Dendritic cells in Melanoma
Polak, M.E.
Citation
Polak, M. E. (2008, September 16). Dendritic cells in Melanoma. Retrieved from https://hdl.handle.net/1887/13100
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Chapter I
General Introduction
Melanoma
Melanomas originate from melanocytes, pigment-producing neural crest cells, which migrate to peripheral tissues during embryonic development [1]. Melanomas can therefore develop in any tissue that contains melanocytes; the best known melanomas are the ones that occur in the skin (cutaneous melanomas) and the eye (uveal
melanomas). Melanomas occur much more often in Caucasians than in Africans or Asians [2, 3]. Both are not the most common tumours in their specific locations, but they are the most deadly malignancies of the eye and the skin, with mean survival times after metastasis formation of 10 and 6 months, respectively [4, 5].
Despite sharing the same progenitor cell, and a deregulation of similar molecular pathways, the epidemiology, biology, immunology and response to treatment of these two tumours differ significantly.
Epidemiology of cutaneous melanoma
Cutaneous melanoma affects around 60,000 patients each year in Europe, resulting in around 16,000 deaths [6, 7]. Unlike most other cancers, melanoma is frequent among young and middle-aged adults. Identified risk factors include skin phenotype and exposure to UV radiation. In particular sun exposure in early childhood may result in melanoma formation; however, tumorigenesis might also be initiated after single severe sunburn in older age [6, 8]. The incidence of cutaneous melanoma is still rising [6, 8, 9], and is associated with the latitude, increasing when closer to the Equator in white populations [9]. In Western European countries, though, a reverse trend is observed, with the highest occurrence of melanoma noted in the North. This may be explained by the lighter skin type of Northern Europeans and their life style,
promoting intense intermittent sun exposure during holiday periods [8, 10].
The most important prognostic factors for cutaneous melanoma include melanoma thickness, body site, histological type of the melanoma, gender of the patient and ulceration [8, 10]. The prognosis of the patient with a melanoma depends highly on the stage of the disease: while the 5-year survival is close to 90% for localized malignancies, it drops dramatically to less than 20% for lesions with distant metastases [11]. Thanks to the increase in awareness and early diagnosis, allowing
successful surgical treatment of thin primary tumours, mortality rates of cutaneous melanoma are currently levelling off, in particular in populations with high incidence rates [8].
Even though intermittent exposure to UV radiation, especially during childhood, has been postulated to be the main risk factor for melanoma, the development of
cutaneous melanoma on non-sun-exposed body parts, such as acral sites and mucosa, suggests that these tumours may arise through two pathways, one associated with melanocyte proliferation and genetic instability and the other with chronic exposure to sunlight and mutations caused by this [12].
Epidemiology of uveal melanoma
Uveal melanoma is much less common than cutaneous melanoma, with about 7 new cases per million people each year, but has a higher mortality rate, approaching 50%
[13]. In a recent study by the EUROCARE Working Group, the incidence of uveal melanoma in Europe strictly correlated with latitude, ranging from less than 2 cases per million in southern countries like Italy and Spain, to more than 8 cases per million in Norway and Denmark [14].
Development of uveal melanoma is not dependent on UV exposure, although recent reports suggest a correlation with exposure to blue light [15, 16]. Identified risk factors include a light skin and iris colour but not a specific hair colour [17], even though a study on melanocortin-1 receptor gene (MC1R) variants expression showed that MC1R variants do not play a role in the susceptibility to uveal melanoma [18].
The median age of uveal melanoma patients is above 60 [5, 19]. A number of risk factors have been identified for uveal melanoma: the presence of microvascular loops and networks, loss of one chromosome 3, and large tumour diameter (LTD) were identified to be strong indicators of a poor prognosis [20, 21]. Other important prognostic factors for uveal melanoma include the mitotic rate, the presence of epithelioid cells, the presence of extrascleral growth, older age and tumour-induced glaucoma [22]. Infiltration of macrophages and T lymphocytes are associated with a higher mortality rate [23, 24]. Once metastases have developed, survival is dismal.
However, a correlation can be seen between better survival and age younger than 60 years, lung/soft tissue as only site of first metastasis, treatment with surgery or
intrahepatic therapy, female sex, and a longer interval from initial diagnosis to metastatic disease [5].
Key stages in tumorigenesis.
Cancerous transformation of a normal cell includes a sequence of genetic and epigenetic events, enabling the cell to proliferate independently and infinitely, to migrate and escape senescence and apoptosis mechanisms, and avoid immune recognition [25]. In cutaneous melanoma, this is reflected histologically in the creation of first a benign lesion, followed by hyperplasia, dysplasia, neoplasia and eventually metastatic cancer.
