SCIENTIFIC REVIEW

Dendritic cells (II): role and therapeutic implications in cancer

S. SATTHAPORN* and O. EREMIN*#
*Section of Surgery, E Floor, West Block, Queen’s Medical Centre, University of Nottingham and #Department of Surgery, Lincoln County Hospital, Lincoln, UK

Introduction Immune surveillance Dendritic cells and cancer

Dendritic cells and anti-cancer therapy Conclusion References

The potential to harness the effectiveness and specificity of the immune system underlies the growing interest in cancer immunotherapy. One such approach uses bone marrow-derived dendritic cells (DCs), phenotypically distinct and very potent antigen-presenting cells, to present tumour-associated antigens (TAAgs) and, thereby, generate tumour-specific immunity. Support for this strategy comes from animal studies that have demonstrated that DCs, when loaded ex vivo with tumour Ags or pulsed with peptides and administered to cancer-bearing hosts, can elicit T cell-mediated cancer destruction. These observations have led to clinical trials designed to investigate the immunological and clinical effects of Ag-pulsed DCs administered as a therapeutic vaccine to patients with cancer. In the design and conduct of such trials, important considerations include Ag selection, methods for introducing TAAgs into MHC class I and II processing pathways, methods for isolating and activating DCs, and route of administration. Although current DC-based vaccination methods are cumbersome and complex, promising preliminary results from clinical trials in patients with malignant lymphoma, melanoma, and prostate cancer suggest that immuno-therapeutic strategies, that take advantage of the unique properties of DCs, may ultimately prove both efficacious and widely applicable treatment in patients with cancer.

Keywords: Cytotoxic cells, dendritic cells, tumour-associated antigens, vaccination immuno-therapy

J.R.Coll.Surg.Edinb., 46, June 2001, 159-167 

INTRODUCTION

The interaction between tumour cells and the host immune system are complex, involving a multitude of cell types and mediators.^1-3 Several lines of evidence suggest that the immune system has the potential to eliminate neoplastic cells, as evidenced by rare but well documented instances of spontaneous remissions (with no or inadequate treatment) in renal cell carcinoma and melanoma. Also, chronically and severely immunosuppressed individuals (transplantation recipients, congenital immune deficiency states and AIDS patients) exhibit an increased incidence of putative virallyinduced neoplasms; presence of AIDS-associated tumours correlate with the degree of immunosuppression.^4-8,9 An intriguing piece of evidence relates to in vivo tumour-related immune responses in patients with paraneoplastic neurological disorders that led to the discovery of onconeural Ags. Paraneoplastic neurological disorders are a rare group of neuronal degenerative diseases that develop as remote effects of malignancies.^10,11 The discovery of onconeural antibodies (Abs) led to the proposal that paraneoplastic cerebellar degeneration, associated with breast and ovarian cancer, is an autoimmune disorder mediated by the humoral arm of the immune system.

Induction of effective tumour immunity can be viewed as a three-step process that includes firstly, appropriate presentation of tumour-associated antigens (TAAgs), secondly, selection and activation of TAAg-specific T cells as well as non-Ag-specific effectors and, lastly, homing of TAAg-specific T cells to the tumour site and effective elimination of malignant cells expressing the TAAgs.^12-14 Cancers may escape immune surveillance due to changes in and modulation of these various processes. The establishment of an effective anti-tumour response is a complex process. Initially, peptides associated with malignant cells must be located and recognised by T cells circulating in the blood stream and permeating tissues. Most solid cancers express small amounts of TAAgs, which may also be cryptic and not readily available for recognition by rare T cell clones, through a low affinity T cell receptor (TCR) complex. Moreover, tumour cells tend to lack co-stimulatory molecules that drive clonal expansion of T cells, the production of key regulatory cytokines, and development into tumour cell specific cytotoxic T lymphocytes (CTLs).

