• dendritic cells;
  • immunotherapy;
  • malignancy.

In the last few years, a number of factors have led to a renewed and intense interest in cancer immunotherapy. At the level of basic science, these include the characterization of a large number of tumour-associated antigens (Pardoll, 1998), the identification of existing (although mainly ineffective) cellular immune responses to these antigens in patients (Nanda & Sercarz, 1995), and an increasing appreciation of the complex interactions between antigens and effector cells (T cells and NK cells) that determine whether tolerance or destruction of antigen-bearing cells is the result. Many animal models of effective anti-tumour immunity have been described and there is good clinical evidence of a T cell-mediated anti-leukaemic effect (graft-vs.-leukaemia; GVL) in man. Initially observed following allogeneic bone marrow transplantation for leukaemia (Antin, 1993), this phenomenon also underlies the beneficial effects of donor lymphocyte infusion (DLI) therapy (Kolb et al, 1995; Lokhorst et al, 1997), as well as the low intensity (‘mini’) transplant regimes for leukaemia and other malignant diseases (Slavin et al, 1998).

The effective presentation of antigen by professional antigen-presenting cells (APCs) is crucial to the induction of positive T-helper and cytotoxic T-cell responses (Croft, 1994). In some instances, however, antigen presentation may instead result in the development of tolerance and even long-lasting anergy (Sallusto & Lanzavecchia, 1999). The very growth of tumours in patients itself implies host unresponsiveness, but the question of whether this is the result of true anergy to tumour antigens or simply of failure to recognize these antigens is largely unknown (Grabbe et al, 1995; Chen, 1998). In this regard it is relevant that the structure of many known tumour antigens is either identical or closely related to self-antigens to which the patient might be expected to be tolerant. This has led to a realization that the modulation of tumour antigen presentation may be an approach that could terminate unresponsiveness and thereby result in therapeutic benefit.

The most potent APCs are dendritic cells (DCs, reviewed in Hart, 1997; Banchereau & Steinman, 1998). Although B cells and macrophages can present antigen efficiently to memory T cells, they are far less efficient than DCs in initiating immune responses in naïve T cells (Croft, 1994). Because they are present in such minute numbers in tissues, it has only recently become possible to isolate and properly characterize these cells. The development of techniques to generate DCs in large numbers in vitro from peripheral blood monocytes or haemopoietic progenitors has recently led to new approaches to cancer immunotherapy that will be the subject of this review.

Immune responses to human tumour antigens

  1. Top of page
  2. Immune responses to human tumour antigens
  3. Dendritic cells: an available source of potent apcs for immunotherapy
  4. Clinical and preclinical studies with in vitro-generated DCs
  5. DCs In leukaemia
  6. Conclusion
  7. References

There are many different classes of antigen associated with human malignancy, some totally unique to each patient, some that are mutation-specific to a particular cancer type, while others are mutations of a more general nature seen in many different malignancies (Boon & van der Bruggen, 1996; Disis & Cheever, 1996; Pardoll, 1998). In many common tumours, however, so-called tumour antigens are no more than a variation of expression of an otherwise normal ‘self’ protein. An example of unique antigen is the idiotypic protein in myeloma and lymphoma – its specificity provides an attractive tumour target for vaccination, but it also entails laborious procedures for vaccine preparation. The bcr/abl gene product in chronic myeloid leukaemia (CML) is an example of a disease-specific mutation with a well-characterized tyrosine kinase protein product. To this class of tumour antigen belong the viral peptides associated with Epstein-Barr virus (EBV) and human papilloma virus (HPV) that characterize certain lymphoproliferative diseases and cervical carcinoma respectively. Mutations (single-base substitutions) in certain oncogenes such as ras or in the tumour suppressor gene p53 are quite common in many tumours. In the latter case, the overexpression of p53 that results from this provides a ‘wild antigen’ target for potential vaccination strategies (Yanuck et al, 1993).

One of the earliest characterized classes of human tumour antigens was identified by defining CD8+ T cells that recognize cloned genes encoding melanoma antigens. This family of MAGE antigens is expressed on normal testes (although in the absence of class I MHC molecules) but, although therefore not truly tumour-specific, the antigens are widely expressed in melanoma (Boon & van der Bruggen, 1996). Very recently, their expression has also been noted in advanced myeloma and seems to reflect increased DNA demethylation of promoter CpG dinucleotides resulting in gene derepression (van Baren et al, 1999). Other melanoma-associated antigens are tyrosinase, gp100 and Mart-1/melan-A, all of which are normal products expressed in pigment biosynthesis. Another normal tissue antigen that is overexpressed on 30% of breast cancers and on ovarian and pancreatic tumours is the her-2/neu protein (Brossart et al, 1998; Disis et al, 1999). This is a growth factor receptor (a membrane tyrosine kinase) and its membrane localization makes it an attractive vaccination target. MUC-1 is an abundantly expressed and abnormally glycosylated mucin also found on these same tumour types and it too may be a target for the induction of tumour immune responses (Houghton & Lloyd, 1998).

Immune stimulation versus immune evasion

There are many studies that have demonstrated the presence of precursor T cells that recognize some of the above-mentioned tumour-associated antigens (TAAs) in human cancer patients. These have been identified in the circulation (Lee et al, 1999), in tumours as tumour-infiltrating lymphocytes (Rosenberg, 1997) and within melanoma-infiltrated lymph nodes (Romero et al, 1998). Following cytokine-mediated expansion in vitro, these cytotoxic T lymphocytes (CTLs) may be capable of killing tumour cells. However, a recent provocative study (Lee et al, 1999) has shown that such populations, when freshly isolated by tetramer sorting, lack the capacity for killing and demonstrate features consistent with anergy (blunting of activation responses such as CD69 expression and failure of cytokine production). This may be important as it suggests that a considerable expansion of tumour peptide-specific T-cell populations may occur in patients (up to 2% of total CD8+ cells), but that these cells may subsequently be rendered anergic. How tumour peptides are taken up and presented by the immune system and the factors determining a positive response rather than tolerance or long-term anergy are issues crucial to the development of effective immunotherapy strategies for human tumours.