For cutaneous melanoma, these stages have been long recognised, and described by Clark in a comprehensive model used for diagnosis [26], including six lesional steps to be recorded during melanoma tumorigenesis:
(1) common acquired melanocytic naevus (BN);
(2) melanocytic naevus with lentigenious melanocytic hyperplasia;
(3) melanocytic naevus with aberrant differentiation and melanocytic nuclear atypia, melanocytic dysplasia; (MDN)
(4) radial growth phase of primary melanoma (RGP-CMM);
(5) vertical growth phase of primary melanoma (VGP-CMM);
(6) metastatic melanoma.
A transition to a vertical growth phase is critical for cutaneous melanoma progression, and unavoidably results in acquisition of an invasive phenotype. Cutaneous melanoma disseminates via lymphatics, and involvement of sentinel lymph nodes is one of the most important risk factors for melanoma prognosis.
The determination of the exact stages of uveal melanoma cancerogenesis is more problematic, since this tumour arises in a site that is not easily accessible for histological follow-up. In the majority of cases, a diagnosis is made at a relatively advanced stage of tumour development. Uncertainty exists, whether the benign choroidal naevus represents a hyperplastic/dysplastic stage of uveal melanoma, or
whether it is an independent risk factor. According to one study, the estimated frequency for uveal melanoma development from a presumed naevus is 1 in 5 [27].
Even though clinical and pathological examination of early stages of uveal melanoma is a conundrum, three rate-limiting steps required to develop a primary tumour were hypothesized [28] and two of them were identified as growth and angiogenesis. Only one additional rate-limiting step is required for a primary tumour to proceed to metastatic disease, the nature of which has not been identified so far, but the likely candidates are mutations in micrometastases and their vascularisation [28]. Due to the lack of lymphatics in the eye, uveal melanoma spreads almost exclusively via the blood stream, and the liver is the usual first site of metastasis. A long-time controversy exists regarding the exact mechanism and timing at which metastatic spread takes place. The strongest hypothesis states, that the micrometastases occur at a very early stage of uveal melanoma development, but they are fairly quiescent or proliferate at a very low rate [28]. Evidence from a recent autopsy study supports this view (Borthwick et al; unpublished).
Molecular mechanisms
A model constructed by Hussein [29] for cutaneous melanoma pathogenesis correlates to stage progression with sequentially acquired abilities of independent growth and proliferation, aberrant differentiation and metastasis, and further on with genetic changes conditioning the transformation. His hypothesis implies a focal progression of only a few cells at each stage of transformation, which eventually lead to the creation of a malignant tumour. In vivo, these transformed naevus cells must acquire many characteristics, such as autonomous growth, a capacity to evade immune surveillance processes, and the capacity to invade the surrounding tissues. Genetic modifications are achieved by way of chromosomal aberrations, microsatellite instability and hypermutability of tumour suppressor genes, oncogenes and mismatch repair (MMR) genes.
Chromosomal aberrations
In cutaneous melanoma, the most common loss of heterozygocity (LOH) involves chromosomes 1p, 6q, 9p or 10q, 11q, and 17q. As shown in Table 1, the chromosomal aberrations in progression of cutaneous melanoma (chromosome 1p) begin as early as
the transformation of a normal melanocyte to a benign and dysplastic naevus.
Subsequent losses on other chromosomes correlate with further stages of cutaneous melanoma progression, and the accumulation of allelic losses, rather than the order in which they occur, is crucial for cutaneous melanoma development [29].
Chromosomal aberrations in uveal melanoma most often include chromosome 3, 6 and 8. Genetic profiling of uveal melanoma revealed the existence of two separate groups of low and high-risk tumours, with the latter expressing highly significant clusters of down-regulated genes on chromosome 3 and up-regulated genes on chromosome 8q [30]. Down-regulation of neural crest/melanocyte genes and an upregulation of epithelial genes was strictly correlated with an aggressive phenotype and the losses in chromosome 3 encoded for genes responsible for adherence (catenin- E), regulation of cell proliferation (RASS1) protein translation (eIF2a), signal
transduction (PIK3R4) and p53-dependent growth regulatory pathway (WIG1), while the gains in chromosome 8 encoded for genes responsible for signal transduction (PRK and WNT signalling pathways), expression of metalloproteinases (TAF2) and increased cell metabolism of nucleic acids and fatty acids (RRM2B and FABP5 respectively) [30]. Amongst tumorigenesis-related gene alterations in uveal
melanomas on chromosomes other than 3 and 8, several abnormalities were reported, including KIT, ERBB3, EDNRB, SPP1, TFAP2A, and SCDGF-B [30, 31].
Chromosomal aberrations result in alteration of fundamental molecular pathways, controlling cell growth, proliferation, cell to cell contact, motility and sensitivity to cell death signals. A reductionist model put together by Dahl and Goldberg [32]
explores the molecular pathways which are affected in melanocytic tumorigenesis, identifying the key genes involved in cutaneous melanoma genesis and progression (Table 1).
