Safety, feasibility and clinical benefit of localized chemotherapy using microencapsulated cells for inoperable pancreatic carc

Cancer Therapy Vol 1, 393-405, 2003.

Dendritic cell-mediated immunosuppression in malignant melanoma

Review Article

Marta E Polak¹, Nicola J Borthwick², Martine J Jager³, Ian A Cree¹

1Translational Oncology Research Centre, Department of Histopathology, Queen Alexandra Hospital, Portsmouth PO6 3LY, UK; ²Department of Pathology, Institute of Ophtalmology, Bath Street, London EC1V 9EL; ³Department of Ophthalmology, Leiden University Medical Center, University of Leiden, Leiden, The Netherlands

__________________________________________________________________________________

*Correspondence: Marta E Polak, Translational Oncology Research Centre, Department of Histopathology, Queen Alexandra Hospital, Portsmouth PO6 3LY, UK; Tel: 0044 2392 286 000 x 5381; Fax: 0044 2392 286 27; e-mail: marta.polak@porthosp.nhs.uk

Key Words: Melanoma, dendritic cell, immunosuppression, vaccine, adjuvant

Abbreviations: Antigen Presenting Cell, (APC); Cluster of Differentiation, (CD); Cytotoxic T Lymphocytes, (CTL); Dendritic cells, (DC); Dinitrophenyl, (DNP); Delayed Type Hypersensitivity, (DTH); Fas Ligand, (FasL); Granulocyte-Macrophage Colony Stimulating Factor, (GMCSF); Human Leukocyte Antigen, (HLA); Heat Shock Protein, (HSP); Interferon, (IFN); Interleukin, (IL); Keyhole Limpet Hemocyanin, (KLH); Langerhance Cells, (LC); Natural Killer Cells, (NK); Peripheral Blood Lymphocytes, (PBL); Tumor Associated Antigen, (TAA); T cell receptor, (TCR); Tumor Growth factor, (TGF); Tumor Necrosis Factor, (TNF); World Health Organisation, (WHO)

Received: 22 December 2003; Accepted: 31 December 2003; electronically published: December 2003

Summary

Melanomas are immunogenic tumors, presenting a range of tumor-associated antigens (TAA), and cases of spontaneous tumor regression indicate that immune control over melanoma growth can be achieved. Evasion of the immune response is a critical part of tumor development and melanomas can avoid recognition by a variety of mechanisms such as impaired expression of HLA molecules, shedding TAA or secretion of immunosuppressive factors. To enhance antigen presentation and prime effector T lymphocytes, a range of dendritic cell (DC) based melanoma vaccines have been developed. A variety of strategies have been employed using autologous DC to stimulate tumor specific immune responses. Although all of these were apparently successful in vitro, when used in patients the responses were disappointing and they ultimately failed to destroy the tumor in the majority of patients. This may reflect observations that melanoma cells suppress immune responses in vitro, and may prevent the generation of effector cells following DC vaccination. Mature DC are normally potent activators of immune responses. However, when immature, they are often immunosuppressive. The DC found in melanoma and in the sentinel lymph nodes invaded by tumor are of an immature phenotype and therefore may suppress the anti-tumor immune response. We suggest, that a successful vaccine for melanoma must include either mechanisms to reverse in situ DC suppression or increase immune stimulation.

I. Introduction

A great deal of effort has been put into developing a vaccine for the treatment of melanoma. However none of the current approaches have addressed immune suppression at the tumor site. It is possible that unless this melanoma-derived immune suppression is reversed immunotherapy will be unsuccessful. Melanomas, unlike most other tumors, can be immunogenic, and can present a range of tumor-associated antigens (TAA). Cases of spontaneous tumor regression have been reported even in very advanced disease (Szekeres and Daroczy, 1981; Ralfkiaer et al, 1987; Tefany et al, 1991), and these reports have encouraged efforts towards anti-tumor immunotherapy. Despite the presence of potent anti-tumor immune cells in their blood, more than 95% of patients gain no benefit from anti-tumor immune therapy. The co-existence of anti-tumor immunity and tumor progression in the same individual remains one of the major paradoxes of melanoma immunology.

II. Escaping immune surveillance

It has been recognised for some time that the immune system plays a crucial role in the removal of malignancies arising through somatic mutation. Successful malignancies must survive this surveillance and are therefore subject to selection pressure resulting in the evolution of escape variants, that can no longer be recognised by either T lymphocytes or NK cells (Burnet, 1970; Festenstein and Garrido, 1986). Since the recognition is based on antigen presentation, the 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 tumors, but are also highly variable. So far seven different major modifications of HLA class I phenotypes have been described in different tumor types. These 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 (Adrian Cabestre et al, 1999).

Loss of HLA class I is often attributable to structural alterations in the proteins involved in antigen processing leading to impaired HLA loading, and therefore surface antigen presentation (Seliger et al, 2001). Melanomas can also express HLA class II proteins, whose expression is generally restricted to APC and activated T cells. This ability does not enhance tumor immune sensitivity, but on the contrary interferes with normal T helper function due to the absence of co-stimulatory molecules such as B7 on the tumor (Becker et al, 1991; Hersey et al, 1994; Becker and Brocker, 1995; Denfeld et al, 1995). Antigen recognition and a successful immune reaction is additionally impeded by heterogeneity in surface protein expression, even within the same tumor (Dalerba et al, 1998). Moreover melanoma cells can shed antigens, which may abrogate anti-tumor cytotoxic cell function or express and release FasL, which causes apoptosis of T lymphocytes and secrete immunosuppressive cytokines (Becker et al, 1991; Ekmekcioglu et al, 1999; Gray et al, 2002; Redondo et al, 2002, 2003; Sombroek et al, 2002; Wolfl et al, 2002; Peguet-Navarro et al, 2003).

III. Dendritic cell based immune vaccines

As the generation of successful anti-tumor immune responses would greatly benefit patients with this aggressive tumor, a number of approaches have been taken to initiate protective immunity. Many of these exploit function of dendritic cells, which act as potent immune response stimulators. Dendritic cells migrate from blood to nearly every tissue in the body, take up antigens and process them. They then migrate to spleen and lymph nodes and deliver the antigens for presentation to lymphocytes. As professional APC they express both HLA class I and II, and can additionally therefore activate both helper and cytotoxic T lymphocytes. They can cross-process antigens between these two pathways and in this way switch the immune response type and evoke cytotoxic reactions against endogenous tumor antigens (Albert et al, 1998a, 1998b; Banchereau and Steinman, 1998; Inaba et al, 1998) (Figure 1). Numerous vaccine strategies have either utilised DC directly or used a variety of mechanisms to stimulate them.

To find suitable antigens for vaccine purposes, melanoma proteins have been screened in search of peptides with potent immunostimulatory characteristics, presented by both HLA class I and II, to activate both cytotoxic and helper T lymphocytes. Studies have identified HLA class I binding peptides, and several peptides presented in the context of multiple HLA-DR alleles and recognisable by CD4+ T cells (Table 1).

Peptides derived from known melanoma associated antigens have been used to load DC generated in vitro from blood or bone marrow precursors or monocytes from both melanoma patients and healthy donors. In order to enhance antigen presentation several peptide modifications have been tested. For example, the proteins were fused with TAP targeting sequence to facilitate antigen processing (Minev et al, 2000) or with heat shock protein to assist antigen delivery into dendritic cells. (Noessner et al, 2002). In one particularly successful approach, fusion of melanoma derived antigen with recombinant HIV trans-activating fusion proteins allowed enhancement of the protein incorporation rate to 95% (Table 2).

One of the most important disadvantages of peptide-based vaccines is the lack of non-self antigens shared by all melanoma cells. A single epitope is seldom sufficient for the induction of a potent immune response, therefore an ideal vaccine should contain a variety of epitopes and proteins. One way of avoiding these difficulties is to use whole tumor cells, or vesicles secreted by them, as a source of antigen. It has been shown that dendritic cells can phagocytose necrotic (Abdel-Wahab et al, 1998) and apoptotic (Soruri et al, 2001) tumor cells, however in the latter case DC maturation strongly depends on the presence of pro-inflammatory cytokines in the environment (Jenne et al, 2000; Labarriere et al, 2002). One approach to increase vaccine immunogenity exploits the technique of cell fusion. Hybridomas of melanoma cells and dendritic cells have been shown to preserve the features of dendritic cells vital for their function (expression of HLA-A, B, C; HLA-DR; CD40, CD54, CD80, CD83, CD86, and the pro-inflammatory cytokine interleukin-12) and expression of melanoma-associated antigens (Holmes et al, 2001; Soruri et al, 2001; Jantscheff et al, 2002).

