# Physiological Role of Plasmacytoid Dendritic Cells and  Their Potential Use in Cancer Immunity

Authors: Jorge Schettini; Pinku Mukherjee
Journal: Clinical and Developmental Immunology
 (2008)

---

## Abstract

Dendritic  cells (DCs) play a pivotal role in the control of innate and adaptive immune responses. They are a heterogeneous cell population, where plasmacytoid dendritic cells (pDCs) are a unique subset capable of secreting high levels of type I IFNs. It has been demonstrated that pDCs can coordinate events during the course of viral infection, atopy, autoimmune diseases, and cancer. Therefore, pDC, as a main source of type I IFN, is an attractive target for therapeutic manipulations of the immune system to elicit a powerful immune response against tumor antigens in combination with other therapies. The therapeutic vaccination with antigen-pulsed DCs has shown a limited efficacy to generate an effective long-lasting immune response against tumor cells. A rational manipulation and design of vaccines which could include DC subsets outside “Langerhans cell paradigm” might allow us to improve the therapeutic approaches for cancer patients.


---

## Body

## 1. Introduction

There is not a clear answer why
tumor immunity is not effectively mounted in most tumor-bearing hosts.  Early
mouse studies,  as well as clinical experience, indicate that the immune system
can recognize and reject tumors [1–11]. On the contrary,
immune-deficient mice and patients have an augmented incidence of cancer which
suggests a relevant role for the immune system [12, 13]. Immunotherapeutic
protocols based on these findings have been developed; however, the results are
variable and limited [14–19]. As observed
in melanoma and other tumors, there is an absence of specific cytotoxic T
lymphocytes (CTLs) expansion in cancer patients. This suggests that
tumor-antigens may not overcome the threshold on the surface of DCs needed to
trigger CTL proliferation (passive factor). In addition, immunoregulatory
factors are involved in downregulating T cell proliferation and inducing T
regulatory cells (active factors), secreted by tumor cells [14]. Thus, DCs play
a critical role in inducing and regulating the immune responses [20, 21].DCs constitute a heterogeneous cell
population, which are classified according to cluster of differentiation (CD)
expression, functionality, and localization, playing a pivotal role in the
control of innate and adaptive immune responses [22]. Generally, DCs’ life
cycle is based on a model commonly referred to as the “Langerhans cells
paradigm” [23]. Immature DCs are strategically located in peripheral and
interstitial spaces of most tissues, and from their location, and always in
surveillance mode, DCs constitutively take up antigens from the environment,
which will be associated with the MHC molecules. Coordinately, DCs mature by
cessation of phagocytosis and endocytosis and move toward the draining lymphoid
nodes (LNs) due to upregulation of chemokine receptor CCR7, thereby, acquiring
responsiveness to a chemotactic gradient of CCL21(-Leu/-Ser) and CCL19
expressed by initial and terminal lymphatic vessels and by mature DCs,
respectively [24, 25].After arriving at the draining
lymphoid nodes, DCs are able to present antigens in the context of MHC and
costimulatory molecules to antigen-specific T cells. This induces a cellular
immune response which drives T cells to differentiate to effectors cells
[26, 27]. Moreover, DCs are important in starting adaptive and innate immunity,
by activating naïve and memory B cells, natural killer, and natural killer T
cells [28–31].Due to the antigen capturing and
presenting properties of DCs, ex vivo delivery of tumor-antigen to DCs has been
used as a strategy to guarantee successful antigen presentation to T cells
[14]. However, the efficacy of this approach to therapeutic vaccination has
been limited in both preclinical and clinical settings [19, 32]. This suggests that we need to better
understand and refine the parameters to establish the optimal conditions for
vaccination against cancer.Recent progress in the
identification of distinct DC subsets has been done. Analysis of the DC population in
several lymphoid organs has shown a considerable heterogeneity, where some
subsets of DCs follow the “Langerhans cell paradigm”, but not all of them
[33, 34]. Unfortunately, the heterogeneity of the human DC network is poorly
understood compared with the mouse DC network. At present, there are two main
pathways of differentiation in mouse DCs. The myeloid pathway generates two
subsets: Langerhans cells and interstitial DCs, whereas the lymphoid pathway
generates plasmacytoid DCs (pDCs) [22, 28, 35]. In contrast to the many studies
in mouse DCs, there are very few studies on mature human DCs from tissue. Human
blood DCs are heterogeneous in their expression of markers, but this may
reflect differences in the activation or maturation states of DCs rather than
separate lineages [36]. However, from in
vitro studies, it is possible to deduce pathways of human dendritic cell
development. Similar to mouse DCs, the myeloid pathway in humans generates
Langerhans cells and interstitial DCs. Blood monocytes, named precursors DC1
(pDC1), are the most commonly used precursor cells for generating human DCs in
culture. In the presence of GM-CSF and IL-4, pDC1 can generate DCs called DC1.
Maturation of these cells is achieved by stimulating cytokines or microbial
products [22, 37–39]. The human
lymphoid pathway also generates pDCs, termed pDC2. These cells are type I IFN
producing cells (IPCs) and they were discovered before their mouse
counterparts. The pDC2 responds to viral and microbial stimuli by producing
type I IFNs [35]. Both human and mouse pDCs can be maturated with bacterial
stimuli or viruses. Upon maturation, human pDC2, named DC2, lacks typical myeloid
markers, such as its
precursor, but displays
the characteristic of mature DCs [40, 41].Although most studies have focused
on the role of pDCs in antiviral immunity, several new lines of evidence have
suggested that pDCs are also involved in tumor immunity, as well as in
promoting peripheral tolerance [42–47].
Interestingly, pDCs can synthesize large amount of functional indoleamine 2,3-dioxygenase
(IDO), which requires autocrine release of type I IFN, upon Toll-like receptor-9
(TLR9) and CD200R ligands stimulation. IDO secretion by pDCs promotes T-cell
death at T-cell areas of secondary lymphoid organs. Notably, through the
upregulation of inducible T-cell costimulator ligand (ICOSL), pDCs have the
ability to generate regulatory T cells [48, 49]. Gathering together, this
evidence suggests that pDCs represent a key effector cell in both innate and
adaptive immunity regulation [35, 50–53]. In this
review, we focus on the characterization, physiology, and potential roles of
pDCs in the antitumor responses.