Cytogenetics
The first genetic changes are observed at the stage of the dysplastic naevus, when after mutation of BRAF/NRAS or KIT genes, the RAS/RAF/MEK/ERK pathway, responsible for the cells’ response to growth factors, becomes constitutively activated and the melanocyte gains the ability of uncontrolled proliferation. Interestingly, the mutations of BRAF and c-Kit are mutually exclusive, and the latter are most common
in non-sun exposed lesions [33]. A mutation in BRAF is often observed in benign naevi; however, only after the mutation of p16/CDK4 in p16/CDK4/RB pathway the cell can avoid another proliferation control mechanism, senescence [32]. Mutations in TP53 and ARF enable cells to avoid apoptosis and at the same time impede DNA repair [32]. The final modification, activation of the PI3K pathway by mutation in PTEN, seems ultimately to enable (melanoma) cells to invade other tissues [32].
Table 1.
Adapted and extended from [29, 32].
A comparison of uveal and cutaneous melanoma tumorigenesis, including molecular mechanisms and physiological pathways impaired during this process.
Cutaneous melanoma Stage of tumorigenesis
process
Uveal melanoma
1p or 9p and/or 10q Monosomy 3 Chromosomal
aberration
NRAS/BRAF/MEK/ERK Genetic
pathway
NRAS/BRAF or KIT
melanocyte proliferation
Mutations
9p, 10q, and 6q Chromosomal
aberration
P16/CDK4/RB Genetic
pathway
P16/CDK4
dysplasia senescence
Mutations
3,6,8 Chromosomal
aberration
P53/ARF BRAF/ERK
Ras P16/CDK
P53/RB
Genetic pathway
P53/ARF
Neoplasia apoptosis
cKit HGF-cMet P16/cyclin D1 MDM2, Bcl2,p53,RB
Mutations
1p, 11q, 17q 3,6,8,1 Chromosomal
aberration
PI3K-AKT PI3K-AKT Genetic
pathway
PTEN
Metastatic cancer
Invasion IGF1-IGF1R
CXCR4 - CXCL12
Mutations
Epigenetic regulation in uveal melanoma
As summarized in Table 1, even though uveal melanoma tumorigenesis involves deregulation of the same molecular pathways as in cutaneous melanoma, mutation
screening and deletion mapping did not reveal modifications in respective gene targets (e.g. BRAF) [34-36]. The current understanding of uveal melanoma tumorigenesis is that the tumour suppressor genes are inactivated due to the post-translational
mechanism, methylation of CpG islands, which represses transcription, and prevents expression of encoded proteins [37]. Inactivation of the p16 promoter by
hypermethylation was shown to be the major mechanism for loss of cell growth control in 50% of uveal melanoma cell lines and 30% of primary uveal melanomas [38]. Other cell cycle control mechanisms inactivated by hypermethylation include RASS1a, RASEF and TIMP [39-41].
Even though gene mutations have been identified very rarely at the early stages of uveal melanoma, this year, Bakalian and colleagues have described molecular mechanisms mediating metastasis of uveal melanoma to the liver [42]. The authors describe deregulation of exactly the same genetic pathways which were demonstrated to regulate the progression of cutaneous melanoma (Table 1), even though the
mutations reported are not necessarily in the same genes.
Microsatelite instability
Microsatelite instability (MSI) is an alternative mechanism to the loss of
heterozygosity in the genesis of cutaneous melanoma. MSI is present at all stages of melanoma progression, from the melanocyte onwards, with its frequency increasing with the stage of tumour [29, 43]. The presence of MSI indicates defective DNA repair mechanisms, resulting in changes during replication and a gradual decrease in expression of MMR proteins in melanocytic lesions during the transition from BN to MDN to CMM [29].
The findings of the only two studies of MSI in uveal melanoma are contradictory, one reporting the presence of MSI in almost 50% of examined cases [44], while the other states that MSI is a very rare event in uveal melanoma [45].
Adaptation of melanoma to the microenvironment
The distinct molecular characteristics in the pathogenesis of melanomas of different tissues may reflect a site-specific aetiology of melanoma, in particular the balance between the impact of environmental factors and genetic instability of melanocytes.
It is however difficult to argue that these molecular differences are not also caused by the influence of the local microenvironment, and the developmental pressure it puts on a growing melanoma, which must adapt to local conditions in order to progress and acquire distinct phenotypic features to be able to break both biochemical and physical restrictions.
Interactions between melanoma and the immune system
Melanomas are immunogenic tumours, and the interaction with the immune system plays a major part in melanoma development.
