Stem Cells 2003;21:495-505
www.StemCells.com
© 2003 AlphaMed Press
Advances in the Use of Dendritic Cells and New Adjuvants for the Development of Therapeutic Vaccines
Stefano M. Santini,
Filippo Belardelli
Laboratory of Virology, Istituto Superiore di Sanità, Rome, Italy
Key Words. Immunotherapy • Cancer • Infectious diseases • Dendritic cells • Adjuvants
Filippo Belardelli, Ph.D., Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy. Telephone: 39-06-4990-3290; Fax: 39-06-4990-2097; e-mail: belard{at}iss.it
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ABSTRACT
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The recent advances in immunology and biotechnology have opened new perspectives for the development of immunotherapy strategies against cancer and infectious diseases. The understanding of the pivotal role of dendritic cells in the initiation and regulation of the immune response has led to an ensemble of preclinical studies and pilot clinical trials, which have provided some evidence on the potential advantages of using dendritic cells as cellular adjuvants for the development of therapeutic vaccines against infectious diseases and malignancies. Current research efforts are focused on the definition of optimal protocols for dendritic cell-based therapies in patients. An additional area of emerging importance in the field of immunotherapy is the identification of safe, selective, and more powerful adjuvants, capable not only of enhancing immune protection against pathogens, but also of breaking tolerance against certain tumor-associated antigens, which is the critical issue for the development of cancer vaccines. The recent recognition of the key role of certain cytokines, such as type I interferons, in linking the innate and adaptive immunity through their action on dendritic cells opens new perspectives for using these natural factors as adjuvants for the development of therapeutic vaccines. We review some of the emerging research aspects in immunotherapy, with special attention to the perspectives of using new adjuvants and dendritic cell-based vaccines for the treatment of cancer and infectious diseases.
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INTRODUCTION
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Attempts to modulate the immune system to induce protection against pathogen-induced diseases and malignancies have represented a considerable part of the biomedical research during the past century. Examples of remarkable success include the eradication or the effective control of some viral and bacterial infections, while many research efforts are still focused on the development of prophylactic and therapeutic vaccines against other life-threatening infectious diseases.
Prophylactic and therapeutic vaccines are based on a different rationale. Instead of preventing a disease, therapeutic vaccines function as substitutes or adjuncts for existing therapies and represent an active immunotherapy treatment for both chronic infectious diseases and cancer. In the case of viral chronic infections, while there is an urgent need for an effective HIV vaccine, vaccines against hepatitis B virus (HBV) and human papillomavirus (HPV) are the most feasible applications of therapeutic immunization, which may represent an antiviral and cancer prevention double-faceted approach. Regarding cancer vaccines, excluding immune prevention against viruses causing human cancers, the main research efforts are directed to the development of vaccination protocols in cancer patients, with the main aim of breaking tolerance towards self-tumor-associated antigens (TAAs). In contrast, prophylactic anticancer vaccination based on the use of TAAs expressed in premalignant phases is still in an early stage of preclinical evaluation. Even though preventive immunization strategies may represent a future intervention option for certain patients with familial cancer history or at high risk for cancer development, the current definition of cancer vaccines generally refers to therapeutic immunization in patients with cancer.
Efforts in developing effective immunotherapy treatments in cancer patients have represented a constant goal of many researchers and clinicians over the years. In the 1890s, William Coley’s observation that treatment of cancer patients with bacterial extracts (Coley’s toxins) could activate general systemic immunity, a portion of which was assumed to be directed against the tumor [1], represented the first indication that manipulation of the immune system could promote an effective antitumor response. One hundred years later, the studies showing that the administration of interleukin-2 (IL-2) to patients with metastatic melanoma or kidney cancer could mediate tumor regression further strengthened this concept [2].
There is no doubt that certain cytokines (interferon [IFN]-
, IL-2, and GM-CSF) currently represent valuable tools in the immunotherapy of some cancer patients [3, 4]. However, no response or poor responses have been obtained in a large number of patients with malignancies. This partly reflects intrinsic characteristics of the outcome of host-tumor interactions, including defects in antigen processing/presentation, escape from immune recognition through reduction or loss of immunogenic peptides in association with major histocompatibility complex (MHC) antigens, lack of costimulatory signals, and secretion of immunosuppressive cytokines. Moreover, the infusion of large amounts of cytokines in clinical immunotherapy studies often results in severe toxicity. Thus, major efforts are currently focused on the development of novel, safe, and more effective immunotherapeutic interventions.