Endogenous expression of antigen by DC offers the potential advantage of prolonged presentation of antigens in the context of both HLA classes, and potentially extends the repertoire of immune stimulation. Nucleic acid-based immunization provides an attractive method for the delivery of protein antigens and adjuvants, without the need to know the sequence of immunogenic epitopes in advance. Additionally it allows the function of multiple restriction elements for the presentation of the same antigen Kim et al, 1997 and the generation of CD8(+) T cells against multiple class I-restricted epitopes within the antigen (Alijagic et al, 1995; Lapointe et al, 2001; Larregina et al, 2001).

Figure 1 Role of dendritic cells in immune system activation. Dendritic cells reside in the majority of tissues, and continuously acquire and process the antigens form the environment (1). The antigen uptake gives them a primary signal for maturation. They start production of proteins necessary for antigen presentation and they migrate via blood to deliver the antigens to spleen and lymph nodes (2). Being antigen-presenting cells, they express both HLA class I and II, and can therefore activate both helper and cytotoxic T lymphocytes. Furthermore, they can cross-process antigens between these two pathways and thus switch the immune response type and evoke cytotoxic reactions against endogenous tumour antigens presented in their HLA proteins. Providing the secondary activation signal (accessory molecules and cytokines), they prevent lymphocyte anergy, resulting in the generation of an army of sensitised cytotoxic and helper, effector and memory lymphocytes (3).

Table 1. Examples of defined epitopes suitable for anti-melanoma immune vaccine therapy.

Protein	HLA-I binding peptides	HLA-II binding peptides	Authors
Cancer-testis antigens
NY-ESO-1		+ 119-143 119-143	Jager et al, 2000; Zarour et al, 2002, 2000b
MAGE-3	EVDPIGHLY	TQHFVQENYLEY	Schultz et al, 2000, Schultz et al, 2001
Melanocyte differentiation antigens
gp-100	gp100[9(87)] gp100[10(86)		Kawashima et al, 1998 Cochlovius et al, 1999 Cochlovius et al, 2000 Kierstead et al, 2001
MelanA/MART-1	GILTVILGV ALMDKSLHV	51-73	van Elsas et al, 1996 Zarour et al, 2000a
Widely expressed antigens
HER2/neu		776-788	Sotiriadou et al, 2001

Table 2. In vitro immune response mediated by melanoma antigens loaded dendritic cells.

Antigen source	Antigen presentation	Inducted cells	Cytotoxic reactivity anti:	References
Defined peptide (MAA)	+++	CTL, NK, Th lymphocytes	Peptide loaded target cells Tumor cell lines Normal melanocytes Autologous tumor cells
Melanoma cell lysates	+++ cross-presentation	CTL, Th lymphocytes	Autologous tumor cells
Hybridomas
Genetically modified cells	+++	CTL, Th lymphocytes (1 study)	Cells presenting MAA antigens Autologous tumor cells	; ; ;

IV. In vitro efficacy of dendritic cells vaccines

Overall, the majority of the in vitro approaches described have been successful, with regards to antigen incorporation/transfection rate, protein production and presentation, and T-lymphocyte activation. Experiments in vitro have proved that dendritic cells are able to process and present melanoma-specific antigens derived from whole melanoma cells, synthesised or purified peptides or when they are genetically modified to produce tumor antigens (Table 2). Moreover, synergistic effects of viral transfection and DC maturation have been observed (Rea et al, 2001; Temme et al, 2002). Transfected DC synthesised the desired product, and the antigen expression remained detectable for at least 7 days. Also DC loaded with killed tumor cells can induce HLA class I- and class II-restricted proliferation of autologousCD8+ and CD4+ T cells, and are therefore able to cross-present tumor cell-derived antigens. In all cases they presented a broad range of tumor antigen epitopes in the context of multiple HLA alleles and stimulated several types of lymphocytes reactive against multiple melanoma antigens.

In the vast majority of studies both proliferative and cytotoxic responses were reported. Lymphocytes co-cultured with genetically modified DC produced Th1 type cytokines and showed multiple antigen specific cytotoxic responses, against melanoma cell lines, HLA-matched B cell lines pulsed with peptide and, most importantly, autologous tumor (Table 2). Induction of not only cytotoxic and helper lymphocytes, but also clones of NK cells have been reported. Tumor-specific T cells with NK activity are potentially of great clinical significance as they provide a mechanism for lysis of tumor cells that have lost HLA expression (Saeterdal et al, 1998)

It is the ability of in vitro expanded lymphocytes to recognize naturally processed and presented epitopes that illustrates the potential use of dendritic cells for vaccination in human cancer. Unfortunate therefore that, despite these encouraging results, so far none of these strategies has found direct effective translation to patient care.

V. Response of patients to DC vaccination

Despite dendritic cells being increasingly used for the immunotherapy of melanoma only a few tumor remissions due to vaccination have been reported (Table 3). Several phase I/II clinical studies have shown that DC vaccines are non-toxic (no grade 3 or 4 WHO scale toxicities), that vaccine injections are well tolerated, and that DC derived in vitro are viable after injection and can mediate biologic activity in situ (Table 3). Both adjuvant therapies and dendritic cell based vaccines caused infiltration of immune cells (both dendritic cells and lymphocytes as well as numerous other types) into the site of vaccination, and the cytotoxic tests on patients immune cells obtained after one or several courses of vaccine administration have given encouraging results. Peripheral blood lymphocytes from patients recognised melanoma cells in vitro, produced pro-inflammatory cytokines and

Table 3. Clinical outcome of DC based vaccines

	No Patients	DC infiltration	T lymphocytes infiltration	Objective response rate — overall percentage			References
				Complete response	Partial response	Disease stabilisa-tion
DC targeted adjuvants
GM-CSF	72	5/5 studies	4/5 studies	1%	16.7%		;
GM-CSF + Other adjuvants	51	1/2 studies		22,5%	27%
Antigen modified dendritic cells
Autologous DC injected into tumor site	7	1/1 studies	1/1 studies	0	57%
Peptide loaded	172	3/13 studies	4/13 studies	2%	12%	3%	;
Melanoma lysates loaded	66		2/4 studies	12%	13.6%	1.5%
Genetically modified	14	1/3 studies	1/3 studies	0%	0%	0%

even killed melanoma cells from cell lines or autologous tumors. Nevertheless, the vaccination was ultimately unsuccessful in most cases, since the melanoma survived and the patient died (Table 3).

A. Adjuvants

Adjuvants stimulate DC and in this way enhance immune response. An earlyattempt to induce clinical inflammatory response in vivo using the dinitrophenyl (DNP) -conjugated melanoma cell immunization of DNP-pre-sensitised patients resulted in cutaneous DTH. In half of the patients the inflammatory reaction was confirmed and caused regression of metastases within 2-4 months. The inflammatory response was associated with the infiltration of CD8+ T cells, enhanced expression of ICAM-1 and HLA-DR by melanoma cells and the presence of numerous HLA-DR+, CD4+, CD1-, Leu-1- dendritic cells (Murphy et al, 1993).