Uveal melanomas arise in an immuno-privileged site, and can develop unharmed by antigen-specific immune cells. In contrast, cutaneous melanomas develop in the skin, which is constitutively surveyed by a network of dendritic cells and infiltrating T lymphocytes. While cutaneous melanoma usually spreads locally via the lymphatic system, uveal melanomas spread haematogenously, as the eye lacks lymphatics. This dichotomy is reflected in their immunology. For example, in uveal melanoma,
metastasis is associated with an increase in HLA Class I expression and tumour antigenicity, whereas in cutaneous melanoma the reverse is true [46, 47]. A study on uveal melanoma immunogenetics showed that this tumour does not share genetic HLA determinants as risk factors with cutaneous melanoma [18]. Although down- regulation and allelic loss of HLA Class I expression is present in up to 40% of melanoma lesions and about 60% of uveal melanoma cell lines [48, 49], HLA polymorphisms do not contribute to an increased susceptibility to this tumour [50].
Moreover, quantitative studies show that loss or down-regulation of HLA Class I expression indicates a favourable rather than a poor prognosis [46], while in cutaneous melanoma, down-regulation of HLA Class I expression correlates with melanoma progression and reduced survival [51]. Similarly to cutaneous melanoma, however, a higher in situ expression of HLA Class II is correlated with poor prognosis and is associated with death of 60% of uveal melanoma HLA-DR-positive patients [23, 52, 53].
Metastatic melanoma of either type is largely chemoresistant and there is an urgent need for new treatments. The fact that both cutaneous and uveal melanomas express tumour-specific antigens, such as Melan A/MART-1 [54-56] that can be recognized
by cytotoxic T lymphocytes (CTL) in association with MHC class I [57][58], raises hope that immunotherapy might be a therapy of choice as treatment of these
malignancies. Well-documented cases of long-term complete remissions of cutaneous melanoma metastases exist [59-62], correlating with specific anti-tumour immune responses [63], expression of pro-inflammatory cytokines [64] and T lymphocytic infiltrates [65, 66].
However, unlike viral infections, CTL found naturally in cancer patients are fewer and functionally inefficient [67], and the patient’s immune response only rarely causes the tumour to regress for long periods [66]. Even though in the majority of vaccination schemes against cutaneous melanoma and uveal melanoma [68-70] the in vitro outcome was successful (with regards to antigen incorporation/transfection rate, protein production and presentation, and T-lymphocyte activation) numerous attempts to induce high numbers of efficient cutaneous melanoma-specific CTL responses by vaccination have failed [71]. Even when vaccination results in high numbers of circulating effector CTL, melanoma-specific CTL are not found in the tumour and the clinical impact is limited [72], [73]. Cutaneous melanoma patients fail to generate efficient anti-tumour CTL and even when induced by vaccination, these CTL cannot home to the tumour and kill it (or, conceivably, do home to the tumour, but are killed in the presence of immunosuppressive cytokines and interactions between Fas and Fas ligand). Similarly, the presence of natural active anti-tumour immune responses and partial or complete regressions of primary melanomas do not ensure a favourable prognosis and often occur in the presence of metastasis [74].
The reasons for the failure of vaccine therapies to date in melanoma are not clear but there is growing evidence that the microenvironment within the tumour may suppress the immune response at multiple levels. It is known that the following mechanisms are important, but their relative contribution to the lack of efficacy of melanoma vaccines remains largely unknown.
Why melanoma avoids recognition - current theories
The complex interactions between the immune system and developing malignancies have been studied by many. The coexistence of tumour-specific immunity with a progressing tumour as observed in most experimental systems and the fact that immunogenic tumours, like melanoma, develop in otherwise immuno-competent patients remain amongst the major paradoxes of tumour immunology [75]. While uveal melanoma develops in an immuno-privileged site, and is therefore protected from tumour-specific immunity, cutaneous melanoma grows under profound
immunological pressure, and must constantly avoid recognition by tumour-specific T lymphocytes. Among several theories which have been constructed to explain why tumours develop in otherwise immuno-competent patients, the main are Immune Surveillance [76], recently updated to Immunoediting [77], Immune Ignorance [78, 79] and the Danger model [80]. The first hypothesis presumes that cancer cells constantly develop in the body, but the majority of them are specifically detected and eliminated by constant surveillance of the immune system, on the basis of their
expression of tumor-specific antigens or molecules induced by cellular stress [76, 81].
This theory was recently developed into the concept of immunoediting, regarding immunosurveillance as a phase of a more complex process which leads to
development of immuno-resistant tumour variants, escaping immune recognition [77, 82]. Since this recognition is based on antigen presentation, loss of HLA molecules and impaired antigen presentation are the most obvious mechanisms of escape from destruction by cytotoxic T lymphocytes (CTL). Alterations in HLA expression are ubiquitous among tumours and include complete loss of any HLA allele, significant down-regulation of one or more alleles, expression of altered HLA alleles or
immunosuppressive HLA alleles, and altered responsiveness to activation signals such as type I interferons [83]. Loss of HLA class I leads to impaired HLA loading, and therefore inefficient surface antigen presentation [84]. Antigen recognition and a successful immune reaction are additionally impeded by heterogeneity in surface protein expression, even within the same tumour [85].
