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Intranodal Administration of Peptide-Pulsed Mature Dendritic Cell Vaccines Results in Superior CD8⁺ T-Cell Function in Melanoma Patients

Isabelle Bedrosian, Rosemarie Mick, Shuwen Xu, Harvey Nisenbaum, Mark Faries, Paul Zhang, Peter A. Cohen, Gary Koski, Brian J. Czerniecki

From the Harrison Department of Surgical Research and the Departments of Surgery, Biostatistics and Epidemiology, Radiology, and Pathology, University of Pennsylvania, Philadelphia, PA; and the Center for Surgery Research, Cleveland Clinic, Cleveland, OH.

Address reprint requests to Brian J. Czerniecki, MD, PhD, Department of Surgery, University of Pennsylvania, 4 Silverstein, 3400 Spruce St, Philadelphia, PA 19104; e-mail: brian.czerniecki{at}uphs.upenn.edu.

	ABSTRACT

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Purpose: We evaluated the feasibility, safety, and immunogenicity of mature, peptide-pulsed dendritic cell (DC) vaccines administered by different routes.

Patients and Methods: We performed a randomized, phase I, dose-escalation study in 27 patients with metastatic melanoma receiving four autologous peptide-pulsed DC vaccinations. Patients were randomly assigned to an intravenous (IV), intranodal (IN), or intradermal (ID) route of administration (ROA). For each route, primary end points were dose-limiting toxicity, maximum-tolerated dose, and T-cell sensitization. Sensitization was evaluated through tetramer staining, in vitro peptide recognition assays, and delayed-type hypersensitivity (DTH) responses.

Results: Twenty-two (81.5%) of 27 patients completed all four vaccinations. Vaccinations were well tolerated; a few patients exhibited grade 1 to 2 toxicities including rash, fever, and injection site reaction. All routes of administration induced comparable increases in tetramer-staining CD8⁺ T cells (five of seven IV, four of seven IN, and four of six ID patients). However, the IN route induced significantly higher rates for de novo development of CD8⁺ T cells that respond by cytokine secretion to peptide-pulsed targets (six [85.7%] of seven IN patients v two [33%] of six ID patients v none [0%] of six IV patients; P = .005) and de novo DTH (seven [87.5%] of eight IN patients v two [33.3%] of six ID patients v one [14.3%] of seven IV patients; P = .01) compared with other routes.

Conclusion: Administration of this peptide-pulsed mature DC vaccine by IN, IV, or ID routes is feasible and safe. IN administration seems to result in superior T-cell sensitization as measured by de novo target-cell recognition and DTH priming, indicating that IN may be the preferred ROA for mature DC vaccines.

	INTRODUCTION

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DENDRITIC CELL (DC) vaccines show promise for treating patients with malignancies.^1–3 DCs exist in peripheral tissues in an immature state but begin the process of maturation on receipt of signals that may indicate infection or inflammatory tissue damage. Conventional methods for culturing DCs from CD14⁺ monocyte precursors use granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 to derive immature DCs in approximately 1 week.⁴ Treatment with tumor necrosis factor alpha, bacterial lipopolysaccharide, CD40L, or monocyte-conditioned medium for an additional 1 to 2 days completes maturation.^5–8 We have developed another method, based on an alternate DC activation pathway, that uses calcium-mobilizing agents to drive differentiation of fully mature DCs from CD14⁺ monocytes in only 2 days.^9,10 Collectively, these methods provide options for using either immature or mature DCs for vaccines.

Although most clinical trials have used immature DCs,^2,11–14 recent evidence indicates they may actually be tolerizing.¹⁵ Others have shown that mature DCs may have an advantage for activating CD8⁺ T cells,¹⁶ further indicating that more studies with mature DCs should be undertaken.