Today, the recent advances in biotechnology and immunology permit the design of new cell-based immunotherapy strategies, which include the ex vivo manipulation of selected cell types derived directly from individuals, and their exposure to one or more cytokines prior to their reinfusion, with apparently minimal or absent toxicity in patients. For instance, on the basis of results of studies in animal models and of preliminary data from clinical trials, an emerging interest is currently focused on the use of dendritic cells (DCs) for the development of cancer vaccines [5–7]. Recent studies suggest that different types of DCs, because of their cytokine-mediated plasticity, can be used not only as ideal cellular adjuvants for therapeutic vaccines against cancer and severe infections, but also, under special conditions, in transplantation and autoimmune diseases. Moreover, now that we understand the key role of DCs in the priming and regulation of the immune response, we have new opportunities to identify novel and powerful adjuvants capable of selectively orienting the immune response towards protection. The identification of safe and effective adjuvants inducing a correct DC activation and antigen presentation is an urgent need not only for enhancing the immunogenicity of vaccines against infectious agents, but also for breaking tolerance against self-TAAs in patients with certain malignancies, which is the major prerequisite for the development of effective cancer vaccines.
The perspectives in the field of immunotherapy and the potential translation of new discoveries from basic research into clinical applications have been discussed in a dedicated session during the First International Workshop "Cell Therapy: Filling the Gap Between Basic Science and Clinical Trials", held at the Istituto Superiore di Sanità in Rome (October 15–17, 2001). In this article, we review the main topics addressed during the session, with special attention to some emerging areas of immunotherapy research, such as DC-based therapies and new adjuvants, discussing selected critical issues regarding the development of therapeutic vaccines against neoplastic and infectious diseases.
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DCS AND THEIR POTENTIAL USE IN IMMUNOTHERAPY
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Plasticity of the DC System and Attempts to Develop DC-Based Therapies
Considerable research efforts are currently focused on the biology of DCs in view of their possible clinical use as cellular adjuvants in the treatment of chronic infectious diseases and tumors. DCs are the key regulators of the adaptive immune system [8–10], as they act as unique cell players in priming naïve T cells and in the initiation of the immune response to pathogens. DCs originate from pluripotent stem cells in the bone marrow, enter the blood stream, and localize into almost all organs. DCs are responsible for directing different types of T-cell responses from thymic negative selection to the generation of effector and memory cells, as well as to the induction of peripheral tolerance. Recent studies indicate that the extent of DC recruitment into inflamed tissues and migration to lymph nodes, the nature of maturation stimuli, and the kinetics of activation have a quantitative and qualitative impact on T-cell stimulation and response.
Some types of DCs and DC precursors have recently attracted major interest by immunologists. Based on the relative expression of a series of surface markers, different subsets of DCs or DC precursors can be identified in peripheral blood, including a major CD1a+/CD11c+ and CD1a-/CD11c+ population, expressing the CD13, CD33, and GM-CSF-receptor (referred to as myeloid DCs), and a CD1a-/CD11- population expressing high levels of CD123 (IL-3R), originally called lymphoid DCs and recently renamed plasmacytoid DCs, which represent the major type I IFN producers upon virus challenge [11, 12]. The myeloid DCs are found in deep interstitial and epithelial tissues, whereas in the skin, dermal and epithelial DCs can be distinguished [13]. In healthy individuals, blood DCs represent 0.5%–1.5% of peripheral blood mononuclear cells, thus rendering it difficult to obtain clinically relevant cell numbers. However, blood DC counts have been shown to increase following cytokine administration. For example, G-CSF and Flt-3L are used to mobilize blood DCs, with an average increase of 40-fold or 10-fold in myeloid or lymphoid subsets, respectively [14, 15], while G-CSF has been shown to selectively favor expansion of the CD123+ subset over the myeloid population [16]. Alternatively, relevant numbers of DCs can be obtained from CD34+ stem cells, expanded, and differentiated in vitro upon exposure to cytokine cocktails, including GM-CSF, IL-4, and tumor necrosis factor (TNF)-, although with low reproducibility and variable final DC yield.