In contrast, the theory of immune ignorance states, that despite the existence of numerous tumour-specific antigens, they are not recognized by cytotoxic T lymphocytes because of the lack of a co-stimulatory signal [78, 79]. Antigen
presentation without a correct second signal may also result in anergy of antigen- specific T lymphocyte and eventual immuno-tolerance. For example, melanomas can also express an HLA class II protein, whose expression is generally restricted to APC and activated T cells. This ability does not enhance tumour immune susceptibility, but instead interferes with normal T helper function due to the absence of co-stimulatory molecules such as B7 on the tumour [86-89]. In opposition to this theory where active self-nonself discrimination is important for immune surveillance or ignorance, the Danger model implies that unresponsiveness is a natural state of immune response, which gets activated only upon receiving appropriate stimulation, i.e. the “danger signal”. Accordingly, tumours can safely arise in immuno-competent organisms, unless an additional dangerous situation occurs, like surgical trauma, or infection [80].
To enable successful tumour elimination, this danger signal must be delivered in a form of microbial pattern receptors or heat shock proteins, which play a critical role in cross-priming, i.e. presentation of antigens produced by one cell on the surface of another cell, preferably professional APC. Additionally, the activation of an immune response must be continuous, not allowing the cytotoxic T lymphocytes to rest [80].
However diverse these hypotheses are, they all acknowledge the importance of proper antigen presentation, activation of antigen-specific T lymphocytes and maintaining the anti-tumour immune response. All these aspects of immune response are under the control of dendritic cells (DC), professional antigen-presenting cells, which are able to activate naïve T lymphocytes, support activation of memory T lymphocytes and at the same time capable of modulating immune reactions and rendering antigen-specific lymphocytes unresponsive. Considering the prominent function of dendritic cells in the regulation of immune responses, special attention should be given to their role in the interaction between tumours and the immune system, their involvement in successful tumour elimination and, conceivably, in tumour immune escape.
DCs: their role in the control of the immune system
Dendritic cells orchestrate the primary antigen-specific immune response. In man, several different subsets of cells exist that share the dendritic phenotype, and DCs isolated form bone marrow, blood or tissues are composed of a mixture of various haematopoietic lineage representatives. Although nearly all known dendritic
subfamilies (except for follicular dendritic cells) originate from one common
precursor (the CD 34+ bone marrow stem cell), their developmental pathways differ in terms of progenitors and intermediate stages, cytokine requirements, surface marker expression and biological function. The classic view on DC development, linking their biological function with cell origin, describes at least three clearly distinct DC developmental pathways. Two types of DC progress from myeloid precursors, and become either myeloid dendritic cells (MDC) or Langerhans cells (LC), and one originates from a lymphoid precursor, creating a third subclass:
plasmacytoid dendritic cells (PDC) [90], [91].
According to the phenotype-function theorem, DCs of myeloid origin activate immune responses by expression of Th1 type immunostimulatory cytokines, while PDC modulate immune responses by expressing the Th2 cytokine spectrum [90]. The current understanding of DC biology is however more complex, taking into account their plasticity [91] and the possibility of the generation of various functional subsets of DC by stimulation with appropriate cytokines, independent of their origin [91, 92].
In summary, the function of DCs depends on their origin, maturation, activation status, interaction with other cells and influence from the microenvironment.
Immature DC
Immature and non-dividing DCs colonize most tissues, where their precursors from bone marrow have migrated to via blood vessels. Their further development depends on possible antigen capture supported by cytokines and growth factor stimulation. In humans, immature DCs have been observed in most organs, including skin, liver, kidney and heart, and tend to be associated with vascular structures. An interdigitating sentinel epithelial network of DCs has been described in the mucosa of the oral cavity, intestinal tract and the respiratory tract [93]. Immature dendritic cells reside in lymph nodes and other secondary lymphoid organs. All skin-draining lymph nodes contain two DC populations: immature resident DC and more mature DC - immigrants from the skin. In the epidermis, above the basal layer of proliferating keratinocytes, reside not less than 109 Langerhans cells [94]. These ubiquitous cells act as sentinels against pathological alterations and infectious invasion. Despite their immature phenotype, DCs located in the peripheral tissues are well prepared for their role. As cells
specialized in antigen capture they can take up larger particles and microorganism by phagocytosis. They can also monitor the biochemical content of their environment via
pino- and macrocytosis and receptor-mediated endocytosis, as they are equipped with C-type lectine receptors as well as FcJ and FcH receptors.