## 2. Differentiation and Trafficking Patterns of pDCs

The growth factor fms-like tyrosine
kinase 3 ligand (FLT3-L) has been described as a key differentiation and trafficking factor for
human and mouse pDCs from hematopoietic progenitor cells (HPCs). FLT3-L
injection in humans causes an increase of both myeloid DCs (mDCs) and pDCs in
the blood. In mice, FLT3-L injection induces the generation of mDCs and pDCs in
blood, lymphoid tissues, liver, and lung [54–59]. In vitro,
mDC and pDCs can be generated from FLT3-L-supplemented BM culture system
[60, 61]. Recently, Fancke et al.
have also shown that M-CSF is capable of driving pDCs from bone marrow precursor cells in vitro and in vivo [62].pDCs account for less than 1% of
total peripheral blood mononuclear cells (PBMCs) and can be isolated through
removal of lineage-positive cells andCD123+ (IL-3R). The identification of two
markers on human (BDCA-2 and BDCA-4)  and one in the mouse (PDCA-1) has
facilitated the isolation of pDCs from PBMC or lymphoid organs by positive
selection with magnetic beads coupled with specific monoclonal antibodies
[63, 64].In human and mice, pDCs have been
found circulating in the blood and cord blood of neonates [65–67].
Interestingly, human pDCs have
been found in fetal liver, thymus, and bone marrow suggesting that pDCs develop
from CD34+ human stem cells (HSCs) within these primary lymphoid tissues [68].
Moreover, pDCs can be located in lymphoid nodes, spleen, tonsils, and Peyer’s
patches.Similar to B and T cell migration
patterns, pDCs leave the bone marrow and migrate into the T cell rich areas of
the secondary lymphoid tissues, through high-endothelial venules (HEVs) in the
lymph nodes, mucosa-associated lymphoid tissues, and through marginal zones of
the spleen under steady-state conditions [69–73]. This unique
migration pattern of pDCs among DCs appears to be connected with their
expression of CD62L and CCR7, which allows the pDCs ligate L-selectin ligands expressed by HEV and
chemokines CCL19 and CCL21 expressed by HEVs and stromal cells within the T-cell
rich areas, respectively [73, 74].The expressions of chemokine
receptors on circulating blood mDCs and pDCs are similar. However, the level of
CCR5, CCR7, and CXCR3 expressions is clearly divergent in these two subsets,
being higher on pDCs than on mDCs [74]. Among these two subsets, pDCs are also
the only to migrate in response to the homeostatic chemokine SDF-1/CXCL12, the
ligand of CXCR4, which is expressed on dermal endothelial cells, in HEVs of
lymphoid nodes, and in malignant cells [44]. This evidence suggests that pDCs
may reach lymph nodes using CXCR4, and also explains their fundamental
localization in the secondary lymphoid organs [70].Interestingly, human pDCs have been
found to infiltrate primary and malignant melanoma, head and neck carcinoma,
ovarian carcinoma, and breast cancer [42–46, 75], as
well as cutaneous inflammatory lesions, which may be dependent on their ability
to express CLA, which binds to E-selectin on dermal endothelial cells and may
enhance their recruitment to the inflammatory site [76].