Another unresolved question in DC vaccine therapy is the optimal route of administration (ROA). Introduction by the intravenous (IV) ROA is attractive because of convenience and the ability to administer large numbers of cells. Subcutaneous or intradermal (ID) administration is another option that may lead to improved T-cell responses compared with DC administered IV,¹¹ although efficiency of migration to lymph nodes may be problematic. A third ROA option is intranodal (IN) administration, which may further improve T-cell activation and enhance tumor responses.¹⁷ This ROA requires additional technical expertise, but it sidesteps problems of migration and allows delivery of a known quantity of DCs to the precise anatomic region where T-cell–dependent immune responses are generated.

The current study was undertaken to establish the dose-limiting toxicity, maximum-tolerated dose (MTD), and immunizing efficacy of peptide-pulsed mature DCs activated using calcium-signaling agents and administered to metastatic melanoma patients using three different ROAs.

	PATIENTS AND METHODS

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Reagents
Calcium ionophore A23187 was purchased from Sigma Chemical Co (St Louis, MO), and a certificate of analysis was provided. Clinical-grade recombinant IL-12 was a generous gift from Genetics Institute (Cambridge, MA). Clinical-grade recombinant IL-2 was a gift from Cetus Corporation (Menlo, CA). Recombinant GM-CSF was purchased from Immunex (Seattle, WA). The clinical-grade melanoma peptides pulsed on DC vaccines MART-1 (27–35), gp100 (209M, 209–216), and tyrosinase (370D, 369–377) were provided by the National Cancer Institute (NCI) Cancer Therapeutics Evaluation Program (Bethesda, MD). Fluorescein-isothiocyanate-(FITC-), cytocrome 5-(Cy-5-), or phycoerythrin (PE)-conjugated mouse antihuman CD86, CD83 (PharMingen, San Diego, CA), CD3, CD14, CD20, CD56, CD80, CD8 alpha and beta, and subclass-matched controls (Becton Dickenson, Mountain View, CA) were used. For in vitro experiments, peptides derived from the tumor antigens MART-1 (27–35), gp100 (209M, 209–216), gp100 (154–162), tyrosinase (370D, 369–377), and negative-control HLA-A2 peptides derived from p53 (149 to 157, STPPPGTRV) and the colon cancer antigen GA733 (174 to 182, YQLDPKFIT) were generated at the University of Pennsylvania Cancer Center Protein Chemistry Laboratory (Philadelphia, PA). MART-1 and gp100 (209M) tetramers were purchased from Coulter Scientific (San Diego, CA).

Study Design
We conducted a randomized, dose-escalation phase I clinical trial using mature CD83⁺ peptide pulsed DC to vaccinate patients with metastatic melanoma (Fig 1A). Patients were randomly assigned to receive the vaccine by IV, IN, or ID ROAs, with each patient receiving all four vaccines by the same ROA. The following three dose levels were planned: level 1, 5 million cells; level 2, 50 million cells; and level 3, 500 million cells. The highest dose level proved to be unfeasible because of high cell numbers requiring multiple leukapheresis and was not investigated. Five patients were to be treated at each dose level. If fewer than two patients experienced dose-limiting toxicity, then the dose was escalated. Otherwise, the MTD had been reached. NCI common toxicity criteria were used to grade toxicities. Dose-limiting toxicity was defined as any grade 3 or higher hematologic or nonhematologic toxicity occurring within 14 days of the final vaccination, excluding alopecia and skin vitiligo. Any evidence of retinal depigmentation was considered a dose-limiting toxicity. For each ROA, dose-limiting toxicity, MTD, and T-cell sensitization rates were determined.

View larger version (34K): [in this window] [in a new window]   Fig 1. Clinical trial design and dendritic cell (DC) vaccine generation. (A) Trial design; (B) overview of vaccine processing; (C) DC activation and peptide pulsing; and (D) vaccine schema. IV, intravenous; ID intradermal; IN, intranodal; IL, interleukin; SFM, serum-free monocyte macrophage medium; GM-CSF, granulocyte-macrophage colony-stimulating factor; DTH, delayed-type hypersensitivity.