Additional receptors involved in antigen recognition by DCs are Toll-like receptors (TLR), cytokine receptors, and CD40. Pathogens are recognized by particular TLR on the base of specific molecular patterns, i.e. lipopolysaccharide via TLR4 and dsRNA via TLR3 [95, 96]. DC have a distinct response pattern of phenotypic differentiation and cytokine production in response to different TLR ligands corresponding to their expression of TLRs [97, 98], [99-101], nevertheless, all these effects are evoked via the NFkB induction pathway, either by MyD88 dependent or independent cascade.
The ultimate effect of specific TLR activation depends upon the particular pathway, and additional molecules involved in pathway regulation [102, 103]. Immature DC have a low T cell stimulatory capacity, do not express many MHC proteins on their surface (although they are abundant in intracellular compartments MIIC, ready to be exposed after the proper maturation signal) and express many proteins on their surface: integrins E1 and 2, and immunoglobulines CD2, CD50, CD54, CD58 [94, 104].
Mature DCs
Capturing a pathological antigen gives DCs the first signal for maturation. They loose E-cadherins, and start migrating to secondary lymphatic organs, via the blood or lymphatic vessels. They also acquire their unique mature phenotype, extend numerous processes that facilitate migration and later, in the lymph nodes, cell-to-cell contact.
During migration, DCs upregulate chemokine receptors (for C-C type chemokines, MIP D, E) [93] and begin to expose antigen-loaded MHC class I and II on their surface. They also start expressing co-stimulatory molecules, CD80 and CD86 (B7-1 and -2, respectively) and CD40 necessary for a proper dialogue with T lymphocytes [94, 104].
Acquiring an immunogen is the first, but not sufficient signal for DC maturation.
Immature peripheral DCs constantly monitor their environment, and additional signals are necessary to activate them properly. If a bacterial or viral pathogen invades a tissue, it will cause the described sequence of events, and will also provoke inflammation. The biochemical environment will change, and this alteration will support DC activation. Bacterial cell wall components like lipopolysaccharides,
lipoteichic acid and proteins, CpG-rich DNA sequences [94, 104, 105] and viral double stranded RNA [104, 106] have been reported as powerful immune response stimuli. Other important DC activators are proinflammatory cytokines (TNFD, IL1 D
and E, IFNJ) produced by macrophages, granulocytes and lymphocytes present at the site of inflammation [104, 107, 108]. All these signals influence dendritic cells sequentially or even simultaneously, and thus the synergistic effect is incredibly strong. In this phase, pre-activated dendritic cells have developed a fully mature phenotype, produce substantial amount of cytokines, and evoke potent immune responses.
Interaction between DC and lymphocytes
The interactions between dendritic cells and lymphocytes are the key events for immune-response induction. Once the DC presents antigen to a lymphocyte, it is stimulated in return, and the maturation process can be completed. Activated T cells release CD40L, a major stimulant for DC. A CD40-CD40L reaction results in an additional increase in the expression of CD80/86, cytokine release, and up-regulation of OX40-L, a member of the TNF-R family, responsible for B-lymphocyte activation and supporting DC-T lymphocyte contact. Other compounds produced or released in this vigorous biochemical conversation are: CXCR L, CXCR-5R, 4-1BB-L, CD6L and SLAM L. Since the contact between DC and T lymphocytes is still being investigated, it is likely that this list is not yet complete.
As has been proved by Galluci et al [109], the presence of exogenous pathogens is not indispensable for activation of DC, as they can also be activated upon stress, by the presence of initially healthy cells, killed via necrosis [109, 110]. Necrotic death seems to be fundamental for DC induction, as it results in the release of cell content and causes inflammation. Apoptotic cells might also be phagocytosed, but this does not result in DC maturation [109]. However, the ultimate hallmarks for immunogenic cell death are exposure of calreticulin [111, 112] and release of the HMGB1 molecule [113].
DCs and immune tolerance
In addition to their function in activating immune responses, DCs play a key role in the development of immune tolerance. Mature PDCs are thought to act during the negative selection of T lymphocytes in the thymus [114], while immature MDC and mature and immature PDC also take part in evoking so-called peripheral tolerance, elimination of self-reactive T lymphocytes emerging in the periphery [115-117].
Under physiological conditions, the PDC would small amount of self-antigens expose on their cell surface and present them to T lymphocytes. However, since the
presenting DCs remain immature, they do not express co-stimulatory molecules, and that results in impaired lymphocyte activation, which causes anergy. Alternatively, self-antigens might be presented by professional tolerogenic DC, expressing the inhibitory receptors ILT3 and ILT4, which suppress T lymphocytes upon antigen presentation and cause generation of antigen-specific suppressive CD8+ T lymphocytes [117]. This mechanism leads to elimination of self-sensitive lymphocytes, and prevents autoimmune aggression.