Dendritic cells (DCs) are the crucial cells providing the necessary components for initiating and developing effective cell-mediated immune (CMI) responses (Satthaporn and Eremin, 2001).¹⁵ Dendritic cells, located in most tissues of the body, capture and process Ags, which are then displayed as MHC-peptide complexes on the DC surface.^16,17 Essential co-stimulatory molecules are upregulated on DCs as they migrate to secondary lymphoid organs (the spleen and lymph nodes) where they liaise with naïve T cells, inducing the activation and proliferation of Ag specific CTLs.^1-3 Thus, effective DC function in cancer involves several interlinked biological processes that occur in sequence: (a) TAAg presentation and recognition in tissues which involves proteolytic intracellular cleavage and peptide surface representation, (b) DC activation and trafficking to regional tumour-draining lymph nodes (LNs), and interaction with CD4+ T cells via the TCR and associated co-stimulatory molecules (CD40, CD80 and CD86), resulting in the generation of Ag-specific CTLs, and (c) migration of CTLs to the tumour site and induction of cancer cell death.

IMMUNE SURVEILLANCE

Eliciting an effective anti-cancer response and removal of malignant cells is a complex biological process. Failure of this process is poorly understood and is believed to be multifactorial, as shown in Table 1.

Table 1: Factors believed to be responsible for failure of immune surveillance

Specific cytokines may have important inhibitory effects on DC functions. In particular, IL-10, has been shown to inhibit the differentiation of DCs from circulating precursors.^18,19 Also, it has been shown to down regulate the expression of key co-stimulatory molecules and to block the production and secretion of the important DC and T cell regulatory molecules.^20,21 The consequences of these various effects are to convert DCs from an immunogenic to a tolerogenic role.^22-24 In addition, IL-10 may inhibit DC accumulation within tumours. Production of IL-10 by monocytes/macrophages and/or tumour cells, thus, may be an important mechanism by which malignant cells escape a protective host immune response and proliferate in an uncontrolled manner.^25,26

Tumour-associated Ag presentation to naïve T cells, without concomitant co-stimulatory signals, not only fails to active T cells but also may induce tolerance towards the Ags concerned. In the normal setting, this mechanism may protect the host from autoimmune-directed tissue damage; in malignancy, however, it affords a mechanism of evading the immune response despite appropriate presentation of tumour-specific Ag epitopes. In animal models, it has been demonstrated that the introduction of co-stimulatory molecules into tumour cells by gene transfection can restore immunogenicity and elicit specific and effective anti-tumour responses.^27,28

The influence of B-7 and CD28 co-stimulation in inducing tolerance or rejection of TAAgs was illustrated in a recent study using Ag presented by mouse EL4 lymphoma cells.²⁹ Because the CTL epitopes presented by EL4 cells had not previously been identified, it was necessary to isolate and purify MHC class I-associated peptides by a combination of affinity chromatography and reversed-phase high-pressure liquid chromatography. Cytotoxic T lymphocytes induced by B7^-1 transfected EL4 cells indicated that at least six different epitopes were presented by the parental EL4 cells, but the CTLs induced by the parental EL4 cells recognised a single dominant epitope. Thus, these results suggested that insufficient stimulation contribute to Ag ‘silencing’ and induction of tolerance. It is worth nothing that the induction of CTLs against the dominant epitopes of B7- tumour cells were also dependent on B7 co-stimulation provided by B7+ host cells. The administration of CTLA-4-Ig, a fusion protein that blocks B7-CD28 interaction, eliminated all CTL responses in mice challenged with cells from the B7-P815 tumour.²⁹

Several recent experiments have elegantly demonstrated that there is a critical threshold of TCR molecules to be engaged with peptide-MHC in order for the triggering of a detectable T cell specific response and that the requisite number of occupied TCRs can be significantly decreased if B7-CD28 co-stimulation is provided.^30-32 Thus, lack of sufficient co-stimulation through the B7 (on DCs) - CD28 (on T cells) interaction will induce ‘silencing’of the TAAgs. The consequences of this are T cell anergy and failure of immune surveillance and removal of proliferating cancer cells.^30-32