Preparation of CD83⁺ DC Vaccines
Vaccines were prepared under Food and Drug Administration investigational new drug 1578 (Fig 1C). Under Good Laboratory Practice, monocytes were placed in serum-free monocyte macrophage medium (Gibco, Grand Island, NY) in the presence of 10 ng/mL of recombinant human GM-CSF overnight for 14 to 16 hours. The following day, calcium ionophore A23187 (375 ng/mL), IL-2 (300 IU), and IL-12 (10 ng/mL) were added. The DC were cultured an additional 18 hours, and 2 hours before harvest, they were pulsed with 50 ng/mL of clinical-grade peptides derived from MART-1 27–35 gp100 (209M, 209–216), and tyrosinase (370D, 369–377), which were supplied by the NCI Cancer Therapeutics Evaluation Program. Two hours later, the activated DC were harvested, washed three times, and resuspended in sterile saline for administration. Samples of vaccine were sent for microbiologic analysis for clearance before patient administration. Viability of the DC vaccine was determined by trypan blue exclusion. The vaccines had to have more than 60% viability for clinical use. The average viability of the vaccines administered was 87% (range, 63% to 98%). The vaccines that were to be administered ID and IN were reconstituted in 1 mL of sterile saline, and the vaccines administered IV were reconstituted in 50 mL of sterile solution in a bag for infusion.

Administration of Peptide-Pulsed DC Vaccine: Clinical Follow-Up and Response Assessment
Patients were admitted to the National Institutes of Health–approved Clinical Research Center at the Hospital of the University of Pennsylvania for vaccine administration; and after the initial vaccine was administered, they were observed overnight. For subsequent vaccines, the patients were observed for 2 hours and then discharged if free of complications. IN vaccines were administered into unaffected groin nodes using direct ultrasound guidance provided by the sonologist (H.N.). Patients received one vaccination every other week for four vaccinations (Fig 1D). Before each vaccination, a complete physical examination was performed, and laboratory values, including complete blood count, electrolyte panel, and liver function tests, were evaluated to assess potential vaccine toxicity. Approximately 2 weeks after the last vaccination, patients underwent laboratory evaluation, including a reimaging of head, chest, abdomen, and pelvis. Patients also underwent delayed-type hypersensitivity (DTH) skin testing and leukapheresis for immune studies. Thereafter, patients were observed every 3 months. Clinical responses (complete, partial, or minor response and stable or progressive disease) were evaluated using Response Evaluation Criteria in Solid Tumors.¹⁸

DC Quality Analysis and Cell-Surface Fluorescence Activated Cell Sorting (FACS) Analysis
Quality of the DC vaccines was monitored after administration by evaluating surface expression of the maturation marker CD83⁺ as well as the costimulatory molecules CD80 and CD86 through FACS analysis, as described previously.⁹

Preparation of T-Lymphocyte Subsets
Cryopreserved lymphocyte-rich elutriation fractions were used to prepare CD8⁺ cells using negative depletion columns as directed by the manufacturer (R&D Systems, Minneapolis, MN).

Quantitation of MART-1 and gp100 Tetramer-Reactive CD8⁺ T Cells
Elutriated lymphocyte-rich fractions from pre- and postvaccination samples were thawed and washed once with sterile phosphate-buffered saline. The cells were stained with tetramers MART-1 PE and anti-CD8 FITC or gp100 (209M) PE and anti-CD8 FITC for 30 minutes at room temperature. Cells were then washed once with FACS buffer and resuspended in phosphate-buffered saline with 0.5% paraformaldehyde. The proportion of tetramer-positive CD8⁺ T cells was determined through FACS analysis using a FACSscan apparatus running CellQuest software (Beckton Dickinson, San Jose, CA) by gating on the CD8⁺ T cells and assessing tetramer-staining cells in this population. Tetramer positivity in individual patients was defined as a two-fold or greater increase in the percentage of tetramer-positive and CD8⁺ cells that was greater than 1% of the CD8⁺ T-cell population between pre- and postvaccine samples.