Antigen presenting cells and tumour immune responses – the importance of antigen cross-presentation Naïve T lymphocytes first encounter and are sensitized to foreign antigens within secondary lymphoid organs (lymph nodes and spleen). Antigens reach these sites within dendritic cells that have generally taken-up these antigens by endocytosis in peripheral sites (Fig 1) and subsequently migrate and simultaneously mature into potent antigen-presenting cells (Banchereau & Steinman, 1998).


Figure 1. Dendritic cell maturation, phenotype and function. Following antigen exposure, immature DCs in peripheral sites migrate to lymph nodes under the influence of regulatory chemokines and simultaneously undergo maturation to effective antigen-presenting cells. CCR, chemokine receptor; LPS, lipopolysaccharide.

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Exogenous antigens are taken up into APCs via the endocytic pathway, processed within endosomes and then presented in the context of class II MHC to CD4+ helper T cells. The activation of CTLs, however, requires class I presentation of antigen which classically results from endogenously synthesized (e.g. viral) proteins. How then might tumour-derived antigens gain access to this class I pathway of presentation that is crucial for the induction of a CTL response?

This dilemma is resolved by the phenomenon of cross-presentation. It is now clear that exogenous protein can be taken up by APCs and presented not just on class II but also on class I molecules (Bennett et al, 1997; Albert et al, 1998; Carbone et al, 1998; Heath & Carbone, 1999). This ‘secondary’ or indirect presentation of antigen would be of crucial importance in the case of tumour cells expressing neo-antigens but lacking MHC or other accessory molecules needed for effective primary or direct antigen presentation to effector cells. It has been shown to occur in experimental tumours in which effective responses, resulting in tumour regression, were shown to require the involvement of bone marrow-derived DCs (Huang et al, 1994).

Two types of evidence have recently shed light on the mechanisms implicated in this process. It has been shown that DCs are capable of taking-up debris from apoptotic cells and, in the case of virally infected cells, presenting their antigens to induce CTLs (Albert et al, 1998). Other studies, however, have shown that the uptake of necrotic cells rather than apoptosed cells results in activation of APCs (Gallucci et al, 1999) or even that these observations might be artifactual, resulting from mycoplasmal infection of DCs by the cell lines (Salio et al, 2000). Although the issue is unresolved, it may be that, under normal circumstances, cross-presentation of normal apoptotic material by APCs is responsible for the ongoing maintenance of peripheral tolerance to self antigens (Kurts et al, 1997; Adler et al, 1998).

Tolerance versus immunity (Fig 2) It has been proposed that an essential part of the immune system's ability to recognize and react to foreign as opposed to self antigens depends on the prevalence of ‘danger’ signals that accompany encounters with the former, but not the latter, antigens. Inflammatory products such as lipopolysaccharide (LPS) or viral antigens (Matzinger, 1994) or heat shock proteins (Suto & Srivastava, 1995) may be such signals that induce maturation and activate APCs for more effective antigen presentation. This theory would also be consistent with the above-mentioned observations (Gallucci et al, 1999) that only the uptake by DCs of necrotic, but not apoptotic, cells can trigger CTL responses. An alternative explanation, however, may be that tolerance is associated with a particular lymphoid DC subset that, at least in mice, can delete CD4+ T cells through a fas/fas-ligand interaction (Suss & Shortman, 1996).

The induction of CD4+ T-helper responses (MHC class II-dependent) is known to be critical for CTL proliferation (Bennett et al, 1997) and these responses by the two classes of T cells need to occur on the same APC (Fig 2). There appears to be considerable significance for CD4+ cell activation in the induction of a sustained CTL response (Hung et al, 1998; Zajac et al, 1998). If this is absent or deficient, it may result in the emergence of anergic T-cell clones (Lee et al, 1999). Although CD4+ CD8+ T-cell interactions were originally thought to involve simultaneous presentation to both cell types, this is probably not the case. There appears to be a major role for ligation of the CD40 molecule on DCs by its ligand (CD40L), which is itself upregulated on activated CD4+ T helper cells (Bennett et al, 1998; Ridge et al, 1998; Schoenberger et al, 1998). CD40 ligation primes the APCs and permits effective presentation of antigen to CTLs through the class I pathway. For this reason, CD40 ligation has been the focus for a number of studies that have proposed a role for this molecule in the induction of clinically effective anti-tumour immunity (Schultze et al, 1997; Buhmann et al, 1999; Labeur et al, 1999).


Figure 2. Pivotal role of DCs in the induction of CD4+ and CD8+ T-cell responses. DC maturation and activation by viruses, HSP or inflammatory products result in positive T-cell responses. DC immaturity, the absence of ‘activation’ signals or the presence of tumour products may induce a defective T-cell response or long-term anergy. CD40:CD40L, ligation of membrane CD40; HSP, heat shock protein; VEGF, vascular endothelial growth factor.

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Thus, the potency of DCs for antigen presentation is affected by a number of aspects related to their maturity and state of activation (e.g. by CD40L). Maturation of DCs (Fig 1) is characterized by high expression of class II molecules, of the B7 molecules, CD80 and CD86 (which activate T cells via their CD28 ligand, Schultze et al, 1996), of CD40 and by the production of cytokines such as interleukin 12 (IL-12) that direct TH1 cell differentiation (Macatonia et al, 1995). Competing for these B7 molecules is a close homologue of CD28, CTLA-4, that only appears on already activated T cells and which probably has an opposite and negative regulatory influence on T-cell expansion (Krummel & Allison, 1995). Recently, a third member of the B7 family (B7-H1) has been described that does not bind to CD28 and this may also have a negative regulatory activity on T cells (Abbas & Sharpe, 1999; Dong et al, 1999).