DENDRITIC CELLS AND CANCER

Escape from immune surveillance is believed to be a fundamental biological feature of malignant disease in man, which contributes to uncontrolled tumour growth, eventually leading to death of the host.³³ Defects in immune response in patients with a variety of tumours and in tumour-bearing animals have been well documented. These defects have been ascribed mostly to suppressor cell function.^34,35 However, a key element in the induction of the anti-cancer immune response, namely TAAg presentation to T cells in a tumour-bearing host (human and animal), has been poorly documented and inadequately studied. Some authors have shown defective function of macrophages in malignant disease (see review by Al-Sarireh and Eremin (2000)].^36-38 Zou et al (1992), however, reported normal and even increased function of Ag presenting cells (APCs), mostly macrophages in tumour-bearing mice.³⁹ In recent studies, it has been shown that a distinct subset of IA+ epidermal APCs appear capable of inducing tolerance to tumour Ags and that activated macrophages may induce structural abnormalities of the TCR-CD3 complex.^40,41 Data has also been presented suggesting that bone marrow-derived APCs play an important role in the presentation of TAAgs, a function previously assigned predominately to tumour cells.^42,43 Therefore, elucidation of the role of such APCs, in particular DCs (the key APCs), may help to better understand the mechanisms underlying anti-tumour immune responses and, perhaps, improve the effectiveness of anti-cancer immunity in tumour-bearing hosts.

Some studies in humans with solid cancers have investigated DC trafficking in peripheral blood. Radmyar et al (1995) demonstrated in their study that substantial numbers of DCs could be obtained from the peripheral blood of patients with renal cell carcinoma.⁴⁴ Phase contrast microscopy revealed the typical cytoplasmic processes, whilst phenotypic analysis confirmed expression of DC-associated molecules, including MHC class II, CD1a, CD80 and CD86 and absence of T cell, B cell and monocyte markers.¹⁵ Functionally, these DCs, when cultured in vitro, were found to be very potent costimulators of the phytohaemagglutinin-induced proliferation of autologous tumour infiltrating lymphocytes.⁴⁴ Almand et al (2000), however, showed that DCs in the peripheral blood of patients with head and neck cancer were significantly immunosuppressed.⁴⁵ There was also an increased intratumoural presence of the immunosuppressive CD34+ progenitor cells. Culturing CD34+ cells with stem cell factor (c-kit ligand) and granulocyte/monocyte-colony stimulating factor (GM-CSF) resulted in the appearance of a substantial number of cells expressing phenotypic markers characteristic of DCs.⁴⁶ Patients with head and neck squamous cell carcinoma also had increased levels of the immunosuppressive peripheral blood CD34+ cells. However, these latter cells were capable of differentiating into DCs in vitro.⁴⁷

Gabriolovich et al (1997) evaluated T cell responses to mitogens and to defined Ags in patients with breast cancer. Defects in response to tetanus toxoid and influenza virus were observed in patients with advanced breast cancer. Dendritic cells isolated from patients with breast cancer demonstrated a significantly decreased ability to stimulate control allogeneic T cells but stimulation of the patients T cells with control allogeneic DCs resulted in normal T cell responses. These data suggest that reduced DC function could be a major cause for the observed defect in cellular immunity documented in the patients with breast cancer in this study. In addition, stem cells from these patients could give rise to functional DCs after in vitro growth with GM-CSF and IL-4. Normal levels of control allogeneic and tetanus toxoid dependent T cell proliferation were observed when DCs obtained from progenitor cells were used as stimulators.⁴⁸