The cytokine GM-CSF stimulates DC maturation in vitro and has been used to stimulate DC activation in vivo. (Mortarini et al, 1997; Chen et al, 2001). Direct sub- or intra-cutaneous injections (Schachter et al, 1998; Janik et al, 1999; Nasi et al, 1999) and the use of genetically modified melanoma cells (Soiffer et al, 1998; Zehntner et al, 1999; Chang et al, 2000; Kusumoto et al, 2001) have been used for vaccine administration either alone or in combination with other cytokines (Janik et al, 1999) and cytotoxic agents (Schachter et al, 1998). Vaccine evaluation was based on immunohistochemical staining of vaccine site biopsies, peripheral blood analysis and functional tests in vitro, as well as clinical outcome. In most cases, despite DC and lymphocyte influx into metastatic tumor sites and the successful specific activation of anti-tumor T lymphocytes, the clinical outcome was far from satisfactory. In two studies achieved remission in only one patient (Chang et al, 2000; Kusumoto et al, 2001), and in three, no anti-tumor effects were observed. In the study performed by Soiffer and colleagues, extensive tumor destruction was observed, but no durable complete remission was reported. (Soiffer et al, 1998). The adjuvant vaccine worked well however when combined with chemotherapy, giving a response rate over 50%. Nevertheless the drug regimen, including cytokines, was very toxic, and this strategy has not been explored further (Schachter et al, 1998). These results are consistent with adjuvant clinical trial studies, where IL2, IFN g, and GM-CSF did not result in an improved clinical outcome (McClay, 2002).

B. DC vaccines

Several clinical trials of DC-based anti-melanoma vaccines have been performed (Table 3). In a study by Triozzi and colleagues the biologic activity of dendritic cells injected directly into tumors was examined. This pilot study demonstrated that DC derived in vitro were viable after intratumoral injection and could mediate biologic activity in situ. (Triozzi et al, 2000) Whether applied intravenously or intradermally, DC can easily migrate to lymphoid organs and tumor sites (Mackensen et al, 1999; Thomas et al, 1999). Many T cell anti-tumor responses were measured, and in 7 out of 9 trials at least transient tumor-specific PBL activity was observed. (Table 3). When keyhole limpet hemocyanin (KLH) was administered, activation of helper T lymphocytes was reported; with DTH directed both against KHL and tumor cells (Nestle et al, 1998; Toungouz et al, 2001; Chang et al, 2002).

Unfortunately, despite the high in vitro anti-tumor activity of patients' PBL, the clinical outcome was not very successful. A maximum overall response rate of 25.6% has been reported with a12% complete response rate (Table 3). Interestingly, the most potent immune response was induced when autologous tumor material was used (Andersen et al, 2001; Thomas et al, 2001; Krause et al, 2002; O'Rourke et al, 2003; Smithers et al, 2003)

VI. Reasons for the failure of DC vaccination

Given that all of the strategies tested are equally successful in vitro, that their application routes in general do not differ, and that they are all based on autologous dendritic cells obtained either from patients blood, generated from CD34+ precursors ex vivo, or monocytes, the reason for the failure to eradicate the tumor is probably independent of the methods of vaccination. Since anti-tumor PBL activity has been shown, this suggests patients' immune systems are capable of producing a wide range of cytotoxic cells, potentially able to recognise tumor antigens. It appears, that although the immune response against melanoma tumors has been induced, in patients its effector phase is not carried through to completion.

There are several possible explanations for the failure of DC vaccinations to eliminate tumor. The simplest explanation would be that the modified cells or pre-sensitised CTL might have been unable to penetrate the tumor or that the antigen specificity of the CTL may have been too narrow. Studies have however confirmed the generation of potent anti-tumor CTL and their successful migration into the tumor site (Table 3). Alternatively, the CTL may be suppressed or killed at the site of the tumor and therefore unable to perform any anti-tumor activity. It is well known that tumors are immune privileged sites and that they create an immunosuppressive environment around themselves, preventing inflammatory responses. This is thought to be achieved by the secretion of a range of immunosuppressive cytokines, such as IL-10, IL-19, IL-6, TGF b1 and 2, macrophage migration-inhibitory factor, gangliosides, heavy chain ferritin, ICAM-1 and prostaglanoids. In addition tumors are not only resistant to TNF receptor pathway mediated apoptosis, but can also express and secrete FasL, which causes apoptosis of activated lymphocytes (Ekmekcioglu et al, 1999; Gray et al, 2002; Redondo et al, 2002; Sombroek et al, 2002; Peguet-Navarro et al, 2003; Redondo et al, 2003; Wolfl et al, 2002).

VII. Modulation of immune responses by dendritic cells

Since inappropriate immune responses can be dangerous (if e.g. induced against healthy tissue), they must be carefully regulated. DC subsets play crucial roles in the selection process in the thymus as well as regulatory roles in lymph nodes and the periphery.

One of the most characteristic features of dendritic cells is the transformation of their phenotype during maturation. DC function is highly dependent on their level of maturation, and cells in various stages of development differ not only in their morphology but also completely alter their surface antigen expression. In humans, the presence of immature DC has been reported in most organs, including liver, kidney and heart, where they tend to be associated with vascular structures (Hart, 1997; Banchereau and Steinman, 1998). An interdigitating sentinel epithelial network of DC has been described in the mucosa of the oral cavity, intestinal tract and the respiratory tract (Hart, 1997). It is increasingly believed that tissue-residing immature dendritic cells constantly incorporate and process various proteins from their environment. Under physiological conditions, they express few self-antigens on their surface for presentation to T lymphocytes. However, since the dendritic cells are immature, they do not express co-stimulatory molecules, and what results is impaired lymphocyte activation, and anergy. This simple mechanism eliminates self-reactive lymphocytes, and prevents autoimmunity (Hart, 1997; Banchereau and Steinman, 1998; Lutz and Schuler, 2002). Tissue resident immature dendritic cells can also phagocytose apoptotic bodies formed when neighbouring cells die by apoptosis. Normally this will not result in an immune response, however, if apoptosis was the result of a viral infection then additional signals at the site of infection (e.g. IFNa, HSP) induce dual activation and maturation of dendritic cells, and launch an immune reaction (Hart 1997; Banchereau and Steinman, 1998; Lutz and Schuler, 2002).

It is not only immature tissue-resident dendritic cells that anergise T lymphocytes. The presence of "semi-matured" dendritic cells circulating in the blood of healthy donors was described by Lutz and Schuler (2002). These cells are loaded with self-antigen, and express antigens associated with a mature phenotype, but do not release cytokines, and therefore do not provide sufficient activation signals for lymphocytes. They react with CD4+ lymphocytes, inducing a subset of regulatory helper lymphocytes, which remain in the organism as memory cells, providing a mechanism that supports peripheral tolerance.

VIII. Melanoma derived, DC mediated immune control

A. Altered phenotype of lymph nodes invaded by melanoma

The induction of T lymphocyte anergy in tissues and the creation of a population of regulatory cells are two distinct pathways leading to tolerance to self-antigens. Thanks to these control mechanisms, severe autoimmune reactions can be avoided. If however dendritic cells are kept artificially immature, it creates a potential hazard for the function of immune system.

Many groups have reported alterations in cell ratio and function in lymph nodes invaded by melanoma. Several authors observed the recruitment of immature plasmacytoid dendritic cells, and T lymphocytes with a suppressive phenotype. (Fernandez-Bussy et al, 1983; Lana et al, 2001; Salio et al, 2003; Vermi et al, 2003). A comparison between sentinel and non-sentinel lymph nodes showed a quantitative reduction in dendritic cell markers, in the sentinel lymph node. This suggests a loss of mature DC and a concomitant decrease in total DC content (Essner and Kojima, 2002)

Histological studies show a profound decrease in the number of antigen-presenting cells expressing HLA class II in the epidermis above the melanoma, with zonal immune suppression in involved lymph nodes. There are decreased numbers of DC in the paracortex of the lymph node, and the majority of remaining LC and DC are phenotypically immature (Fernandez-Bussy et al, 1983; Cochran et al, 1987; Toriyama et al, 1993; Garcia-Plata et al, 1995; Barbour and Coventry, 2003) (Figure 2). In vitro assays confirmed the suppressed functional characteristics of cells derived from melanoma-invaded sentinel lymph nodes or exposed to conditioned supernatants from melanoma cell cultures (Hoon et al, 1987a, 1987b; Farzad et al, 1997; Chang et al, 1999).

