• Dendritic cells;
  • Tumor antigens;
  • Vaccinations


  1. Top of page
  2. Abstract
  3. Introduction
  4. Definition of DCs and Methods for DC Generation
  5. The Choice of Antigen Source
  6. Routes of DC Delivery
  7. Immune Response to Vaccination
  8. Clinical Studies with DCs
  9. Conclusions and Perspectives
  10. Acknowledgements
  11. References

Dendritic cells (DCs) are specialized antigen-presenting cells whose immunogenicity leads to the induction of antigen-specific immune responses. DCs can easily be generated ex vivo from peripheral blood monocytes or bone marrow/circulating hematopoietic stem cells cultured in the presence of cytokine cocktails. DCs have been used in numerous clinical trials to induce antitumor immune responses in cancer patients. The studies carried out to date have demonstrated that DCs pulsed with tumor antigens can be safely administered, and this approach produces antigen-specific immune responses. Clinical responses have been observed in a minority of patients. It is likely that either heavy medical pretreatment or the presence of large tumor burdens (or both) is among the causes that impair the benefits of vaccination. Hence, the use of DCs should be considered in earlier stages of disease such as the adjuvant setting. Prospective applications of DCs extend to their use in allogeneic adoptive immunotherapy to specifically target the graft versus tumor reaction. DCs continue to hold promise for cellular immunotherapy, and further investigation is required to determine the clinical settings in which patients will most benefit from the use of this cellular immune adjuvant.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Definition of DCs and Methods for DC Generation
  5. The Choice of Antigen Source
  6. Routes of DC Delivery
  7. Immune Response to Vaccination
  8. Clinical Studies with DCs
  9. Conclusions and Perspectives
  10. Acknowledgements
  11. References

The first demonstration that tumor rejection antigens exist goes back to the late 1980s when tumor-infiltrating lymphocytes from melanoma patients were shown to lyse HLA-matched melanoma cell lines, suggesting the existence of shared melanoma antigens [1]. In the subsequent years, the first genes encoding tumor antigens (such as tyrosinase, gp-100, the MART and MAGE genes) were cloned, and the immunogenic epitopes were identified. These and subsequent studies pointed out that tumors often upregulate the expression of molecules that are normally suppressed or expressed at much lower levels in adult tissues. T lymphocytes capable of recognizing these antigens usually exist in the periphery, possibly due to the lack of presentation of these antigens during thymic selection or lower avidity of the T-cell receptor (TCR) [1,2]. However, in most cases, the immune system fails to recognize and destroy tumor cells that may give rise to clinically relevant malignancies. The tumor escape mechanisms include the inefficiency of tumor cells as antigen-presenting cells (APCs) and the lack of efficient contact between immune system and tumor cells [3,4].

There is also evidence that antitumor immune responses can extinguish established tumors, especially in patients affected by melanoma or renal cell carcinoma. Infiltration of the primary tumor with lymphocytes has been associated with a better prognosis in different types of malignancies [5]. Similarly, immune-mediated paraneoplastic syndromes characterized by an immune response directed against antigens shared by tumor and normal tissues (such as the central nervous system) have been associated with a better clinical outcome and even with spontaneous tumor regressions [6]. However, such spontaneous immune responses are rare and still remain largely elusive. Thus, the goal of modern tumor immunotherapy is to trigger the immune system in order to mimic such rejection events and improve clinical outcome.

Particularly, the induction CD8+ cytotoxic T lymphocytes (CTLs) directed against tumor epitopes in vivo is the desirable effect of a specific immunotherapy approach, given that these immune effectors are mainly responsible for tumor rejection [1]. These lymphocytes recognize via the TCR 8–11 amino acids–long peptide epitopes in the context of the HLA class I molecules. Upon encounter with cells that express the target antigen, the CTLs activate their lytic machinery and kill the cells. The induction of CD4+ helper T cells also plays a major role in antitumor immunity, and immunization strategies should probably take into account providing immunogenic epitopes for these lymphocytes.

The first immunization strategies for cancer patients often involved the administration of tumor lysates or irradiated tumor cells together with immunological adjuvants such as bacillus Calmette-Guérin (BCG) [2]. This vaccination method has recently been reported by Vermorken et al. [7] to be possibly associated with a protection from relapses in patients with stage-II colorectal cancer, but this study needs further confirmation by other groups. Such an approach is limited by the requirement of sufficient amount of tumor material and by potential concerns related to the administration of autologous tumor cells, though irradiated, to patients in clinical remission of disease.

Recent advances in the knowledge of the immune system have opened new perspectives for the development of antitumor immunization strategies. In particular, the administration of immunogenic APCs such as dendritic cells (DCs) loaded with tumor antigens is now considered one of the most promising approaches to the specific cancer immunotherapy and is being evaluated in many cancer centers for different malignancies and in different clinical settings.