Bell et al (1999) have analysed the presence of immature and mature DCs within adenocarcinoma of the breast using immunohistochemistry.⁴⁹ Immature DCs were defined by expression of CD1a- and intracellular major histocompatibility complex class II-rich vesicles. Mature DCs were defined by the expression of CD83 and DC-Lamp. These workers demonstrated two levels of heterogeneity of DCs infiltrating breast carcinoma tissue: (a) immature DCs were localised to the tumour bed and (b) mature DCs were confined to peri-tumoural areas.⁴⁹ Lespagnard et al (1999) evaluated 142 primary breast carcinomas for the presence of DCs, using immunohistochemistry and anti-s100 protein antibody. They showed that 42% of breast carcinomas contained tumour infiltrating DCs expressing S100+ (S100+ TiDCs) and the number of S100+ TiDCs varied according to the grade of the tumours, being highest in grade III cancers. An association was also found between S100+TiDCs and tumour size, lymph node involvement, eostrogen/ progesterone receptor status and age.⁵⁰ Another study identified a population of CD1A+ cells within the lymphoid cell infiltrate in human breast cancer. In the majority of cases the infiltrate was low, compared with the number of macrophages and T cells. There was no correlation between the number of CD1A+ cells and tumour grade, with all tumour grades expressing similar numbers of infiltrating CD1A+ cells. However, the CD1A+ cells were closely associated with tumour cells. It is likely that CD1A+ cells have a role in Ag capture and presentation in human tumours.⁵¹

An investigation in patients with melanoma by Enk et al (1997) has shown a differential DC function.⁵² Patients whose melanoma were responding (rM) to chemotherapy had DCs which were five times more potent inducers of allogeneic T cell proliferation than those patients whose tumours were progressing (pM). Phenotypic analysis showed a marked depression of CD86 expression on DCs in the latter patients. Culture supernatants from pM showed production of a TH2-type cytokine profile (IL-10), whereas a TH1-type cytokine profile (IL-2, IL-12 and interferon-gamma (IFN-gamma) was found predominantly in patients whose melanomas had responded to treatment.52 A recent study by Cayeux et al (1999) have shown that the mixed lymphocyte response in vitro (measure of CMI) was inhibited by anti-CD80, anti-CD86, anti-HLADR, anti-HLA class I antibodies and supernatants of melanoma cultures. The characteristics of DCs generated from patients with melanoma were comparable with those obtained from healthy donors. Dendritic cell function was inhibited by soluble factors present in melanoma cell cultures.⁵³

Ninomiya et al (1999), showed that DCs from patients with hepatocellular carcinoma had significantly lower capacity to stimulate allogeneic T cell proliferation, compared with DCs isolated from patients with liver cirrhosis and normal controls.⁵⁴ In patients with hepatocellular carcinoma, DCs expressed significantly lower levels of HLA-DR and induction of IL-12 production. On the other hand, DCs from such donors produced significantly higher levels of nitric oxide and tumour necrosis factor-alpha (TNF-alpha) compared with DCs from donors with liver cirrhosis and normal controls. These results confirm a defect of DC maturation in patients with established hepatocellular carcinoma and probably during carcinogenesis and tumour induction.⁵⁴

Although a dysfunction of DCs is predictable in malignancies and, in fact, is being documented in a number of solid cancers in man, the biological mechanisms underlying these defects of DCs are poorly defined and appear to be heterogeneous in different cancers.

DENDRITIC CELLS AND ANTI-CANCER THERAPY

Dendritic cells are potentially good candidates for immune-based therapies for a variety of reasons. In particular, the following aspects are important, (a) their ability to migrate through tissues and infiltrate into tumours, where they encounter TAAgs which they capture, digest, and re-express for effective induction of a CMI response, (b) their capacity to activate naïve T cells in regional lymph nodes and their differentiation into CTLs, specifically able to interact with cancer cells and lead to tumour cell damage and death, and (c) their role as APCs and capacity to process and present a spectrum of different Ags simultaneously that allows for the induction of a broad repertoire of anti-tumour immune responses to occur.

The ability of DCs to generate anti-tumour immune responses in vivo has been documented in a number of animal tumour models.^55,56 Most of these experiments have involved in vitro isolation of DCs, followed by loading of the DCs with tumour Ags and injection of the Ag-bearing DCs into syngeneic animals as a cancer vaccine. Dendritic cells loaded with tumour lysates, tumour Ag-derived peptides, synthetic MHC class I-restricted peptides and whole proteins, have all been demonstrated to generate tumour-specific immune responses and anti-tumour activities.^57-60 Furthermore, Ag-loaded DCs can be used therapeutically to induce regression of preexisting tumours.⁶¹ Dendritic cells loaded with appropriate TAAgs can induce either protection or rejection of malignant cells in various animal models.^62-65