Several studies have examined the tolerizing influence of melanoma cells on the maturation and function of DC and show that under the influence of melanoma, DC acquire an immunosuppressive phenotype and cause the generation of anergic T lymphocytes. The immunostimulatory function of DC obtained from progressing and regressing melanoma metastases show a significant difference in the abilities of each population. In addition, monocyte-derived DC exposed to tumor supernatant failed to acquire full allo-stimulatory activity and rapidly underwent apoptosis (Enk et al, 1997; Steinbrink et al, 1999; Kiertscher et al, 2000; Steinbrink et al, 2002).

In the presence of melanoma cells or tumor conditioned media, CD 80, 86 and HLA class I and II are up-regulated on the DC surface, even though expression of immature DC-related antigens like E-cadherin is retained, and the DC maturity factor, CD 83, is not expressed (Remmel et al, 2001; Padovan et al, 2002).

These cells therefore have a phenotype allowing antigen presentation by immature cells, leading to the suppression of antigen-specific immune responses.

It may be worth considering whether the characteristic dissemination of melanoma via the lymphatics and primary metastatic spread into the lymph nodes is coincidental.

B Dendritic cells as a key to immune escape

Melanoma is a tumor recognisable to the immune system and cannot grow and develop in the presence of a competent immune system. In the early stages of tumor development melanoma acquire an "invisible" phenotype following the selection pressure of the immune system. This however might be not enough to ensure further tumor cell survival. The tumor needs a more secure and permanent strategy. Lymph nodes are the nerve centres of the immune response, places where antigens are presented to lymphocytes and where decisions about immune responses are made. By invading these, melanoma creates an immune-privileged site in the centre of immune reaction. What can be harmful to infiltrating cytotoxic cells must also influence to at least the same degree any regulatory cells residing in vicinity. Dendritic cells act as the key component in immune reaction regulation. Under normal circumstances they are able to stimulate populations of lymphocytes against danger (e.g. tumor cells), however if their maturation is halted and their phenotype switched into modulatory mode, instead of immune stimulation, they will induce immune tolerance. Melanoma cells have the potential to keep dendritic cells in an immature state (Figure 2), impaired and suppressed, yet able to control and suppress other components of the immune response. By invading lymph nodes, melanoma acquire a potent strategy of immune evasion. The hunted transforms into the hunter — instead of escaping the immune system, in effect the melanoma takes control and deletes the tumor sensitive lymphocytes at the command centre of immune reactivity (Figure 3).

Functional alterations in lymph nodes invaded by melanoma should be considered when attempting immune therapy. If our hypothesis is correct, any external immune intervention is unlikely to result in tumor destruction, despite the induction of immunocompetent cells. Tumor specific cytotoxic cells will migrate into the lymph nodes and instead of being activated they will be anergized and may undergo apoptosis due to the interaction with dendritic cells modulated by the melanoma. To obtain a successful anti-melanoma vaccination, the immune suppression in draining lymph nodes must be overcome.

Figure 2. Presence of immature DC within the lymphoid tissue (Immunohistochemistry): FXIIIa staining of cells without protrusions.

Figure 3. Suppression of immune system managed by melanoma-derived altered maturation of dendritic cells. Antigen presentation by immature dendritic cells is one of the immune control mechanisms. Dendritic cells kept in the immature state by cytokines released by melanoma are capable to modulate immune response and anergise antigen specific T lymphocytes. This mechanism can be potentially used by melanoma to avoid immune recognition and to suppress the immune reaction.

Acknowledgements

We are grateful to Ms Penny Johnson for expert technical assistance. The study was funded by Wessex Cancer Trust UK.

References

Abdel-Wahab Z, DeMatos P, Hester D, Dong XD, Seigler HF (1998) Human dendritic cells, pulsed with either melanoma tumor cell lysates or the gp100 peptide (280-288, induce pairs of T-cell cultures with similar phenotype and lytic activity. Cell Immunol 186, 63-74.

Adrian Cabestre F, Moreau P, Riteau B, Ibrahim EC, Le Danff C, Dausset, J, Rouas-Freiss N, Carosella ED, Paul P (1999) HLA-G expression in human melanoma cells, protection from NK cytolysis. J Reprod Immunol 43, 183-193.

Albert ML, Pearce SF, Francisco LM, Sauter B, Roy P, Silverstein RL, Bhardwaj N (1998a) Immature dendritic cells phagocytose apoptotic cells via alphavbeta5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J Exp Med 188, 1359-1368.

Albert ML, Sauter B, Bhardwaj N (1998b) Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature, 392, 86-89.

Alijagic S, Moller P, Artuc M, Jurgovsky K, Czarnetzki BM, Schadendorf D (1995) Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation. Eur J Immunol 25, 3100-3107.

Andersen MH, Keikavoussi P, Brocker EB, Schuler-Thurner B, Jonassen M, Sondergaard, I, Straten, PT, Becker, JC, Kampgen E (2001) Induction of systemic CTL responses in melanoma patients by dendritic cell vaccination, cessation of CTL responses is associated with disease progression. Int J Cancer 94, 820-824.

Andre F, Schartz NE, Movassagh M, Flament C, Pautier P, Morice P, Pomel C, Lhomme C, Escudier B, Le Chevalier T, Tursz T, Amigorena S, Raposo G, Angevin E, Zitvogel L (2002) Malignant effusions and immunogenic tumour-derived exosomes. Lancet 360, 295-305.

Bakker AB, Marland G, de Boer AJ, Huijbens RJ, Danen EH, Adema GJ, Figdor CG (1995) Generation of antimelanoma cytotoxic T lymphocytes from healthy donors after presentation of melanoma-associated antigen-derived epitopes by dendritic cells in vitro. Cancer Res 55, 5330-5334.

Banchereau J, Palucka AK, Dhodapkar M, Burkeholder S, Taque N, Rolland A, Taquet S, Coquery S, Wittkowski KM, Bhardwaj N, Pineiro L, Steinman R, Fay J (2001) Immune and clinical responses in patients with metastatic melanoma to CD34 (+) progenitor-derived dendritic cell vaccine. Cancer Res 61, 6451-6458.

Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392, 245-252.

Barbour AH, Coventry, BJ (2003) Dendritic cell density and activation status of tumour-infiltrating lymphocytes in metastatic human melanoma, possible implications for sentinel node metastases. Melanoma Res 13, 263-269.

Bateman AR, Harrington KJ, Kottke T, AhMed A, Melcher AA, Gough MJ, Linardakis E, Riddle D, Dietz A, Lohse CM, Strome S, Peterson T, Simari R, Vile RG (2002) Viral fusogenic membrane glycoproteins kill solid tumor cells by nonapoptotic mechanisms that promote cross presentation of tumor antigens by dendritic cells. Cancer Res 62, 6566-6578.

Becker JC, Brocker, EB (1995) Lymphocyte-melanoma interaction, role of surface molecules. Recent Results Cancer Res 139, 205-214.

Becker JC, Dummer R, Hartmann AA, Burg G, Schmidt RE (1991) Shedding of ICAM-1 from human melanoma cell lines induced by IFN-gamma and tumor necrosis factor-alpha. Functional consequences on cell-mediated cytotoxicity. J Immunol 147, 4398-4401.

Berard F, Blanco P, Davoust J, Neidhart-Berard EM, Nouri-Shirazi M, Taquet N, Rimoldi D, Cerottini JC, Banchereau J, Palucka AK (2000) Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells. J Exp Med 192, 1535-1544.

Bettinotti MP, Kim CJ, Lee KH, Roden M, Cormier JN, Panelli M, Parker KK, Marincola FM (1998) Stringent allele/epitope requirements for MART-1/Melan A immunodominance, implications for peptide-based immunotherapy. J Immunol 161, 877-889.

Burnet FM (1970) The concept of immunological surveillance. Prog Exp Tumor Res 13, 1-27.

Chang AE, Li Q, Bishop DK, Normolle DP, Redman BD, Nickoloff BJ (2000) Immunogenetic therapy of human melanoma utilizing autologous tumor cells transduced to secrete granulocyte-macrophage colony-stimulating factor. Hum Gene Ther 11, 839-850.

Chang AE, Redman BG, Whitfield JR, Nickoloff BJ, Braun TM, Lee PP, Geiger JD, Mule JJ (2002) A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced cancer. Clin Cancer Res 8, 1021-1032.