Yet another subset of DC that supports self-tolerance consists of semi-matured DC circulating in the blood [118]. Cells from this small population are self-antigen
loaded, show surface antigen expression associated with a mature phenotype, but they do not release cytokines, and thus also do not provide sufficient activation signals for lymphocytes. In reaction with CD4+ lymphocytes, they induce a subset of regulatory helper lymphocytes, which remain as memory cells. The development of suppressive T lymphocytes in tissues and creating a population of regulatory T cells are two distinct ways of inducing immune tolerance to self-antigens.
All these data indicate that DC may act as homeostasis guardians, eliciting immune responses in reaction to danger signals, of exogenous or endogenous origin, but refraining from stimulation in physiological, potentially harmless, situations.
Influence of microenvironment on DC biology
The decision about eliciting either an anti-tumour immune response or tolerance depends solely on the manner of antigen presentation. If an antigen is presented by a DC with a tolerogenic phenotype, or if an immature DC does not recognise the
antigen as potentially dangerous, i.e. in the absence of a danger signal, this will lead to lymphocyte suppression, anergy or unresponsiveness. Recently, the significance of the local microenvironment has been brought into the picture. Since DC act as environmental sensors guarding the homeostasis of the organism, they must be very sensitive to any changes ongoing in their close vicinity. In order to execute their functions they undergo a complex transformation, including changes in shape, size, motility, and also complete alterations of cell surface antigen expression, in reaction to exogenous factors. A pluri-potent immature DC can differentiate into an activating or a tolerizing phenotype, depending on the signals they receive at the time of antigen uptake. In peripheral tissues, they are prone to become tolerogenic, as too sudden immune activation is not favorable for the organism. Indeed, the presence of immunoregulatory DCs has been shown in peripheral tissues, and their role in the regulation of immune responses has been proven.
This immune regulation is particularly important in tissues where acute inflammatory reactions can cause severe damage, for instance in the eye, where the state of
hyporesponsivenes is achieved by a number of mechanisms, including the secretion of immunosuppressive cytokines, particularly two isoforms of TGFE [119, 120]. This immune privilege is so pronounced that presentation of an antigen in the anterior chamber of the eye results in peripheral tolerance [121]. Numerous mechanisms are involved in regulation of this anterior chamber associated immune deviation
(ACAID), but the impaired presentation of antigens is a critical event [122, 123].
Confirming the influence of the ocular microenvironment on DC function, it has been shown that aqueous fluid changes the DC antigen-presenting skills [124], and DC residing in the uveal tract acquired the ability of antigen presentation only after a period of culture [125].
Mucosal surfaces
Mucosal surfaces of the intestinal and respiratory tract, which guard the interior of the organism against entry of numerous pathogens, are another example of a location where immune compromise is vital for healthy functioning. DC reside in all mucosal tissues, e.g. in the pharynx, the epithelium of tonsils, encircling both the alimentary and the respiratory tract openings, in the gut, where multiple DC populations reside in
the Peyer’s patches, colonic lymphoid follicles, maesenchymal lymph nodes and the lamina propria.
The main function of the mucosal immune system is to discriminate between
potentially infectious pathogens and innocuous food antigens [126, 127]. The balance between immune tolerance and an active immune response in the mucosa is delicate, and if not maintained, may result in life-threatening disorders. An abrogated function of the mucosal immune system may lead to inflammatory bowel disease (IBD) [128], as well as cancers of the gastrointestinal tract [129, 130]. In order to ensure
hyporesponsiveness, the gut has evolved effector mechanisms, which specifically prevent excessive inflammation and tissue injury. This state has been associated with the local production of suppressive molecules (for example IL-10, TGFE1,
prostaglandin 2), insufficient delivery of co-stimulatory signals, and synthesis of low molecular weight non-protein mediators with oxidative capacities [131]. Similarly, maintenance of immune tolerance is crucial in the respiratory tract and ear mucosa, where abnormal activation of T lymphocytes may lead to allergy and asthma [132, 133] or chronic otitis [134]. Mucosal DC regulate specific innate or adaptive immune responses to help distinguish between commensal microbiota, pathogens and self antigens. They are responsible for oral tolerance to food antigens [135], directly sample bacteria through the epithelial layer in an in vitro model [136], are involved in T cell activation and silencing [137], and mediate homing of T lymphocytes to gut [138]. Extensive evidence proves that the function of mucosal DC is strongly influenced by their immunosuppressive environment [139, 140] , and in return, mucosal DC further regulate local immune responses [128, 141-143].