In conclusion, lack of an efficient cytotoxic T-cell response to tumour cells could occur through a number of different mechanisms, some of which may relate to the uptake of antigens by APCs that remain immature in the absence of the products of inflammation or cell necrosis. There is also accumulating evidence that it may be a result of direct inhibition by products of the tumour cells themselves (Fig 2). These include vascular endothelial growth factor, VEGF (Gabrilovich et al, 1996, 1997, 1998), cytokines such as IL-10 (Steinbrink et al, 1999), as well as other as yet unspecified factors recently described in leukaemic cell lines (Buggins et al, 1999).

Dendritic cells: an available source of potent apcs for immunotherapy

  1. Top of page
  2. Immune responses to human tumour antigens
  3. Dendritic cells: an available source of potent apcs for immunotherapy
  4. Clinical and preclinical studies with in vitro-generated DCs
  5. DCs In leukaemia
  6. Conclusion
  7. References

Phenotype and function in immature and mature DCs (Fig 1)

It is clear that DCs are far from being a single homogeneous population (reviewed in Hart, 1997; Reid, 1997; Banchereau & Steinman, 1998; Reid et al, 2000). Probably all DCs ultimately derive from CD34+ bone marrow stem cells (Caux et al, 1992; Reid et al, 1992; Santiago-Schwarz et al, 1992), but understanding of their phenotypic and functional differences must take into account two factors. Firstly, that much of the reported data relates to the properties of DCs cultured in vitro from different precursor populations and under a variety of cytokine influences. The properties of such cultured cells bear an unclear relationship to those of fresh DCs isolated from blood or various tissue sites. Secondly, both tissue DCs and those cultured in the laboratory can be classed as either immature or mature. Immature cells (those located in peripheral organs such as Langerhans cells in the skin) are specialized for antigen uptake and processing, but have lower levels of class II molecule expression and of accessory molecules CD80, CD86 and CD40, all of which are important in presenting antigen to T cells and inducing a proliferative T-cell response (Cella et al, 1997). They are also deficient in the production of IL-12 which directs TH1 cell differentiation (Macatonia et al, 1995).

In contrast, having encountered and processed antigens, DCs lose the capacity for antigen uptake, migrate centrally to secondary lymphoid organs and simultaneously undergo maturation (Fig 1). In so doing, they increasingly express the above-mentioned molecules and develop a unique capacity for antigen presentation and the induction of CD4+ and CD8+ T-cell responses. Chemokines and their receptors (CCRs) probably determine migration patterns and CCR6 (and other CCRs) on immature cells are replaced by CCR7 in maturing cells (Dieu et al, 1998; Sallusto et al, 1999). Both MIP-3β and secondary lymphoid chemokine (SLC) are constitutively expressed in secondary lymphoid tissues and are ligands for CCR7 that appears on migrating, mature DCs (Gunn et al, 1999). Mature DCs themselves also produce chemokines and this may prove to be important in self-regulation of their migratory behaviour, as well as in interactions between DCs themselves (Sallusto et al, 1999).

Blood and tissue DCs (Table I)

Table I.  Tissue distribution, phenotype and function of human dendritic cell populations.
  1. acc. mol, accessory molecule expression (CD80, CD86, CD40, IL-12); IL-3R, expression of interleukin 3 receptor-α (CD123); IDC, interdigitating dendritic cells.

Peripheral blood
 MyeloidCD11c+ CD83 CD33+ IL-3Rlo (DC1)Stimulate Th1 cells
 LymphoidCD11c CD83 CD33 IL-3Rh (DC2)Stimulate Th2 cells
Lymph node & secondary lymphoid tissue  
 IDCCD11c+ CD83+ CD33+ IL-3Rlo acc. mol.++Stimulate Th1 cells
 Germinal centreCD11c+ CD4+Stimulate memory T cells: B-cell responses
 PlasmacytoidCD11c CD83 CD33 IL-3Rh? Stimulate Th2/? self tolerance
Skin & peripheral tissues
 EpidermalCD1a+ acc. molloAntigen uptake and processing
 DermalCD1a ± CD32+ acc. molloAntigen uptake and processing

Mature DCs are found in secondary lymphoid tissue as interdigitating dendritic cells in the paracortical area of lymph nodes (IDC presenting to naïve T cells, Steinman et al, 1997), in the germinal follicle (presenting to memory T cells, Grouard et al, 1996) and around high endothelial venules (plasmacytoid DCs, Grouard et al, 1997; Olweus et al, 1997). The nature of these plasmacytoid cells is particularly intriguing as their exact function is unknown. They may correspond to the so-called DC2 subpopulation in peripheral blood, are CD11c- and interleukin 3 receptor-α-positive (IL-3R+), and may be of lymphoid or myeloid origin (Olweus et al, 1997). They produce large amounts of interferon α and induce a TH2 CD4+ T-cell response (Siegal et al, 1999). In contrast, CTL responses are triggered by TH1 CD4+ cells that are induced by CD11c+ cells of myeloid origin (termed DC1), which are also found in peripheral blood (Robinson et al, 1999) and can be derived from cultured monocytes in vitro. A negative feedback loop to the immune response has been proposed in which the IL-4 generated in the Th2 response may stimulate DC1 maturation yet kill DC2, thus ensuring a balanced Th1/Th2 response (Rissoan et al, 1999). Understanding the contrasting effects upon effector T-cell populations of these two peripheral DC types may be very important for research into DC therapies (Reid et al, 2000). An example of this has recently been provided by Arpinati et al (2000) who showed a fivefold increase in DC2 in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs). They speculate that a resulting shift from TH1 to TH2 responses might account for the observed low rate of acute graft-vs.-host disease (GVHD) in PBSC transplants and might be exploited deliberately to induce immune deviation in stem cell or organ transplants.