DCs are leukocytes that are highly specialized in the capture and presentation of antigens to T cells [8,9]. They are presently believed to control the induction (and, possibly, the suppression) of antigen-specific immune responses in vivo [10]. DCs for clinical use can be generated in sufficient numbers from circulating precursors, including peripheral blood CD14+ monocytes and CD34+ stem cells [11,12]. Injection of DCs loaded with tumor-associated antigens (TAAs) into patients was shown to break tolerance and to induce antitumor cytotoxic immune responses in vivo [1115]. The DC-based clinical trials performed so far have demonstrated that this form of immunotherapy is feasible and safe [1115]. Moreover, some studies reported cases of tumor regression or growth arrest following DC administration.

Definition of DCs and Methods for DC Generation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Definition of DCs and Methods for DC Generation
  5. The Choice of Antigen Source
  6. Routes of DC Delivery
  7. Immune Response to Vaccination
  8. Clinical Studies with DCs
  9. Conclusions and Perspectives
  10. Acknowledgements
  11. References

The current model of antigen presentation places DCs at the center of immunity, since these cells are viewed as those APCs in charge of capturing antigens in peripheral tissues and presenting them to T lymphocytes in the secondary lymphoid tissues. Recent evidence suggests that DCs not only present dangerous antigens for the induction of protective immunity but also acquire “self” products and constitutively present them in a tolerogenic fashion. This phenomenon is presently believed to contribute to the maintenance of self-tolerance (Fig. 1) [10,16]. The factors determining the immunogenicity of DCs, and thus the outcome of antigen presentation, are still under investigation. These factors are also a matter of great interest from the clinical perspective, given the necessity to present TAAs in an immunogenic manner. It seems likely that the degree of maturation achieved by the DCs plays a key role in this context [17], whereby the influence of factors encountered in the peripheral tissues (both pathogen-derived products and autologous cytokines and prostaglandins) may be crucial [18,19]. Regulatory T cells probably also act at the interface between DCs and T lymphocytes and contribute to avoid the expansion of autoreactive T-cell clones [20].

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Figure Figure 1.. Immune outcomes of antigen presentation by dendritic cells (DCs). Bone marrow (BM)–derived DC precursors migrate via the bloodstream to the peripheral tissues. From these, DCs migrate to the afferent lymphoid tissues, where they present antigens to lymphocytes. It is believed that, in the presence of danger signals such as inflammatory products or necrosis, DCs acquire enhanced immunogenicity, thereby leading to the stimulation of an antigen-specific immune response. Conversely, in the absence of infection, inflammation, or necrosis, the DCs reaching the lymphoid organs tolerize the immune system to self antigens.

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No DC-specific marker has been described so far, hence DCs are typically defined based on a combination of parameters that include morphology, phenotype, cytokine secretion, immunostimulatory capacity, chemokine and chemokine receptor pattern, and migration in response to chemotactic stimuli. Human DCs are characterized by the surface expression of high amounts of major histocompatability complex (MHC) class II molecules and the absence of lineage markers. The DC phenotype varies, depending on the stages of maturation and differentiation. CD1a is preferentially expressed on human immature myeloid DCs, whereas CD83 is typically upregulated in response to activation stimuli such as tumor necrosis factor alpha (TNF-α), Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS], cytidylyl-2p,5p-phosphoryl guanosine [CpG], double-stranded RNA [dsRNA]), prostaglandin E 2 [PGE2], or T cell–derived signals including CD40 ligand and interferon-gamma (IFN-γ) [8]. Recent data indicate that CD83 is likely to be involved in T-cell stimulation and may be downregulated by viruses such as herpes simplex virus 1 [21]. DCs also express adhesion molecules, including CD11a, CD11c, CD50, CD54, and CD58, as well as the costimulatory molecules CD80 (B7.1), CD86 (B7.2), dectin, and CD40 [8,22]. Importantly, in response to activation stimuli, DCs express CCR7, the corresponding receptor for the chemokine macrophage inflammatory protein 3 alpha (MIP3β), that directs DC migration to the afferent lymph nodes. DCs are also characterized by potent immunostimulatory capacity, which can be detected in mixed leukocyte reaction (MLR) and by the ability to prime antigen-specific lymphocytes, both in vitro and in vivo [2325]. These functional properties are enhanced upon exposure to activating stimuli. Finally, DC immunogenicity is largely determined by the capacity to secrete cytokines such as TNF-α, interleukin 6 (IL-6), IL-12, IL-15, and IL-18, which contribute to activate lymphocytes and prime the subsequent immune response [8].

DCs are divided into myeloid and plasmacytoid DCs. Although these two cell types share several morphological, phenotypical, and functional properties, they exert different functions [8]. The plasmacytoid DCs, also named DC2s, are characterized by positivity for the IL-3 receptor α, as well as expression of the TLR 9, TLR 10, and of the pre-T-cell receptor α chain. They are important producers of IFN-α in response to viral infections (including HIV), CpG dinucleotides, and CD40 ligand, and recently it has been suggested that they provide key help for antibody-mediated immune responses [8,26]. DC2s acquire potent allostimulatory capacity upon culture with IL-3 or microbial stimuli, though they fail to produce IL-12, which is important for initiating cell-mediated immune responses. Given their still elusive role and immunogenicity, this APC subtype has not been employed in clinical studies so far.