In Vitro Analysis of CD8⁺ T Cells for Peptide Reactivity
Autologous monocytes from the patients were pulsed with MART-1 (27–35), gp100 (209M, 209–217), or tyrosinase (370D, 369–377) peptides at 10 ng/mL 2 hours before harvest. Harvested cells were washed twice and plated in fresh RPMI, 5% AB serum, and 30 IU of IL-2 with purified pre- and postvaccine CD8⁺ T cells at a T-cell to DC ratio of 20:1. After 1 week, the T cells were harvested and tested against peptide-pulsed HLA-A2⁺ T2 target cells (a gift of Steve Rosenberg, MD; NCI, Bethesda, MD), as described previously.^9,19 In vitro reactivity of pre- and postvaccine CD8⁺ T cells to melanoma antigen was determined by cytokine enzyme-linked immunosorbent assay, as described previously.¹⁹ Cultures were considered positive for peptide reactivity if there was at least 100 pg/mL of specific interferon gamma (IFN-) secretion and there was at least a two-fold increase in specific cytokine release compared with the control cultures of target cells pulsed with two irrelevant HLA-A2 binding peptides. The criterion for determining whether a patient developed a previously absent peptide specificity included the requirement for nonreactivity of CD8⁺ T cells to peptide before vaccination and the development of positive reactivity, as defined above, after vaccination.

Measurement of DTH
DTH testing was performed both before and 2 weeks after vaccination (Fig 1D). In sterile saline, 100 µg of MART-1 (27–35), gp100 (209M, 209–217), or tyrosinase (370D, 369–377) peptides were administered ID. In addition, standard mumps and Candida skin tests were placed as control for DTH. Forty-eight hours later, DTH was assessed by measuring the area of erythema and induration using two-dimensional measurements. DTH was scored positive if the area of erythema and induration was greater than 4 mm.

Histopathology
The skin punch biopsy specimen was fixed in 10% neutrally buffered formaldehyde and embedded in paraffin. Four-µm thick sections were cut off the paraffin block for histologic and immunohistochemical evaluation. Immunohistochemical staining was performed using antibodies against CD45rb (LCA; clone pd7/26 & 2b11, 1:150; DAKO, Copenhagen, Denmark), CD3 (polyclonal, 1:150; DAKO), CD4 (clone 1f6, 1:10; NeoMarker, Fremont, CA), CD8 (clone c8/144b, 1:20; DAKO), and CD20 (clone L26, 1:150; DAKO) with avidin-biotin complex method.

Statistical Analysis
The intent of this study was to determine the dose-limiting toxicity and MTD for each ROA. DTH positivity, peptide reactivity, tetramer positivity rates, and 95% exact CIs were also estimated for each ROA. All patients are included in the toxicity analysis. A patient had to receive all four vaccinations to be included in the analyses of immunologic response. Patients were randomly assigned to ROA to avoid selection bias and to impose balance and patient comparability among the arms. The study was neither designed nor powered to detect prespecified differences among the arms. The comparisons described are exploratory analyses and should be considered for hypothesis generation. With modest sample sizes per arm, only large differences are detectable. The comparisons of DTH positivity, peptide reactivity, and tetramer positivity rates among the arms were performed by Fisher’s exact test using StatXact software (Cytel Software, Cambridge, MA). All quoted significance levels are two-sided.

	RESULTS

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Patient Characteristics and Vaccinations
Twenty-seven patients with metastatic melanoma provided informed consent for an IRB-approved protocol to receive four vaccinations with a mature peptide-pulsed DC vaccine (Table 1). Patients were randomly assigned to receive the vaccine by IV, ID, or IN ROAs. The first 15 patients were assigned to receive 5 million cells, and the subsequent 12 patients were assigned to receive 50 million cells. In all patients, CD83⁺, CD80⁺, CD86⁺, and CD14^dim mature DC were generated by treating CD14⁺ monocytes in serum-free monocyte macrophage medium with GM-CSF, calcium ionophore, IL-2, and IL-12 (an example is shown in Fig 2). For IN administration, it required only a few minutes to identify target nodes, and these injections were well tolerated by the patients. We injected the DC vaccine in 1.0 mL divided into each of two nodes to minimize disruption. Figure 3 demonstrates the injection of the vaccine into a groin lymph node for a representative patient.