It is important to note that different and even conflicting findings have been reported in mice in which myeloid DCs were found to induce Th2 responses but lymphoid DCs had Th1-stimulating properties (Pulendran et al, 1999). A CD8α+, DEC-205+ lymphoid-derived DC population in mice has been shown to induce T-cell tolerance through a fas/fas ligand interaction (Suss & Shortman, 1996; Ardavin, 1997). A thymic DC precursor has been identified in man – this appears to be a common precursor for T, B and natural killer (NK) cells but not for myeloid cells (Galy et al, 1995). Whether these DCs, as in mice, mediate T-cell death or unresponsiveness is an important but still unanswered question.

In vitro generation of DCs

It is possible to grow large numbers of DCs in vitro either from CD34+ progenitors (bone marrow, blood or cord blood) or from peripheral blood monocytes. The conditions of culture differ for these different precursor populations and has recently been extensively reviewed by Young (1999). Granulocyte-macrophage CSF (GM-CSF), stem cell factor (SCF) and flt-3 ligand, together with tumour necrosis factor alpha (TNFα), are all factors known to be active in CD34+ cell cultures (Caux et al, 1992; Reid et al, 1992; Santiago-Schwarz et al, 1992), whereas GM-CSF and IL-4 seem to be sufficient for monocyte cultures (Bender et al, 1996; Romani et al, 1996). In the absence of TNFα or CD40 ligand (CD40L), lipopolysaccharide or a monocyte-conditioned medium, the cultured DCs are of immature phenotype. Capable of active endocytosis, they express CD1a, but only low amounts of the B7 accessory molecules CD80 and CD86 or CD83 or CD40. Because of the ease of procurement, the majority of recent tumour vaccine studies have taken the monocyte route for DC production. It should be emphasized that the conditions for antigen loading and for presentation by cultured DCs are different. If the objective is to obtain tumour antigen-loaded cells capable of inducing T-cell responses, there is a need to ensure maturation of these cells by one or other of the aforementioned factors.

Presentation of tumour antigens by dendritic cells

The identification of DCs within tumour tissue begs the question whether they may play any part in an immune response. A recent report of mature as well as immature DCs in and around certain breast tumours suggests they may indeed have such a role (Bell et al, 1999). The high levels of the chemokine MIP-3α within tumour tissue may attract immature DCs to the tumour bed (Dieu et al, 1998). Conversely, malignant cells appear to inhibit DC development from their precursors, an effect that may be mediated by vascular endothelial growth factor (VEGF) and by IL-10 (Gabrilovich et al, 1996, 1998; Steinbrink et al, 1999). In breast cancer patients, DCs have been shown to have an impaired capacity for antigen presentation (Gabrilovich et al, 1997) and such factors may, as already mentioned, reduce the likelihood of effective CTL responses.

The unique properties of DCs have suggested to investigators that they may have the ability to reverse the immunological unresponsiveness generally seen in malignant disease (Young & Inaba, 1996; Schuler & Steinman, 1997). This might occur through the more effective presentation of tumour epitopes with low affinity for the T-cell receptor, such as certain self-antigens. Under ex vivo conditions, such antigens or indeed potentially immunogenic neo-antigens introduced into DCs, might be more efficient inducers of helper and cytotoxic T cells in the absence of tumour-derived inhibitory factors. This could have significant clinical potential for tumour vaccination, however, antigen loaded DCs may themselves become targets for destruction by expanded CTL clones. Survival and anti-tumour efficacy of antigen-loaded murine DCs were both markedly reduced following previous immunization (Hermans et al, 2000), an observation which may be significant in the design of tumour vaccine strategies.

There is no doubt that, in animals, both tumour protection and even regression of established tumours can be effectively induced by DCs. Strategies have involved pulsing bone marrow-derived DCs with whole tumour cell lysates (Knight et al, 1985; Grabbe et al, 1995), defined tumour peptides (Mayordomo et al, 1995) or with a range of peptides that have been acid-eluted from the tumour cell membrane, followed by i.v or s.c. administration to tumour-bearing animals (Zitvogel et al, 1996). It can be shown that the protective immune response involves TAA presentation both on Class I and Class II MHC (Huang et al, 1994) and is mediated both by specific CD4+ and CD8+ CTLs (Porgador & Gilboa, 1995; Zitvogel et al, 1996; Toes et al, 1999). In the case of malignancies of low immunogenicity, it seems that CD40 ligation prior to antigen exposure is important for maximal tumour protection (Labeur et al, 1999). Although other agents (lipopolysaccharide) could also induce high levels of MHC class II, CD80 and CD86, and IL-12 production by DCs, the CD40L stimulus was paramount for successful vaccination.

In human patients with melanoma, DCs generated from peripheral blood and charged with known TAA peptides (Melan A/MART-1) were able to elicit CTLs that killed melanoma cells in vitro in an MHC class I-restricted manner (Bakker et al, 1995). MUC-1 is an antigen overexpressed in many tumours (breast, ovarian and pancreatic tumours, and in myeloma and B-cell lymphoma) and its ubiquity makes it an attractive vaccination target (Houghton & Lloyd, 1998). Non-MHC-restricted T-cell responses to tandem repeat sections of the protein core are observed in patients and these probably occur through cross-linking of the T-cell receptors (TCRs). The Tübingen group have recently shown that HLA-A2 binding peptides can be synthesized, loaded on to DCs and can induce potent MHC-restricted killing of cell targets that include breast, pancreatic and renal cell carcinomas (Brossart et al, 1999). Their finding that this was enhanced by including a pan-HLA-DR binding peptide emphasizes the importance of CD4+ T-helper cell epitopes in the induction of these CTL responses. Similar findings are reported in the case of her-2/neu-derived peptides (E75 and GP2). These peptides also show HLA-A2-restricted binding, however, CTLs induced by peptide-loaded DCs showed a wide range of tumour targets that included colon, breast and renal cell carcinomas (Brossart et al, 1998). The HLA selectivity of these peptides is a potential disadvantage for vaccine design, however, some studies have shown that other peptides from protein antigens such as her-2/neu and carcinoembryonic antigen (CEA) may bind to HLA-A3 and be equally effective in CTL generation (Kawashima et al, 1999).