## 3. Activation of Plasmacytoid DCs

Virtually, all cell types are able
to produce type I IFNs in response to viral exposure. The amount, kinetics, and
types of IFN will depend on the cell type. However, pDCs are considered the
professional type I IFN producing cells [35]. pDCs can produce 100–1000 times more
type I IFN than the other blood cell types upon activation [35], or the
equivalent of 10 pg/cell [77]. Myeloid DCs can also secrete type I IFN in
response to RNA viruses, but less efficiently than pDCs [78].It is important to note that not
all viruses can activate pDCs to produce IFNs. Also, pDCs do not require to be
infected to secrete type I IFN [79, 80]. Once secreted, type I IFNs induce MxA,
an IFNα-inducible intracellular protein [75], oligoadenylate synthetase, and
double-stranded RNA-(dsRNA-)-dependent protein kinase (PKR). Together, these
proteins have the biological role in inducing cellular resistance by blocking
viral replication, and, therefore, viral spread [81].Moreover, type I IFN modulates
several aspects of the immune response, including pDC survival, mDCs
differentiation [82], modulation of Th1 and CD8+ T-cell responses,
cross-presentation and cross-priming independent of CD4+ T helper cells [83],
upregulation of MHC and costimulatory molecules, activation of NK cells, and
induction of primary antibody responses [84].pDC activation with pathogens or oligodeoxynucleotides
(ODNs) with multiple unmethylated CpG dinucleotides induces the secretion of
several other cytokines and chemokines, such as TNFα, IL-1, and IL-6. In mouse,
but not in humans, pDCs have the capacity to synthesize bioactive IL-12,
although this capacity still remains controversial [85–87]. Virally, stimulated
pDC produces chemokines, such as CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES),
CXCL8 (IL-8), and CXCL10 (IP-10) which stimulate Th1, and NK cells homing to
site of infection through IP-10 and CCL4, respectively [88, 89].


## 4. Regulation of Type I IFN Synthesis on pDCs

This unique subset of DCs can
secrete type I IFNs faster than other cells to a wider range of viral and
nonviral stimuli. Moreover, pDCs express a broader profile of IFNA genes than
other antigen-presenting cells (APCs). In humans, the type I IFN family
consists of 13 IFNα subtypes, one IFNβ, one IFN-ω, one IFN-κ, and one IFN-τ.
IFNα1 is the major subtype expressed by pDCs, but other subtypes are also
secreted, including IFNα2, -α5,
-α8, -α10, and -α14 and a recently described family of IFNλ1-3 (also named
IL-29, IL-28A, and IL-28B, resp.) [90, 91].What makes pDCs synthesize type I
IFN faster than other cells? Recently, it has been shown that transcription
factors of the family of interferon regulatory factors (IRFs) play an important
role in the regulation of interferon gene transcription. Nine mammalian IRF
family members have been identified to guide the induction of IFN production,
as well as to regulate and differentiate various cells types [92]. Expression
of IRF-3 supports induction of IFNβ and IRF-5 or IRF-7 is sufficient to
stimulate IFNα genes expression. Unlike other cells, pDCs have been shown to express
constitutively higher levels of IRF-5, -7, and -8 mRNA, which might explain why
this particular subset of DCs secrete faster and large quantities of type I
IFNs than other cell types [93, 94].