Chang JW, Vaquerano JE, Zhou YM, Peng M, Leong SP (1999) Characterization of dendritic cells generated from peripheral blood of patients with malignant melanoma. Anticancer Res 19, 1815-1820.

Chen B, Stiff P, Sloan G, Kash J, Manjunath R, Pathasarathy M, Oldenburg D, Foreman KE, Nickoloff BJ (2001) Replicative response, immunophenotype, and functional activity of monocyte-derived versus CD34 (+)-derived dendritic cells following exposure to various expansion and maturational stimuli. Clin Immunol 98, 280-292.

Chinnasamy N, Chinnasamy D, Toso JF, Lapointe R, Candotti F, Morgan RA, Hwu P (2000) Efficient gene transfer to human peripheral blood monocyte-derived dendritic cells using human immunodeficiency virus type 1-based lentiviral vectors. Hum Gene Ther 11, 1901-1909.

Cochlovius B, Linnebacher M, Zewe-Welschof M, Zoller M (1999) Recombinant gp100 protein presented by dendritic cells elicits a T-helper-cell response in vitro and in vivo. Int J Cancer 83, 547-554.

Cochlovius B, Stassar M, Christ O, Raddrizzani L, Hammer J, Mytilineos I, Zoller M (2000) In vitro and in vivo induction of a Th cell response toward peptides of the melanoma-associated glycoprotein 100 protein selected by the TEPITOPE program. J Immunol 165, 4731-4741.

Cochran AJ, Pihl E, Wen DR, Hoon DS, Korn EL (1987) Zoned immune suppression of lymph nodes draining malignant melanoma, histologic and immunohistologic studies. J Natl Cancer Inst 78, 399-405.

Dalerba P, Ricci A, Russo V, Rigatti D, Nicotra MR, Mottolese M, Bordignon C, Natali PG, Traversari C (1998) High homogeneity of MAGE, BAGE, GAGE, tyrosinase and Melan-A/MART-1 gene expression in clusters of multiple simultaneous metastases of human melanoma, implications for protocol design of therapeutic antigen-specific vaccination strategies. Int J Cancer 77, 200-204.

Denfeld RW, Dietrich A, Wuttig C, Tanczos E, Weiss JM, Vanscheidt W, Schopf E, Simon JC (1995) In situ expression of B7 and CD28 receptor families in human malignant melanoma, relevance for T-cell-mediated anti-tumor immunity. Int J Cancer, 62, 259-265.

Dhodapkar MV, Young JW, Chapman PB, Cox WI, Fonteneau JF, Amigorena S, Houghton AN, Steinman RM, Bhardwaj N (2000) Paucity of functional T-cell memory to melanoma antigens in healthy donors and melanoma patients. Clin Cancer Res 6, 4831-4838.

Drexler I, Antunes E, Schmitz M, Wolfel T, Huber C, Erfle V, Rieber P, Theobald M, Sutter G (1999) Modified vaccinia virus Ankara for delivery of human tyrosinase as melanoma-associated antigen, induction of tyrosinase- and melanoma-specific human leukocyte antigen A*0201-restricted cytotoxic T cells in vitro and in vivo. Cancer Res 59, 4955-4963.

Ekmekcioglu S, Okcu MF, Colome-Grimmer MI, Owen-Schaub L, Buzaid AC, Grimm EA (1999) Differential increase of Fas ligand expression on metastatic and thin or thick primary melanoma cells compared with interleukin-10. Melanoma Res 9, 261-272.

Enk AH, Jonuleit H, Saloga J, Knop J (1997) Dendritic cells as mediators of tumor-induced tolerance in metastatic melanoma. Int J Cancer 73, 309-316.

Essner R, Kojima M (2002) Dendritic cell function in sentinel nodes. Oncology (Huntingt, 16 (1 Suppl 1, 27-31.

Farzad Z, McBride WH, Ogbechi H, Asnong-Holthoff C, Morton DL, Cochran AJ (1997) Lymphocytes from lymph nodes at different distances from human melanoma vary in their capacity to inhibit/enhance tumor cell growth in vitro. Melanoma Res 7 Suppl 2, S59-65.

Fernandez-Bussy R, Cambazard F, Mauduit G, Schmitt D, Thivolet J (1983) T cell subsets and Langerhans cells in skin tumours. Eur J Cancer Clin Oncol, 19, 907-913.

Festenstein H, Garrido F (1986) MHC antigens and malignancy. Nature, 322, 502-503.

Firat H, Zennou V, Garcia-Pons F, Ginhoux F, Cochet M, Danos O, Lemonnier FA, Langlade-Demoyen P, Charneau P (2002) Use of a lentiviral flap vector for induction of CTL immunity against melanomaPerspectives for immunotherapy. J Gene Med 4, 38-45.

Garcia-Plata D, Lessana-Leibowitch M, Palangie A, Gulliemette J, Sedel D, Mendez L, Mozos E (1995) Immunophenotype analysis of dendritic cells and lymphocytes associated with cutaneous malignant melanomas. Invasion Metastasis, 15, 125-134.

Gray CP, Arosio P, Hersey P (2002) Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells. Blood, 99, 3326-3334.

Hart DN (1997) Dendritic cells, unique leukocyte populations which control the primary immune response. Blood, 90, 3245-3287.

Hersey P, Si Z, Smith MJ, ThomasWD (1994) Expression of the co-stimulatory molecule B7 on melanoma cells. Int J Cancer, 58, 527-532.

Holmes LM, Li J, Sticca RP, Wagner TE, Wei Y (2001) A rapid, novel strategy to induce tumor cell-specific cytotoxic T lymphoctye responses using instant dentritomas. J Immunother, 24, 122-129.

Hoon DS, Bowker RJ, Cochran AJ (1987a) Suppressor cell activity in melanoma-draining lymph nodes. Cancer Res 47, 1529-1533.

Hoon DS, Korn EL, Cochran AJ (1987b) Variations in functional immunocompetence of individual tumor-draining lymph nodes in humans. Cancer Res 47, 1740-1744.

Housseau F, Lindsey KR, Oberholtzer SD, Gonzales MI, Boutin P, Moorthy AK, Shankara S, Roberts BL, Topalian SL (2002) Quantitative real-time RT-PCR as a method for monitoring T lymphocyte reactivity to full-length tyrosinase protein in vaccinated melanoma patients. J Immunol Methods 266, 87-103.

Imro MA, Manici S, Russo V, Consogno G, Bellone M, Rugarli C, Traversari C, Protti MP (1999) Major histocompatibility complex class I restricted cytotoxic T cells specific for natural melanoma peptides recognize unidentified shared melanoma antigen (s). Cancer Res 59, 2287-2291.

Inaba K, Turley S, Yamaide F, Iyoda T, Mahnke K, Inaba M, Pack M, Subklewe M, Sauter B, Sheff D, Albert M, Bhardwaj N, Mellman I, Steinman RM (1998) Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells. J Exp Med 188, 2163-2173.

Jager E, Jager D, Karbach J, Chen YT, Ritter G, Nagata Y, Gnjatic S, Stockert E, Arand M, Old LJ, Knuth A (2000) Identification of NY-ESO-1 epitopes presented by human histocompatibility antigen (HLA)-DRB4*0101-0103 and recognized by CD4 (+) T lymphocytes of patients with NY-ESO-1-expressing melanoma. J Exp Med 191, 625-630.

Janik JE, Miller LL, Kopp WC, Taub DD, Dawson H, Stevens D, Kostboth P, Curti BD, Conlon KC, Dunn BK, Donegan SE, Ullrich R, Alvord WG, Gause BL, Longo DL (1999) Treatment with tumornecrosis factor-alpha and granulocyte-macrophage colony-stimulating factor increases epidermal Langerhans' cell numbers in cancer patients. Clin Immunol 93, 209-221.

Jantscheff P, Spagnoli G, Zajac P, Rochlitz CF (2002) Cell fusion, an approach to generating constitutively proliferating human tumor antigen-presenting cells. Cancer Immunol Immunother 51, 367-375.

Jenne L, Arrighi JF, Jonuleit H, Saurat JH, Hauser C (2000) Dendritic cells containing apoptotic melanoma cells prime human CD8+ T cells for efficient tumor cell lysis. Cancer Res 60, 4446-4452.