Monocyte- or CD34-derived DCs for antigen presentation? As mentioned above, there are two techniques for obtaining mature DCs for peptide loading – from peripheral blood monocytes and from CD34+ progenitors. A comparison of these cell populations showed that they appear to have equal potential for the activation of CTLs from their precursors in vitro where such precursors (CTLp) are present in high numbers, as in the case of a recall antigen such as influenza matrix peptide. In melanoma patients with very low numbers of CTLp against a Melan-A/Mart-1 peptide, the CD34+-derived DCs were more effective than monocyte-derived cells (Mortarini et al, 1997). As this may reflect different requirements for antigen presentation for memory as opposed to naïve CTLp, confirmation of this finding may have importance for tumour vaccine development.

A link between innate and adaptive immunity to tumours All these studies have concentrated on T-cell responses, however, an intriguing murine study suggests that DCs may also trigger innate NK-mediated anti-tumour immunity (Fernandez et al, 1999). Flt-3 ligand given to tumour-bearing mice expanded DC numbers and resulted in tumour regression that was confirmed as being dependent upon the presence of NK cells. In vitro-generated DCs were also shown to trigger NK killing of an NK-sensitive cell line in culture. This has suggested that, at least in experimental models, tumour cell killing might be a two-stage process initiated by a non-specific innate NK cell-dependent mechanism. Resulting necrotic or apoptotic tumour bodies would be taken-up by DCs and result in a cognate T-cell immune response through cross-presentation.

Clinical and preclinical studies with in vitro-generated DCs

  1. Top of page
  2. Immune responses to human tumour antigens
  3. Dendritic cells: an available source of potent apcs for immunotherapy
  4. Clinical and preclinical studies with in vitro-generated DCs
  5. DCs In leukaemia
  6. Conclusion
  7. References

The issues that will probably have an impact on the success or failure of cell-based immunotherapy in cancer are the following. What is the preferred source of DCs and which conditions of in vitro culture are most favourable for vaccination? To where do administered DCs travel following administration and what should be the favoured site of vaccination? What mode of presentation of tumour antigen on DCs is most effective in provoking a sustained CTL response with resulting clinical efficacy?

Sources of DCs and routes of administration

The homing capacity of ex vivo-generated DCs has been examined in mice (Eggert et al, 1999) and recently also in cancer patients (Morse et al, 1999). In these, the fate of the indium-labelled monocyte-derived DCs was followed after s.c., intradermal (i.d.) or i.v injection. Following i.v injection, the cells homed to lungs, liver and spleen (as seen in the mice), but administration by this route or even the s.c route did not result in any cells being identified in the lymph nodes. Only after i.d. injection were a few cells seen in regional nodes (0·4%) at 48 h. In order to investigate the ability of autologous monocyte-derived DCs to induce immune responses in volunteers, Dhodapkar et al (1999) gave s.c injections of these cells pulsed with tetanus toxoid, influenza matrix peptide or keyhole limpet haemocyanin (KLH) and followed CD4+ and CD8+ T-cell responses. Both primary and secondary responses were observed as early as 7 d after immunization and persisted beyond 90 d. The homing properties of these DCs were not reported, however, it may be important that DCs were matured prior to administration in monocyte-conditioned medium (MCM) and that the antigens studied, unlike most tumour antigens, were strong imunogens.

It is as yet unclear whether homing of administered DCs to the T-cell areas of regional lymph nodes is essential to the generation of an effective immune response. An alternative route of DC delivery is the by the intranodal injection of antigen-pulsed cells into regional lymph nodes. This has already been exploited with beneficial clinical effects in patients with advanced malignant melanoma (Nestle et al, 1998).

Clinical immunization strategies – non-DC

This review concentrates on the manipulation of DCs and tumours or their products ex vivo. It is important, however, to set this in the context of other strategies that have taken a different route to the same objective of engendering a host-vs.-tumour response. Attempts have been made to engineer the malignant cells themselves to express co-stimulatory molecules that they lack and thereby to directly trigger T-cell responses (Wu et al, 1995; Huang et al, 1996). In the case of myeloma, retroviral gene transfer successfully induced expression of the B7-1 molecule on tumour cells resulting in CTL induction, although this was not possible with cytokines or CD40L (Tarte et al, 1999). Alternatively, cells may be transduced with genes encoding cytokines, as recently described for GM-CSF in human melanoma (Soiffer et al, 1998) or IL-12 in murine leukaemia (Dunussi-Joannoppoulos et al, 1999). In these cases, effective responses may result either from activation of DCs or though directing effector helper T cells along the Th1 pathway.

Tumour antigens have also been administered directly, relying upon their uptake and processing in vivo to achieve tumour regression. The most readily available and unique antigens are the idiotype (Id) proteins from myeloma and lymphoma patients. In the series from Stanford, both humoral and cellular immune responses were seen in patients with non-Hodgkin's lymphoma (NHL) vaccinated with idiotype protein together with the strong immunogen KLH (Kwak et al, 1992). These responses were, however, dependent upon the use of an additional adjuvant. A similar strategy was adopted in the vaccination of normal donors of bone marrow to myeloma patients (Kwak et al, 1995). In this case, immunity was transferred with the bone marrow to the recipient, although it is unclear whether plasma cells can actually be targeted by such a strategy. A more recent study demonstrated idiotype-specific T-cell responses in myeloma patients given purified M-component, together with GM-CSF, by the intradermal route (Osterborg et al, 1998). In breast and ovarian cancer patients, a her-2/neu peptide vaccine given i.d together with GM-CSF resulted in cellular responses to both the peptides, as well as to the her-2/neu protein itself, through the process of ‘epitope spreading’ (Disis et al, 1999). None of these studies has so far demonstrated incontrovertible evidence that the observed response translates into clinical benefit.

