Dendritic Cell Immunotherapy for the Treatment of Neoplastic ... - Core

Biology of Blood and Marrow Transplantation 12:113-125 (2006) 䊚 2006 American Society for Blood and Marrow Transplantation 1083-8791/06/1202-0001$32.00/0 doi:10.1016/j.bbmt.2005.09.003

Dendritic Cell Immunotherapy for the Treatment of Neoplastic Disease William K. Decker, Dongxia Xing, Elizabeth J. Shpall Department of Blood and Marrow Transplantation, University of Texas M.D. Anderson Cancer Center, Houston, Texas Correspondence and reprint requests: William K. Decker, PhD, Department of Blood and Marrow Transplantation, University of Texas M.D. Anderson Cancer Center, Box 65, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: [email protected]). Received June 25, 2005; accepted September 7, 2005

ABSTRACT It has long been promised that dendritic cell immunotherapy would revolutionize the treatment of neoplastic disease. Now, more than 10 years since the publication of the first clinical data, a firmer understanding of immunology and dendritic cell biology is beginning to produce interesting clinical results. This article reviews the clinical trials that established many of the concepts with which today’s investigators are achieving improved results, discusses issues in dendritic cell immunotherapy that are currently unresolved, and offers a perspective on the strategies that the authors believe will be important for the design of future vaccine trials, including the use of Toll-like receptor agonists as maturation agents, the accessory use of the plasmacytoid dendritic cell subset, and the maximization of T-cell help. © 2006 American Society for Blood and Marrow Transplantation

KEY WORDS Dendritic cell

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Immunotherapy

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Vaccine

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Clinical trial

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T-cell help

INTRODUCTION

MYELOID VERSUS PLASMACYTOID LINEAGES

In the 10 years that have elapsed since the publication of the first dendritic cell (DC) immunotherapy trial [1], progress has been made toward the goal of using DCs as a legitimate therapy for the treatment of neoplastic disease. This painstaking progress has been based somewhat on empirical trial and error but also on important advances in basic immunology and DC biology that have provided a fundamental understanding of the cellular and molecular interactions that govern adaptive immune responses. As newly established concepts are collectively accepted and assimilated, reports of efficacy become more common and, importantly, become increasingly accompanied by immune correlates that can validate bona fide vaccine responses. We begin with a discussion of issues important to the generation of effective DC vaccines and a summary of significant or insightful trials that have used this approach. This discourse is followed by the authors’ perspective on the promising strategies that may produce meaningful clinical results in the future.

In vivo, there exist 2 subpopulations of DCs that seem to derive independently from myeloid or lymphoid committed precursors [2-4]. DCs that express myeloidspecific lineage markers, also known as DC1, seem to be best suited for the generation of antigen-specific effector T cells and are the subset that has been used exclusively in published vaccine trials [2-4]. DCs that express lymphoid-specific lineage markers, called plasmacytoid DCs or DC2, may serve as accessory cells that aid in the immune response by secreting large amounts of type I interferons (IFNs; eg, IFN-␣) in response to viral infection and other types of inflammation [2-7]. As the principal component of DC vaccines, myeloid DCs have been extensively characterized and reviewed [4,8,9]. Figure 1 summarizes the life cycle of a myeloid DC from generation in the bone marrow to immature immune sentinel in the periphery to mature mediator of T-cell responses in the lymph node. Figure 2 outlines major differences between the myeloid and plasmacytoid subsets.

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Figure 1. The life cycle of a myeloid (DC1) dendritic cell (DC). Immature DCs are very efficient in antigen uptake and processing. Antigens are taken up by scavenger receptors or by macropinocytosis (a). Next to the common pathways for MHC class I and II–restricted antigen presentation, DCs are unique in that exogenous antigens may access the class I pathway (b). This process is referred to as cross-presentation. Inflammatory agents induce the migration of the immature dendritic cell into the T-cell areas of secondary lymphoid organs. This migratory process (c) is controlled by different subsets of chemokine receptors and is aided by the secretion of metalloproteases, which degrade the extracellular matrix. T-cell activation units (d), consisting of peptide-loaded MHC class II and co-stimulatory molecules such as B7, are displayed after maturation on the DC, where they may constitute part of the immunologic synapse. Mature DCs also secrete several chemokines and cytokines, whose activity is modulated by proteases and protease inhibitors (e). ER indicates endoplasmic reticulum; MHC, major histocompatibility complex; MIIC, MHC class II compartment; TAP, transporter associated with antigen processing. Figure reproduced from Hartgers et al. [9] with permission.

Consistent with an apparent role in viral defense, plasmacytoid DCs express intracellular Toll-like receptors (TLR)–7 and -9, which are important in the recognition of microbial nucleic acids [2,3,6,10], but do not express other TLRs, such as TLR-1, -2, -3, -4, -5, or -8 [2,3]. In general, plasmacytoid DCs do not express high levels of major histocompatibility complex (MHC) class II [2,11-13], do not have the phagocytic capacity of their myeloid counterparts [2,13], do not present antigens particularly well [2,14], and are poor T-cell stimulators [2,3]. Unlike myeloid DCs, which begin life in circulation but typically migrate to peripheral tissues as they differentiate, fully differentiated plasmacytoid DCs may circulate in the blood, where they comprise approximately 0.1% of circulating white cells [3,15], and can be identified as rare CD4⫹/CD3⫺ cells that also express CD123 and HLA-DR [2,3,5,16]. In response to activation by TLR 114

ligation, inflammatory chemokines, or both, plasmacytoid DCs migrate to lymphoid organs or to sites of inflammation, where they can secrete up to 10 pg of IFN-␣ per cell in situ [2-4,7,17,18]. It is interesting to note that upon activation, plasmacytoid DCs may adopt the characteristics of their myeloid counterparts, including upregulation of MHC class II and costimulatory molecules, interleukin (IL)–12 secretion, effective antigen presentation, and the ability to stimulate antigen-specific T lymphocytes [2-4,13,19,20]. Such reports have somewhat obfuscated the issue of whether the myeloid and plasmacytoid DC phenotypes represent true lineage specificity or, despite lineage specific marker expression, actually represent different stages of maturation or development among a single committed population [2,3]. Moreover, though the preponderance of evidence suggests that plasmacytoid DCs enhance immu-

Dendritic Cell Clinical Trials

nating plasmacytoid DCs might be found in myeloid preparations derived from adherence or elutriation procedures, it is expected that they would be absent in preparations derived from CD14⫹ monocyte selection. Maintenance of such contaminating populations might also have to be supported by the addition of Flt-3 ligand. Adding to this set of unknown variables, the degree by which plasmacytoid DC participation might enhance adaptive immune responses has not yet been determined; ie, such participation might not necessarily impart a critical level of immune enhancement in vivo, thus allowing the contribution of the plasmacytoid subset to be ignored in the context of the myeloid-dependent response. Therefore, it remains to be determined whether the optimal generation or perpetuation of an adaptive immune response will require the participation of accessory plasmacytoid DCs in support of antigen-loaded myeloid DCs (Figure 3).