## 5. Differential Expression and Function of TLRs in pDCs

This unique ability of pDCs to
secrete large amounts of type I IFN depends on cellular receptors able to sense
several types of nucleic acid. TLR is a family of 11 pattern recognition
receptors (PRRs) which mediate the recognition of many pathogens through the
detection of distinct pathogen-associated molecular patterns (PAMPs) [95, 96].pDCs and mDCs each has a different
TLR expression profile. In humans, mDCs can express TLR-1, -2, -3, -4, -5, -7,
and -8, while pDCs express mainly TLR7 and -9 [97, 98]. Uniquely, TLR-7, -8, and
-9 detect PAMPs in endosomal/lysosomal compartments followed by acidification [99, 100].
Transcriptional regulation of IFNβ and IFNα genes on pDCs is controlled mainly
by IRF-3 and IRF-5/7. IRF-3 can be activated by TLR-3 and TLR-4, but there is
no evidence of this pathway on pDCs. Instead, IRF-7 has a constitutively high
expression in pDCs and it is recruited by myeloid differentiation primary
response gene 88 (MyD88) through the adaptor molecule TRAF6 when TLR-7 or -9 is
triggered [101].Many studies have shown that
exposure to synthetic TLR-7 or -9 agonists (e.g., imidazoquinoline, CpG ODN)
induces pDCs to secrete IFNα and proinflammatory cytokines (IL-8 and TNFα),
maturation, which heighten their T-cell stimulatory capacity [97, 102–104].Interestingly, endogenous antigens,
such as DNA from necrotic cells, may be taken up by pDCs and signal through
TLR-9 in autoimmune diseases [105]. TLR-9 agonist has a therapeutic potential
and it has been used to induce innate and adaptive immune responses. Synthetic
TLR-9 agonists are currently being tested in multiple phase II and phase III
human clinical trial as adjuvants to cancer vaccine and in combination with
conventional chemotherapy and others protocols [106–108].


## 6. pDCs Can Link Innate and Adaptive Immunity via Type I IFNs

There are abundant studies in human
and mice showing the importance of type I IFN to regulate inflammation and link
innate and adaptive immunity [113–115]. IFNα and -β
are considered as important components of innate immunity together with their
well-known antiviral activity [114]. Type I IFN released by human pDCs activates
NK cell cytolytic activity, and also induces IFNγ production in NK cells through IL-12 secretion
[116, 117]. Although with different molecular mechanisms in human and mice, type
I IFN secreted by pDCs, upon stimulation, can affect T-cell functions.  Thus,
activated pDCs can induce T cells to make IL-10 and IFNγ [113, 118], and also induce a Th1 polarization
[119]. It has also been reported that type I IFN can induce early activation
markers (CD69) on T cells, long-term survival [120], and generation of a
long-term antitumor immune response [121]. Recently, several studies have
provided important evidence for a role of type I IFN in the differentiation of
the Th1 subset [122], in the generation and activity of CTLs, as well as in
supporting in vivo
proliferation and survival of T cells [123, 124]. Altogether, these studies have
led to the recognition of an important role of this cytokine in linking innate
with adaptive immunity [115, 125].On the other hand, murine pDCs can
also inhibit certain mDCs functions. Upon infection, mice pDCs are the primary
source of IFNα and IL-12, and type I IFNs they produce inhibit the
synthesis of IL-12 from mDCs, a critical immunostimulatory cytokine of the T-cell-mediated
immunity [79]. In human, the production of IL-12 by pDCs is still
controversial, but some studies claimed the contrary [98, 126].Interestingly, pDCs are critical
for the generation of plasma cells and antibody responses. It appears that the
depletion of pDCs from human blood abrogates the secretion of IgGs in response
to viral infection.  Furthermore, activated pDCs can induce activated B cells to
differentiate plasma cells. Through Type I IFN and IL-6 secreted by pDCs, B cells are induced to develop into plasmablast and differentiate into antibody-secreting plasma cells [29].