Caution in the application of peptide vaccination has been urged on the basis of an animal model which demonstrated the superiority of DC-based presentation of adenoviral oncogenes compared with the peptide alone (Toes et al, 1998). The peptides alone induced tolerance in contrast with the peptide-loaded DCs. Not only the mode of administration but also the actual dose of tumour antigen delivered may be crucial in avoiding tolerance. This point was made over 15 years ago in the first report of DC-mediated tumour regression in a mouse sarcoma model (Knight et al, 1985).

Clinical immunization strategies using DCs

Protein-based. The simplest approach to loading DCs with antigens in vitro is to incubate them with either the tumour cells themselves or more often with cell lysates or eluates (i.e unselected whole protein molecules). If the tumour antigen is known, defined peptides can be prepared and even ‘tailored’ to bind with high affinity to specific HLA molecules. There are numerous preclinical examples of effective tumour immunity induced in this way (Mayordomo et al, 1995; Zitvogel et al, 1996; Fields et al, 1998). A number of reports also show the clinical feasibility of this technique. In a study from Stanford, idiotype (Id)- and KLH-pulsed peripheral blood DCs were given i.v to four B-cell follicular lymphoma patients on three occasions, followed by booster s.c injections of KLH and idiotype protein (Hsu et al, 1996). Some tumour regression was observed. In myeloma, the unique tumour antigen is also an Id protein and has been shown to be cross-presented on DCs to Id-specific CD4+ T cells (Dembic et al, 2000). Several studies show that it is feasible to induce both humoral and cellular responses to Id protein by the administration of peptide-loaded DCs to patients with this disease. Cull et al (1999) observed a T-cell proliferative response in two patients after immunization with Id-loaded monocyte-derived DCs together with GM-CSF, however, CTLs were not detected and no clinical response was seen. A similar strategy in 11 patients but using CD34+-derived DCs also elicited T-cell and humoral responses, but the clinical response was limited (Titzer et al, 2000). The Stanford group have reported results in 12 myeloma patients who received a regime of Id-loaded DCs followed by Id protein vaccination several months after high-dose therapy and autologous stem cell transplantation (Reichardt et al, 1999). Most developed clear T-cell proliferative responses to KLH, but only two developed an Id-specific response and one a cytotoxic response. Clinical benefit could not be evaluated.

In a study of 16 patients with advanced melanoma, monocyte-derived DCs were loaded with either tumour lysate, HLA-A2 binding peptides (tyrosinase, Melan-A/Mart-1 or gp-100) or HLA-A1 binding peptides (MAGE-1/MAGE-3) in conjunction with KLH (Nestle et al, 1998). Intranodal injection of these vaccines resulted in the development of delayed-type hypersensitivity responses (DTH) to KLH in all patients and to peptide-pulsed DCs in 11 patients. Furthermore, CTLs with activity against peptide-pulsed T2 cell targets were identified at the DTH challenge sites.

In prostate cancer, the proteins prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) and peptides derived from them are being studied in phase I and phase II trials. Patients with advanced disease have been immunized with peripheral blood DCs pulsed with the peptides PSM-P1 and -P2 (Lodge et al, 2000), or with a fusion protein of GM-CSF and PAP (Burch et al, 2000). In some cases, DCs have been followed by booster injections of the proteins themselves. Disease regression has been observed in up to 30% of cases in one study (Lodge et al, 2000), although the correlation with in vitro T-cell responses to these antigens has been variable. The limited response to defined peptide epitopes requiring presentation on a limited range of HLA molecules (mainly HLA-A2) has prompted the investigation of alternative RNA or DNA approaches to vaccine preparation.

DNA- and RNA-based strategies The approach using selected peptides obviates the need for antigen processing but has the potential disadvantage of restricting presentation and, therefore, the response to a narrow range of epitopes which may be of low immunogenicity. A technically more demanding approach is to introduce DNA or RNA molecules encoding tumour antigens into DCs. This allows the presentation of a wider range of epitopes and, furthermore, increases the potential for both class I and class II presentation of peptides. The need to select peptides with specific HLA binding is also avoided. Successful tumour regression has been seen in animal models with these techniques (Specht et al, 1997; Boczkowski et al, 2000) and clinical trials are ongoing using both RNA- and DNA-transfected DCs in myeloma and solid tumours. Encouraging preclinical studies show very wide-ranging CTL responses, for example to RNA encoding PSA. It is suggested that this approach may overcome some of the limitations of protein- or peptide-based therapies (Heiser et al, 2000). A potential drawback of whole cell mRNA vaccination may be the risk of inducing autoimmunity, although preliminary evidence from this recent study suggests this may not be a problem.

DC tumour-cell hybrids A development which shows considerable promise is the formation of DC tumour-cell hybrids. These heterokaryons, which may be generated by polyethylene glycol or electrofusion, probably express the full repertoire of tumour cell antigens and acquire all the antigen-presenting capacity of DCs. This has been demonstrated in the case of a MUC-1-expressing tumour in mice and it was shown that the hybrid cells could break tolerance even in human MUC-1 transgenic animals and result in tumour regression (Gong et al, 1997, 1998). A very recent report describes the application of this technique in a clinical vaccine study of 17 patients with advanced renal cell carcinoma (Kugler et al, 2000). Tumour cells were fused with third-party donor monocyte derived DCs in order to promote an allogeneic ‘helper’ T-cell response and were given as s.c injections over several months. The 41% response rate (with four complete regressions) was highly encouraging and was accompanied by emerging clones of tumour-specific CTLs with MUC-1 specificity, as well as a positive DTH response. If this procedure proves to be more widely applicable, it would not only circumvent the need for tumour antigen identification but would also have the desirable effect of ensuring that the broadest range of epitopes would be presented to the immune system (Kufe, 2000).

