INTRODUCTION

IMMUNE SURVEILLANCE

DENDRITIC CELLS AND CANCER

DENDRITIC CELLS AND ANTI-CANCER THERAPY

Indications

INTRODUCTION

Induction of effective tumour immunity can be viewed as a three-step process that includes：

• appropriate presentation of tumour-associated antigens (TAAgs),
• selection and activation of TAAg-specific T cells as well as non-Ag-specific effectors and
• homing of TAAg-specific T cells to the tumour site and effective elimination of malignant cells expressing the TAAgs.

Dendritic cells (DCs) are the crucial cells providing the necessary components for initiating and developing effective cell-mediated immune (CMI) responses. 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. Essential co-stimulatory molecules are upregulated on DCs as they migrate to secondary lymphoid organs (the spleen and lymph nodes) where they liaise with na?ve T cells, inducing the activation and proliferation of Ag specific CTLs. Thus, effective DC function in cancer involves several interlinked biological processes that occur in following sequence:

• TAAgpresentation and recognition in tissues which involves proteolytic intracellular cleavage and peptide surface representation,
• 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
• 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, shown as below:

• Cryptic or poorly expressed TAAgs on malignant cells
• Shedding and/or internalisation of Ag-Ab complexes, resulting from generation of a humoral and ineffective anti-cancer response
• Defective TAAg processing and/or presentation, e.g. down regulation and defective TAAg capture, processing and surface re-expression with MHC class I molecules
• Down modulation of T cell activity and CTL tumour cell lysis due to inappropriate secretion of immunosuppressive factors, e.g. prostanoids (prostaglandin E2 (PGE2)), cytokines (interleukin 10 (IL-10)) and transforming growth factor B (TGF B)
• Lack of the necessary DC costimulatory molecules - B7-family (B7.1/CD80 or B7.2/CD86), and CD40-ligand interaction -resulting in anergy of CD4+/CD8+ T cells and the establishment of tolerance towards the malignant cell

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.

Some studies in humans with solid cancers have investigated DC trafficking in peripheral blood. It was showed that DCs in the peripheral blood of patients with head and neck cancer were significantly immunosuppressed. There was also an increased intratumoural presence of the immunosuppressive CD34+ progenitor cells. Patients with head and neck squamous cell carcinoma also had increased levels of the immunosuppressive peripheral blood CD34+ cells.

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.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.

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.There is evidence that shows that dendritic cell function was inhibited by soluble factors present in melanoma cell cultures.

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. 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.

Dendritic cell progenitors give rise to myeloid ( monocytes and CD11c +DCs ) and lymphoid (CD11c-plasmacytoid DCs) precursors. Upon interaction with inflamed endothelium, monocytes differentiate into CD11+ blood DCs which give rise to langerhans cells, interstitial DCs, and macrophages. Differentiation of plasmacytoid DCs from CD34+progenitors can be blocked by Id2 and Id3 overexpression, suggesting their lymphoid origin.

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---

• 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;
• Their capacity to activate native T cells in regional lymph nodes and theirdifferentiation into CTLs, specifically able to interact with cancer cells and lead to tumour cell damage and death;and
• 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.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. Furthermore, Ag-loaded DCs can be used therapeutically to induce regression of preexisting tumours. Dendritic cells loaded with appropriate TAAgs can induce either protection or rejection of malignant cells in various animal models.

Several systems have been used to deliver TAAgs to DCs, including

• Defined peptides of known sequences,
• Undefined acid-eluted peptides from autologous tumours,
• Whole tumour lysates,
• Retroviral and adenoviral vectors,
• Tumour cell-derived RNA,
• Fusion of DCs with tumour cells, and
• Exosomes derived from DCs pulsed with tumour peptides (subcellular structures containing high levels of MHC molecules and peptides).

Several observations have established the rationale for evaluating tumour Ag-bearing DCs as therapeutic vaccines in humans.For example:

Table 2: Summary of dendritic cell clinical trials

The unique ability of DCs to induce and sustain primary immune responses makes them optimal candidates for vaccination protocols in cancer.

Clinical application of DCs as therapeutic vaccines involves:

• Source of DCs
• Methods for isolating and activating DCs
• 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).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.

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

In generating DCs for use as cellular vaccines, regardless of source, the infused cells should possess a stable as well as anactivated 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.

DC-based immunisation requires that the cells present one or more tumour Ags to the host 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.

Although the results, to date, of the various DC trials are exciting and look promising, the current procedures used for isolating and activating DC 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.

In addition to questions regarding the best source of DCs foruse in clinical trials, the choice of tumour Ag which is delivered to the DCs is almost certainly going to have a profound influence on clinical outcome. There are following Ag choices:

• Combinations of Ags will be used to reduce the risk of generating Ag-loss variants that evade the immune response.
• 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.

Indications：What types of cancer can be treated with DC vaccine?

In certain types of cancer, such as the skin cancermalignant melanoma, it is known that patients can mount an immune response to the developing tumour. However, in many cases this response ultimately fails. Beefed-up dendritic cells help to maintain this response in these patients.

The patients with breast cancer, non-Hodgkin lymphoma,renal cell cancer, glioma, parathyroid cancer,who have not responded to conventional treatments and have metastatic skin lesions, can response to DC therapy.

Dendritic cell therapy alone may also prove useful for patients at high risk of recurrence after a primary tumour has been removed by surgery, or for healthy people at risk of developing familial types of cancer.Apart from above tumour,other tumours in thatPromising preliminary results were receivedfrom clinical trials include: hepatocellular carcinoma, lung cancer, colonrectal cancer, nasopharygeal cancer, esophageal cancer and thyroid cancer.