Typically, circulating immature DCs enter tissues in response to inflammatory chemoattracting cytokines. After having ingested and processed incoming pathogens, they switch their chemokine receptor set and migrate to regional lymph nodes in response to lymphoid chemokines, which also direct their position within lymphoid tissues, so that DCs can efficiently present processed antigens to lymphocytes, priming them for specific immune response.
It is generally thought that a better understanding of the dynamics of CD8 T cells and their differentiation into effector and central memory T cells will have important implications in immunotherapy, with a major impact on the development of antimicrobial and antitumoral vaccines. This issue has been elegantly pointed out by A. Lanzavecchia (Bellinzona, Switzerland) during his lecture at the meeting, describing two distinct memory T-cell subsets. Although memory T cells have been traditionally viewed as effector cells that have reverted into a quiescent state, they have recently been shown to be heterogenous cell populations comprising at least two cell subsets, endowed with different migratory capacity and effector functions. The "central memory T cells" are characterized by L-selectin and CCR7 expression as naïve T cells and lack immediate effector function. "Effector memory T cells" rapidly produce IFN-
or IL-4 or release prestored perforin upon encounter with antigen, migrate into inflamed tissues, and are characterized by the lack of the lymph node-homing receptors, L-selectin and CCR7. Upon restimulation in secondary lymphoid organs, central memory T cells proliferate and differentiate to effector cells [17, 18]. DCs and cytokines directly or indirectly produced by DCs and affecting their functions are crucial for maintaining constant levels of protective memory, further emphasizing the plasticity of these cells and their importance in ensuring long-term immune responses. Regarding the maintenance of humoral response, Lanzavecchia has recently shown that basically two distinct types of B-cell memory can be distinguished: an antigen dependent "short-term serological memory" relying on B-cell differentiation into short-lived plasma cells and in some long-lived memory cells, and a "long-term serological memory" resulting from antigen-independent polyclonal activation of memory B cells. It has been pointed out how selectively targeting one memory compartment "may open new ways of effective vaccination" [19].
For cancer immunotherapy protocols, as well as for research purposes, large numbers of immature myeloid DC-like cells are currently generated in vitro from CD14+ monocytes exposed to GM-CSF and IL-4 or IL-13. These DCs are weak antigen-presenting cells (APCs) due to low/moderate surface expression of costimulatory molecules. However, their activation/terminal maturation can be promoted by exposure to bacterial components, such as lipopolysaccharide; staphylococcus aureus cowan I strain; macrophage-conditioned medium (MCM); and cytokines including IL-1ß and TNF- or recombinant soluble CD40L, which induce the expression of the maturation marker CD83 and a marked upregulation of costimulatory molecules [9, 10, 20].
With regard to possible clinical applications of DC-based vaccines, after pulsing with the appropriate antigen(s) with or without exposure to cytokines, DCs are enabled to stimulate the host immune system and can be reinfused back into the donor or used for in vitro expansion of lymphocyte populations for patient injections. Notably, there is also growing evidence indicating that special types of DCs, prepared under particular conditions, can serve to induce and maintain tolerance [21]. This activity is largely dependent on DC ontogeny, maturation stage, and exposure to different cytokines such as TGF-ß or IL-10. In fact, exposure of immature DCs to IL-10 results in a switch in their phenotype, enabling them to favor the induction of anergic T cells, characterized by reduced IL-2 and IFN- production and low CD25 expression [22, 23], while antigen presentation by immature DCs can induce regulatory T cells, which produces large amounts of IL-10 [24, 25]. It has been argued that the induction of regulatory T cells by immature DCs could be therapeutically exploited in patients with autoimmune diseases, while the injection of allogeneic immature DCs could promote tolerance to transplanted organs [21, 26].
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CHARACTERIZATION OF A NOVEL TYPE OF DCS OBTAINED AFTER EXPOSURE OF HUMAN MONOCYTES TO TYPE IIFN: POTENTIAL ADVANTAGES FOR DC-BASED IMMUNOTHERAPY STRATEGIES
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We briefly review here an ensemble of studies recently performed by our group [27, 28] and extensively presented and discussed at the meeting (S.M. Santini, Rome, Italy) regarding the characterization of a new type of DCs generated from human monocytes in the presence of type I IFN.