GENERATION PROTOCOLS

Figure 2. Subsets of human dendritic cells (DCs). Blood DCs, mobilized by Flt-3 ligand, contain both CD11c⫹ myeloid DCs (MDCs) and CD11c⫺ plasmacytoid DCs (PDCs). Most clinical studies to date have been performed with DCs made by culturing monocytes with GM-CSF and IL-4. These preparations contain cells that resemble interstitial DCs and are devoid of Langerhans cells. These DCs are immature and require exogenous factors for maturation. Myeloid DCs can also be generated by culturing CD34⫹ hematopoietic precursor cells (HPCs) with GM-CSF and TNF-␣, thus allowing the derivation of each myeloid DC subset. A distinct subset of precursors, CD34⫹CD45RA⫹, gives rise in vitro to plasmacytoid DCs upon culture with Flt-3 ligand. Figure reproduced from Banchereau et al. [8] with permission.

nity, some reports suggest that plasmacytoid DCs, both mature and immature, may instead promote tolerance and immune suppression [21-24]. In a clinical setting, plasmacytoid DCs may be unnecessary components of a myeloid-based vaccine that can use the patient’s own plasmacytoid subset in vivo. These in situ plasmacytoid DCs might require activation via the application of inflammatory cytokines or TLR-7/TLR-9 agonists (eg, unmethylated CpG dinucleotides) [10]. Alternatively, contaminating plasmacytoid DCs in a vaccine preparation could be preactivated by the inflammatory cytokines used in maturation, thereby abrogating the need for the generation of systemic inflammation. Although contami-

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Most clinical protocols have generated myeloid DCs by the adherence of peripheral blood mononuclear cells (PBMCs) to tissue culture plasticware and subsequent incubation in a cytokine cocktail containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, as first reported by Sallusto and Lanzavecchia in 1994 [25] and later modified by Romani et al. in 1996 [26] and Heiser et al. in 2000 [27]. There are, however, many other methods by which myeloid DCs may be generated for vaccine therapy, and it is not known whether any of these alternative methods imparts a degree of clinical efficacy superior to that of the standard approach. Few protocols have deviated from the simple formula of differentiating adherent monocytes with GM-CSF and IL-4. Some trials that have experimented with the standard protocol are listed in Table 1. Myeloid DCs may be generated from either monocytic CD14⫹ precursors or CD34⫹ hematopoietic progenitor cells [28,29]. Although monocytes may be collected in far greater numbers, progenitor cells maintain the ability to divide and may be expanded exponentially in culture. Of 3 recent studies that used CD34⫹ progenitors, 1 demonstrated remarkable efficacy [30], whereas the other 2 did not [31,32]; however, the studies differed in many other aspects, and any meaningful comparison of a single variable would be difficult. After activation of DC precursors by GM-CSF, DC differentiation may be influenced by a variety of cytokines, including IL-4 [4,33], IL-15 [4,34], tumor necrosis factor (TNF)–␣ [4,35], IFN-␣ [4,36-38], and thymic stromal lymphopoietin [4,39]. Some preclinical studies have indicated that DCs generated with IL-15 are highly efficient in cytotoxic T lymphocyte (CTL) priming [4,34], and DCs generated with thy115

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Figure 3. Loading and presentation of MHC class I and MHC class II antigens by dendritic cells. Antigens external to the dendritic cell are phagocytosed or taken up by receptor-mediated endocytosis. Once internalized, these exogenous antigens are degraded in the lysosomal compartment, loaded onto empty MHC class II molecules, and presented on the cell surface, where they may prime a cognate CD4⫹ T cell (left). Dendritic cells also possess the ability to present some exogenously derived class II antigens on MHC class I by a poorly characterized mechanism termed cross-presentation. Internal dendritic cell antigens, such as those present endogenously or, eg, by mRNA transfection or viral infection, are constitutively degraded at a basal level by the proteasome. Degradation products are processed in the endoplasmic reticulum, loaded onto MHC class I, and transported to the cell surface for priming of cognate CD8⫹ T cells (right). During the loading of dendritic cell vaccines with peptides, the peptide antigens are thought to mainly bind empty MHC class I and class II molecules on the cell surface without being internally processed by the dendritic cell. MHC indicates major histocompatibility complex; MIIC, MHC class II compartment; TAP, transporter associated with antigen processing.

mic stromal lymphopoietin elicit mainly an allergictype (T-helper type 2 [Th-2]) response [4,39]; however, the vast majority of published clinical trials have used GM-CSF for activation and either IL-4 or TNF-␣ as the agent of differentiation. Coculture with

IL-4 generates a homogenous population of immature myeloid DCs that most resemble interstitial DCs, whereas coculture with TNF-␣ yields a heterogeneous population of interstitial DCs and Langerhans cells that exhibit a mature phenotype [4,35]. From a

Table 1. Clinical Trials That Have Generated Immature Dendritic Cells by Methods Other Than the Incubation of Adherent Monocytes in GM-CSF and IL-4

Reference Banchereau [30] Barrou [42]

Bedrosian [85]

Di Nicola [32] Mackensen [31]

Clinical Responses

Length of Incubation

CD34ⴙ selection of mobilized PBMCs Culture of total PBMCs followed by elutriation for DC purification Elutriation of neutrophil-depleted PBMCs CD34ⴙ selection of mobilized PBMCs CD34ⴙ selection of mobilized PBMCs

8d

GM-CSF, Flt-3L, TNF-␣

Yes

41% (7/17)

7d

GM-CSF, IL-13

No

0% (0/24)

GM-CSF, calcium ionophore A23187, IL-2, IL-12 GM-CSF, TNF-␣, SCF, Flt-3L IL-3, IL-6, SCF for expansion, then GM-CSF, IL-4 GM-CSF, IL-4 ⴙ patients received low-dose IL-2 SC for 12 d after DC administration GM-CSF, IL-4 ⴙ 6 patients received IFN-␥ 12 h before DC administration

Yes

Stift [63]

CD14ⴙ selection of PBMCs

Stift [64]

CD14ⴙ selection of PBMCs

SC, subcutaneously; SCF, stem cell factor. 116

Direct Generation of Mature DCs?

Source of Precursor Cells

36 h

12 d 7 d for expansion then 21 d for differentiation 5d

5d

Cytokine Cocktail

Yes

0% IV 10% ID 22% IN 17%

(0/8) (1/10) (2/9) (1/6)

No

14% (2/14)

No

0% (0/20)

No

40% (4/10)


Table 2. Approaches to Loading Dendritic Cell Vaccines and the Associated Combined Clinical Efficacy of Selected Studies

References

Loading Approach

Single/Multiple Antigen

No. of Studies

1, 40, 41 42 43-45, 121-123 46, 47 32 30, 31, 52-56 57-67 68, 69 70 72 73 Total

Peptide Whole protein Idiotype mRNA Viral vector Peptide mix Tumor lysate Tumor mRNA Whole tumor cells Tumor heat shock proteins Tumor dendritic cell fusion

Single Single Single Single Single Multiple Multiple Multiple Multiple Multiple Multiple

3 1 6 2 1 7 11 2 1 1 1 36

Any Clinical Response 3.1% 0.0% 22.9% 0.0% 16.6% 21.7% 12.9% 5.6% 31.6% 6.7% 8.7% 14.4%

(1/32) (0/24) (11/48) (0/19) (1/6) (25/115) (18/140) (1/18) (6/19) (1/15) (2/23) (66/459)

Durable CR 3.1% 0.0% 6.3% 0.0% 16.6% 5.2% 2.1% 0.0% 15.8% 0.0% 0.0% 3.7%

(1/32) (0/24) (3/48) (0/19) (1/6) (6/115) (3/140) (0/18) (3/19) (0/15) (0/23) (17/459)

CR indicates complete response.

functional standpoint, it will likely be difficult to determine with certainty the full relevance of these different DC subsets to the generation of an optimal immune response in vivo. There has been much interest in the potential of the partially mature DCs that may be generated by culturing adherent PBMCs in GM-CSF and IFN-␣ for 3 days [4,36-38], although this strategy has been reported only preclinically in the literature. Biologically, it is unclear at what stage DC function might be best influenced by IFN-␣. In vivo, immature myeloid DCs might come into contact with IFN-␣–secreting plasmacytoid DCs just before maturation near sites of inflammation. In such instances, IFN-␣ might serve as a signal for maturation and migration to the peripheral lymphoid organs. Alternatively, myeloid DCs might encounter the plasmacytoid subset after maturation near the high endothelial venules of the peripheral lymphoid organs [2,3]. In either case, plasmacytoid DCs might be providing additional cytomodulatory signals, including signals that require cell-to-cell contact, that can modify the myeloid subset or naive T lymphocytes in a functional manner that amplifies specific antiviral responses. ANTIGENIC PREPARATION AND DC LOADING A wide variety of antigens and antigenic preparations (Table 2) have been used to load DCs or otherwise elicit an immune response. The strategies for generating an antigen-specific immune response include the loading of DCs with single antigens in the form of tumor-specific peptides [1,40,41], proteins [42] (including idiotype protein for immunotherapy of myeloma [43-45]), and single messenger RNA (mRNA) transcripts [46,47], as well as attempts to express tumor antigens endogenously in DCs by the use of plasmid or viral constructs [32]. In contrast to a total antigen approach, the single-antigen approach is specific and avoids the theoretical problem of priming