Promising preliminary data has been reported in patients with cancer.^66-69 Several systems have been used to deliver TAAgs to DCs, including (a) defined peptides of known sequences, (b) undefined acid-eluted peptides from autologous tumours, (c) whole tumour lysates, (d) retroviral and adenoviral vectors, (e) tumour cell-derived RNA, (f) fusion of DCs with tumour cells, and (g) exosomes derived from DCs pulsed with tumour peptides (subcellular structures containing high levels of MHC molecules and peptides).^1-3 These observations have established the rationale for evaluating tumour Ag-bearing DCs as therapeutic vaccines in humans. The unique ability of DCs to induce and sustain primary immune responses makes them optimal candidates for vaccination protocols in cancer (see Table 2).^2,70,71

Table 2: Summary of dendritic cell clinical trials

However, DC-mediated induction of immunity represents a major therapeutic challenge, and several parameters need to be considered to ensure the optimal outcome of DC-based vaccination protocols including (a) the source of DCs, (b) methods for isolating and activating DCs and (c) route of administration.

Many groups have generated DC-like cells by culturing CD14+ monocyte-enriched PBMCs in vitro. When cultured for 1-2 weeks with media supplemented with GM-CSF and IL-4, monocytes give rise to large numbers of cells that are morphologically and phenotypically similar to the ‘classical’ density-purified DCs (see Figure 1a and 1b). ^72,73 These cytokine-generated DCs require additional maturation in vitro with TNF-alpha or INF-alpha in order to fully stimulate in an allogeneic MLR or prime Ag-specific T cell responses in vitro and in vivo.^74-76

Figure 1: Light microscopic morphology of DCs. Activated DCs (Figure 1b) were generated from adherent mononuclear cells and incubated with cytokineconditioned medium for 7 days at 37 °C, in a 5% CO2 incubator. DCs show characteristic morphology with irregular shape and cytoplasmic projections or veils

a

b

Moreover, without this additional maturation step the DC phenotype can revert to that of a monocyte. Human DCs can also be enriched as circulating precursors from the blood by density-based purification techniques.⁷⁷ After a period of in vitro culture and maturation, DC precursors become larger and less dense. Gradient solutions lacking potentially immunogenic proteins such as bovine serum albumin,⁷⁸ have been employed and have included Percoll,⁷⁹ Nycodenz,⁸⁰ and metrizamide.⁸¹

These different fluids are osmotically active, to varying degrees, and have additional stimulatory properties as well. The use of density-based isolation, however, is limited by the low frequency of DC precursors in blood, representing around 1% of peripheral blood mononuclear cells (PBMCs).⁷⁷  As a result, leukopheresis has been performed in order to isolate sufficient numbers of DCs for therapeutic vaccination in humans.

In generating DCs for use as cellular vaccines, regardless of source, the infused cells should possess a stable as well as an activated phenotype. In addition to expressing the requisite MHC and co-stimulatory molecules to prime T cells, the cells should express appropriate adhesion molecules and chemokine receptors to attract the DCs to secondary lymphoid organs for priming. Otherwise, ineffective priming may occur, particularly if the DCs are administered systemically rather than locally into the relevant draining lymph nodes. Intra-lymphatic or intra-nodal injection of DCs may be used to deliver DCs directly to secondary lymphoid organs, although these routes of administration have not, as yet, been shown conclusively to produce more effective vaccination.^82,83

DC-based immunisation requires that the cells present one or more tumour Ags to the host’s T cells. Truly tumour-specific Ags (TSAgs) offer theoretical advantages as immunotherapy targets. The immune response induced by such Ags presumably would be limited to malignant cells bearing the antigenic epitopes, thereby, limiting the risk of collateral damage to normal tissues.^84,85 If such proteins arise in the tumour following thymic development, clones of T cells reactive with such peptides may exist as they probably avoided apoptosis and thymic deletion. Nevertheless, the immunogenicity of these Ags may be limited by the number of epitopes contained within the protein. Moreover, TSAgs may vary between individuals with the same tumour type. Viral tumour Ags can represent desirable immune targets given the inherent immunogenicity of many viral proteins.⁸⁶