As illustrated in Figure 1A
, a frequently used method for generating large numbers of DCs for clinical studies involves the generation of DCs from monocytes after two steps of in vitro treatment: A) induction of immature DCs after several days of culture in the presence of GM-CSF and IL-4, and B) induction of mature DCs after further treatment with different maturation agents. It might be argued that DCs generated after several days of in vitro exposure of monocytes to high levels hardly reflects the possible scenario of physiological exposure of monocytes to cytokines induced in the course of a natural infection.
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Figure 1. Two main pathways for the generation of human DCs from monocytes in the presence of different cytokines. A) Exposure to IL-4 (alternatively IL-13) and GM-CSF drives monocytes to differentiate into immature DCs. This phenotype is lost upon cytokine deprivation or diverted upon exposure to different cytokines. Immature DCs are weak APCs due to low/moderate surface expression of costimulatory molecules. Their activation/terminal maturation can be promoted by the addition of stimuli such as bacterial or viral components or products; recombinant soluble CD40L; MCM; and cytokines, including IL-1ß and TNF-, which induce the expression of the maturation marker CD83 and a marked upregulation of costimulatory molecules. B) Exposure of monocytes to type I IFN and GM-CSF rapidly induces their differentiation into partially mature DCs (IFN-DCs) expressing higher levels of costimulatory molecules and low-moderate levels of CD83 [27, 28]. The possible advantages of using IFN-DCs as cellular adjuvants for the development of therapeutic vaccines are discussed in the text.
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We have demonstrated that highly active, partially mature DCs can be generated within 3 days after stimulation of monocytes with type I IFN and GM-CSF [27, 28]. These cells (IFN-DCs) were characterized by loss of adhesion to the substrate and long dendritic-like processes. A 3-day exposure of blood monocytes to IFN- resulted in a marked upregulation of HLA-DR and the costimulatory molecules CD80 and CD86 together with CD40 and the intercellular adhesion molecule, ICAM-1 (CD54). IFN-DCs also displayed intermediate levels of CD123 expression. Even though the majority of DCs generated in the presence of type I IFN displayed features of immature DCs, markers of activated/mature DCs, such as CD83 and CD25, could be detected in a variable percentage (12%–40%) of the cells, with very high costimulatory molecule expression. Consistently with the appearance of DC activation markers [29], IFN-DCs produced significant IL-15 amounts in culture supernatant without any further stimulus [27, 28]. IFN-DCs were found to markedly express the novel apoptosis-inducing molecule TRAIL (TNF-related apoptosis-inducing ligand). As a consequence of TRAIL expression, they were capable of specifically killing TRAIL-sensitive tumor cells [27]. With respect to migratory behavior, IFN-DCs not only expressed very high levels of CCR5, but also exhibited an enhanced migration in response to its ligands, the inflammatory chemokines regulated upon activation, normal T-cell expressed and secreted, Mip-1, and especially, Mip-1ß. Consistently with their partially mature phenotype, a remarkable fraction of the IFN-DCs proved to express CCR7 and showed a migratory response to Mip-3ß [28], a chemokine regulating DC trafficking to secondary lymphoid organs [30–33]. Interestingly, IFN-DCs expressed considerable levels of the chemokine Mip-3ß. When injected intravenously into severe combined immunodeficient (SCID) mice, IFN-DCs exhibited an enhanced migratory behavior with respect to immature DCs generated in the presence of IL-4, rapidly localizing within mouse skin, as demonstrated by the detection of human DNA sequences by polymerase chain reaction. Notably, IFN-DCs potently stimulated the proliferative response of allogenic T cells and the production of high levels of IFN- production in mixed lymphocyte reactions even at very low stimulator/responder ratios [27].