View larger version (14K): [in this window] [in a new window]   Fig 2. Qualitative assessment of dendritic-cell (DC) activation. Mature DC (MDC) activated with calcium ionophore, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-2, and IL-12 demonstrate elevated levels of CD83, CD80, and CD86, and diminished CD14 expression compared with immature DC (iDC). Solid bar represents isotype control antibody; open bar represents the antibody against the surface molecule indicated.

View larger version (128K): [in this window] [in a new window]   Fig 3. Intranodal administration of peptide-pulsed mature dendritic cells (DC). Five million activated dendritic cells in 1.0 mL were injected into noninvolved groin nodes by ultrasound guidance. The figure shows the node expanding with the vaccine being injected. The needle can be seen in place in the node.

Peptide-Pulsed Mature DC Vaccines Were Well Tolerated
The DC vaccines in this protocol were well tolerated, and there were no dose-limiting toxicities (Table 2). In the IV group, one of five patients who received 5 million DC developed a grade 1 rash, and one of three patients who received 50 million DC experienced grade 1 fever. Of the five patients who received 5 million DC IN, three demonstrated grade 1 fever. In the ID arm, two of five patients who received 5 million cells experienced grade 1 rash, and two of five patients who receive 50 million DC demonstrated toxicity (one patient with grade 2 rash and one patient with a grade 2 reaction at the injection site). There were five adverse events reported to the IRB that were deemed not related to the vaccine but, rather, were considered related to the patients’ underlying melanoma.

All ROAs Lead to an Increase in Tetramer-Reactive CD8⁺ T Cells
It was apparent that some patients exhibited a considerable proportion (3% to 6%) of CD8⁺ T cells that were either MART-1 (27–35) or gp100 (209M) tetramer staining before vaccination. One patient actually presented with approximately 20% tetramer-positive cells before vaccine (Fig 4A). The existence of these naturally primed tetramer-staining T cells did not, however, prevent appreciable enhancements in response to vaccination. Overall, 65% of assessable patients (13 of 20 patients) developed an increase in peptide antigen-specific CD8⁺ T cells as assessed by tetramer staining for MART-1 and gp100 209M. Tetramer responses by the ROA and cell dose are listed in Table 3. For rate estimation, the results of both doses have been pooled. There was no difference (P = .99) in the development of tetramer-reactive CD8⁺ T cells among the ROA measured for MART-1 or gp100 (209M). Five of seven patients who were administered the vaccine IV (71.4%; 95% CI, 29% to 96.3%), four of seven patients who were administered IN (57.1%; 95% CI, 29% to 96.3%), and four of six patients who were administered ID (66.7%; 95% CI, 22.2% to 95.7%) demonstrated an increase in tetramer-reactive CD8⁺ T cells after vaccination. Several patients (patients 17a, 14a, 16a, 25a, 13a, and 7a) demonstrated fairly high levels (10%) of MART-1 tetramer-positive cells in the peripheral blood after vaccination (Fig 4A). A similar group of patients also had more than 10% tetramer-positive cells develop to gp100 (209M) as well (Fig 4A). There were a few patients in whom the percentage of tetramer-reactive T cells declined slightly after vaccination (Fig 4A). There was also no difference in the relative increase in tetramer-reactive T cells developing in any of the ROAs for the patients who developed an increase in tetramer-reactive CD8⁺ T cells (Fig 4B). An example of tetramer-reactive CD8⁺ T cells for two patients is shown in Fig 4C and 4D.

View larger version (48K): [in this window] [in a new window]   Fig 4. Measurement of tetramer-reactive CD8+ T cells before and after vaccination. Percentage of CD8+ tetramer-positive cells (A) and relative increase after vaccination (B) calculated from fluorescence activated cell sorting analysis. Each line/symbol represents individual subject. Tetramer plots from two patients (C, D). IN, intranodal; IV, intravenous; ID, intradermal.