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an autoimmune response against normal cellular antigens. Moreover, single-antigen or single-epitope immune responses are easier to document and quantify, particularly by staining of antigen-specific T cells with HLA-/peptide-specific tetramers. On the downside, single-antigen/-epitope approaches suffer from a variety of deficiencies, including tumor immune escape, HLA specificity (in the case of peptides), and a lack of T-cell help via the failure to load either MHC class II (peptides and mRNA) or MHC class I (protein) with a matched or linked epitope [48-51]. Most importantly, the single-antigen approach does not allow the immune system to choose the epitopes from the total antigen pool that will be most useful for the promulgation of an effective response. It should be recognized that the most effective epitopes will vary from patient to patient according to HLA type, genetic background, and the antigen pool of each individual tumor. Many different trials have also used a multipleantigen approach, including the use of peptide mixes [30,31,52-56], whole tumor lysates [57-67], total tumor mRNA [68,69], whole tumor cells [70], tumor apoptotic bodies [71], tumor-derived heat shock proteins [72], and tumor/DC fusions [73]. In addition, 2 trials, currently under way, use unloaded DCs derived from leukemic blasts [74] (G. Ossenkoppele, personal communication, Vrije University Medical Center, 2005), a strategy originally described by Choudhury et al. [75] and modified subsequently by Westers et al. [76] and Houtenbos et al. [77]. Most of these strategies can theoretically present a full complement of tumor-specific antigens to the patient’s immune system, yet all suffer from a common shortcoming. These strategies have the potential to load either MHC class I (tumor mRNA, cell fusions, and leukemia-derived DCs) or MHC class II (tumor lysates, tumor cells, tumor apoptotic bodies, and heat shock protein preparations), but not both. When loading DCs by phagocytosis, investigators typically cite the 117

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phenomenon of cross-presentation as a mechanism by which both MHC class II and MHC class I may be loaded (Table 3). Cross-presentation is a bona fide biological phenomenon by which MHC class II antigens are also presented by MHC class I; however, several recent reports have demonstrated suboptimal efficiency of cross-presentation in vivo, and some investigators now question its physiologic relevance [7880]. To generate an optimal immune response, we would hypothesize that both MHC class I and MHC class II should be efficiently loaded with tumor-specific antigens, because this approach might lead to a maximization of T-cell help in support of all relevant CD8⫹ effectors. To address recent questions about the efficiency of cross-presentation, we have compared the T cell–stimulatory abilities of DCs loaded with either class I or class II antigens with those of DCs doubly loaded with both class I and class II antigens. Our preliminary data support the contention that cross-presentation can be suboptimal in its efficiency (unpublished data). MATURATION PROTOCOLS The manner by which a DC vaccine is matured is likely to be of critical importance in the generation of robust, antigen-specific immune responses. Most published trials that have used matured DCs cite the use of TNF-␣ or ITIP (a combination of the inflammatory cytokines IL-1␤, TNF-␣, IL-6, and the CCR7inducing steroid compound prostaglandin E2) as the agent of maturation. One of the most widely used maturation cocktails is ITIP [81-83]. Other immunomodulatory or inflammatory agents that have been used either clinically or preclinically include CD40 ligand (CD40L) [84], IFN-␣ [36-38], IFN-␥, Ca2⫹ ionophore, IL-2, IL-12 [85], and TLR agonists [86]. Each of these compounds has its own unique and useful biological effects, and if improvements to the current gold standard are to be forthcoming, such improvements will likely be generated by a cocktail approach rather than the use of a single agent. Promising maturation molecules include CD40L and TLR

agonists. CD40L can upregulate DC immunocompetence by mimicking the effects of T-cell help, and the use of CD40L as a single agent of DC maturation has been cited clinically (D. Gabrilovich, personal communication, University of South Florida, H. Lee Moffitt Cancer Center, 2003). TLRs recognize nonspecific pathogen–associated molecular patterns such as lipopolysaccharide, double-stranded RNA, or unmethylated CpG dinucleotides, and TLR agonism can promote DC maturation in the absence of other inflammatory agents [86]. We have demonstrated that the addition of the synthetic RNA analogue and TLR agonist poly I:C to the ITIP cocktail in vitro leads to an impressive upregulation of DC IL-12 secretion (unpublished data). Similarly, Cisco et al. [87] have demonstrated that transfection of DCs with mRNA encoding a constitutively active TLR-4 allows the generation of antigen-specific CTL activity superior to that of DCs matured with either ITIP or lipopolysaccharide. Although highly promising, these types of strategies have, as yet, been described only preclinically. Of note, Bedrosian et al. [85] used a unique maturation cocktail of Ca2⫹ ionophore, IL-2, and IL-12 to generate mature DCs. Although the use of Ca2⫹ ionophore to rapidly generate mature DCs from peripheral blood monocytes was originally reported by Czerniecki et al. [88] in 1997, this strategy does not often appear in the clinical literature. It is likely that additional agents such as Ca2⫹ ionophore, CD40L, or TLR agonists will eventually become more commonplace in the clinical literature, perhaps in conjunction with ITIP, as investigators seek to improve current protocols by generating in vitro–derived mature DCs that more closely rival the functional efficacy of their in vivo–derived counterparts. ROUTE OF DC ADMINISTRATION During the normal processes of immune homeostasis, the immature DC maintains itself in peripheral tissues, where it constantly samples the antigenic milieu of its surroundings. Upon detection of a

Table 3. Studies in Which Clinical Outcome Can Be Significantly Correlated with Antigen-Specific Immunologic Phenomena

Reference

Disease

Clinical Response

Banchereau [30]

Melanoma

7/17

Butterfield [40]

Melanoma

1/13

Yamanaka [62]

Glioblastoma

2/10

Yu [65]

Glioblastoma

3/9

DTH indicates delayed-type hypersensitivity. 118

Immune Correlate Integrated direct and recall responses of 4 peptide antigens (8 total assays) to obtain an indexed score, which correlated with clinical outcome (P < .015) Demonstrated that IFN-␥– and IL-4–secreting CD3ⴙ/MART-1 tetramerⴙ cells were vastly higher in single responders than among all other nonresponders 2/2 responders were DTH positive to tumor lysate, but only 1/4 nonresponders was DTH positive 3/3 patients alive at >200 wk after vaccination exhibited significantly increased IFN-␥– secreting tumor antigen–specific (by tetramer staining) T cells after vaccination. Only 1/6 nonresponders exhibited a similar phenomenon


“danger” signal, such as a TLR agonist or an inflammatory cytokine, the DC matures, upregulates CCR7, and migrates to the T-cell areas of the peripheral lymphoid organs—the sites at which T-cell priming will occur [80,89,90]. However, an oft-cited study by de Vries et al. [83] demonstrated that migration of mature, in vitro–derived human DCs to peripheral lymphoid organs is of dubious efficiency, on the order of only 1% to 4% (but this rate is statistically better than the 0.3% migration rate observed with immature DCs). These data were later corroborated by Ridolfi et al. [91], who produced essentially identical results in a trial that involved 8 cancer patients. Lending credence to the hypothesis that in vitro– derived DCs migrate suboptimally in vivo, Bedrosian et al. [85] demonstrated superior peptide-specific delayed-type hypersensitivity and IFN-␥ responses when mature DCs were administered intranodally in comparison to intradermally (90% of vaccinees intranodally versus 30% of vaccinees intradermally). This same study also demonstrated that immune responses were virtually absent when DCs were delivered intravenously [85]. Most recent studies cite intranodal administration as the delivery method of choice, and this cumbersome procedure will likely remain necessary until such time that in vitro– derived DCs can be induced to migrate in a more efficient manner.

IMMUNOMONITORING A key issue facing DC immunotherapy has been the inconsistent ability of investigators to correlate vaccine-specific or antigen-specific immune responses with clinical observations. Frequently, a delayedtype hypersensitivity response may be weakly correlated with efficacy [61,92], but too often it is reported that antigen-specific assays such as ELISpot (EnzymeLinked Immunospot), proliferation, or CTL lysis cannot be correlated with clinical observations [40,42,58,60,63,92]. Conversely, some investigators have demonstrated a good correlation between immune responses and clinical remissions. Some of the trials discussed subsequently are well known precisely because clinical and immunologic data are well correlated (Table 3); however, oncology still lacks a breakthrough trial with wholly unambiguous correlations, such as the recent publication of Lu et al. [93], which correlated the reduction of human immunodeficiency virus (HIV) viral loads with HIV-specific T-cell responses in patients receiving DC immunotherapy for chronic HIV infection.

CLINICAL TRIALS In 1995, Mukherji et al. [1] published the results of the first clinical trial that sought to establish the fea-

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sibility and safety of MAGE-1 peptide–loaded HLAA1⫹ DCs for the treatment of metastatic melanoma. After this preliminary report, 3 high-profile publications demonstrated that DC immunotherapy could induce objective clinical responses as well. Hsu et al. [43] claimed a clinical response rate of 75% (2 complete responses [CRs] and 1 partial response [PR] out of 4 patients) after the intravenous administration of blood-isolated, idiotype-pulsed DCs to patients with follicular B-cell lymphoma. Patients also received subcutaneous injections of soluble idiotype. After a successful phase I trial, Tjoa et al. [52] demonstrated a clinical response rate of 27% (9 PRs out of 33 patients) after the intravenous administration of prostate-specific membrane antigen (PSMA) peptide–pulsed immature DCs to patients with metastatic prostate cancer. DCs in this study were generated by the culture of adherent PBMCs in GM-CSF and IL-4. Nestle et al. [94] cited a 38% clinical response rate (2 CRs, 3 PRs, and 1 minor response (MR) out of 16 patients) after the intranodal administration of peptide (MART-1, tyrosinase, and glycoprotein [gp]100 or MAGE-1 and MAGE-3, depending on patient HLA type) or lysate-pulsed immature DCs to patients with metastatic melanoma. As with Tjoa et al., DCs were generated by culture of adherent PBMCs in GM-CSF and IL-4. Despite these apparent successes, many basic questions about the underlying biology of human DCs were poorly understood and still remain to be answered in vivo. In 1999 and 2001, Dhodapkar et al. published 2 important studies demonstrating that DC maturation was required for the induction of antigenspecific immune responses by the administration of both mature [95] and immature [96] DCs to healthy volunteers. Most importantly, they demonstrated that the administration of immature DCs could result in the development of antigen-specific tolerance [96]. Four years later, these observations were underscored in a review article that cited a 14.9% (21/141) clinical response rate to immature vaccines and a 22.8% (18/ 79) response rate to mature vaccines [92]. Other reviews on this topic were also published contemporaneously [97]. At the same time, Mackensen et al. [98] and Morse et al. [99] demonstrated that either immature or mature DCs injected intravenously localized initially to the lungs and subsequently to liver, spleen, and bone marrow, with no evidence of lymph node localization. These authors further demonstrated that matured DCs injected intralymphatically could localize to regional lymph nodes for at least 24 hours, and immature DCs administered subcutaneously remained essentially in situ with very low levels of migration to regional lymph nodes (0.1%-0.4%, in agreement with 119

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de Vries et al. [83]). Fong et al. [100] would later demonstrate a lack of antigen-specific Th-1 responses in prostate cancer patients receiving antigen-pulsed DCs intravenously, but not intradermally or intralymphatically. Given this new understanding of human DC biology, it might be tempting to view the results of previous studies in a somewhat ambiguous manner, and this issue warrants further discussion. Immature DC vaccines are not completely without beneficial therapeutic effects [92], and it is generally assumed that some differential level of maturity may be imparted to human DCs simply by routine handling and culture, as is commonly observed with mouse Langerhans cells after short-term culture [101,102]. This simple assumption helps to mitigate the major criticism of Nestle et al. [94] and of Tjoa et al. [52]. Although it is more difficult to reconcile Tjoa and associates’ intravenous vaccination schedule with clinical efficacy, it is not entirely without precedent. In the pivotal study of Fong et al. [100], 55% of patients who received intravenous administration of DCs developed antigen-specific antibody titers typical of a Th-2 type response. Therefore, in pretreated prostate cancer patients with minimal residual disease, it is not difficult to envision transient tumor susceptibility to PSMA antibodies in much the same way that circulating lymphomas may be susceptible to rituximab (Rituxan Genentech, Inc, San Francisco, CA) [103] and that solid breast tumors may be susceptible to trastuzumab (Herceptin Genentech, Inc, San Francisco, CA) [104]. The success reported by Hsu et al. [43] is tempered somewhat by the study’s small sample size and by a perceived difficulty in separating immune responses due to DC vaccination and immune responses due to the administration of soluble idiotype protein. Nevertheless, the efficacy of this approach continued to be excellent in a larger follow-up study by Timmerman et al. [44], and it seems likely that some unique features of B-cell lymphomas or of this particular vaccination approach are contributing to clinical success in a manner not entirely analogous to that with other DC-based therapeutic strategies. In 2001, Banchereau et al. [30] published a landmark study against which future studies will be compared. In this trial, CD34⫹ progenitor-derived DCs pulsed with 4 HLA-A2 melanoma peptides (MART-1, tyrosinase, MAGE-3, and gp100) were delivered subcutaneously to 18 patients with stage IV metastatic melanoma. The vaccine induced antigen-specific IFN-␥ secretion in response to at least 1 of the 4 peptides in 17 of the 18 patients, as assayed by ELISpot. In general, the number of antigens to which the patient responded and the magnitude of each response correlated well with the overall clinical outcome. In addition to excellent immune correlates, the trial produced impressive clinical results, with 3 CRs and 4 120

PRs out of 17 evaluable patients (41%). The durability of these remissions, however, was not discussed. The following year, Heiser et al. [46] pursued a novel strategy in the generation of a DC vaccine for prostate cancer. DCs derived by the incubation of adherent PBMCs in GM-CSF and IL-4 were coincubated with in vitro– derived prostate-specific antigen (PSA) mRNA. DCs were then cultured overnight to allow expression, processing, and presentation of the PSA antigen, as well as, the authors claim, slight maturation by a nonspecific, mRNA-dependent mechanism. The vaccine was delivered both intravenously and intradermally to 13 patients with metastatic prostate cancer. Strikingly, in all 8 patients evaluated by ELISpot and all 10 patients evaluated by chromium 51 release cytotoxicity, responses against PSA were significantly higher after vaccination. Unfortunately, clinical results were difficult to assess and were not durable. As many investigators have noted [47,105,106], transfection of DCs with mRNA alone does not allow for robust priming of CD4⫹ lymphocytes, thus leading to a response that is largely devoid of T-cell help. Very recently, Su et al. [47] implemented an elegant strategy designed to address the lack of T-cell help that plagues mRNA-based trials. After the generation of DCs by the incubation of adherent monocytes in GM-CSF and IL-4, the authors electroporated the DCs with a human telomerase reverse transcriptase (hTRT) lysosome-associated membrane protein fusion mRNA. hTRT encodes for the catalytic subunit of human telomerase, an antigen preferentially expressed in most tumors. To allow presentation of hTRT epitopes by MHC class II for the generation of antigen-specific CD4⫹ T cells, the hTRT transcript was fused to the lysosomal targeting signal of lysosome-associated membrane protein 1. After electroporation, the DCs were matured with ITIP and delivered intradermally to patients with metastatic prostate cancer. In comparison to patients whose DC vaccine was transfected with the native hTRT transcript, those who received the fusion transcript demonstrated an 8-fold increase in hTRT-specific CD4⫹ lymphocytes after vaccination, as assayed by IFN-␥ ELISpot. It is interesting to note that upregulation of hTRT-specific CD4⫹ lymphocytes did not seem to alter the quantity or quality of hTRTspecific CD8⫹ lymphocytes; moreover, impressive clinical responses were not observed. By using a unique method of total antigen loading, O’Rourke et al. [70] reported improved efficacy for the treatment of metastatic melanoma by using a DC vaccine that was loaded with whole, irradiated tumor cells. After maturation by culture in monocyte-conditioned medium and intradermal administration, O’Rourke et al. reported a clinical response rate of 32% (6/19), including 3 durable CRs that involved the resolution of lesions in lymph node, kidney, adrenal gland, me-