## 7. Plasmacytoid DCs and Their Role in Cancer Immunity

Before the maturation of pDCs, they
have a poor T-cell stimulation capacity. Early experiments reported that CD40L
in combination with IL-3-stimulated pDCs develop into a functionally
distinct DCs type that promotes the development of IL-4-secreting Th2 cells
[40]. Also, pDCs can prime Th1 or Th0 allogeneic responses
[118, 127, 128]. Furthermore, pDCs mature following exposure to influenza virus
and exhibited an equivalent efficiency to expand the repertoire of
anti-influenza  virus cytotoxic T lymphocytes and Th1 CD4+ T cells [104, 129].It is clear now that immature mDCs
and pDCs infiltrate solid tumor and lack the ability to induce T-cell activation
[75]. However, they still present tumor antigens and induce IL-10-producing
CD4+/CD25+ regulatory T cells that inhibit antitumor immunity [130].
Nevertheless, using an anti-IL-10 mAb and CpG ODN, it is possible to induce a
robust antitumor CTL response and tumor rejection in vivo [111]. Recently,
murine pDCs have been described to have the ability to elicit in vivo, in naïve mice, an
antigen-specific CD8+ T cell response against endogenous antigens, as well as
exogenous peptides, but not against exogenous antigens, and were capable of
protecting mice from tumor challenge [131].It has also been reported that
human tumor antigens pulsed pDCs in
vitro can prime IFNγ-secreting melanoma-specific CTLs [42]. Synergy
among DC subsets has not been fully explored in the development of antitumor
immunity. An interesting study has shown
that immunizations with a mixture
of matured pDCs plus mDCs resulted in increased levels of antigen-specific CD8+
T cells and an enhanced antitumor response compared with immunization with
either dendritic cell subset alone [109]. Altogether, these studies suggest
that it is possible to re-establish and/or maximize an antitumor immune
response when pDCs are taken in the regimen [132–137] (Table 1).Table 1TumorSystemDC sourceProtocolReferencesEG7 T-cell lymphomaMurineExpanded in vivo (FLt3L),
and sorted
from BMCpG-activated OVAp-pulsed pDCs/mDCsLou et al. [109] (2007)K17-35-OVA melanomaMurineIsolated
TIDCs from K17-35
melanomaOVA-pulsed
TIDCsPreynat-Seauve et al. [110]
(2006)C26
colon CarcinomaMurineIsolated
TIDCs from C26
tumorTIDCs
activated with CpG +
anti-IL-10R (i.p.)Vicari et al. [111] (2002)M3 MelanomaMurine—Topical
application ImiquimodPalamara et al. [46] (2004)Melanoma cell
linesHumanSorted
from PBMCpDCs
activated with CD40L-transfected
J558Salio et al. [42] (2003)Melanoma stage
IIIb/c, IVHuman—CpG-7909
(s.c.) (ProMune)Pashenkov et al. [112] (2006)DCs,
dendritic cells; pDCs, plasmacytoid DCs; BM, bone-marrow; OVAp, OVA peptide; TIDCs,
tumor-infiltrating DCs (myeloid and plasmacytoid); PBMC, peripheral blood
mononuclear cells.


## 8. Clinical Significance of pDCs

There is evidence that pDCs are
located in several types of tumors: head and neck cancer, ovarian cancer,
primary melanoma cancer, and breast cancer [42–46, 75].
Secreted factors by tumor cells may inhibit pDCs function, such as TGFβ,
vascular endothelial growth factor β (VEGFβ), and IL-10.On the contrary, other studies have
reported that pDCs and tumor-infiltrating DC (TIDC) are functional and fully
competent APCs. Isolation of TIDC showed an intermediate maturation phenotype
and the capacity to take up particles, as well as produce IL-12 and maintain
its migratory capacity. Infiltrating pDCs are capable of producing IFNα, as well as inducing
complete regression or significant reduction of melanomas after a topically
treatment of imiquimod (a small synthetic immune response modifier recognized
by TLR7) [46, 110, 138, 139]. In
addition, intratumoral stimulation of pDCs with TLR7 and -9 agonists has been
successfully used in the clinic to treat basal cell carcinoma, human
papillomavirus-infected warts, and condylomata accuminata [140, 141]. TLR
signaling on pDCs can be used to induce type I IFNs and possibly protect DCs
from tumor-derived inhibitory factors (such as VEGFβ or IL-10), as well as support
T-cell survival, therefore, improving vaccination efficacy [112, 142–147].Thus, it will be critical to
evaluate if stimulation of pDCs may overcome tumor-mediated inhibitory effects
and can enhance a local antitumor immunity.


## 9. Conclusions

DCs are a heterogeneous cell
population, where plasmacytoid dendritic cells (pDCs) are a unique subset
capable of secreting high levels of type I IFNs. It has been demonstrated that
pDCs can coordinate events during the course of viral infection, atopy,
autoimmune diseases, and cancer. Therefore, pDCs as a main source of type I IFN
is an attractive target for therapeutic manipulations of the immune system to
elicit a powerful immune response against tumor antigens in combination with
others therapies.A rational manipulation and design
of vaccines which could include DCs subsets outside “Langerhans cell paradigm”
might allow us to improve the therapeutic approaches for cancer patients.



---

*Source: 106321-2009-01-26.xml*