As the ability of effectively priming naïve T cells, inducing de novo immune response is a peculiar feature of professional APCs; as a second step, IFN-DCs were loaded with viral antigens for performing studies of T-cell priming activity. In vitro primary stimulation of autologous T cells with IFN-DCs pulsed with inactivated HIV virions induced vigorous lymphocyte proliferation and a Th1 polarized response, as revealed by the virtual absence of IL-4 in culture supernatant in the presence of high amounts of IFN- after restimulation with antigen-pulsed DCs. As evidenced by enzyme-linked immunospot assay, antigen presentation by IFN-DCs resulted in higher numbers of IFN--producing cells as compared with conventional immature DCs. We then evaluated the in vivo activity of DC-based vaccination in the human-peripheral blood lymphocyte (PBL)-SCID mouse model [27, 28]. SCID mice reconstituted with human PBLs were immunized according to a vaccination schedule involving repeated injections of autologous DCs pulsed with inactivated HIV. IFN-DC-based vaccination provided experimental evidence of enhanced human humoral response toward the whole spectrum of HIV-1 proteins, with antibodies belonging mainly to the IgG1 isotype, as compared with the response elicited by conventional immature monocyte-derived DCs. Of interest, human CD8+ T cells recovered from the vaccinated xenochimeras exhibited specific responses toward HIV-1 antigens (manuscript in preparation). The ensemble of these results suggest that partially mature DCs generated from monocytes after short-term exposure (i.e., 3 days) to type I IFN could be valuable candidates to be used as powerful cellular adjuvants for the preparation of DC-based vaccines in patients with severe chronic infections (e.g., HIV-1, hepatitis C virus [HCV], and HPV) and certain types of cancer. Moreover, these data underline the important role of type I IFNs as natural cytokines capable of inducing a rapid link between innate and adaptive immunity in response to infections [34].
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IDENTIFICATION OF NEW ADJUVANTS: IMPORTANCE OF THERAPEUTIC VACCINE DEVELOPMENT
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The development of vaccines has often been hampered by the poor immunogenicity of the relevant antigenic components, which require potent adjuvants in order to efficiently promote a protective immune response. Adjuvants are chemical or natural compounds that, when administered with a vaccine, enhance immune response to immunogens by acting on the cells involved in the initiation and maintenance of the immune response. Major research efforts are now focused on designing more selective adjuvants capable of eliciting a Th-1 type of immune response, which is thought to be an important correlate of protection for certain human diseases. Unfortunately, the few adjuvants currently used in humans show weak activity and/or poor selectivity in shaping the immune response towards the putative protection patterns, while safety concerns have limited the utilization of new substances proven to be effective in animals. In the past decade, the dissection of crucial events leading to the triggering of the immune response has provided new clues for understanding how adjuvants, whose mechanism of action has remained elusive for years, may function in vivo. Chemical adjuvants, such as immmune-stimulatory adjuvants and Freund’s adjuvant (IFA) among others, have been tested in humans and have shown to significantly enhance response to vaccination to various levels. New and potentially more efficient adjuvants are currently being designed and evaluated for human use, including bacterial toxins and their derivatives, unmethylated CpG-rich DNA, chemokines, and cytokines. Recent studies have demonstrated that the effects of some adjuvants are mostly mediated by cytokines, which are the key mediators of the immune response. Moreover, certain cytokines act as potent stimulators of DCs, which are the main cell players in the initiation of the immune response, thus representing ideal targets for adjuvant activity. Recent studies have pointed out a potential interest in using type I IFN as an adjuvant of vaccines against infectious diseases. In fact, these cytokines potently enhance both T-cell and antibody responses to a soluble protein and promote immunological memory by acting on DCs [35]. Furthermore, recent studies have shown that endogenous type I IFN is indispensable for the action of several Th1-promoting adjuvants, and administration of this cytokine as an adjuvant of the human influenza vaccine results in a remarkable enhancement of vaccine immunogenicity in mice, comparable or even superior to that obtained with the most powerful adjuvants [36]. The challenge will now be to evaluate whether such remarkable adjuvant activity can also be demonstrated in humans.
The identification of powerful immune adjuvants is even more essential for the development of effective strategies of anticancer immunotherapy, as the critical issue in cancer patients is how to break the immune tolerance against selfTAAs without causing adverse effects on the host. The most recent advances in immunotherapy of cancer suggest that cytokines such as IFN-, GM-CSF, IL-2, and IL-12 can represent valuable adjuvants for the development of cancer vaccines, even though the optimal modalities for their clinical use have still to be defined.
If the use of cytokines, alone or in combination, is regarded as a promising approach for the development of effective cancer vaccines, a remarkable interest is still focused on the identification of other synthetic or natural substances endowed with powerful and possibly selective adjuvant activity to be used in cancer immunotherapy. In his lecture, A. Shimosaka (Gunma, Japan) extensively reviewed the mechanism of action of the immunopotentiating glycosylceramide compound, KRN7000, its biologic activities, and its use in tumor immunotherapy. KRN7000 was originally isolated from ocean sponge Agelas mauritianus, which can be found in southern Japan; this compound now can be totally synthesized and has been shown to stimulate the immune system (especially natural killer [NK] cells, T cells, and macrophages) through DC activation. After being found active against experimental tumors in murine models [37, 38], KRN7000 has been demonstrated to be safe when administrated to healthy volunteers and cancer patients and to increase NK cells, thus leading to clinical studies aimed at exploring its use as an adjuvant in DC-based vaccination or in patients with decreased NK cells.
It is reasonable to assume that now that we understand much more about the important steps in the initiation and regulation of the immune response, novel and more selective and effective immune adjuvants will be identified in the near future, thus leading to new perspectives for the development of prophylactic and therapeutic vaccines against infectious diseases and cancer.
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IMMUNOTHERAPY OF CANCER AND THE ATTEMPTS TO DEVELOP CANCER VACCINES
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The current clinical use of some cytokines (especially IFN-, IL-2, and GM-CSF) to activate host antitumor immune reactions in cancer patients represents a successful translation of the progress in immunology and cytokine research into clinical practice. Cytokines can be used as immune adjuvants of an antitumor immune response to improve antigen presentation and stimulation of adaptive immunity via expansion of specific T cells. IL-2 administration has been employed in directly enhancing tumor-specific T-cell response in melanoma patients, whereas GM-CSF administration has been shown to improve antigen presentation by promoting expansion and differentiation of monocytes, granulocytes, and DCs, however, with some contrasting results, possibly due to different vaccination schedules [39]. In spite of the promising results obtained in animal models, the possible advantage of a systemic administration of IL-12 in cancer patients is still controversial, since it may exert limited clinical efficacy and considerable toxicity, suggesting the necessity to design protocols minimizing side effects without affecting adjuvant activity [40]. Much more has to be learned about the use of cytokines with immune adjuvant activity in boosting antitumor immune response in patients. Studies in mouse tumor models are useful in defining mechanisms of action of cytokines and in inspiring testing of novel modalities for their use in patients. For example, lessons from animal models and from recent clinical studies have shown how IFN- can induce an antitumor response and how these cytokines could be more selectively used in patients, alone or in combination with other therapeutic interventions [41]. New modalities of using IFN- in cancer patients include the utilization of these cytokines as adjuvants of cancer vaccines [34, 41].
Today, the recent progress in immunology and biotechnology offers new opportunities for the development of cancer vaccines, a challenge that has been pursued by many investigators over the years. During the past decade, many tumor antigens have been identified and molecularly characterized, thus rendering feasible the design of specific vaccinations against several human cancers. There have been many attempts to develop effective therapeutic vaccination of melanoma patients by utilizing peptides, recombinant antigens, cellular lysates, and other immunization approaches (Table 1) [42–55]. One critical issue is represented, however, by the fact that the immune system often responds poorly and fails to control tumor development, as cancer cells easily evade immune response because of the lack of an appropriate antigen presentation step, which may lead to tolerance of TAAs.
G. Parmiani (Milan, Italy) presented an extensive, updated review of the results and response rates of clinical trials for cancer vaccination, which were initiated following supportive experimental evidence stemming from studies in animal tumor models. He reviewed the clinical studies of genetically modified tumor cells expressing cytokines, such as IL-2 and IL-4, and of peptides from TAAs in melanoma patients [56]. Possible reasons explaining the limited clinical responses might include the nature of the adjuvants and the clinical settings, which have generally included patients with advanced disease. Parmiani also suggested possible improvements in the construction of gene-modified cancer vaccines and provided an overview of the critical issues regarding the development of DC-based vaccines for cancer treatment.
DCs are now regarded as ideal cellular adjuvants for the development of therapeutic cancer vaccines. Of interest, it has been reported that neoplastic progression is frequently associated with a compromised DC function, suggesting a link between defects in DC activities and tumor development [57, 58]. Today, synthetic peptides carrying tumor epitopes can be used for pulsing DCs, which are then injected into patients in order to induce antitumor immunity. However, an effective immune response to tumors can be hampered by the low immunogenicity of tumor-associated epitopes. Thus, to overcome this limitation, certain amino acid residues in the anchor sites of some of these peptides can be conveniently modified to alter their binding to MHC class I molecules for enhancing cytotoxic T lymphocyte response stimulation. The majority of tumor peptides are associated to MHC class I molecules; few of them are presented in the context of MHC class II. Notably, cancer patients show a weak CD4 T-helper response to class II HLA-restricted antigens, which can, at least in part, explain the impaired immune response to tumors. Since a discrete number of HLA class II restricted epitopes have now been identified, they should be included in the peptide mixtures used to load DCs for antitumor immunization in order to possibly support a more effective immune response. A combination of distinct peptides is generally preferable to broaden the T-cell repertoire necessary for achieving an antitumor response, thus limiting escape mutation and evasion from immune response. Alternatively, recombinant full-length proteins can be used to avoid HLA restriction favoring the response to different epitopes. In addition, allogenic or autologous tumor cell lysates and apoptotic bodies from neoplastic cells may represent a valuable source of known and unidentified cancer antigens for antitumor vaccination. However, other approaches are being evaluated, including the introduction of tumor RNA into DCs or the use of tumor-DC cell hybrids as well as DC-derived exosomes. While normal monocyte-derived DCs are generally loaded with tumor antigens and subsequently used to immunize cancer patients, DCs differentiated from malignant cells and naturally expressing the relevant tumor antigen, as in the case of chronic myeloid leukemia or acute myeloid leukemia, can be directly reinfused into patients. Over the past few years, the results from animal models and the development of protocols to generate sufficient numbers of human DCs have led to some encouraging data in pilot clinical studies and to phase I/II trials aimed at verifying the efficacy of these strategies in patients with some malignancies, such as melanoma, renal, and prostate cancer. Comparative studies with DCs generated by different experimental procedures, as those described in Figure 1
, will allow us to understand the optimal modalities for the development of DC-based cancer vaccines.
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ATTEMPTS TO DEVELOP THERAPEUTIC VACCINES AGAINST SEVERE CHRONIC INFECTIONS: MAIN CHALLENGE OF IMMUNOTHERAPY
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During this century, vaccination strategies have led to the eradication of some life-threatening infectious diseases, such as smallpox, and to the development of some effective vaccines, whose current use has exerted a marked impact on human health. Nevertheless, the control of certain persistent viral infections, such as those sustained by HBV, HCV, and HIV, still remains a major challenge despite numerous efforts to develop effective antiviral treatment and therapeutic vaccines. As in a large percentage of individuals, the immune system cannot mediate an effective viral clearance, and there is a growing number of chronic HBV and HCV carriers at high risk for developing liver cancer. Likewise, persistent infection with HPV 16 and 18 has been linked to development of vulvar and cervical carcinomas in women. The risk is dangerously increased in immunosuppressed patients, transplant recipients, and HIV-infected individuals. The attempts to stimulate the immune system for effectively fighting these persistent virus infections have often been discouraging, mostly because of the difficulty to revert pathogen-induced immunosuppression and the lack of powerful immune adjuvants. Recently, an increasing interest has been focused on the efforts to define immunotherapy strategies in patients with severe chronic infectious diseases in combination with the current antiviral therapies, which are often ineffective in inducing a long-term response. However, a specific knowledge of the pathogenesis of a given infection is essential in order to identify the optimal timing and conditions that may favor antiviral immune interventions. Starting from the knowledge that HIV-1 can cause an extensive immune activation at certain stages of the natural infection, during which CD4+ T-cell activation supports massive HIV-1 production, G. Pantaleo (Lausanne, Switzerland) presented an immunomodulatory strategy based on combining the use of the immunosuppressive drug cyclosporin A (CsA) with the conventional treatment of the so-called highly active antiretroviral therapy (HAART) during primary HIV-1 infection. He described the immune-modulating effects observed in a group of adult subjects with primary HIV-1 infection when the patients were treated with CsA along with HAART. Pantaleo concluded that the rapid shutdown of T-cell activation in the early phases of primary HIV-1 infection due to CsA treatment can have long-term beneficial effects and establish a more favorable set-point for immune interventions, which may be important in achieving a long-lasting control of viral infections and, possibly, HIV-1 eradication [59, 60]. Several immunotherapy strategies recently have been evaluated in HIV-1 infected patients [61–67]. On the basis of our preclinical studies in chimeric models of SCID mice reconstituted with human PBL [27, 28, and manuscript in preparation (Lapenta C, Santini SM, Logozzi M et al.)], we may visualize that DCs generated from monocytes in the presence of IFN- can represent valuable cellular adjuvants from DC-based immunotherapy in patients chronically infected with HIV-1 as well as with other viruses such as HCV, HBV, and HPV. However, it should be pointed out that, even though the chimeric SCID mice reconstituted with human cells represent unique animal systems for studying the efficacy of vaccines based on the use of human DCs, these models do not fully mimic the natural HIV infection occurring in humans. Only the results stemming from well-controlled clinical trials with DC-based vaccines in virus-infected patients will provide a clear-cut answer in regard to the possible advantage of using certain types of DCs for the development of therapeutic vaccines.
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FINAL REMARKS
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The recent progress in basic immunology and biotechnology has opened new perspectives for developing selected strategies of immunotherapy in patients with cancer and some severe infections. The emerging knowledge on the mechanisms regulating the immune response provides new clues for identifying novel powerful adjuvants, including certain cytokines, to be selectively used for the development of therapeutic vaccines. In recent years, different classes of genetic vaccines have been developed. Recombinant DNA technology has permitted the introduction of antigens or epitopes into viral vectors, including poxvirus, adenovirus, alphavirus, and poliovirus vectors, allowing the targeting of specific tissues or professional APCs. Genes encoding costimulatory molecules and/or cytokines can also be included into such vectors to enhance antigen presentation and immune priming. Direct infection of APCs may have the advantage of resulting in endogenous processing of antigens and cross-priming by improved antigen presentation in class I HLA context. In addition, distinct bacteria can be genetically engineered in order to target in vivo APCs, especially when administered by enteric route to stimulate mucosal immunity. However, one possible main disadvantage of this approach is represented by the development of immune reactivity against the viral particles used to deliver the genetic material. An alternative approach is represented by direct injection of nucleic acids, DNA or RNA.
In light of the complex biology of DCs and in the view of an expanding clinical use of DCs for immunotherapy, a priority for successful clinical immune intervention will be represented by the definition of standardized procedures for both DC generation and cell quality controls, as well as by the definition and availability of clinical grade reagents, preservatives, additives, cytokines, media and suitable plastic ware, and equipment. Other important issues to be addressed are the definition of the sufficient DC number to be injected to obtain a favorable clinical response and the optimal treatment schedule. The ideal injection route is also far from being identified, and systematic comparative studies in clinical trials are needed. The choice of the injection site can be linked to the standardization of the procedures of DC preparation, as DCs obtained by different methods are likely to express different adhesion molecules and chemokine receptors and exhibit functional characteristics, including the migration activity in response to distinct sets of chemokines. However, in spite of the several still unclear aspects regarding the optimal modalities for preparing and using DC-based vaccines, the research progress in this field is so rapid that we predict that, in the near future, cell therapies based on the use of DCs will represent a valuable treatment option for certain patients with cancer or infectious diseases. Likewise, the emerging knowledge on DC biology will allow us to identify new and more selective adjuvants for the development of therapeutic vaccines.
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ACKNOWLEDGMENT
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Work in our laboratory was supported in part by grants from the Italian Association for Cancer Research and from the Italian Ministry of Health (special project on "Cytokines as Vaccine Adjuvants" and the Italian National Project on AIDS). We thank A. Lanzavecchia, G. Parmiani, A. Shimosaka, and G. Pantaleo, whose excellent lectures at the First International Workshop "Cell Therapy: Filling the Gap Between Basic Science and Clinical Trials" (October 15–17, 2001, Rome) inspired us in writing this review. We are grateful to Cinzia Gasparrini and Anna Ferrigno for excellent secretarial assistance.
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Received September 16, 2002;
accepted for publication January 31, 2003.
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Abstract
Introduction