Conversely, the so-called myeloid DCs have been fairly well characterized and largely used as a cellular adjuvant in immunotherapy studies. Typically, these include the Langerhans cells and the interstitial DCs [8]. The isolation of these cell types from peripheral tissues is difficult and only yields trace amounts of DCs. However, large numbers of cells with the DC phenotype and functional properties can be obtained by exposing bone marrow precursors (CD34+) or peripheral blood monocytes to cocktails of cytokines that typically include GM-CSF, TNF-α, IL-4 or IL-13, and Flt-3 ligand [27]. This cocktail of cytokines induces hematopoietic stem cells to differentiate into DCs, part of which exhibit a phenotype of Langerhans cells [8]. The use of stem cell–derived DCs is particularly appealing in the setting of bone marrow and peripheral blood progenitor cell (PBPC) transplantation when part of the marrow harvest or apheresis product could be used for the generation of vaccine [28]. In this context, one should take into account that the cytokines commonly used for stem cell mobilization have immunological effects; hence, it may be preferable to use GM-CSF instead of G-CSF, which appears to preferably expand DC2s [29,30]. GM-CSF and IL-4 (or IL-13) are sufficient to induce peripheral blood monocytes (CD14+, HLA-DR+, CD11c+, CD1) to differentiate into DCs in an immature state, which are believed to resemble the features of interstitial DCs [23,8]. These APCs are efficient in antigen uptake but show reduced capacity to stimulate T-cell proliferation and to induce antigen-specific CTLs [8, 24, 31]. The immunogenicity of these DCs can be enhanced by exposing them to activation stimuli such as TNF-α, TLR ligands, CD40 ligand, monocyte-conditioned medium (MCM), or the MCM mimic containing IL-1β, TNF- IL-6, and PGE2 [813]. Some authors have reported that stem cell–derived DCs may be better CTL inducers than monocyte-derived DCs [32]. Unfortunately, a proper comparison of the biological and functional properties of these two different types of DCs is still lacking. Only three published clinical trials have made use of stem cell–derived DCs [3335], and no study has ever directly compared them with monocyte-derived DCs with respect to their capacity to induce antigen-specific immunity in vivo. Therefore, given the reduced number of cytokines required to generate monocyte-derived DCs, these still remain the most accessible APCs used for these kinds of studies.

Alternatively, some groups have made use of peripheral blood DC precursors (CD14, HLA-DR+, CD11c+, CD1+) enriched via subsequent density gradients centrifugation steps [3640]. Interestingly, these APCs can be expanded several fold via in vivo administration of Flt-3 ligand, thus increasing DCs yields [14,39]. A recent paper comparing this type of DCs with the monocyte-derived DCs found that the peripheral blood DCs are more susceptible to maturation stimuli and are equally effective as the monocyte-derived DCs in MLR and in antigen presentation [41]. However, the peripheral blood DCs have increased migratory capacity and reduced cytokine production. The clinical results obtained with these DCs are encouraging and indicate that these circulating DCs represent a suitable type of APC for cancer immunotherapy.

Recent data suggest that PGE2 may be necessary to determine DC responsiveness to MIP3β, which attracts them to the afferent lymph nodes from the injection site [42]. This requirement may particularly apply to monocyte-derived DCs, whereas circulating CD1+ DCs may not need this prostaglandin in order to migrate [41]. In light of this evidence, addition of PGE2 to the culture medium before DC injection may help improve vaccination efficacy, especially when DCs are generated from monocytes.

Finally, some groups have reported that immature DCs are less immunogenic than mature DCs in vivo and possibly induce antigen-specific tolerance instead of immunity [43,44]. In fact, this point still remains controversial, since immature DCs were shown to rapidly migrate from the site of injection to the afferent lymph nodes [45], and some of the clinical studies that have employed these APCs reported positive results [40, 4648]. The upcoming results of the ongoing clinical trials will probably help clarify this issue.

In several studies, monocytes have been isolated by plastic adherence in tissue culture plates and further cultured in medium containing the necessary cytokines. However, the CD14+ monocytes can also be efficiently isolated by positive or negative selection [13]. Monocytes can be obtained by repeated blood draws, so that freshly generated DCs are available for each vaccine injection. From 100 ml blood, up to 20 million monocyte-derived DCs can easily be obtained [49]. Alternatively, the DC precursors (monocytes or PBPCs) can be collected by a single apheretic procedure and frozen down in aliquots, either before or after differentiation into DCs has been induced [13]. Several cell factories are now commercially available which allow generating DCs in closed systems and thus match the increasingly demanding good manufacturing practice (GMP) guidelines. Berger et al. [50] reported the generation of an average of more than 300 million monocyte-derived DCs from a single apheresis in a closed system, and similar results have been obtained by Motta et al. [51].