Kawashima I, Tsai V, Southwood S, Takesako K, Celis E, Sette A (1998) Identification of gp100-derived, melanoma-specific cytotoxic T-lymphocyte epitopes restricted by HLA-A3 supertype molecules by primary in vitro immunization with peptide-pulsed dendritic cells. Int J Cancer, 78, 518-524.

Kierstead LS, Ranieri E, Olson W, Brusic V, Sidney J, Sette A, Kasamon YL, Slingluff CL, Jr, Kirkwood JM, Storkus WJ (2001) gp100/pmel17 and tyrosinase encode multiple epitopes recognized by Th1-type CD4+T cells. Br J Cancer 85, 1738-1745.

Kiertscher SM, Luo J, Dubinett SM, Roth MD (2000) Tumors promote altered maturation and early apoptosis of monocyte-derived dendritic cells. J Immunol 164, 1269-1276.

Kikuchi A, Nieda M, Schmidt C, Koezuka Y, Ishihara S, Ishikawa Y, Tadokoro K, Durrant S, Boyd A, Juji T, Nicol A (2001) In vitro anti-tumour activity of alpha-galactosylceramide-stimulated human invariant Valpha24+NKT cells against melanoma. Br J Cancer 85, 741-746.

Kim CJ, Cormier J, Roden M, Gritz L, Mazzara GP, Fetsch P, Hijazi Y, Lee KH, Rosenberg SA, Marincola FM (1998) Use of recombinant poxviruses to stimulate anti-melanoma T cell reactivity. Ann Surg Oncol 5, 64-76.

Kim CJ, Prevette T, Cormier J, Overwijk W, Roden M, Restifo NP, Rosenberg SA, Marincola FM (1997) Dendritic cells infected with poxviruses encoding MART-1/Melan A sensitize T lymphocytes in vitro. J Immunother 20, 276-286.

Krause SW, Neumann C, Soruri A, Mayer S, Peters JH, Andreesen R (2002) The treatment of patients with disseminated malignant melanoma by vaccination with autologous cell hybrids of tumor cells and dendritic cells. J Immunother 25, 421-428.

Kusumoto M, Umeda S, Ikubo A, Aoki Y, Tawfik O, Oben R, Williamson S, Jewell W, Suzuki T (2001) Phase 1 clinical trial of irradiated autologous melanoma cells adenovirally transduced with human GM-CSF gene. Cancer Immunol Immunother 50, 373-381.

Labarriere N, Bretaudeau L, Gervois N, Bodinier M, Bougras G, Diez E, Lang F, Gregoire M, Jotereau F (2002) Apoptotic body-loaded dendritic cells efficiently cross-prime cytotoxic T lymphocytes specific for NA17-A antigen but not for Melan-A/MART-1 antigen. Int J Cancer 101, 280-286.

Lana AM, Wen DR, Cochran AJ (2001) The morphology, immunophenotype and distribution of paracortical dendritic leucocytes in lymph nodes regional to cutaneous melanoma. Melanoma Res 11, 401-410.

Lapointe R, Royal RE, Reeves ME, Altomare I, Robbins PF, Hwu P (2001) Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100. J Immunol 167, 4758-4764.

Larregina AT, Watkins SC, Erdos G, Spencer LA, Storkus WJ, Beer Stolz D, Falo LD, Jr (2001) Direct transfection and activation of human cutaneous dendritic cells. Gene Ther 8, 608-617.

Lau R, Wang F, Jeffery G, Marty V, Kuniyoshi J, Bade E, Ryback ME, Weber J (2001) Phase I trial of intravenous peptide-pulsed dendritic cells in patients with metastatic melanoma. J Immunother 24 (, 66-78.

Linette GP, Shankara S, Longerich S, Yang S, Doll R, Nicolette C, Preffer FI, Roberts BL, Haluska FG (2000) In vitro priming with adenovirus/gp100 antigen-transduced dendritic cells reveals the epitope specificity of HLA-A*0201-restricted CD8+ T cells in patients with melanoma. J Immunol 164, 3402-3412.

Lotze MT, Hellerstedt B, Stolinski L, Tueting T, Wilson C, Kinzler D, Vu H, Rubin JT, Storkus W, Tahara H, Elder E, Whiteside T (1997) The role of interleukin-2, interleukin-12, and dendritic cells in cancer therapy. Cancer J Sci Am 3 Suppl 1, S109-114.

Lotze MT, Shurin M, Esche C, Tahara H, Storkus W, Kirkwood JM, Whiteside TL, Elder EM, Okada H, Robbins P (2000) Interleukin-2, developing additional cytokine gene therapies using fibroblasts or dendritic cells to enhance tumor immunity. Cancer J Sci Am 6 Suppl 1, S61-66.

Lutz MB, Schuler G (2002) Immature, semi-mature and fully mature dendritic cells, which signals induce tolerance or immunity? Trends Immunol 23, 445-449.

Mackensen A, Herbst B, Chen JL, Kohler G, Noppen C, Herr W, Spagnoli GC, Cerundolo V, Lindemann A (2000) Phase I study in melanoma patients of a vaccine with peptide-pulsed dendritic cells generated in vitro from CD34 (+) hematopoietic progenitor cells. Int J Cancer 86, 385-392.

Mackensen A, Krause T, Blum U, Uhrmeister P, Mertelsmann R, Lindemann A (1999) Homing of intravenously and intralymphatically injected human dendritic cells generated in vitro from CD34+ hematopoietic progenitor cells. Cancer Immunol Immunother 48 (2-3, 118-122.

McClay EF (2002) Adjuvant therapy for patients with high-risk malignant melanoma. Semin Oncol 29, 389-399.

Minev BR, Chavez FL, Dudouet BM, Mitchell MS (2000) Synthetic insertion signal sequences enhance MHC class I presentation of a peptide from the melanoma antigen MART-1. Eur J Immunol 30, 2115-2124.

Mortarini R, Anichini A, Di Nicola M, Siena S, Bregni M, Belli F, Molla A, Gianni AM, Parmiani G (1997) Autologous dendritic cells derived from CD34+ progenitors and from monocytes are not functionally equivalent antigen-presenting cells in the induction of melan-A/Mart-1 (27-35)-specific CTLs from peripheral blood lymphocytes of melanoma patients with low frequency of CTL precursors. Cancer Res 57, 5534-5541.

Motta I, Andre F, Lim A, Tartaglia J, Cox WI, Zitvogel L, Angevin E, Kourilsky P (2001) Cross-presentation by dendritic cells of tumor antigen expressed in apoptotic recombinant canarypox virus-infected dendritic cells. J Immunol 167, 1795-1802.

Murphy GF, Radu A, Kaminer M, Berd D (1993) Autologous melanoma vaccine induces inflammatory responses in melanoma metastases, relevance to immunologic regression and immunotherapy. J Invest Dermatol 100, 335S-341S.

Nair SK, Morse M, Boczkowski D, Cumming RI, Vasovic L, Gilboa E, Lyerly HK (2002) Induction of tumor-specific cytotoxic T lymphocytes in cancer patients by autologous tumor RNA-transfected dendritic cells. Ann Surg 235, 540-549.

Nasi ML, Lieberman P, Busam KJ, Prieto V, Panageas KS, Lewis JJ, Houghton AN, Chapman PB (1999) Intradermal injection of granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with metastatic melanoma recruits dendritic cells. Cytokines Cell Mol Ther 5, 139-144.

Nestle FO (2000) Dendritic cell vaccination for cancer therapy. Oncogene 19, 6673-6679.

Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadendorf D (1998) Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 4, 328-332.

Noessner E, Gastpar R, Milani V, Brandl A, Hutzler PJ, Kuppner MC, Roos M, Kremmer E, Asea A, Calderwood SK, Issels RD (2002) Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells. J Immunol 169, 5424-5432.

Nouri-Shirazi M, Banchereau J, Bell D, Burkeholder S, Kraus ET, Davoust J, Palucka KA (2000) Dendritic cells capture killed tumor cells and present their antigens to elicit tumor-specific immune responses. J Immunol 165, 3797-3803.

O'Rourke MG, Johnson M, Lanagan C, See J, Yang J, Bell JR, Slater GJ, Kerr BM, Crowe B, Purdie DM, Elliott SL, Ellem KA, Schmidt CW (2003) Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine. Cancer Immunol Immunother 52, 387-395.

Padovan E, Terracciano L, Certa U, Jacobs B, Reschner A, Bolli M, Spagnoli GC, Borden EC, Heberer M (2002) Interferon stimulated gene 15 constitutively produced by melanoma cells induces e-cadherin expression on human dendritic cells. Cancer Res 62, 3453-3458.

Panelli MC, Wunderlich J, Jeffries J, Wang E, Mixon A, Rosenberg SA, Marincola FM (2000) Phase 1 study in patients with metastatic melanoma of immunization with dendritic cells presenting epitopes derived from the melanoma-associated antigens MART-1 and gp100. J Immunother 23, 487-498.

Peguet-Navarro J, Sportouch M, Popa I, Berthier O, Schmitt D, Portoukalian J (2003) Gangliosides from human melanoma tumors impair dendritic cell differentiation from monocytes and induce their apoptosis. J Immunol 170, 3488-3494.

Philip R, Alters SE, Brunette E, Ashton J, Gadea J, Yau J, Lebkowski J, Philip M (2000) Dendritic cells loaded with MART-1 peptide or infected with adenoviral construct are functionally equivalent in the induction of tumor-specific cytotoxic T lymphocyte responses in patients with melanoma. J Immunother 23, 168-176.

Prabakaran I, Menon C, Xu S, Gomez-Yafal A, Czerniecki BJ, Fraker DL (2002) Mature CD83 (+) dendritic cells infected with recombinant gp100 vaccinia virus stimulate potent antimelanoma T cells. Ann Surg Oncol 9, 411-418.

Ralfkiaer E, Hou-Jensen K, Gatter KC, Drzewiecki KT, Mason DY (1987) Immunohistological analysis of the lymphoid infiltrate in cutaneous malignant melanomas. Virchows Arch A Pathol Anat Histopathol, 410, 355-361.

Rea D, Havenga MJ, van Den Assem M, Sutmuller RP, Lemckert A, Hoeben RC, BoutA, Melief CJ, Offringa R (2001) Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgene-encoded antigen presentation to cytotoxic T cells. J Immunol 166, 5236-5244.

Redondo P, Sanchez-Carpintero I, Bauza A, Idoate M, Solano T, Mihm MC, Jr (2003) Immunologic escape and angiogenesis in human malignant melanoma. J Am Acad Dermatol 49, 255-263.

Redondo P, Solano T, B VA, Bauza A, Idoate M (2002) Fas and Fas ligand, expression and soluble circulating levels in cutaneous malignant melanoma. Br J Dermatol 147, 80-86.

Reeves ME, Royal RE, Lam JS, Rosenberg SA, Hwu P (1996) Retroviral transduction of human dendritic cells with a tumor-associated antigen gene. Cancer Res 56, 5672-5677.

Remmel E, Terracciano L, Noppen C, Zajac P, Heberer M, Spagnoli GC, Padovan E (2001) Modulation of dendritic cell phenotype and mobility by tumor cells in vitro. Hum Immunol 62, 39-49.

Russo V, Tanzarella S, Dalerba P, Rigatti D, Rovere P, Villa A, Bordignon C, Traversari C (2000) Dendritic cells acquire the MAGE-3 human tumor antigen from apoptotic cells and induce a class I-restricted T cell response. Proc Natl Acad Sci U S A, 97, 2185-2190.

Saeterdal I, thor Straten P, Myklebust JH, Kirkin AF, Gjertsen MK, Gaudernack G (1998) Generation and characterization of gp100 peptide-specific NK-T cell clones. Int J Cancer 75, 794-803.

Salio M, Cella M, Vermi W, Facchetti F, Palmowski MJ, Smith CL, Shepherd D, Colonna M, Cerundolo V (2003) Plasmacytoid dendritic cells prime IFN-gamma-secreting melanoma-specific CD8 lymphocytes and are found in primary melanoma lesions. Eur J Immunol 33, 1052-1062.

Schachter J, Rakowsky E, Sulkes A, Adler A (1998) A sequential four-drug chemotherapy and biotherapy with interferon alpha and GM-CSF--an innovative protocol for the treatment of metastatic melanoma. Cancer Biother Radiopharm 13, 155-164.

Schuler-Thurner B, Dieckmann D, Keikavoussi P, Bender A, Maczek C, Jonuleit H, Roder C, Haendle I, Leisgang W, Dunbar R, Cerundolo V, von Den Driesch P, Knop J, Brocker EB, Enk A, Kampgen E, Schuler G (2000) Mage-3 and influenza-matrix peptide-specific cytotoxic T cells are inducible in terminal stage HLA-A2.1+ melanoma patients by mature monocyte-derived dendritic cells. J Immunol 165, 3492-3496.

Schuler-Thurner B, Schultz ES, Berger TG, Weinlich G, Ebner S, Woerl P, Bender A, Feuerstein B, Fritsch PO, Romani N, Schuler G (2002) Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells. J Exp Med 195, 1279-1288.

Schultz ES, Lethe B, Cambiaso CL, Van Snick J, Chaux P, Corthals J, Heirman C, Thielemans K, Boon T, van der Bruggen P (2000) A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes. Cancer Res 60, 6272-6275.

Schultz ES, Zhang Y, Knowles R, Tine J, Traversari C, Boon T, van der Bruggen P (2001) A MAGE-3 peptide recognized on HLA-B35 and HLA-A1 by cytolytic T lymphocytes. Tissue Antigens 57, 103-109.

Seliger B, Ritz U, Abele R, Bock M, Tampe R, Sutter G, Drexler I, Huber C, Ferrone S (2001) Immune escape of melanoma, first evidence of structural alterations in two distinct components of the MHC class I antigen processing pathway. Cancer Res 61, 8647-8650.

Smith SG, Patel PM, Porte J, Selby PJ, Jackson AM (2001) Human dendritic cells genetically engineered to express a melanoma polyepitope DNA vaccine induce multiple cytotoxic T-cell responses. Clin Cancer Res 7, 4253-4261.

Smithers M, O'Connell K, MacFadyen S, Chambers M, Greenwood K, Boyce A, Abdul-Jabbar I, Barker K, Grimmett K, Walpole E, Thomas R (2003) Clinical response after intradermal immature dendritic cell vaccination in metastatic melanoma is associated with immune response to particulate antigen. Cancer Immunol Immunother 52, 41-52.

Soiffer R, Lynch T, Mihm M, Jung K, Rhuda C, Schmollinger JC, Hodi FS, Liebster L, Lam, P, Mentzer S, Singer S, Tanabe KK, Cosimi AB, Duda R, Sober A, Bhan A, Daley J, Neuberg D, Parry G, Rokovich J, Richards L, Drayer J, Berns A, Clift S, Dranoff G, et al (1998) Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte-macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma. Proc Natl Acad Sci U S A, 95, 13141-13146.

Sombroek, CC, Stam AG, Masterson AJ, Lougheed SM, Schakel MJ, Meijer CJ, Pinedo HM, van den Eertwegh AJ, Scheper RJ, de Gruijl TD (2002) Prostanoids play a major role in the primary tumor-induced inhibition of dendritic cell differentiation. J Immunol 168 4333-4343.

Soruri A, Fayyazi A, Gieseler R, Schlott T, Runger TM, Neumann C, Peters JH (1998) Specific autologous anti-melanoma T cell response in vitro using monocyte-derived dendritic cells. Immunobiology 198, 527-538.

Soruri A, Fayyazi A, Neumann C, Schlott T, Jung T, Matthes C, Zwirner J, Riggert J, Peters JH (2001) Ex vivo generation of human anti-melanoma autologous cytolytic T cells by dentritic cell/melanoma cell hybridomas. Cancer Immunol Immunother 50, 307-314.

Sotiriadou R, Perez SA, Gritzapis AD, Sotiropoulou PA, Echner H, Heinzel S, Mamalaki, A, Pawelec G, Voelter W, Baxevanis CN, Papamichail M (2001) Peptide HER2 (776-788) represents a naturally processed broad MHC class II-restricted T cell epitope. Br J Cancer 85, 1527-1534.

Steinbrink K, Graulich E, Kubsch S, Knop J, Enk AH (2002) CD4 (+) and CD8 (+) anergic T cells induced by interleukin-10-treated human dendritic cells display antigen-specific suppressor activity. Blood 99, 2468-2476.

Steinbrink K, Jonuleit,H, Muller G, Schuler G, Knop J, Enk AH (1999) Interleukin-10-treated human dendritic cells induce a melanoma-antigen-specific anergy in CD8 (+) T cells resulting in a failure to lyse tumor cells. Blood 93, 1634-1642.

Sumimoto H, Tsuji T, Miyoshi H, Hagihara M, Takada-Yamazaki R, Okamoto S, Ikeda Y, Takahashi T, Kawakami Y (2002) Rapid and efficient generation of lentivirally gene-modified dendritic cells from DC progenitors with bone marrow stromal cells. J Immunol Methods 271, 153-165.

SzekeRes L, Daroczy J (1981) Electron microscopic investigation on the local cellular reaction to primary malignant melanoma. Dermatologica 163, 137-144.

Tanaka Y, Dowdy SF, Linehan DC, Eberlein TJ, Goedegebuure PS (2003) Induction of antigen-specific CTL by recombinant HIV trans-activating fusion protein-pulsed human monocyte-derived dendritic cells. J Immunol 170, 1291-1298.

Tefany FJ, Barnetson RS, Halliday GM, McCarthy SW, McCarthy WH (1991) Immunocytochemical analysis of the cellular infiltrate in primary regressing and non-regressing malignant melanoma. J Invest Dermatol 97, 197-202.

Temme A, Morgenroth A, Schmitz M, Weigle B, Rohayem J, Lindemann D, Fussel M, Ehninger G, Rieber EP (2002) Efficient transduction and long-term retroviral expression of the melanoma-associated tumor antigen tyrosinase in CD34 (+) cord blood-derived dendritic cells. Gene Ther 9, 1551-1560.

Thomas R, Chambers M, Boytar R, Barker K, Cavanagh LL, MacFadyen S, Smithers M, Jenkins M, Andersen J (1999) Immature human monocyte-derived dendritic cells migrate rapidly to draining lymph nodes after intradermal injection for melanoma immunotherapy. Melanoma Res 9 5, 474-481.

Thomas R, Padmanabha J, Chambers M, McFadyen S, Walpole E, Nielssen G, Smithers M (2001) Metastatic lesions in the joint associated with acute inflammatory arthritis after dendritic cell immunotherapy for metastatic melanoma. Melanoma Res 11, 167-173.

Thurner B, Haendle I, Roder C, Dieckmann D, Keikavoussi P, Jonuleit H, Bender A, Maczek C, Schreiner D, von den Driesch P, Brocker EB, Steinman RM, Enk A, Kampgen E, Schuler G (1999) Vaccination with mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med 190, 1669-1678.

Tjandrawan T, Martin DM, Maeurer MJ, Castelli C, Lotze MT, Storkus, WJ (1998) Autologous human dendriphages pulsed with synthetic or natural tumor peptides elicit tumor-specific CTLs in vitro. J Immunother 21, 149-157.

Toriyama K, Wen DR, Paul E, Cochran AJ (1993) Variations in the distribution, frequency, and phenotype of Langerhans cells during the evolution of malignant melanoma of the skin. J Invest Dermatol 100, 269S-273S.

Toungouz M, Libin M, Bulte F, Faid, L, Lehmann, F, Duriau, D, Laporte, M, Gangji, D, Bruyns C, Lambermont M, Goldman M, Velu T (2001) Transient expansion of peptide-specific lymphocytes producing IFN-gamma after vaccination with dendritic cells pulsed with MAGE peptides in patients with mage-A1/A3-positive tumors. J Leukoc Biol 69, 937-943.

Triozzi PL, Khurram R, Aldrich WA, Walker MJ, Kim JA, Jaynes S (2000) Intratumoral injection of dendritic cells derived in vitro in patients with metastatic cancer. Cancer 89, 2646-2654.

Tsao H, Millman P, Linette GP, Hodi FS, Sober AJ, Goldberg MA, Haluska FG (2002) Hypopigmentation associated with an adenovirus-mediated gp100/MART-1-transduced dendritic cell vaccine for metastatic melanoma. Arch Dermatol 138, 799-802.

Tuting T, Wilson CC, Martin DM, Kasamon YL, Rowles J, Ma DI, Slingluff CL, Jr, Wagner SN, van der Bruggen P, Baar J, Lotze MT, Storkus WJ (1998) Autologous human monocyte-derived dendritic cells genetically modified to express melanoma antigens elicit primary cytotoxic T cell responses in vitro, enhancement by cotransfection of genes encoding the Th1-biasing cytokines IL-12 and IFN-alpha. J Immunol 160, 1139-1147.

van Elsas A, van der Burg SH, van der Minne CE, Borghi M, Mourer JS, Melief CJ, Schrier PI (1996) Peptide-pulsed dendritic cells induce tumoricidal cytotoxic T lymphocytes from healthy donors against stably HLA-A*0201-binding peptides from the Melan-A/MART-1 self antigen. Eur J Immunol 26, 1683-1689.

Vermi W, Bonecchi R, Facchetti F, Bianchi D, Sozzani S, Festa S, Berenzi A, Cella M, Colonna, M (2003) Recruitment of immature plasmacytoid dendritic cells (plasmacytoid monocytes) and myeloid dendritic cells in primary cutaneous melanomas. J Pathol, 200, 255-268.

Whiteside TL, Gambotto A, Albers A, Stanson J, Cohen EP (2002) Human tumor-derived genomic DNA transduced into a recipient cell induces tumor-specific immune responses ex vivo. Proc Natl Acad Sci U S A 99, 9415-9420.

Wolfl M, Batten WY, Posovszky C, Bernhard H, Berthold F (2002) Gangliosides inhibit the development from monocytes to dendritic cells. Clin Exp Immunol 130, 441-448.

Yang S, Kittlesen D, Slingluff CL, Jr, Vervaert CE, Seigler HF, Darrow TL (2000) Dendritic cells infected with a vaccinia vector carrying the human gp100 gene simultaneously present multiple specificities and elicit high-affinity T cells reactive to multiple epitopes and restricted by HLA-A2 and -A3. J Immunol 164, 4204-4211.

Yang S, Linette GP, Longerich S, Haluska FG (2002) Antimelanoma activity of CTL generated from peripheral blood mononuclear cells after stimulation with autologous dendritic cells pulsed with melanoma gp100 peptide G209-2M is correlated to TCR avidity. J Immunol 169, 531-539.

Zarour HM, Kirkwood JM, Kierstead LS, Herr W, Brusic V, Slingluff CL, Jr, Sidney J, Sette A, Storkus WJ (2000a) Melan-A/MART-1 (51-73) represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4 (+) T cells. Proc Natl Acad Sci U S A 97, 400-405.

Zarour HM, Maillere B, Brusic V, Coval K, Williams E, Pouvelle-Moratille S, Castelli F, Land S, Bennouna J, Logan T, Kirkwood JM (2002) NY-ESO-1 119-143 is a promiscuous major histocompatibility complex class II T-helper epitope recognized by Th1- and Th2-type tumor-reactive CD4+ T cells. Cancer Res 62, 213-218.

Zarour HM, Storkus WJ, Brusic V, Williams E, Kirkwood JM (2000b) NY-ESO-1 encodes DRB1*0401-restricted epitopes recognized by melanoma-reactive CD4+ T cells. Cancer Res 60, 4946-4952.

Zehntner S, Townsend W, Parkes J, Schmidt C, Down M, Bell J, Mulligan R, O'Rourke M, Ellem K, Thomas R (1999) Tumor metastasis biopsy as a surrogate marker of response to melanoma immunotherapy. Pathology 31, 116-122.

Dr. Marta E Polak