diastinum, pleuropericardium, and lung. The authors were also able to correlate clinical success with disease burden, demonstrating that 5 of 6 clinical responders entered the study with serum S-100B levels of ⬍0.36 ␮g/mL, whereas only 2 of 8 nonresponders entered the study with S-100B levels below this threshold. Yu et al. [65] attempted to address central nervous system immunoprivilege by the generation of a vaccine for malignant glioblastoma multiforme. DCs were generated by the incubation of adherent PBMCs in GM-CSF and IL-4 and then loaded by overnight incubation with autologous tumor lysate and injected subcutaneously. Although antigen-specific immune responses were difficult to evaluate, the authors did demonstrate the expansion of IFN-␥–secreting HER-2 (c-erb-b2)–specific CD8⫹ T cells in 3 patients, as well as expansion of IFN-␥–secreting CD8⫹ T cells specific for 3 different glioblastoma-associated antigens in a single patient (HER-2, gp100, and MAGE-1) by tetramer analysis. In 2 patients who underwent craniotomy during the study, the authors also reported T-cell infiltrates composed of both CD4⫹ and CD8⫹ lymphocytes. Most impressive was the study’s reported median survival of 133 weeks (versus 30 weeks for cited historical controls and perhaps 65 weeks under the best of circumstances [107]); moreover, 3 (38%) of 8 patients were alive at ⱖ200 weeks after vaccination. All 3 surviving patients had significantly expanded IFN-␥–secreting, tetramer-specific T-cell populations after vaccination, whereas only 1 of 6 patients who did not survive demonstrated a similar expansion. The small sample size, however, precludes definitive analysis of efficacy. Despite the selected reports of clinical efficacy described previously, most DC immunotherapy trials have not reported significant clinical responses. Of 98 trials published through mid 2003, only 17 trials documented a complete remission. These 17 trials shared no common characteristic that separated them from trials without a CR, and the combined rate of CR was only 12% (33 CRs out of 273 patients vaccinated) [108]; moreover, clinical responses were rarely durable, and relapse was commonplace. Our own analysis of 36 recent trials (Table 2) suggests even more pessimistic results. Because of this suboptimal clinical picture, investigators continue to look for new strategies that can increase the efficacy of DC vaccination. One of the most relevant “new” strategies has been documented for nearly 25 years [48-51]: the optimal generation of T-cell help.

portant to consider the sequence of events that occur in its absence. Upon detection of a “danger” signal, DCs in vivo can efficiently coordinate the elimination of cells presenting foreign antigens by the priming of CD8⫹ effectors that recognize such antigens. Equally as important, in the absence of danger signals, DCs maintain self-reactive T cells in a state of nonresponsiveness. Although T-cell precursors that recognize self-antigens with high affinity are deleted during development in the thymus as part of the process of central tolerance, medium- and low-affinity self-reactive CD8⫹ T-cell precursors are normally maintained in an unresponsive state by the mechanisms of peripheral tolerance. Effective priming of circulating CD8⫹ T cells not only requires DC presentation of the cognate peptide antigen in the context of MHC class I, but also requires CD4⫹ T-cell help [109,110]. Possible mechanisms of T-cell help include the upregulation of costimulatory molecules on the antigen-presenting cell (APC) surface after CD40/CD40L signaling between the helper T cell and the APC [106,111-113]. Other investigators, however, have reported the transient expression of CD40 on the surface of CD8⫹ CTLs and suggest that T-cell help involves direct signaling between the CD4⫹ cell and the CD8⫹ cell. In the latter model, the APC assumes the additional function of bringing CD4⫹ and CD8⫹ cells into close proximity for proper cross talk to occur [114-116]. CD4⫹ helper T cells are generated, not by antigens endogenous to the APC, but by exogenous antigens that are engulfed by APCs and presented on the surface by the MHC class II complex. In the absence of appropriate T-cell help, activated CD8⫹ clones tend to become lethargic and may not respond to future activation stimuli [109,117,118]. This is one of several mechanisms by which the immune system is prevented from recognizing self antigens and inducing an autoimmune response [119]. Our data have indicated that the double loading of DC MHC class I and MHC class II positively affects primary and recall T-cell responses through the maximization of CD40L-mediated T-cell help. In addition to describing this phenomenon empirically, we provide preliminary evidence to suggest it mechanistically [120], and we are optimistic that continued focus on this aspect of immune stimulation could result in the development of DC vaccines with significantly enhanced efficacy.

THE IMPORTANCE OF T-CELL HELP IN THE GENERATION AND PERPETUATION OF THE ADAPTIVE IMMUNE RESPONSE

NONBIOLOGICAL IMPEDIMENTS TO SUCCESS: COST AND REGULATORY OVERSIGHT

To appreciate the importance of T-cell help in the activation of the adaptive immune response, it is im-

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In addition to the biological impediments that have hindered a wider implementation of DC immunotherapy, regulatory oversight and cost now add ad121

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ditional levels of complexity to a treatment that may be only marginally effective in a select population of individuals. These costs include many thousands of dollars for patient apheresis, monocyte enrichment, the expensive cytokines and growth media required to generate and mature DCs, and (optionally) various means of vaccine administration. Labor costs and the expense of maintaining a Food and Drug Administration (FDA)–compliant current Good Manufacturing Practice (cGMP) facility for complex DC manipulations can be substantial. Investigators working with an established cGMP facility might expect to incur costs of $4,000 to $16 000 per patient, depending on a broad host of variables. Investigators seeking to establish and maintain their own FDA-compliant facility should expect costs to be appreciably greater. Even though most DC immunotherapy trials are phase I in scope, regulatory requirements have become considerable. Similar regulatory oversight is required in Canada, Europe, Australia, and many other countries where clinical cellular therapy trials are being conducted. This process requires approval of the clinical protocol and validated cell-manufacturing procedures by the FDA or equivalent state-recognized regulatory agency. The use of clinical-grade reagents is always most desirable. If such reagents are not available, procurement of certificates of analysis to document the sterility of the major reagents is required. The costs and regulatory requirements of current DC studies underscore the importance of conducting well-designed trials with evaluable clinical and laboratory end points so that maximal information can be obtained from each study.

CONCLUSION Despite a persistent lack of consensus in almost every important aspect of DC immunotherapy, a modicum of progress has been achieved over the last 10 years. Many investigators now accept that DCs must be matured and must reach the T-cell areas of the peripheral lymphoid organs to function as advertised. In the coming years, we are optimistic that investigators will begin to embrace other important concepts, such as the use of TLR agonists in the maturation process, the loading of vaccines with multiple antigens, and the maximization of T-cell help. Also, with continued empirical progress, it seems plausible that DC immunotherapy may one day provide realistic treatment opportunities for distinct subsets of patients with neoplastic disease. Moreover, despite the slow progress and, at times, ambiguous results, those in the field continue to remain highly committed to the development of therapeutic strategies that will be of benefit to broader cross-sections of individuals as well. 122

REFERENCES 1. Mukherji B, Chakraborty NG, Yamasaki S, et al. Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells. Proc Natl Acad Sci U S A. 1995;92:8078-8082. 2. Colonna M, Tinchieri G, Liu YJ. Plasmacytoid dendritic cells in immunity. Nat Immunol. 2004;5:1219-1226. 3. McKenna K, Beignon AS, Bhardwaj N. Plasmacytoid dendritic cells: linking innate and adaptive immunity. J Virol. 2005;79:17-27. 4. Banchereau J, Palucka AK. Dendritic cells as therapeutic vaccines against cancer. Nat Rev Immunol. 2005;5:296-306. 5. Siegal FP, Kadowaki N, Shodell M, et al. The nature of the principle type 1 interferon-producing cells in human blood. Science. 1999;284:1835-1837. 6. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol. 2004;5:987-995. 7. Coccia EM, Severa E, Giancomini D, et al. Viral infection and Toll-like receptor agonists induce a differential expression of type I and lambda interferons in human plasmacytoid and monocyte-derived dendritic cells. Eur J Immunol. 2004;34: 796-805. 8. Banchereau J, Paczesny S, Blanco P, et al. Dendritic cells: controllers of the immune system and a new promise for immunotherapy. Ann N Y Acad Sci. 2003;987:180-187. 9. Hartgers FC, Figdor CG, Adema GJ. Towards a molecular understanding of dendritic cell immunobiology. Immunol Today. 2000;21:542-545. 10. Krug A, Towarowski S, Bristch S, et al. Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12. Eur J Immunol. 2001;31:3026-3037. 11. Chehimi J, Starr SE, Kawashima H, et al. Dendritic cells and IFN-␣-producing cells are two functionally distinct non-B, non-monocytic HLA-DR⫹ cell subsets in human peripheral blood. Immunology. 1989;68:488-490. 12. O’Doherty U, Peng M, Gezelter S, et al. Human blood contains two subsets of dendritic cells, one immunologically mature and the other immature. Immunology. 1994;82:487-493. 13. Grouard G, Rissoan MC, Filgueira L, et al. The enigmatic plasmacytoid T cells develop into dendritic cells with interleukin (IL)-3 and CD40-ligand. J Exp Med. 1997;185:1101-1111. 14. Fiebiger E, Meraner P, Weber E, et al. Cytokines regulate proteolysis in major histocompatibility complex class II-dependent antigen presentation by dendritic cells. J Exp Med. 2001;193:881-892. 15. Ueda Y, Haigihara M, Okamoto A, et al. Frequencies of dendritic cells (myeloid DC and plasmacytoid DC) and their ratio reduced in pregnant women: comparison with umbilical cord blood and normal healthy adults. Hum Immunol. 2003; 64:1144-1151. 16. Kadowaki N, Antoneko S, Lau JYN, Liu YJ. Natural interferon-␣/␤-producing cells link innate and adaptive immunity. J Exp Med. 2000;192:219-226. 17. Fitzgerald-Bocarsly P, Feldman PM, Mendelsohn M, Curl S, Lopez C. Human mononuclear cells which produce interferon-alpha during NK[HSV-FS] assays are HLA-DR positive cells distinct from cytolytic natural killer effectors. J Leukoc Biol. 1988;43:323-334. 18. Cella M, Jarrossay D, Facchetti F, et al. Plasmacytoid mono-


19.

20.

21.

22.

23. 24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

cytes migrate to inflamed lymph nodes and produce high levels of type I IFN. Nat Med. 1999;5:919-923. Cella M, Facchetti F, Lanzavecchia A, Colonna M. Plasmacytoid dendritic cells activated by influenza virus and CD40L drive a potent TH1 polarization. Nat Immunol. 2000;1:305-310. Lore K, Metts MR, Benchley JM, et al. Toll-like receptor ligands modulate dendritic cells to augment cytomegalovirusand HIV-1-specific T cell responses. J Immunol. 2003;171: 4320-4328. Moseman EA, Liang X, Dawson AJ, et al. Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4⫹CD25⫹ regulatory T cells. J Immunol. 2004;173:4433-4442. Martin P, Del Hoyo GM, Anjuere F, et al. Characterization of a new subpopulation of mouse CD8␣⫹B220⫹ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential. Blood. 2002;100:383-390. Bilsborough J, Viney JL. Getting to the guts of immune regulation. Immunology. 2002;106:139-143. Kuwana M, Kaburaki J, Wright TM, Kawakami Y, Ikeda Y. Induction of antigen-specific human CD4⫹ T cell anergy by peripheral blood DC2 precursors. Eur J Immunol. 2001;31: 2547-2557. Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med. 1994;179:1109-1118. Romani N, Reider D, Heuer M, et al. Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability. J Immunol Methods. 1996;196:137-151. Heiser A, Dahm P, Yancey DR, et al. Human dendritic cells transfected with RNA encoding prostate-specific antigen stimulate prostate-specific CTL responses in vitro. J Immunol. 2000;164:5508-5514. Caux C, Dezutter-Dambuyant C, Schmidt D, Banchereau J. GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature. 1992;360:258-261. Caux C, Massacrier C, Dezutter-Dambuyant C, et al. Human dendritic Langerhans cells generated in vitro from CD34⫹ progenitors can prime naive CD4⫹ T cells and process soluble antigen. J Immunol. 1995;155:5427-5435. Banchereau J, Palucka AK, Dhodapkar M, et al. Immune and clinical responses in patients with metastatic melanoma to CD34⫹ progenitor-derived dendritic cell vaccine. Cancer Res. 2001;61:6451-6458. Mackensen A, Herbst B, Chen JL, et al. Phase I study in melanoma patients of a vaccine with peptide-pulsed dendritic cells generated in vitro from CD34⫹ hematopoietic progenitor cells. Int J Cancer. 2000;86:385-392. Di Nicola M, Carlo-Stella C, Mortarini R, et al. Boosting T cell-mediated immunity to tyrosinase by vaccinia virus-transduced, CD34⫹-derived dendritic cell vaccination: a phase I trial in metastatic melanoma. Clin Cancer Res. 2004;10:5381-5390. Peters JH, Xu H, Ruppert J, et al. Signals required for differentiating dendritic cells from human monocytes in vitro. Adv Exp Med Biol. 1993;329:275-280. Mohamadzadeh M, Berard F, Essert G, et al. Interleukin 15 skews monocyte differentiation into dendritic cells with features of Langerhans cells. J Exp Med. 2001;194:1013-1020. Chomarat P, Dantin C, Bennett L, Banchereau J, Palucka AK.

BB&MT

36.

37.

38.

39. 40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

TNF skews monocyte differentiation from macrophages to dendritic cells. J Immunol. 2003;171:2262-2269. Paquette RL, Hsu NC, Kiertscher SM, et al. Interferon-␣ and granulocyte-macrophage colony-stimulating factor differentiate peripheral blood monocytes into potent antigen-presenting cells. J Leukoc Biol. 1998;64:358-367. Luft T, Pang KC, Thomas E, et al. Type I INFs enhance the terminal differentiation of dendritic cells. J Immunol. 1998; 161:1947-1953. Santini SM, Lapenta C, Logozzi M, et al. Type I interferon as a powerful adjuvant for monocyte-derived dendritic cell development and activity in vitro and in Hu-PBL-SCID mice. J Exp Med. 2000;294:1777-1788. Leonard WJ. TSLP: finally in the limelight. Nat Immunol. 2002;3:605-607. Butterfield LH, Ribas A, Dissette VB, et al. Determinant spreading associated with clinical response in dendritic cellbased immunotherapy for malignant melanoma. Clin Cancer Res. 2003;9:998-1008. Ueda Y, Itoh T, Nukaya I, et al. Dendritic cell-based immunotherapy of cancer with carcinoembryonic antigen-derived, HLA-A24-restricted CTL epitope: clinical outcomes of 18 patients with metastatic gastrointestinal or lung adenocarcinomas. Int J Oncol. 2004;24:909-917. Barrou B, Benoit G, Ouldkaci M, et al. Vaccination of prostatectomized prostate cancer patients in biochemical relapse, with autologous dendritic cells pulsed with recombinant human PSA. Cancer Immunol Immunother. 2004;53:453-460. Hsu FJ, Benike C, Fagnoni F, et al. Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat Med. 1996;2:52-58. Timmerman JM, Czerwinski DK, Davis TA, et al. Idiotypepulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients. Blood. 2002;99:1517-1526. Reichardt VL, Milazzo C, Brugger W, et al. Idiotype vaccination of multiple myeloma patients using monocyte-derived dendritic cells. Haematologica. 2003;88:1139-1149. Heiser A, Coleman D, Dannull J, et al. Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest. 2002;109:409-417. Su Z, Dannull J, Yang BK, et al. Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8⫹ and CD4⫹ T cell responses in patients with metastatic prostate cancer. J Immunol. 2005;174:3798-3807. Tucker MJ, Bretscher PA. T cells cooperating in the induction of delayed-type hypersensitivity act via the linked recognition of antigenic determinants. J Exp Med. 1982;155:1037-1049. Bretscher PA. A cascade of T-T interactions, mediated by the linked recognition of antigen, in the induction of T cells able to help delayed-type hypersensitivity responses. J Immunol. 1986;137:3726-3733. Mitchison NA, O’Malley C. Three-cell-type clusters pf T cells with antigen-presenting cells best explain the epitope linkage and noncognate requirements of the in vivo cytolytic response. Eur J Immunol. 1987;17:1579-1583. Shirai M, Pendleton CD, Ahlers J, et al. Helper-cytotoxic T lymphocyte (CTL) determinant linkage required for priming of anti-HIV CD8⫹ CTL in vivo with peptide vaccine constructs. J Immunol. 1994;152:549-556. Tjoa BA, Simmons SJ, Bowes VA, et al. Evaluation of phase 123

W. K. Decker et al.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63. 64.

65.

66.

67.

124

I/II clinical trials in prostate cancer with dendritic cells and PSMA peptides. Prostate. 1998;36:39-44. Schuler-Thurner B, Schultz ES, Berger TG, et al. Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells. J Exp Med. 2002;195:1279-1288. Smithers M, O’Connell K, MacFadyen S, et al. Clinical response after intradermal immature dendritic cell vaccination in metastatic melanoma is associated with immune response to particulate antigen. Cancer Immunol Immunother. 2003;52:41-52. Svane IM, Pedersen AE, Johnsen HE, et al. Vaccination with p53-peptide-pulsed dendritic cells, of patients with advanced breast cancer: report from phase I study. Cancer Immunol Immunother. 2004;53:633-641. Liu KJ, Wang CC, Chen LT, et al. Generation of carcinoembryonic antigen (CEA)-specific T-cell responses in HLAA*0201 and HLA-A*2402 late-stage colorectal cancer patients after vaccination with dendritic cells loaded with CEA peptides. Clin Cancer Res. 2004;10:2645-2651. Geiger JD, Hutchinson RJ, Hohenkirk LF, et al. Vaccination of pediatric solid tumor patients with tumor lysate-pulsed dendritic cells can expand specific T cells and mediate tumor regression. Cancer Res. 2001;61:8513-8519. Chang AE, Redman BG, Whitfield JR, et al. A phase I trial of tumor lysate-pulsed dendritic cells in the treatment of advanced cancer. Clin Cancer Res. 2002;8:1021-1032. Hernando JJ, Park TW, Kubler K, et al. Vaccination with autologous tumor antigen-pulsed dendritic cells in advanced gynaecological malignancies: clinical and immunological evaluation of a phase I trial. Cancer Immunol Immunother. 2002; 51:45-52. Oosterwijk-Wakka JC, Tiemessen DM, Bleumer I, et al. Vaccination of patients with metastatic renal cell carcinoma with autologous dendritic cells pulsed with autologous tumor antigens in combination with interleukin-2: a phase I study. J Immunother. 2002;25:500-508. Iwashita Y, Tahara K, Goto S, et al. A phase I study of autologous dendritic cell-based immunotherapy for patients with unresectable primary liver cancer. Cancer Immunol Immunother. 2003;52:155-161. Yamanaka R, Abe T, Yajima N, et al. Vaccination of recurrent glioma patients with tumor lysate-pulsed dendritic cells elicits immune responses: results of a clinical phase I/II trial. Br J Cancer. 2003;89:1172-1179. Stift A, Friedl P, Dubsky T, et al. Dendritic cell-base vaccination in solid cancer. J Clin Oncol. 2003;21:135-142. Stift A, Sachet M, Yagubian R, et al. Dendritic cell vaccination in medullary thyroid carcinoma. Clin Cancer Res. 2004;10: 2944-2953. Yu JS, Liu G, Ying H, et al. Vaccination with tumor lysate-pulsed dendritic cells elicits antigen-specific, cytotoxic T-cells in patients with malignant glioma. Cancer Res. 2004;64:4973-4979. Pandha HS, John RJ, Hutchinson J, et al. Dendritic cell immunotherapy for urological cancers using cryopreserved allogeneic tumor lysate-pulsed cells: a phase I/II study. BJU Int. 2004;94:412-418. Vilella R, Benitez D, Mila J, et al. Pilot study of treatment of biochemotherapy-refractory stage IV melanoma patients with autologous dendritic cells pulsed with a heterologous melanoma cell line lysate. Cancer Immunol Immunother. 2004;53: 651-658.

68. Caruso DA, Orme LM, Neale AM, et al. Results of a phase 1 study utilizing monocyte-derived dendritic cells pulsed with tumor RNA in children and young adults with brain cancer. Neurooncology. 2004;6:236-246. 69. Caruso DA, Orme LM, Amor GM, et al. Results of a phase I study utilizing monocyte-derived dendritic cells pulsed with tumor RNA in children with stage 4 neuroblastoma. Cancer. 2005;103:1280-1291. 70. O’Rourke MGE, Johnson M, Lanagan C, et al. Durable complete clinical responses in a phase I/II trial using an autologous melanoma cell/dendritic cell vaccine. Cancer Immunol Immunother. 2003;52:387-395. 71. Yannelli JR, Sturgill J, Foody T, Hirschowitz E. The large scale generation of dendritic cells for the immunization of patients with non-small cell lung cancer (NSCLC). Lung Cancer. 2005;47:337-350. 72. Janetzki S, Palla D, Rosenhauer V, et al. Immunization of cancer patients with autologous cancer-derived heat shock protein gp96 preparations: a pilot study. Int J Cancer. 2000; 88:232-238. 73. Avigan D, Vasir B, Gong J, et al. Fusion cell vaccination of patients with metastatic breast and renal cancer induces immunological and clinical responses. Clin Cancer Res. 2004;10: 4966-4708. 74. Ossenkoppele GJ, Stam AGM, Westers TM, et al. Vaccination of chronic myeloid leukemia patients with autologous in vitro cultured leukemic dendritic cells. Leukemia. 2003;17: 1424-1426. 75. Choudhury A, Gajewski JL, Liang JC, et al. Use of leukemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome-positive chronic myelogenous leukemia. Blood. 1997;89:1133-1142. 76. Westers TM, Stam AG, Scheper RJ, et al. Rapid generation of antigen-presenting cells from leukemic blasts in acute myeloid leukemia. Cancer Immunol Immunother. 2003;52:17-27. 77. Houtenbos I, Westers TM, Stam AG, et al. Serum-free generation of antigen presenting cells from acute myeloid leukaemic blasts for active specific immunisation. Cancer Immunol Immunother. 2003;52:455-462. 78. Wolkers MC, Brouwenstijn N, Bakker AH, Toebes M, Schumacher TNM. Antigen bias in T-cell cross-priming. Science. 2004;304:1314-1317. 79. Zinkernagel RM. On cross-priming of MHC class I-specific CTL: rule or exception? Eur J Immunol. 2002;32:2385-2392. 80. Ochsenbein AF, Sierro S, Odermatt B, et al. Roles of tumor localization, second signals and cross priming in cytotoxic T-cell induction. Nature. 2001;411:1058-1064. 81. Lee AW, Truong T, Bickham K, et al. A clinical grade cocktail of cytokines and PGE2 results in uniform maturation of human monocytes-derived dendritic cells: implications for immunotherapy. Vaccine 2002;20(suppl 4):A8-A22. 82. Scandella E, Men Y, Gillssen S, Forster R, Groettrup M. Prostaglandin E2 is a key factor for CCR7 surface expression and migration of monocyte-derived dendritic cells. Blood. 2002;100:1354-1361. 83. de Vries IJM, Krooshoop DJEB, Scharenborg NM, et al. Effective migration of antigen-pulsed dendritic cells to lymph nodes in melanoma patients is determined by their maturation state. Cancer Res. 2003;63:12-17. 84. Caux C, Massacrier C, Vanbervliet B, et al. Activation of human dendritic cells through CD40 cross-linking. J Exp Med. 1994;180:1263-1272.


85. Bedrosian I, Mick R, Xu S, et al. Intranodal administration of peptide-pulsed mature dendritic cell vaccines results in superior CD8⫹ T-cell function in melanoma patients. J Clin Oncol. 2003;21:3826-3835. 86. Pasare C, Medzhitov R. Toll-like receptors: linking innate and adaptive immunity. Microbes Infect. 2004;6:1382-1387. 87. Cisco RM, Abdel-Wahab Z, Dannull J, et al. Induction of dendritic cell maturation using transfection with RNA encoding a dominant positive toll-like receptor 4. J Immunol. 2004; 172:7162-7168. 88. Czerniecki BJ, Carter C, Rivoltini L, et al. Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells. J Immunol. 1997;159:3823-3837. 89. Jenkins MK, Khoruts A, Ingulli E, et al. In vivo activation of antigen-specific dendritic cells. Annu Rev Immunol. 2001;19: 23-45. 90. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol. 1994;12:991-1045. 91. Ridolfi R, Riccobon A, Galassi R, et al. Evaluation of in vivo labelled dendritic cell migration in cancer patients. J Transl Med. 2004;2:27-37. 92. Hersey P, Menzies SW, Halliday GM, et al. Phase I/II study of treatment with dendritic cell vaccines in patients with disseminated melanoma. Cancer Immunol Immunother. 2004;53: 125-134. 93. Lu W, Arraes LC, Ferreira WT, Andrieu JM. Therapeutic dendritic-cell vaccine for chronic HIV-1 infection. Nat Med. 2004;10:1359-1365. 94. Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med. 1998;4:328-332. 95. Dhodapkar MV, Steinman RM, Sapp M, et al. Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells. J Clin Invest. 1999;104:173180. 96. Dhodapkar MV, Steinman RM, Krasovsky J, Munz C, Bhardwaj N. Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med. 2001;193:233-238. 97. Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol. 2003;21:685-711. 98. Mackensen A, Krause T, Blum U, et al. Homing of intravenously and intralymphatically injected human dendritic cells generated in vitro from CD34⫹ hematopoietic progenitor cells. Cancer Immunol Immunother. 1999;48:118-122. 99. Morse MA, Coleman RE, Akabani G, et al. Migration of human dendritic cells after injection in patients with metastatic malignancies. Cancer Res. 1999;59:56-58. 100. Fong L, Brockstedt D, Benike C, Wu L, Engleman EG. Dendritic cells injected via different routes induce immunity in cancer patients. J Immunol. 2001;166:4254-4259. 101. Schuler G, Steinman RM. Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro. J Exp Med. 1985;161:526-546. 102. Girolomoni G, Simon JC, Bergstresser PR, Cruz PD Jr. Freshly isolated spleen dendritic cells and epidermal Langerhans cells undergo similar phenotypic and functional changes during short-term culture. J Immunol. 1990;145:2820-2826. 103. Maloney DG. Preclinical and phase I and II trials of rituximab. Semin Oncol. 1999;26(5 suppl 14):74-78.

BB&MT

104. Sliwkowski MX, Lofgren JA, Lewis GD, et al. Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin). Semin Oncol. 1999;26(4 suppl 12):60-70. 105. Dannull J, Nair S, Su Z, et al. Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA encoding OX40 ligand. Blood. 2005;105:3206-3213. 106. Ribas A, Butterfield LH, Amarnani SN, et al. CD40 crosslinking bypasses the absolute requirement for CD4 T cells during immunization with melanoma antigen gene-modified dendritic cells. Cancer Res. 2001;61:8787-8793. 107. Demuth T, Berens ME. Molecular mechanisms of glioma cell migration and invasion. J Neurooncol. 2004;70:217-228. 108. Ridgway D. The first 1000 dendritic cell vaccinees. Cancer Invest. 2003;21:873-886. 109. Janeway CA, Travers P, Hunt S, Walport M. Immunobiology: The Immune System in Health and Disease. 3rd ed. New York: Garland Publishing Inc; 1997. 110. Bennett SRM, Carbone FR, Karamalis F, Miller JFAP, Heath WR. Induction of a CD8⫹ cytotoxic T lymphocyte response by cross-priming requires cognate CD4⫹ T cell help. J Exp Med. 1997;186:65-70. 111. Bennett SRM, Carbone FR, Karamalis F, et al. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature. 1998;393:478-480. 112. Schoenberger SP, Toes REM, Van der Voort EIH, Offringa R, Melief CJM. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature. 1998;393: 480-483. 113. Van Mierlo GJD, Boonman ZFHM, Dumortier HMH, et al. Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8⫹ CTL to cause tumor eradication. J. Immunol. 2004;173:6753-6759. 114. Behrens G, Li M, Smith CM, et al. Helper T cells, dendritic cells and CTL immunity. Immunol Cell Biol. 2004;82:84-90. 115. Rocha B, Tanchot C. Towards a cellular definition of CD8⫹ T-cell memory: the role of CD4⫹ T-cell help in CD8⫹ T-cell responses. Curr Opin Immunol. 2004;16:259-263. 116. Bourgeois C, Rocha B, Tanchot C. A role for CD40 expression on CD8⫹ T cells in the generation of CD8⫹ T cell memory. Science. 2002;297:2060-2063. 117. Tham EL, Shrikant P, Mescher MF. Activation-induced nonresponsiveness: a Th-dependent regulatory checkpoint in the CTL response. J Immunol. 2002;168:1190-1197. 118. Macian F, Im SH, Garcia-Cozar FJ, Rao A. T-cell anergy. Curr Opin Immunol. 2004;16:209-216. 119. Sprent J, Kishimoto H. The thymus and negative selection. Immunol Rev. 2002;185:126-135. 120. Decker WK, Xing D, Li S, et al. Double loading of dendritic cell MHC class I and MHC class II with an AML antigen repertoire enhances T-cell responses in vitro. Blood. 2004;104:693a. 121. Lim SH, Baily-Wood R. Idiotype protein-pulsed dendritic cell vaccination in multiple myeloma. Int J Cancer. 1999;83: 215-222. 122. Titzer S, Christensen O, Manzke O, et al. Vaccination of multiple myeloma patients with idiotype-pulsed dendritic cells: immunological and clinical aspects. Br J Haematol. 2000; 108:805-816. 123. Maecker HT, Auffermann-Gretzinger S, Nomura LE, et al. Detection of CD4 T-cell responses to a tumor vaccine by cytokine flow cytometry. Clin Cancer Res 2001;7(suppl 3):902s-908s.

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