IN Administration of Peptide-Pulsed DCs Enhances Development of DTH After Vaccination
Patients were skin tested before and after vaccination with 100 µg of MART-1 (27–35), gp100 (209M, 209–217), or tyrosinase (370D, 369–377) peptides in normal saline. Skin reactions were evaluated 48 hours later. All patients had evidence of DTH to at least one of the recall antigens (either Candida or mumps), indicating that the patients were not anergic. There was a statistically significant difference (P = .01) among the arms in the rates of patients who developed a de novo positive DTH reaction to at least one peptide (Table 3). The reactivity rates were 87.5% (95% CI, 47.3% to 99.7%) for IN patients, 33.3% (95% CI, 4.3% to 77.7%) for ID patients, and 14.3% (95% CI, 0.4% to 57.9%) for IV patients (Fig 5A). One IV patient (8a) did not complete the four vaccines but demonstrated the development of DTH to MART-1 after the third and final vaccine (not shown). As with tetramer reactivity and peptide recognition of pulsed T2 targets, the development of DTH response did not correlate with dose of vaccine (Table 3). An example of a positive DTH reaction is shown in Fig 5B. Punch biopsy of the DTH site demonstrated the influx of CD8⁺ T cells with few CD4⁺ cells and no evidence of B cells, indicating that the response was mediated through CD8⁺ T cells (Fig 6).

View larger version (20K): [in this window] [in a new window]   Fig 5. Intranodal administration of peptide-pulsed mature dendritic cells (DC) result in enhanced de novo development of delayed-type hypersensitivity (DTH) reactions. (A) Percentage of patients developing positive DTH reaction to a peptide not present before vaccine by route of DC administration. (B) Example of positive DTH reaction to MART-1 and gp100 (209M) after vaccine. IV, intravenous; IN, intranodal; ID, intradermal.

View larger version (173K): [in this window] [in a new window]   Fig 6. CD8+ T cells infiltrate site of positive delayed-type hypersensitivity (DTH) response. (A) Hematoxylin and eosin staining; (B) lymphocyte infiltration around vessel in skin; (C) anti-CD3 staining; (D) anti-CD4 staining; (E) anti-CD8 staining; and (F) anti-CD20 staining. Results are of a representative biopsy from one patient.

	DISCUSSION

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It should be kept in mind, however, that tetramer staining only reveals the number of T cells that carry a peptide antigen–specific receptor (such as those recognizing the MART-1 or gp100 209M antigen). It tells us nothing about T-cell function. In contrast, in vitro cytokine secretion assays and in vivo DTH responses collectively encompass several important functional qualities, including expansion and recognition of antigen, secretion of cytokines with known antitumor effects (such as IFN-),²⁰ trafficking, and ability to mediate inflammatory effects. We found that the IN ROA clearly induced superior T-cell function in these critical categories. Therefore, these data are consistent with previous reports^2,17 of improved T-cell function with IN administration of DC vaccines. IN administration of calcium ionophore and possibly other mature DC vaccines may, therefore, be the route of choice for optimized T-cell function. However, because the numbers of patients treated in this trial were limited, we cannot discard the possibility that the IV and ID routes would have performed on a par with IN if higher doses of DCs were administered.

The mature DC vaccines administered in this trial were well tolerated with few toxicities, which is consistent with the experience of others. Despite the fact that we did not reach dose-limiting toxicity, we did note that IN ROA at doses as low as 5 million cells results in substantial CD8⁺ T-cell activation as measured by the immunologic parameters assessed in the trial. However, clinical responses were also relatively rare, with only three patients demonstrating evidence of tumor regression. Two of the three patients that showed such responses developed an increase in peptide-recognizing CD8⁺ T cells as assessed by tetramer staining or a dramatic increase in cytokine secretion to a specific peptide (patients 16a and 25a). The third patient demonstrated neither an increase in tetramer nor a significant increase in cytokine secretion, but the patient did develop a positive DTH to gp100 after vaccination. Defining which immune parameters are most correlative with clinical response is unclear. We are continuing to evaluate which immunologic response parameters correlate best with clinical activity in these patients. It is clear, however, that there is not a perfect correlation in individual patients among the immune parameters measured in this study. For instance, an increase in tetramer-reactive cells did not always translate into enhanced peptide reactivity either in vitro, against pulsed T2 targets, or in vivo, in DTH responses. In addition, although the presence of cytokine-secreting cells was always seen in patients with positive DTH, cytokine-secreting cells could be observed in individuals without demonstrable DTH. Such results indicate that the tested immune response parameters can develop and act, to some degree, independent of one another.

It is possible to selectively mature DC with distinct, polarized functional attributes.^19,21–23 So-called DC1 secrete high levels of IL-12 and skew the development of CD4⁺ T cells into high IFN-, low IL-4/IL-5 (Th1) cytokine secretion profiles. DC2 secrete little or no IL-12 and, instead, skew development of CD4⁺ T cells into low IFN-, high IL-4/IL-5 (Th2) patterns. We recently showed that the effects of polarized DCs on CD8⁺ cells had little to do with skewing T-cell cytokine secretion profiles but, instead, induced profound alterations in T-cell antigen sensitivity, or functional avidity, whereby DC1-sensitized CD8⁺ cells could respond to 10- to 100-fold lower antigen concentrations than DC2-sensitized cells.²⁴ This enhanced functional avidity conferred by DC1 cytotoxic T lymphocytes (CTL) usually made the difference between directly recognizing and killing melanoma cells and the failure to do so, indicating profound functional advantages for using DC1. The DC generated for the present trial were predominantly DC2.¹⁹ This means that the functional capacity of CD8⁺ T cells sensitized by calcium-activated DC are probably suboptimal with respect to the important features of high functional avidity and concomitant capacity to directly recognize and kill tumor. This may explain the failure to achieve substantial clinical efficacy in this trial. Whether or not an IN-administered DC1 vaccine will lead to the development of CTL with an enhanced functional avidity and superior clinical results is the subject of a new clinical study.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicated no potential conflicts of interest.

	ACKNOWLEDGMENTS

 
We thank Janine Devine, Robert Sachs, Vickie Sallee, the staff of the Leukapheresis Unit in the University of Pennsylvania Bloodbank, and the staff of the National Institutes of Health General Clinical Research Center for their assistance in performing this trial.

	NOTES

 
Supported by American Cancer Society grant no. RPG 99-0029.

	REFERENCES

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1. Banchereau J, Palucka A, Dhodapkar M, et al: Immune and clinical responses in patients with metastatic melanoma to CD34+ progenitor-derived dendritic cell vaccine. Cancer Res 61:6451–6458, 2001[Abstract/Free Full Text]

2. Nestle FO, Alijagic S, Gilliet M, et al: Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 4:328–332, 1998[CrossRef][Medline]

3. Thurner B, Haendle I, Roder C, et al: Vaccination with Mage-3A1 peptide-pulsed mature monocyte-derived dendritic cells expands specific cytoxic T cells and induces regression of some metastases in advanced stage IV melanoma. J Exp Med 190:1669–1678, 1999[Abstract/Free Full Text]

4. Sallusto F, Lanzavecchia A: Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granuloctye/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179:1109–1118, 1994[Abstract/Free Full Text]

5. Sallusto F, Cella M, Danieli C, et al: Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: Downregulation by cytokines and bacterial products. J Exp Med 182:389–400, 1995[Abstract/Free Full Text]

6. Zhou L, Tedder T: CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc Natl Acad Sci USA 93:2588–2592, 1996[Abstract/Free Full Text]

7. Reddy A, Sapp M, Feldman M, et al: A monocyte-conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells. Blood 90:3640–3646, 1997[Abstract/Free Full Text]

8. Palucka K, Taquet N, Sanchez-Chapuis F, et al: Dendritic cells as the terminal stage of monocyte differentiation. J Immunol 160:4587–4595, 1998[Abstract/Free Full Text]

9. Czerniecki B, Carter C, