7-H2 or ICOSL (B7RP-1), and B7-H4 (B7x).
clonal expansion and differentiation into memory cells and cytokinesecreting effector cells. The strength of the T-cell response is
dependent on many factors, including the concentration of antigen
on the DC, the affinity of the T-cell receptor for the corresponding
pMHC, the state of DC maturation, and the type of maturation
stimulus.65 For example, T-cell stimulation by immature DCs leads
to initial T-cell proliferation but only short-term survival (“abortive
proliferation”), whereas stimulation by mature DCs results in
long-term T-cell survival and differentiation into memory and
effector T cells.65 Enhanced survival following priming, referred to
as T-cell “fitness,” is characterized by resistance to cell death in the
absence of cytokines and by responsiveness to the “homeostatic”
cytokines IL-7 and IL-15, which promote T-cell survival in the
absence of antigen.65,66
Importantly, recent observations indicate that CD4⫹ T-cell help
at the time of priming is required to generate CD8⫹ T-cell
memory.67-69 This effect is thought to be mediated by CD40CD40L interactions between CD4⫹ T cells and DCs.70 Other T-cell
surface molecules are also involved in the generation of long-lived
T-cell responses and T-cell memory and have corresponding
ligands that are up-regulated on activated APCs such as DCs.71
Examples include members of the TNF receptor superfamily,
Figure 3. DC plasticity in response to different maturation stimuli directs Th
polarization. DCs can direct the fate of naive CD4⫹ T cells, depending on the type of
DC maturation stimulus. Following priming, CD4⫹ T cells may differentiate toward
T-helper 1 (Th1) cells, which produce IFN␥ and support CD8⫹ cytotoxic T lymphocyte
(CTL) responses, or toward T-helper 2 (Th2) cells, which produce IL-4, IL-5, and
IL-13, support humoral immunity, and down-regulate Th1 responses. The direction of
Th polarization is determined by the secreted cytokine profile of the stimulating DCs,
which in turn depends on the DC subtype, the anatomic location of the DCs, and the
type of maturation stimulus.45,56 These factors control other characteristics of the
T-cell response as well, such as tolerance induction57 or T-cell homing.58,59 Th1polarizing stimuli such as LPS or flagellin direct a DC differentiation program that
causes the DCs to secrete IL-12p70, which together with IFN␥ potently induce CD4⫹
T cells to differentiate into IFN␥-secreting Th1 effector cells. This T-cell program is
mediated largely by the transcription factors signal transducer and activator of
transcription 4 (Stat4) and T-bet.60,61 Th1 polarization can also be induced in the
absence of IL-12p70 by mechanisms that are not entirely known but may be due in
part to IL-12–related cytokines such as IL-27. Other DC maturation stimuli such as
cholera toxin or schistosome eggs can differentiate DCs that do not produce IL-12p70
and that, in the presence of IL-4, induce naive CD4⫹ T cells to differentiate into
IL-4–secreting Th2 effector cells. It is not clear whether Th2 polarization is induced by
specific DC cytokines or is rather a default program carried out in the absence of a
Th1 polarization signal from the DCs. However, DC secretion of chemokines such as
thymus and activation-regulated chemokine (TARC) and MDC can act to potentiate a
Th2 response by preferentially attracting Th2 cells. The Th2 program in CD4⫹ T cells
is dependent on transcription factors GATA-3 and c-Maf.60,61
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
MANIPULATING DC BIOLOGY FOR IMMUNOTHERAPY
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Table 3. DC chemokine receptors and chemokines
Receptors
CCR1 and CCR5
Receptor expression on DCs
Immature DCs
Ligands
CCL3 (MIP-1␣)
Ligand expression by DCs
Mature DCs (some stimuli only)
CCL4 (MIP-1␤)
Notes
CCL3, 4, and 5 are also secreted by activated
inflammatory cells and activated endothelium
CCL5 (RANTES)
CCR2
Immature DCs
CCL2 (MCP-1)
Not expressed
CCL2 is secreted by activated monocytes,
CCR4
Immature DCs
CCL17 (TARC)
Mature DCs (some stimuli only)
CCR4 is preferentially expressed on Th2 cells, and
CCR6
Immature DCs (some subsets only)
CCL20 (MIP-3␣)
Not expressed
CCL20 is expressed by activated monocytes and
CCR7
Mature DCs
CCL19 (MIP-3␤)
Not expressed
CCL19 and 21 are expressed in T-cell zones of lymph
macrophages and endothelium
CCL22 (MDC)
TARC and MDC can promote Th2 responses.
lymphocytes
CCL21 (SLC)
nodes and spleen, lymphatic endothelium, and
lymph node high endothelial venules
CXCR1 and CXCR2
Immature DCs
CXCL8 (IL-8)
Mature DCs
IL-8 is a mediator of neutrophil recruitment
CXCR3
Not expressed
CXCL10 (IP-10)
Mature DCs (some stimuli only)
IP-10 attracts IFN␥-producing T cells, which express
CXCR4
Mature ⬎ Immature
CXCL12 (SDF-1)
Not expressed
SDF-1 is a chemoattractant for lymphocytes and
CXCR3, and can promote Th1 responses.
monocytes. CXCR4 is also a coreceptor for
T-cell-trophic strains of HIV
CCR indicates CC motif chemokine receptor; CCL, CC motif chemokine ligand; MIP, macrophage inflammatory protein; RANTES, regulated on activation, normally
T-expressed, and presumably secreted; MCP, monocyte chemoattractant protein; TARC, thymus and activation-regulated chemokine; MDC, macrophage-derived chemokine;
SLC, secondary lymphoid chemokine; CXCR, CXC motif chemokine receptor; CXCL, CXC motif chemokine ligand; IP-10, IFN␥-inducible 10-kDa protein; SDF-1, stromal
cell-derived factor 1; HIV, human immunodeficiency virus.
of DCs and can directly interact with DCs to enhance both CD4⫹
and CD8⫹ T-cell responses.82,83
DC induction of immune tolerance
Antigen presentation by immature DCs is considered to be an
important pathway by which tolerance to self-antigens is maintained. Antigens targeted to immature DCs in vivo can induce
tolerance through abortive proliferation and anergy of antigenspecific T cells, whereas simultaneous delivery of a DC maturation
stimulus induces a full effector T-cell response (Figure 4).20,84,96
Immature DCs can also induce tolerance through the induction of
CD4⫹ and CD8⫹ regulatory T (Tr) cells that suppress immune
responses by way of secretion of cytokines such as IL-10 and
TGF␤ (Figure 4).97-99 This is in contrast to “naturally occurring”
CD4⫹ Tr cells produced in the thymus, which constitutively
express CD25 (IL-2R␣), CTLA-4, and Foxp3, and exert their
immunosuppressive effect in a cell contact-dependent manner.84-86,97,100,101 Mature DCs can inhibit naturally occurring Tr cells
Figure 4. Tolerogenic DCs. There are a number of pathways by which immature MDCs can be rendered tolerogenic. Some of these mechanisms may overlap. The 5
mechanisms summarized here (from left to right) include antigen presentation by resting (steady-state) DCs; exposure of DCs to “modulating” cytokines such as IL-10 and
transforming growth factor-␤ (TGF␤) or to other modulating substances such as corticosteroids and vitamin D3; targeted inhibition of the RelB transcription factor (which
controls CD40 expression) or direct inhibition of CD40; DC exposure to CD8⫹CD28⫺ regulatory T (Tr) cells (which have been associated with graft tolerance in patients who
received transplants); and the induction of indoleamine 2,3-dioxygenase (IDO)–expressing DCs (IDO-DCs) by ligating B7-1 and B7-2 molecules on the DC with a cytotoxic
T-lymphocyte–associated antigen 4 (CTLA-4)–immunoglobulin fusion protein or by CD4⫹CD25⫹ Tr cells. Immature DCs (left) can induce tolerance through the induction of
abortive proliferation and anergy, as well as through the induction of IL-10–producing Tr cells.84,85,86 DCs that have been modulated by factors such as IL-10 or TGF␤ (second
from left) may also lead to the inhibition of effector T-cell expansion and the induction of IL-10–secreting Tr cells.87,88 Stat3 signaling within the DC appears to be critical for this
effect.89 IL-10–producing Tr cells are also induced by DCs that are deficient in RelB or CD4090 (center). CD8⫹CD28⫺ Tr cells induce tolerogenic DCs by up-regulating inhibitory
receptors immunoglobulin-like transcript 3 (ILT3) and ILT4 on the DC surface, which ultimately leads to decreased DC expression of B7-1 and B7-2 and T-cell anergy91 (second
from right). Finally, IDO-DCs (right) inhibit T-cell expansion and induce T-cell apoptosis by way of IDO-mediated tryptophan catabolism within the DCs.92-94 Both MDCs and
PDCs can also be rendered tolerogenic by factors secreted by malignant tumors (not shown).95
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O’NEILL et al
through the production of IL-6.102 DC expression of CD40 may be
an important factor in determining whether T-cell priming will
result in immunity or Tr cell–mediated immune suppression.
Antigen-exposed mouse DCs which lack CD40 prevent T-cell
priming, suppress previously primed immune responses, and
induce IL-10–secreting CD4⫹ Tr cells.90
DCs may actively be rendered tolerogenic by a number of
mechanisms. In humans, a subset of monocyte-derived DCs has
been described that expresses indoleamine 2,3-dioxygenase (IDO),
inhibits T-cell proliferation, and induces T-cell death.92 IDO can be
induced in DCs by ligation of their B7 molecules with CTLA-493,94
(Figure 4). Large numbers of “IDO DCs” can be found in
tumor-draining lymph nodes, suggesting that they may be involved
in the immunologic unresponsiveness seen in cancer patients.92
DCs may also be rendered tolerogenic by naturally occurring
CD8⫹CD28⫺ Tr cells, which up-regulate inhibitory receptors on
DCs and disrupt CD40-induced B7-1 and B7-2 expression91
(Figure 4). Finally, DCs can be rendered tolerogenic in culture by
the presence of IL-10, TGF␤, vitamin D3, or corticosteroids (Figure
4).87 DC Stat3 activity may be critical to the induction of
antigen-specific T-cell tolerance. Stat3 is activated by tyrosine
phosphorylation following DC exposure to IL-10 and other factors
produced by tumor cells, and forced expression of activated Stat3
in DCs can result in impaired antigen-specific T-cell responses.89
Manipulation of DCs for cancer
immunotherapy
Current approaches to DC vaccine design
The most common approach to using DCs for vaccines is to prepare
large numbers of autologous mature MDCs ex vivo, load them with
antigens, and inject them back into the subject (Figure 5).103,104
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
Three general methods have been described, involving, respectively (1) differentiating DCs from leukapheresis-derived monocytes with GM-CSF and IL-4105,106 (the most popular approach;
IL-13 has been used by some groups in place of IL-4), (2) GM-CSF
and TNF␣–mediated differentiation of CD34⫹ hematopoietic progenitor cells into mixtures of interstitial DCs and Langerhans
cells107 (Flt3L or stem cell factor may be added to expand DC
progenitors, and differentiation may be skewed toward Langerhans
cells by adding TGF␤ to the culture108), or (3) directly isolating
DCs from leukapheresis products by density gradient centrifugation109 or with commercially available closed systems that use
immunomagnetic beads. The yields of both plasmacytoid and
classic myeloid-type DCs purified from blood can be significantly
enhanced by stimulating patients with Flt3L prior to leukapheresis,110 although pharmaceutical-grade Flt3L is not currently available. All 3 types of DC preparations can stimulate antigen-specific
T-cell responses in human subjects and have been associated with
clinical responses in cancer patients. No direct comparisons have
been performed in clinical trials, although one such trial is
currently in progress.
DCs are frequently matured in culture prior to injection.
Currently, many laboratories using monocyte-derived DCs induce
maturation by the addition of a “cocktail” of IL-1␤, IL-6, TNF␣,
and PGE2.111 Several groups have observed that DCs matured in
this manner do not secrete detectable bioactive IL-12p70, but still
express CCR7 and induce Th1 and CD8⫹ T-cell responses.112,113
How the DCs induce these T-cell responses is currently under
investigation.
The choice of tumor antigen is important to consider (Table 4).
Because vaccines may select for tumor cells that escape immune
detection by loss of target antigen expression, antigens critical to
tumor growth are preferred. MHC-restricted peptide antigens are
frequently used, including altered or enhanced peptides that boost
Figure 5. Clinical DC vaccines. There are many alternative approaches for the preparation and use of DC
vaccines to treat cancer. DCs may be prepared ex vivo
following 3 general methods (lower left), each of which
results in a different mixture of cells. DCs may be matured
and loaded with antigens using a variety of techniques.
Some of these techniques include the addition of DC
survival factors, the use of substances that induce crosspresentation, or the use of stimulators of other innate
immune cells such as NKT cells. DCs loaded with RNA or
DNA can take advantage of sequences encoding cytokines, chemokines, or DC survival factors. Numerous
variables such as antigen dose, cell dose, and route of
administration also need to be optimized. In addition, less
costly and labor-intensive approaches that stimulate and
load DCs with antigen in situ are being explored. There
are many unresolved issues regarding the monitoring of
immune responses and in correlating these responses
with clinical outcome. DC vaccines may prove most
effective in the adjuvant setting or in combination with
other treatments.
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MANIPULATING DC BIOLOGY FOR IMMUNOTHERAPY
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Table 4. Classes of tumor antigens
Category
Cancer-testis (CT) antigens
Examples
MAGE-1 (MAGEA1)
Notes
Expressed in germ cells, germinal tissues, tumor cells.
BAGE
GAGE-1 (GAGE1)
NY-ESO-1 (CTAG1)
Lineage-specific antigens
Melanocyte antigens:
Expressed in specific tissues or cells.
-Tyrosinase (TYR)
-Melan-A/MART-1 (MLANA)
-gp100/Pmel17 (SILV)
Tumor-specific altered gene
HER-2/neu (ERBB2)
Associated with a wide variety of tumors. KRAS2 is mutated in 30% to 40% of
products (amplified,
p53 (TP53)
colorectal cancers, and p53 is mutated in up to 70% of all human cancers. Altered
aberrantly expressed,
Ras genes (KRAS2, HRAS, NRAS)
MUC1 glycosylation is seen in a variety of adenocarcinomas, and these altered
overexpressed or
Mucin 1 (MUC1)
glycopeptides can be presented by DCs to T cells.114 Myeloid leukemia cells can
mutated genes, splice
Beta-catenin (CTNNB1)
potentially be differentiated into DCs to vaccinate against endogenously expressed
variants, gene fusion
MUM1 (IRF4)
leukemia-specific antigens.115
products, etc)
CDK4
BCR-ABL fusion products
N-acteylglucosaminyltranferase V (MGAT5)
Survivin (BIRC5)
TERT
CEA
AFP
Immunoglobulin idiotypes
Multiple myeloma
B-cell lymphoma
Viral antigens
HPV E6 and E7 proteins
EBV (HHV4) LMP1 and LMP2 proteins
Unique, tumor-specific idiotypes because of clonal rearrangements of immunoglobulin
genes. Associated with B-cell malignancies.
May be used for tumors such as cervical cancer that are induced by oncogenic
viruses.
A useful web site with links to current cancer antigen databases may be found at: http://www.cancerimmunity.org/statics/databases.htm. TERT indicates telomerase
reverse transcriptase; AFP, alpha-fetoprotein; HPV, human papillomavirus; EBV, Epstein-Barr virus; LMP1, latent membrane protein 1.
immunity to less immunogenic self-antigens or that improve
antigen presentation or T-cell receptor affinity.3,104,107,110,116,117 A
disadvantage to using peptides is that they must be compatible with
the HLA type of the patient, often restricting peptide vaccination
studies to individuals with common HLA types. In addition, the
half-life of pMHC complexes may be short, and competition may
prevent priming to lower-affinity epitopes when mixtures of
peptides are used.
DCs may be loaded with purified or recombinant proteins,
transduced with nonreplicating recombinant viral vectors, or
transfected with RNA or, less commonly, plasmid vectors encoding
tumor-associated antigens.118-122 All of these approaches allow the
host’s MHC molecules to select epitopes from an antigen’s entire
amino acid sequence (Figure 5). Immunogenicity may be enhanced
by using antigens coupled to or expressing other more immunogenic molecules such as foreign proteins (eg, keyhole limpet
hemocyanin [KLH]), cytokines (IL-12, IL-15),118,123 costimulatory
molecules (B7-2, CD40L), or chemokines (CCL21). DCs may also
be loaded with whole tumor cells or tumor cell lysates or be
transfected with whole tumor RNA, which permit vaccination with
the complete antigenic content of the tumor.103,124-126
Studies to compare routes and frequency of injection, DC dose,
and DC subset will be essential to optimize DC immunotherapy.
DC vaccines may be stored frozen prior to vaccination103,104 and are
typically injected intradermally, subcutaneously, or intravenously
in numbers ranging from 2 to 100 million cells. Route of
administration may directly affect the nature of T-cell priming.
Skin injections may be required to induce immunity to cutaneous
tumors, whereas intravenous injections may be less effective at Th1
induction but more effective at induction of humoral immunity.127,128 Injection into lymph nodes or lymphatics has also been
attempted, because only 5% or fewer DCs may migrate to draining
nodes following subcutaneous injection. Direct injection into
tumors is also being investigated.
Lessons learned from early DC vaccine trials
DC vaccines have minimal side effects and have induced antigenspecific cytotoxic T lymphocyte (CTL) and Th1 responses in
healthy volunteers and in patients with a variety of advanced
cancers.5,9 Most of the trials in cancer patients have focused on the
safety and immunogenicity of DC vaccines and were not designed
to evaluate clinical responses. Larger controlled trials are now
under way to objectively assess clinical efficacy by documenting
responses following standardized criteria such as World Health
Organization (WHO) or Response Evaluation Criteria in Solid
Tumors Group (RECIST) guidelines.129
Some fundamental lessons have been learned from the smaller,
published DC vaccine trials, although they have not led to a
consensus on optimal antigen source or dose, DC dose, DC subset
or frequency, or route of administration. Most investigators now
avoid intravenous administration, as studies have suggested that
subcutaneous or intradermal vaccination leads to improved DC
migration to lymph nodes127 and enhanced Th1 polarization.128
Importantly, several studies indicate that DCs need to be matured to
effectively generate antigen-specific immune responses in humans.
Injection of healthy volunteers with antigen-loaded immature DCs
has been associated with tolerogenic responses,99 and a randomized
trial in patients with metastatic melanoma comparing peptidepulsed immature DCs with peptides administered with adjuvant
and GM-CSF demonstrated significantly lower immunogenicity in
patients receiving the DC vaccine.130 In addition, a direct comparison of peptide-loaded immature and mature DCs in patients with
metastatic melanoma showed that only mature DCs induced
antigen-specific CTL responses.98
The availability of sensitive and specific techniques to monitor
the induction of antigen-specific T-cell responses has provided
insight into the capacity of DCs to induce primary responses to
tumor antigens. For instance, it is clear that DC immunization can
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2242
O’NEILL et al
elicit Th1 and CD8⫹ T-cell responses specific to the immunizing
antigens (as measured by enzyme-linked immunospot [ELISPOT],
lymphocyte proliferation, cytolytic assays, and peptide-MHC tetramer staining), with a suggestion of epitope or antigen spreading
in some cases.131,132 However, immune responses in most studies
have been weak or undetectable, and durability has not been clearly
established. Correlation with tumor regression or disease stabilization has been variable and needs to be established in larger
trials. Furthermore, a general lack of standardization makes
results difficult to assess or compare, especially when we have
little concept of what magnitude of response correlates with
protective immunity.
The results of DC-based clinical trials have been extensively
reviewed5,9; therefore, our comments are limited to more recent
studies. Although it is difficult to compare the results of the DC
vaccine trials published to date, in our opinion the most impressive
objective clinical responses have been associated with the use of
whole proteins, killed tumor cells, or tumor lysates. This may be
because these are exogenous antigen sources that target MHC II to
generate CD4⫹ T-cell help and also target MHC I by way of
cross-presentation to generate CD8⫹ CTLs. Using tumor-specific
idiotype immunoglobulin-pulsed DCs in patients with follicular
lymphoma, Timmerman et al118 reported 2 long-lasting complete
responses (CRs) and 1 partial response (PR) among 10 patients
with measurable disease in the pilot phase of the study. An
additional 25 patients were vaccinated after their best clinical
response was achieved by chemotherapy, and objective tumor
regression was seen in 4 of 18 patients with residual disease. Holtl
et al125 reported a trial of 35 patients with metastatic renal cell
carcinoma who received monthly injections of autologous, mature
monocyte-derived DCs loaded with tumor lysates. Of 27 evaluable
patients, 2 had objective CR (as per WHO), 1 had a PR, and 7 had
stable disease. Objective responses and disease stabilization were
long lasting, ranging from 6 months to 3 years. Durable CRs were
also reported by O’Rourke et al126 in a trial of 17 patients with
metastatic melanoma who received mature monocyte-derived DCs
loaded with autologous irradiated tumor cells. By WHO criteria
there were 3 CRs (with durable remissions of over 3 years) and 3
PRs among 12 patients who completed the vaccinations. One
patient with progressive disease was vaccinated every 6 weeks for
more than 3 years, indicating that maintenance vaccinations may be
useful even for patients with slowly progressive disease. Finally,
another promising trial using autologous tumor lysate pulsed DCs
showed objective responses in patients with refractory cutaneous
T-cell lymphoma after intranodal vaccination.133 Larger studies
will be important to confirm the results of the these trials.
New vaccine strategies that exploit
DC biology
Tumors can evade immunity by a number of mechanisms, including mutations in genes encoding target antigens, loss of antigen
expression, or immunosuppressive maneuvers such as secretion of
TGF␤.1 This may be particularly true of large or metastatic tumors.
Thus, DC vaccines may be most effective in the adjuvant setting for
patients in remission but with a high risk of recurrence. However,
DC biology may be exploited in many ways to generate more
effective immunotherapies, and multimodality approaches may be
used to enhance the effectiveness of these vaccines. Below we
discuss some novel applications studied in murine models or in
preclinical studies using human cells.
BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
Provision of CD4ⴙ T-cell help for CD8ⴙ T cells
Vaccination studies in mice using MHC II–deficient DCs,134 as well
as experiments that demonstrate the importance of CD4⫹ T cell
help to generate CD8⫹ T-cell memory,67-69 call into question
vaccine strategies that target only CD8⫹ T-cell responses. Peptideloaded DC vaccines should incorporate antigens targeting both
CD4⫹ and CD8⫹ T cells, and a polyvalent approach should be
considered. Peptide-loaded dendritic cells can clearly prime CD4⫹
T-cell responses,131 but a more practical approach that circumvents
the problems of HLA-restricted peptides may be to target crosspresentation. For example, targeting antigens to Fc receptors on
DCs using antibody-antigen complexes has been shown to activate
both CD4 and CD8 effector responses and tumor immunity in
mice.135 Coating myeloma cells with anti–syndecan-1 antibody
similarly promotes cross-presentation.136 Pharmaceutical-grade antibodies already in use to treat human cancer (eg, anti-CD20,
anti-HER-2/neu) may act in part through this mechanism and could
be used in the preparation of DC vaccines. Cross-presentation can
also be enhanced by targeting DC surface receptors such as
DEC-205,20 loading DCs with killed cells or cell lysates or by
stimulating DCs with TLR agonists that induce cross presentation49
(Figure 5). Transfected RNA, which primarily targets MHC I, may
also be targeted to MHC II by incubating the transfected DCs with
antisense oligonucleotides to the MHC II–associated Ii protein137
or by using fusion constructs carrying an endosomal/lysosomal
sorting signal.138
Strategies to recruit, mature, and load DCs in situ
Existing DC vaccine methods require expensive facilities and
labor-intensive cell processing. To avoid this, alternative approaches that simultaneously recruit, mature, and pulse DCs with
antigens in vivo are being explored (Figure 5). To recruit DCs,
locally implanted chemokines such as MIP-3␤ may be used to
condition the injection site prior to vaccination.139 To mature DCs,
simple vaccines that take advantage of CpG motif DNA (a TLR9
agonist) coinjected with or conjugated to a protein antigen have
been used.140,141 Vaccination with heat shock protein-peptide
complexes can similarly mature DCs in situ and may induce
immunologic and clinical responses in melanoma patients.142
Another in situ approach uses CpG motif-containing DNA
vaccines that encode tumor antigens. These vaccines can be
engineered to carry xenogeneic antigens143 or to include DCspecific promoters to specifically target antigen expression to DCs.
DNA vaccines may also be designed to drive the expression of
survival factors such as Bcl-xL144 or to encode DC maturation
signals145 or immunostimulatory cytokines.146
Certain microbes directly induce MDC or PDC maturation,
even in nonreplicating form, and are being tested as recombinant
vaccine vectors.3 One advantage of some viral vectors is that IFN␣
generated from virus-stimulated DCs may promote cross-priming
of CD8⫹ T-cell responses.35 Microbial vectors may also be
engineered to express adhesion molecules, costimulatory molecules, or cytokines that direct Th polarization, promote T-cell
activation and longevity, and promote DC survival (Figure 5).
Ex vivo–derived DCs can also be matured in situ by preconditioning the injection site with TLR agonists,147 and DC migration
can be enhanced by preconditioning the injection site with cytokines or with DCs themselves.7,148,149 This approach may be
preferable to ex vivo maturation, because DC cytokines such as
IL-12 are often expressed only briefly after exposure to many
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BLOOD, 15 OCTOBER 2004 䡠 VOLUME 104, NUMBER 8
MANIPULATING DC BIOLOGY FOR IMMUNOTHERAPY
maturation stimuli, and local production of cytokines and chemokines induced by local application of TLR agonists may also
promote DC viability and migration to draining lymph nodes.
Strategies to activate NKT cells
Vaccination with melanoma cells in adjuvant can activate CD1drestricted NKT cells that recognize tumor-associated gangliosides,150 and intravenous delivery of a soluble antigen together with
the synthetic CD1d-binding glycolipid ␣-GalCer can lead to in
vivo activation of NKT cells and induction of antitumor T-cell
immunity.83 Trials to test the activating potential of ␣-GalCer–
pulsed DCs are under way in cancer patients.
Inhibition of immune tolerance
One way to enhance cancer vaccines is to simultaneously block
inhibitory costimulatory molecules or Tr cells. For example,
administering an inhibitory antibody to CTLA-4 in previously
vaccinated cancer patients can result in effective antitumor immunity.151 Possible synergy of CTLA-4 blockade and concomitant
tumor antigen vaccination has been observed in patients with
metastatic melanoma.152 In this study, tumor regressions were
accompanied by significant toxicity, including severe or lifethreatening autoimmunity. Nevertheless, this approach is worth
addressing in conjunction with DC vaccines, using different
dosages or schedules to alleviate toxicity. In mice, blockade of the
inhibitory costimulatory molecule B7-H1 has also been shown to
improve DC-mediated antitumor T-cell responses.51
The activity of cancer vaccines may be enhanced through
depletion or inhibition of Tr cells through the use of cytotoxic
anti-CD25 antibodies or IL-2 coupled to cytotoxic molecules. In
mice with poorly immunogenic tumors, depletion of Tr cells alone
can slow tumor growth but does not efficiently reject the tumor.153
However, immune responses induced by antigen-pulsed mature
DCs are significantly enhanced in CD25-depleted mice.154 The use
of both of CTLA-4 blockade and CD25⫹ cell depletion may further
potentiate the effectiveness of vaccines.155
Combination therapies
Multimodality approaches incorporating tumor vaccination have
also shown promise in animal models, although it may prove
difficult to translate some of these approaches into clinical use. For
example, in one study the combination of vaccination with
adoptively transferred T cells and administration of IL-2 resulted in
tumor regression and long-term cures.156 Using another approach,
Cui et al157 showed that transducing hematopoietic progenitor cells
with a model tumor antigen and transplanting these cells into
irradiated recipient mice resulted in expression of the antigen in
2243
donor-derived DCs in the host’s lymphoid organs. When combined
with systemic agents that generate and activate DCs and adoptive
transfer of donor T cells, this treatment resulted in expansion of
antigen-specific T cells and successful treatment of the antigenbearing tumor. Antitumor vaccination in combination with therapies that target the tumor’s vascular supply have also shown
promise in mouse models,158 as has vaccination during lymphoid
recovery following bone marrow transplantation.159
Use of “regulatory DCs” for the induction of
transplantation tolerance
DC-based immunotherapy may also prove to be a highly selective
way to induce graft tolerance in organ or hematopoietic stem cell
transplantation or to induce tolerance in patients with autoimmune
disease. Studies in mice and humans have shown that tolerogenic
or “regulatory DCs” (rDCs) may be induced ex vivo by culturing
immature DCs in modulating cytokines or growth factors such as
IL-10 and TGF␤. In a mouse model for the treatment of leukemia,
rDCs have been used to treat acute graft-versus-host disease and
leukemia relapse in conjunction with allogeneic bone marrow
transplantation.88
Conclusion
DC-based immunotherapy is still in its infancy. Two-arm trials are
needed to assess the efficacy of DC vaccines compared with other
immunotherapies and to optimize the use of DCs for vaccines.
Until then, it is not truly meaningful to compare DC immunotherapy with standard cancer therapies in large randomized trials.
The greatest clinical benefit of DC immunotherapy for cancer may
be found in the adjuvant setting, although it is hoped that patients
with advanced cancer will also benefit, at the very least, through
disease stabilization. Eventually, it is possible that the most
effective DC therapies may not necessarily involve the ex vivo
manipulation of DCs. Multimodality approaches that include novel
biologic agents may also help achieve effective, durable antitumor
immune responses. With greater understanding of DC biology and
of mechanisms to enhance DC immunogenicity, the answers will
begin to come.
Acknowledgments
We thank Teresita O’Neill for assistance with artwork; Stephen
Schachterle for preparation of dendritic cells; and Giorgio Inghirami, Marie Larsson, Anne-Sophie Beignon, and Mojca Skoberne
for helpful advice.
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2004 104: 2235-2246
doi:10.1182/blood-2003-12-4392 originally published online
July 1, 2004
Manipulating dendritic cell biology for the active immunotherapy of
cancer
David W. O'Neill, Sylvia Adams and Nina Bhardwaj
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