Melanoma as an immunosuppresive microenvironment
The secretion of large quantities of immunosuppressive cytokines and other immunosuppressive factors by tumour cells creates a specific microenvironment which drastically modifies T lymphocyte response, prevents T lymphocyte activation and proliferation and triggers the creation of numerous suppressive and regulatory T lymphocyte clones [144-147]. Even though uveal melanoma develops in an immuno- privileged site, it contributes to the immunosuppression by expressing FasL, and
secreting immunosuppressive cytokines [148-151], macrophage migration inhibitory factor (MIF) which can inhibit natural killer (NK) cell-mediated cytolysis [152] and, induced by IFNJIDO [151]. Cutaneous melanoma developed multiple mechanisms of immunosuppression resulting in creation of an intratumoral immunosuppressive micro-environment, by secreting immunosuppressive cytokines, including TGFE and IL10 [153, 154], expression and release of FasL, which causes apoptosis of T
lymphocytes [155] and shedding antigens, which abrogate anti-tumor cytotoxic lymphocyte function [156]. Secretion of immunosuppressive cytokines is clearly important to melanoma development: not only are IL10, TGFE and IDO present in patients’ blood, primary melanomas and SLN [153, 154, 157-159], but cytokine levels correlate with tumour progression and invasiveness [157, 158]. Similarly, despite growing in an already immunosuppressed environment, uveal melanoma significantly contribute to the local immune-privilege, secreting TGFE [148]. Investigations on melanoma sentinel lymph nodes showed that lymph node immunosuppression was related to the presence of cutaneous melanoma [154] and relative stimulation or suppression correlated with the distance of the node from the nearest deposit of primary or metastatic melanoma [160]. This distance-related immunosuppression has been further confirmed in studies on T lymphocyte activity [161], DC infiltrate [162]
and DC morphology [163].
This evidence suggests that in fact melanoma not only avoid recognition by the immune system, but also actively alters local functioning of the immune system.
Since DCs are sensitive to the signals from the microenvironment and reside in close proximity to melanoma and in sentinel lymph nodes suppressed by the melanoma, it is conceivable that their function is compromised in such an immunosuppressive
microenvironment. As they are the key regulators of immune reactions, this would result in down-regulation of tumour-specific immune responses.
It is therefore important to investigate the immunophenotype and heterogeneity of DCs within the melanoma tumour tissue as well as to define the degree of suppression of dendritic cell function in draining lymph nodes of melanoma.
Scope of the thesis:
The aim of this thesis is to investigate the influence of an immunosuppressive microenvironment on the biological behaviour of tumour and their regional lymph node dendritic cells, As models we used both cutaneous and uveal melanoma.
In Chapter I, the biology of melanomas of the skin and eye is introduced, and a comparison is made, describing differences and similarities in the epidemiology, biology, immunology, molecular pathways and response to treatment of these two tumours. The current understanding of the role of dendritic cells in the control of immune responses and the influence of the local microenvironment on DC phenotype and function is described, as well as a brief insight into melanoma immunology and strategies of melanoma immune escape. As different approaches exist with regard to the use of vaccines, their advantages and limitations are reviewed in Chapter II.
Hypotheses are provided to suggest why current vaccines do not work in patient treatment.
In Chapter III the presence and activation status of DCs within cutaneous melanoma metastases in the regional lymph nodes are investigated. Since the influence of melanoma on DC maturation may result in significant alterations of tumour-specific immune response, the expression of Factor XIIIa (FXIIIa), CD40, CD83 and HLA- DR as well as cell morphology are examined using immunohistochemistry and image analysis techniques.
To investigate in depth the local immunosuppressive microenvironment created during progression of melanoma, we also examined (Chapter IV) the presence of suppressor T lymphocytes and tolerizing dendritic cells (DCs), the expression of immunosuppressive cytokines (IL-10, TGFb1 and TGFb2) and the enzyme
indoleamine 2,3-dioxygenase (IDO). Knowledge of the local microenvironment is essential for understanding the local immune responses.
Since uveal melanoma is an example of an immunogenic tumour growing in a profoundly immunosuppressed site, we also examined (Chapter V) the
microenvironment in the eye by analysing the presence and phenotype of local DCs
which have been exposed to immunosuppressive factors derived both from the tumour and the local immune privilege.
To test whether it is possible to influence the phenotype and function of DCs residing in an immunosuppressive microenvironment, in Chapter VI we investigate the immune response of mixed cultures of PDCs, MDCs and macrophages derived from tonsils, stimulated with commonly used immunoadjuvants: LPS, Poly: IC, IFNy and humanized anti-CD40 antibody. If achieved, the reversal of the immunosuppressive phenotype may have a significant impact on melanoma vaccine development and creation of successful anti-melanoma immunotherapy.
Chapter VII presents the current situation in the field of melanoma vaccine
development, summarising the results of 57 clinical trials reported between 2003 and 2007.
Finally, in Chapter VIII conclusions drawn from the above mentioned studies are summarised and a hypothesis of the role of immunosuppressed dendritic cells in progression of cutaneous melanoma and uveal melanoma is outlined.
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