Breaking self-tolerance

Because of the close relationship of many TAAs to self proteins, there is concern that techniques to enhance immune responses might break self-tolerance. In a murine study of protection against a lymphoblastic leukaemia cell line, syngeneic DCs pulsed with peptides eluted from the cell line also induced severe autoimmune disease (Roskrow et al, 1999). Fortunately, so far there has been little clinical evidence that this will be a serious problem, although skin depigmentation (vitiligo) has occasionally been observed in melanoma patients immunized with melanoma-associated peptides (Mackensen et al, 2000).

Boosting the response to cellular immunotherapy

There are many imponderables regarding the efficacy of cell-based treatments. Ultimately the objective is to promote a CD8+ CTL response and this probably needs to be sustained. As already mentioned, help from CD4+ T cells is probably a vital component of such a response as both this and ‘conditioning’ of the APCs are important factors for triggering CTL proliferation. In older vaccine strategies, the ‘danger signals’ provided by adjuvants may have fulfilled this function. Currently, many strategies include the highly immunogenic antigen KLH, both as a tracer antigen to ensure efficient vaccination and also for its perceived ability to provide CD4+ cell help. The use of cytokines accompanying or following DC vaccination may also have a role. A low, non-toxic dose of IL-2 has been shown to boost the tumour protective response against a weakly immunogenic mouse sarcoma following DC-based vaccination (Shimizu et al, 1999). IL-12 may also be a candidate cytokine as it has the function of enhancing Th1 responses to antigen, as well as increasing NK cell cytotoxicity. In a recent phase I study, IL-12 administration alone to patients with advanced solid tumours was shown to increase previously impaired NK and T-cell proliferative and cytotoxic function (Robertson et al, 1999).

Monitoring the response

It is probable that, if cellular immunotherapy is to be of benefit in clinical oncology, this will be in the setting of minimal residual disease (MRD) following other established therapies. If sensitive techniques are available to monitor MRD, this will be useful for the evaluation of DC-based treatment. Apart from this, however, early information about responses will depend upon laboratory evaluation of the immune response rather than tumour progression or patient survival. There is much debate about the best techniques for achieving this. The skin DTH response to the vaccinating peptide or to peptide-loaded DCs may be of value (Disis et al, 1999). This may correlate with proliferative T-cell responses in vitro and the limiting dilution assay is a useful tool for the estimation of low numbers of proliferating precursors. Cytotoxicity assays that rely on isotope labelling of target cells are commonly employed, but are inconsistent and subject to difficulties with target cell labelling. These may be replaced by dye-based methods (Nociari et al, 1998) or by techniques that depend on an enzyme or cytokine release ‘read-out’. Cytokine release by responding effector cells is measured in the ELISPOT test which has a 1:100 000 cell sensitivity, but depends generally on knowing the identity of the tumour antigen and does not identify the responding cell (Lalvani & Hill, 1998). Alternatively, flow cytometry can identify intracellular cytokine production (e.g. interferon γ) and simultaneously provide sensitive data on both the nature and frequency of the precursor cell (Maino & Picker, 1998). Finally, the clonality of the responding T-cell populations may be assessed by analysing the frequency of TCR molecular rearrangements of the variable (V) sequence genes and, more specifically, by labelling the TCR with antigen-specific tetramer constructs (Altman et al, 1996).

DCs In leukaemia

  1. Top of page
  2. Immune responses to human tumour antigens
  3. Dendritic cells: an available source of potent apcs for immunotherapy
  4. Clinical and preclinical studies with in vitro-generated DCs
  5. DCs In leukaemia
  6. Conclusion
  7. References

It seems probable that most of the GVL effect observed after allogeneic transplantation and DLI is directed against minor histocompatibility antigens, mHAg (HA-1, HA-2), which are expressed on haemopoietic precursors in the context of MHC molecules (HLA-A*0201, Mutis et al, 1999). This phenomenon, like GVHD itself, has been shown to depend on the integrity of the host DCs, although whether donor DCs are also involved remains unresolved (Shlomchik et al, 1999). The peptide structure of the HA-1 and HA-2 minor antigens is known and it has been possible to generate allogeneic CTLs in vitro to these peptides loaded on cultured DCs (Mutis et al, 1999). These cells were lytic to acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) cells, but not to fibroblasts. It may prove possible to scale-up this procedure for adoptive immunotherapy which could be as effective as DLI with a possible reduced risk of non-haemopoietic cell damage, i.e GVHD (Falkenburg et al, 1997).

Leukaemia-specific targets There are a number of candidate gene products that may potentially be recognized as leukaemia specific and therefore targets for autologous vaccine design in leukaemia. These include the bcr–abl protein fusion gene product expressed exclusively in CML cells, as well as overexpression of some other less-specific antigens such as proteinase-3. A peptide, PR-1, derived from this protein was capable of generating a T-cell line that lysed both CML cells and AML blasts. PR-1-specific T cells have also been identified in CML patients responding to either interferon α or allogeneic bone marrow transplantation (Molldrem et al, 1996, 2000). In addition, the PRAME oncogene (Matsushita et al, 1999) and the Wilm's tumour antigen-1 (WT-1) gene product are proving of interest. The transcription factor WT-1 is expressed on normal CD34+ progenitor cells but considerably overexpressed in such cells in AML and CML. It has been possible to generate WT-1 peptide-specific allogeneic CTLs against leukaemic progenitors that leave normal CD34+ cells unaffected (Gao et al, 2000), raising the possibility of adoptive CTL immunotherapy with a reduced risk of GVHD. The bcr–abl gene product has a novel junctional amino acid sequence. This may be presented on cultured DCs in vitro to induce a CD4+ T-cell line which specifically recognizes the b3a2-containing cell lysates (Mannering et al, 1997). In an alternative approach, peptides eluted from AML blasts were screened for binding to HLA-A2 or HLA-B7 molecules and these fractions could induce CD8+ CTL clones in healthy PBMCs which responded exclusively to patient leukaemic cells (Ostankovitch et al, 1998).