The Choice of Antigen Source

  1. Top of page
  2. Abstract
  3. Introduction
  4. Definition of DCs and Methods for DC Generation
  5. The Choice of Antigen Source
  6. Routes of DC Delivery
  7. Immune Response to Vaccination
  8. Clinical Studies with DCs
  9. Conclusions and Perspectives
  10. Acknowledgements
  11. References

Anticancer vaccinations attempt to elicit tumor-directed CD8+ CTLs that lyse tumor cells presenting MHC class I–associated peptides derived from tumor-associated proteins. Several different strategies are currently available to deliver antigens into DCs during the ex vivo manipulation for further presentation to T cells in the recipient. DCs can be pulsed with synthetic peptide epitopes derived from known TAAs such as MUC1, Her-2/neu, survivin, tyrosinase, telomerase, CEA, p53, MAGE, or Melan-A/MART [1, 1214, 5255]. Although most of these peptides are designed to bind HLA-A2, the most common HLA class I molecule among Caucasians, several peptide epitopes have been identified that bind to other HLA class I alleles. Moreover, HLA class II binding peptides have also been reported that either unspecifically trigger CD4+ lymphocyte activation or are derived from tumor antigens and induce antigen-specific CD4+ helper T cells [1, 53, 56]. Many of these peptides are now commercially available and ready to use under GMP conditions. Hence, peptide-based vaccinations are potentially applicable to most patients. In some immunization studies, peptides are injected directly into the patients without previous incubation with DCs ex vivo. For this kind of approach, the peptides are usually coinjected with immune cytokines such as GM-CSF, which favors DC migration and activation in situ, or with incomplete Freund adjuvant [57,58]. Major drawbacks related to the use of peptides are (a) the restriction to some HLA class I alleles, (b) the need to determine the expression of the target antigen by a tumor, and (c) the likelihood that targeting single or few tumor epitopes may impede the detection of tumor cells that downregulate those antigens. However, to some extent, tumor escape may be prevented by the expansion of lymphocytes directed against epitopes other than those used for immunization, a phenomenon named “epitope spreading,” which has already been observed in some clinical studies [49,59].

Another approach is to use recombinant proteins as antigens. These are captured by DCs, then processed and presented in the form of immunogenic peptides in the context of HLA molecules. This approach bypasses the HLA restriction of the peptides and was successfully applied to the treatment of patients with follicular lymphoma [36,37]. In this case, DCs were pulsed ex vivo with tumor-specific idiotype protein to induce antitumor immunity, with encouraging clinical results. The effectiveness of the same approach for the treatment of myeloma is still under investigation [60,61]. Similarly, a recombinant prostatic acid phosphatase (PAP) has been used to load autologous DCs by Fong et al. [38]. An alternative strategy is gene-based delivery of TAAs into DCs. DCs can be transduced with recombinant viruses (retroviral or adenoviral vectors, vaccinia virus) or transfected with RNA encoding for a specific tumor antigen [11, 12, 14, 62].

Several other approaches also exist that, instead of using single or few antigens, make use of whole tumor material as an antigenic source. These approaches use tumor lysates, dead tumor cells (apoptotic bodies, necrotic cells), DCs fused with tumor cells, or total tumor RNA [11, 12, 14]. All of these methods were shown to induce immunity against the parental tumor and are being evaluated in the clinical setting. Importantly, whole tumor–derived materials represent the entire antigenic repertoire of a tumor; thus the resulting immune response simultaneously targets many tumor antigens. In fact, it is likely that preferential expansion of CTLs directed against immunodominant epitopes will happen in some cases; this may be related to the higher frequency of some epitope-specific effectors or to the strong immunogenicity of some tumor-derived peptides (or both) [63,64]. One potential advantage of the use of RNA compared with the other tumor-derived materials cited above is that methods exist for the unspecific amplification of messenger RNA [6567]. This translates into the applicability of this method also in those cases when small tumor specimens such as needle biopsies would not be sufficient to obtain lysates or apoptotic tumor cells for DC pulsing. Moreover, the antigens encoded by the transfected RNA may be processed and presented on both HLA class I and class II molecules, thus inducing CD8+ as well as CD4+ antitumor lymphocytes [64,68]. The elicited immune response, at least according to the in vitro experiments, seems to be restricted to immunodominant tumor epitopes while saving nonmalignant autologous cells [6470]. Thus, RNA transfection of DCs appears as a very attractive approach for the induction of antitumor CTLs in a variety of malignancies.

In vivo DC loading has also been evaluated in preclinical models. In particular, immunization with DNA vaccines by gene gun represents an attractive approach; here gold particles coated with expression plasmid DNA encoding target genes are “bombarded” into the skin [71,72]. This procedure transfects plasmid DNA directly into the DCs present in the skin. Transfected DCs express the encoded antigen and present the processed peptides to the antigen-specific T cells to initiate an immune response in the afferent lymph nodes. In light of the results reported by Sudowe et al. [71] and Garg et al. [72] in the animal model, this approach may reveal as an effective method for antitumor immunity induction.

Routes of DC Delivery

  1. Top of page
  2. Abstract
  3. Introduction
  4. Definition of DCs and Methods for DC Generation
  5. The Choice of Antigen Source
  6. Routes of DC Delivery
  7. Immune Response to Vaccination
  8. Clinical Studies with DCs
  9. Conclusions and Perspectives
  10. Acknowledgements
  11. References

The best route of administration for the ex vivo–generated and manipulated DCs to generate an efficient immune response in vivo is still to be defined. Different studies have used different routes of delivery: DCs have been injected intradermally (i.d.), subcutaneously (s.c.), intravenously (i.v.), and intranodally, and the intratumoral injection of DCs has also been proposed [73]. A recent report by Mullins and coworkers [74] suggests that s.c. injection of antigen-loaded DCs may confer more extensive protection from tumor growth than i.v. delivery because of the induction of memory CD8+ T cells in both spleen and lymph nodes. Studies in humans indicate that i.v.-injected DCs may preferentially localize to the lungs and, afterward, to spleen and liver. Conversely, i.d. injection may result in DC migration to the afferent lymph nodes [75]. A preliminary report that compared s.c., i.d., and i.v. injections of DCs loaded with recombinant PAP showed that Th1 immune responses are more likely induced by i.d. injection than by other delivery methods [76]. Consistent with this, another trial observed stronger immunity after i.d. injection than after i.v. injection of peptide-loaded DCs [59]. However, significant immune responses also have been noticed in studies that made use of s.c. and i.v. injections. DCs injected i.d. or s.c. are normally administered in close proximity of the inguinal, axillary, or cervical lymph nodes. In fact, the rate of DCs reaching the afferent lymph nodes may be small (less than 10%) [13]. Interestingly, it has recently been suggested that DC migration from the site of injection to the lymph nodes and the subsequent immune response may be enhanced by the previous topical administration of inflammatory factors. This should lead to upregulation of the CCR7 ligand CCL21 in the local lymphoid vessels, thus favoring DC traffic through the vessels to the lymph nodes [77]. The intranodal injection bypasses the migration step and sets DCs into direct contact with the lymphoid tissue. Nestle and colleagues [46, 78, 79] were the first to report intranodal DC administration in humans, and they detected tumor regressions in some patients. A recent study by Bedrosian et al. [80] found that intranodal administration of peptide-pulsed DC was superior to the i.v. and i.d. routes for T-cell sensitization and delayed-type hypersensitivity (DTH) priming. However, this way of delivery often necessitates an ultrasonographic visualization of the lymph nodes to deliver the injection, thus implying additional instruments and skill requirements.

Different schedules of DC administration have been employed in the clinical trials. Most of the studies have made use of weekly, biweekly, or monthly injections with at least two vaccine administrations [1214]. In some cases, in the presence of clinical response, booster injections have been administered over several months. In fact, it is still unclear whether the antitumor immunity elicited by vaccination would last in the absence of DC administration or if it necessitates repeated recall vaccine injections. Different DC numbers, including escalating doses of DCs, have been tested [39, 47, 48]. Some data suggest that increasing the number of DCs may improve the outcome of vaccination, though other studies did not find any correlation between the elicited immune response and the number of DCs used [59, 81, 82].

Immune Response to Vaccination

  1. Top of page
  2. Abstract
  3. Introduction
  4. Definition of DCs and Methods for DC Generation
  5. The Choice of Antigen Source
  6. Routes of DC Delivery
  7. Immune Response to Vaccination
  8. Clinical Studies with DCs
  9. Conclusions and Perspectives
  10. Acknowledgements
  11. References

The detection of the immune response to tumor antigens following vaccination represents one of the major endpoints of the clinical vaccination studies. The DTH assay represents a possible approach to this goal. It is usually performed by intradermal injection of tumor-derived material or DCs loaded with tumor antigen(s) before and after the vaccination course [1214]. In the case of tumor regression after vaccine administration, the detection of either infiltrating lymphocytes or inflammatory cells (or both) in tumor specimens, whenever these are easily reachable, should be performed in order to correlate the clinical outcome with the elicited immune response [83]. In some cases, the tumor-infiltrating lymphocytes can be isolated and further characterized [46]. However, in most studies the lymphocytes reacting to the tumor antigens have been detected in the peripheral blood mononuclear cells. The T cells specific for a defined tumor-derived epitope can be tracked via different approaches, which typically include ELISPOT, cytokine secretion assay from Miltenyi, intracellular staining for IFN-γ, tetramers, proliferation assays, ELISAs, cytotoxicity assays, and real-time polymerase chain reaction for IFN-γ [49, 59, 84]. The results obtained with different methods are often, though not necessarily, consistent, and further refining of these techniques is still required [59].

When whole tumor–derived material (tumor lysates, total tumor RNA, fusions, tumor-derived peptides) is used as an antigen source for vaccine preparation, the autologous tumor cells or tumor material, when available, can be used to determine immunoreactivity before and after vaccine administration [1214, 85]. The same DCs loaded with tumor antigens may work as a suitable target in immunological monitoring [48, 6470]. In this kind of approach, the antigens involved in the immune response are often not known. However, in selected patients who express defined HLA alleles and tumor antigens, the immunization against known tumor epitopes could also be evaluated [48].

Some studies found no correlation between the immune response to the antigen used for immunization and the clinical outcome, since some tumor regressions were observed in patients who showed little response to vaccination [13,59]. Besides, different studies have already reported the expansion of lymphocytes specific for different tumor epitopes following vaccination [49], and in the study by Butterfield et al. [59] the only complete clinical remission was induced in a patient showing epitope spreading. These data indicate that immunity versus an array of different tumor antigens, including molecules not present in the vaccine preparation, should possibly be monitored.

An improved characterization (phenotypic and functional) of the antitumor T lymphocytes will also be necessary for a better understanding of the lymphocyte subsets involved in tumor rejections. This goal can be pursued by combining tetramer staining with antibodies for surface markers such as CD45RA, CD45RO, CD27, CCR7, CD28, and CD25 or with intracellular staining for cytokines such as IFN-γ, IL-4, IL-10 [86]. The antigen-specific lymphocytes can be isolated by fluorescence-activated cell sorter (FACS) or magnetic cell sorting (MACS) technology, expanded, and further characterized.

Finally, pulsing DCs with immunogenic epitopes (such as influenza peptides or CD4 epitopes) or antigens (such as keyhole limpet hemocyanin [KLH] or HBsAg) has been performed with the double intent to exploit them as an immune adjuvant and to use them as an immunological tracer to evaluate DC priming efficacy in vivo and responsiveness of the immune system to vaccine administration [33, 43, 49, 87]. The use of these immunogens may be particularly useful in order to compare different vaccine administration routes and schedules or DCs generated according to different protocols.

Clinical Studies with DCs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Definition of DCs and Methods for DC Generation
  5. The Choice of Antigen Source
  6. Routes of DC Delivery
  7. Immune Response to Vaccination
  8. Clinical Studies with DCs
  9. Conclusions and Perspectives
  10. Acknowledgements
  11. References

Since the publication in 1996 of the first DC-based vaccination trial performed by Hsu et al. [36], more than 60 related studies have been published. The most frequently targeted diseases are represented by melanoma (15 reports) and prostate cancer (12 reports). Altogether, the clinical experience has demonstrated that DCs can be administered safely, with no significant side effects, except for a few cases of vitiligo reported in patients vaccinated for melanoma. In most of the clinical studies, an immune response directed against the antigens used for vaccination could be detected, suggesting that DCs loaded with antigens are immunogenic in vivo. Nonetheless, clinical responses were only induced in a minority of patients. These results can partially be explained by the immunosuppressive effects of previous chemotherapy or radiotherapy administration and by the advanced stage of disease with large tumor burdens of the patients selected for the studies. Table 1 lists the immunological and clinical results of the DC-based clinical studies published in the past 2 years. Given space constraints, we specifically present only some of these trials while referring to the respective references for the others.

Table Table 1.. DC–based anticancer vaccination studies
  1. a

    Abbreviations: CEA, carcinoembryonic antigen; imm, immature DCs; mat, mature DCs; RCC, renal cell carcinoma.

AuthorsDiseaseSource of antigenNo. of patientsDendritic cell typeResponse to tumor antigenComplete responsePartial response or mixed responseStable disease
Su et al. [48]RCCTotal tumor RNA10imm6/7   
Marten et al. [97]RCCFusions12mat7/12004
Marten et al. [98]RCCTumor lysates15imm3/13017
Oosterwijk-Wakka et al. [99]RCCTumor lysates12imm0000
Holtl et al. [89]RCCTumor lysates35mat5/6217
O'Rourke et al. [82]MelanomaIrradiated autologous tumor cells17mat 330
Butterfield et al. [59]MelanomaMART peptide18imm18/18102
Smithers et al. [87]MelanomaTumor peptides18imm 121
Krause et al. [100]MelanomaFusions17mat 011
Schuler-Thurner et al. [88]MelanomaPeptides16mat16/16108
Banchereau et al. [33]MelanomaPeptides18mat16/18072
Fong et al. [38]Prostate cancerRecombinant prostatic acid phosphatase21mat21/21006
Heiser et al. [47]Prostate cancerProstate-specific antigen RNA13imm13/1306/7 (tumor marker)0
Maier et al. [78]Cutaneous T-cell lymphomasTumor lysates10mat8/8140
Morse et al. [101]Muir-Torre syndrome colon cancerCEA RNA24imm“in selected patients”1 (tumor marker)23
Sadanaga et al. [102]Gastrointestinal cancersMAGE-3 peptides12imm4/8030
Fong et al. [39]Colon and non-small cell lung cancerCEA peptide12mat7/12212
Timmerman et al. [37]Follicular lymphomaIdiotype10mat8/10220
Reichardt et al. [61]MyelomaIdiotype12mat2/12   
Lin et al. [90]Nasopharyngeal cancerEpstein-Barr virus peptides16mat9/16020
Pecher et al. [103]Solid malignanciesMUC1 DNA10imm4/10000
Chang et al. [104]Solid malignanciesTumor lysates14imm4/10020
Hernando et al. [105]Gynecological cancersTumor lysates8mat2/8020
Geiger et al. [106]Pediatric solid tumorsTumor lysates15imm3/7015
Kikuchi et al. [107]Malignant gliomaFusions10mat6/10020

Several groups have recently reported the efficacy of DC vaccinations for the treatment of melanoma. Butterfield et al. [59] recruited 18 melanoma patients, 10 of whom had measurable disease, and assigned them to receive three biweekly injections of 105, 106, or 107 DCs, which were injected either i.v. or s.c. Thus, a total of six groups of patients can be identified in this study. DCs were generated from adherent peripheral blood mononuclear cells collected in a single leukapheresis and frozen down in aliquots. The DCs used were generated by GM-CSF and IL-4, thus were likely immature DCs, and they were pulsed with a class I restricted MART 1 peptide. The authors made use of different assays to detect the antigen-specific immune response: these included MHC class I tetramer, IFN-γ ELISPOT, IFN-γ and IL-4 intracellular cytokine staining, and cytotoxicity assay. Interestingly, tetramers staining and IFN-γ ELISPOT were revealed as the most sensitive to detect the antigen-specific T lymphocytes in the peripheral blood and demonstrated that the i.d. injection route was more immunogenic than the i.v. one. One complete response and two stabilizations of disease were recorded in this study. Remarkably, in the only patient showing tumor rejection, expansion of lymphocytes specific for other melanoma-associated class I and class II epitopes could be detected. Finally, two cases of vitiligo following vaccine administration were reported in this study. A peptide-based DC vaccine was also used by Schuler-Thurner et al. [88], who demonstrated how mature monocyte-derived DCs loaded with HLA class II–restricted melanoma peptides can induce tumor-specific Th1 cells. In this study, numbers ranging from 12–28 million DCs were injected s.c. over a five-dose course, with immunity to the adjuvant KLH and to the tumor epitopes that emerged rapidly after the first vaccine administrations. Among the 16 fully evaluable patients, eight experienced stabilization of disease and one presented a complete clinical response. Encouraging results were also obtained by O'Rourke et al. [82] in a study employing mature monocyte-derived DC injected i.d. at biweekly intervals for six times and, afterward, at 6-week intervals. DCs were loaded with autologous tumor lysates, and two DC doses were compared: 1 million versus 5 million per injection. Of the initially 19 enrolled patients, 12 completed the treatment: 3 of these 12 achieved durable complete responses, and 3 had partial responses. Disease regression was not correlated with DC dose or with the development of DTH in response to autologous irradiated tumor.

Other trials have focused on the application of DCs to the immunotherapy of lymphoma. In a study published by Maier and colleagues [78], 10 patients with cutaneous T-cell lymphoma (CTCL) were vaccinated with mature monocyte–derived DCs pulsed with KLH and tumor autologous lysates. Patients received intranodal injection of the vaccine once a week for 8 weeks, with additional booster administrations, depending on the clinical response (median of 9.5 DC injections per patient). DTH reactions to DCs loaded with the tumor lysate developed in all of the eight evaluable patients, whereas the tumor lysate induced significant proliferation of the peripheral blood lymphocytes harvested after vaccination in three patients who presented clinical response. Five out of the 10 patients presented clinical responses (one complete response [CR] and four partial responses [PRs]).

Timmerman et al. [37] reported the vaccination of patients with follicular lymphoma by peripheral blood DCs isolated via density-gradient centrifugation. The patients enrolled in the study received three monthly i.v. infusions plus one injection 2–6 months later of DCs pulsed with KLH and the autologous tumor idiotype. Two weeks after each infusion, the patients received subcutaneous injections of tumor idiotype conjugated with KLH. Of the 35 patients included in this study, 23 mounted T-cell or humoral antiidiotype responses. Among the 10 patients with evaluable disease, there were two CRs and one PR, and in one patient with bone marrow–localized disease molecular remission was observed. Also, 25 patients were vaccinated after first chemotherapy. Among 18 patients with residual disease after chemotherapy, four achieved a complete remission after vaccination, and eight had stabilization of disease. Importantly, remissions (two CRs and one PR) of disease could be induced in patients progressing after vaccine administration by booster injections of idiotype-KLH conjugates.

Two different studies have evaluated the use of DCs pulsed with RNA for the treatment of solid tumors. Heiser et al. [47] vaccinated 13 patients with metastatic prostate cancer with three i.v. administrations of escalating doses (107, 3 × 107, and 5 × 107) of immature monocyte–derived DCs at biweekly intervals. A concomitant dose of 107 DCs was injected subcutaneously at each vaccination cycle. DCs were pulsed with prostate-specific antigen (PSA) RNA. Vaccination was associated with significant decrease in the log slope PSA in six of seven subjects; in three evaluable patients a transient molecular clearance of circulating tumor cells was observed. A similar study was conducted by an affiliated group for patients with metastatic renal cell carcinoma. In this case, Su et al. [48] used total tumor RNA for DC pulsing. The authors evaluated the frequency of antitumor lymphocytes by ELISPOT and cytotoxicity assay. In the ELISPOT, autologous DCs transfected with tumor RNA were used as the target. In one patient, the lytic activity of the ex vivo–generated CTLs before and after vaccination was determined against the RNA-pulsed DCs and autologous tumor cells. Out of 15 recruited patients, 10 completed the vaccination course. The authors detected expansion of tumor-specific T cells in six out of seven evaluable patients. Interestingly, an expansion of lymphocytes specific for the TAA hTERT, G250, and oncofetal antigen but not for self-antigens expressed by normal renal tissues could be detected. Since most of the patients underwent subsequent secondary therapies, the clinical outcome of vaccination was not an endpoint of the study. However, the authors refer a low tumor-related mortality (3/10) after a mean follow-up of about 20 months.

Some potential benefits associated with DC immunotherapy in renal cell carcinoma are also suggested by a study performed by Holtl and colleagues [89]. These authors vaccinated 35 patients by monthly i.v. or i.d. administration of mature monocyte–derived DCs loaded with lysates of autologous or allogeneic tumor cells (mean dose of DCs per vaccination: 9 × 106; mean number of vaccinations: 4.6). Enhanced immune responses against oncofetal antigen could be detected in five of six patients tested; two CRs, one PR, and seven stabilizations of disease were recorded among the 27 evaluable patients. Importantly, the two patients achieving CR after completion of the vaccination course were those exhibiting the strongest immune response to the oncofetal antigen as detected by antigen-specific proliferation. For both of these patients autologous metastatic tumor tissue was the antigen source.

Finally, Lin et al. [90] pulsed autologous mature monocyte–derived DCs with Epstein-Barr virus–associated peptides and used them to treat 16 patients with metastatic nasopharyngeal carcinoma, all with local recurrence or distant metastasis after conventional therapies. The patients received four weekly injections of 5–10 × 105 DCs into one inguinal lymph node. The immune response was monitored by ELISPOT, intracellular staining for IFN-γ, and cytotoxicity assay. DC administration was well tolerated, except for transient rigors or swelling at the lesion side of the neck or mild fever, which were recorded in 4 out of the 16 vaccinated patients. Epitope-specific CD8+ T-cell responses were elicited or boosted in nine patients. Peptide-specific CTLs were detected in the peripheral blood lymphocytes after vaccination in patients who were immunized with the HLA-A1101–restricted LMP2 peptide. In two of these patients, this coincided with partial tumor reduction.

Conclusions and Perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Definition of DCs and Methods for DC Generation
  5. The Choice of Antigen Source
  6. Routes of DC Delivery
  7. Immune Response to Vaccination
  8. Clinical Studies with DCs
  9. Conclusions and Perspectives
  10. Acknowledgements
  11. References

The phase I and II clinical studies with DCs are hardly comparable, given that different methods for DC culture, antigen loading, and administration have been used. Altogether, the data reported so far indicate that these ex vivo–generated APCs are immunogenic in vivo and that DC injection was associated with a clinical response in some patients. Phase III studies are necessary to evaluate the potential clinical advantages of DC vaccination and are already ongoing for some diseases, such as melanoma and prostate cancer [13,15]. It is a general conviction that, if any, the clinical benefits associated with this immunotherapeutic approach are more likely to be recorded among patients in remission of disease or with small tumor burden. Meanwhile, it seems probable that the efficacy of DC vaccinations will be improved by the novel methods of antigen loading and by the concomitant administration of cytokines or immunogenic factors such as IL-2, IL-12, or CpG dinucleotides, which should amplify the immune response in vivo.

Particularly appealing is the application of DCs to allogeneic bone marrow and PBPC transplantations. In this context, the recently developed protocols for reduced-intensity conditioning (the so-called mini-allo) have increased the safety of this kind of treatment and extended its applicability in leukemia (also in older patients), Hodgkin and non-Hodgkin lymphoma, myeloma, and nonhematological malignancies such as renal cell carcinoma and breast cancer [9195]. In this context, DCs could be used for the expansion and adoptive transfer of lymphocytes against TAAs or minor histocompatibility antigens [96]. This may help to selectively target the graft-versus-tumor reaction, while possibly minimizing the graft-versus-host effect.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Definition of DCs and Methods for DC Generation
  5. The Choice of Antigen Source
  6. Routes of DC Delivery
  7. Immune Response to Vaccination
  8. Clinical Studies with DCs
  9. Conclusions and Perspectives
  10. Acknowledgements
  11. References

P.B. is supported by a grant from the DFG, SFB 510. A.N. acknowledges an Award 2003 from the Anna Fuller Fund for Research in Molecular Oncology and the FIRB grant RBAU01THPL.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Definition of DCs and Methods for DC Generation
  5. The Choice of Antigen Source
  6. Routes of DC Delivery
  7. Immune Response to Vaccination
  8. Clinical Studies with DCs
  9. Conclusions and Perspectives
  10. Acknowledgements
  11. References
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