Although the results, to date, of the various DC trials (see Table 2) are exciting and look promising, the current procedures used for isolating and ‘arming’DCs are prolonged and problematic and, as yet, are not applicable to routine clinical practice. As discussed, many groups are currently pursuing techniques for in vitro generation of DCs from CD34+ precursors or CD14+ monocytes. These approaches, while expensive, can generate large numbers of cells for use in clinical trials. Administration of growth factors (e.g., GMCSF, IL-4, TNF-alpha and IFN-alpha have all been used in vitro to generate and activate DCs in patients with cancer; this offers another interesting therapeutic approach. However, whether such an approach will circumvent the inhibitory milieu (both systemic and in situ at the tumour site) in cancer patients requires further careful studies.

In addition to questions regarding the best source of DCs for use in clinical trials, the choice of tumour Ag with which to ‘arm’ the DCs is almost certainly going to have a profound influence on clinical outcome. At present, the choices are limited because only a few TAAgs have been identified and are suitable for loading/priming DCs.^87-89 On the other hand, the number of suitable candidate Ags is continually expanding and is expected to accelerate, as a consequence of the intensification of gene mapping and isolation.

Ultimately, combinations of Ags will be used to reduce the risk of generating Ag-loss variants that evade the immune response. The increasing use of microarray technology in assessing tumours may enable individualised Ag combinations to be determined. Antigen delivery also remains to be optimised. Use of protein, whether tumour-derived or produced by recombinant DNA methods, can be cumbersome and potentially limiting, especially at concentrations that may be necessary for MHC class I delivery. Use of specific peptide conjugates or fusion constructs (e.g. with HIV tat) may increase the efficiency of presenting epitopes from these soluble proteins. RNA, DNA, and viral vectors are more easily produced and may offer an alternative approach, although issues of transfection efficiency and DC viability remain unresolved.

The potential benefits of administering cytokines or other DC activators, in combination with DC vaccination, remain relatively unexplored. Clearly, DCs can elaborate their own cytokines for Ag priming, as previously discussed. However, supplementing culture media used for generating DCs in vitro with additional cytokines may enhance the APC functions of these DCs. TNF-• or CD40 ligand are known to activate DCs in vitro and could increase the potency of DC-based therapy.^90,91 The addition of IL-12 may aid in Ag priming and generating TH1 responses in vitro or in vivo.⁹² Synergy between DC vaccination and IL-2 has already been demonstrated in an animal model. ⁹³ No consensus exists on the optimal approach to assessment of immune responses in patients undergoing immunotherapy, let alone DC vaccination. Delayed-type hypersensitivity testing with an Ag challenge injected into the skin has been used to assess gross immune reactivity. Cytokine production by both CD4+ and CD8+ T cells can be detected by cytokine ELISA, ELISPOT, or intracellular cytokine staining. Other techniques for assessing CD4+ T cell responses include measurement of Ag-specific proliferation. CD8+ T cell responses have also been assessed with CTL assays. However, these assays assess the functions of circulating T cells but do not necessarily reflect immune responses in lymphoid organs. Improved immunologic assays that better correlate with clinical outcome will be required before these assays can serve as surrogates for evaluation of tumour status.

CONCLUSION

By isolating and arming DCs with Ag ex vivo, it may be possible to allow the cells to mature in the absence of an inhibitory milieu. If defective maturation of DCs is a common occurrence in malignancy, then identification of agents that induce their maturation, in vivo, may represent an elegant solution to this problem. In the absence of such agents, administration of DCs grown in vitro from circulating DC precursors, which have been activated and induced to mature in vitro, and armed with appropriate tumour Ags, may prove useful in the treatment of a variety of cancers for which existing therapeutic options have limited use and clinical efficacy.

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Copyright date: 26th April 2001

Correspondence: Professor O. Eremin, Section of Surgery, E Floor, West Block, Queen’s Medical Centre, University of Nottingham NG7 2UH, UK

©2001 The Royal College of Surgeons of Edinburgh, J.R.Coll.Surg.Edinb.