Leukaemia-derived DCs It is notable that the infusion of allogeneic donor lymphocytes to patients with leukaemic relapse after bone marrow transplantation is particularly effective in CML. It has been postulated that one reason for this is the potential for CML cells to fully differentiate into functioning mature DCs which, after all, are known to be mainly of pluripotential stem cell origin. It has indeed been possible to grow mature DCs from chronic-phase CML cells and these originate from the leukaemic clone (Choudhury et al, 1997). It was observed that autologous T cells, stimulated by these DCs, were specifically cytotoxic to CML cells and inhibited proliferation of CML progenitors in culture. This suggests not only that the leukaemic DCs may present CML-specific antigen to T cells, but that they may elicit killing of leukaemic but not normal bone marrow cells. Investigators in Leiden have described an allogeneic but leukaemia-specific effect of donor-derived T cells in patients after DLI for relapsed CML (Smit et al, 1998). In this case, T-cell clones that had expanded within the patient, recognized and inhibited the proliferation of a CD34+ CML precursor (PCILp). A subsequent study used this approach first to identify donor PCILp and then to expand these ex vivo, and used the cells for adoptive immunotherapy of a patient in accelerated phase after stem cell transplant. The T-cell clones were CD4+ rather than CD8+ and resulted in a complete molecular remission (Falkenburg et al, 1999).

These encouraging results have been observed in CML and it may be speculated that leukaemia-derived DCs are presenting antigens in an allogeneic setting to elicit CML-specific responses. There is also now evidence that leukaemic blasts in AML may have an analogous potential for antigen presentation. This has been observed in vitro following transfection of blasts with the CD80 accessory molecule (Mutis et al, 1998) and in a murine leukaemia model using cells transfected with the IL-12 gene (Dunussi-Joannoppoulos et al, 1999). In the latter case, protection and regression of established leukaemia was achieved which could not be replicated by systemic IL-12 administration. A simpler approach to the generation of leukaemia-derived DCs in AML is suggested by a number of recent reports that show that AML leukamia blasts may differentiate in culture over 7–14 d under the influence of cytokines known to be important in normal DC development. In over 50% of unselected AML cases, functioning DCs were obtained in GM-CSF + TNFα with either SCF or IL-4 (Robinson et al, 1998; Charbonnier et al, 1999), and also in serum-free conditions with GM-CSF + TNFα+ IL-4 (Cignetti et al, 1999) or with CD40L (Choudhury et al, 1999). Intriguingly, cytokines may not be essential to the process of leukaemic cell differentiation. Engels et al (1999) have shown that CD33+ CML cells undergo very rapid acquisition of DC characteristics when exposed to a calcium ionophore. The leukaemic identity of these cells was confirmed in most cases by the demonstration of a clonal marker. In one report, specific CTLs could be generated against autologous blast cells (Choudhury et al, 1999).

As has been mentioned previously, tumour cells may have the capacity to downregulate T-cell responses through secreted products such as VEGF or IL-10 (Gabrilovich et al, 1996, 1998). In leukaemia, a similar situation may prevail. A leukaemia cell line transfected with CD80 was shown to be capable of inducing an allogeneic T-cell response, but the proliferating T cells were defective in their production of γ-interferon and IL-2 (Buggins et al, 1999). These cytokines are integral to an effective Th1 response, so these in vitro findings are reminiscent of the report mentioned earlier indicating a similar deficiency in the freshly isolated melanoma-specific T cells from cancer patients (Lee et al, 1999). One might therefore conclude that, in patients with malignant disease, tumour- or leukaemia-specific T cells capable of response to tumour antigens may subsequently be rendered anergic through a variety of mechanisms. Such observations lend support to strategies of DC vaccination that seek to enhance immune responses to tumour vaccines by the concurrent administration of cytokines (Shimuzu et al, 1999).


  1. Top of page
  2. Immune responses to human tumour antigens
  3. Dendritic cells: an available source of potent apcs for immunotherapy
  4. Clinical and preclinical studies with in vitro-generated DCs
  5. DCs In leukaemia
  6. Conclusion
  7. References

There is now good evidence that malignant cells of many tumour types may be recognized and eliminated by the immune system. The availability of DCs and their capacity to enhance tumour antigen recognition is just one of a number of promising approaches to immunotherapy in malignant disease. As has been described, the boundary between tolerance and activation is a fine one and those involved in this endeavour need to be mindful of the possibly crucial role of peptide selection, route and timing of vaccine delivery and techniques to ensure sustained helper, as well as cytotoxic, T-cell responses. In haematological malignancy, it may be that a major significance of such therapies will be in the context of the allogeneic presentation of tumour antigens. Reliable methods of measuring patient responses to DC vaccines are still not optimal and this is particularly the case with those diseases in which the tumour antigens remain poorly characterized.


  1. Top of page
  2. Immune responses to human tumour antigens
  3. Dendritic cells: an available source of potent apcs for immunotherapy
  4. Clinical and preclinical studies with in vitro-generated DCs
  5. DCs In leukaemia
  6. Conclusion
  7. References
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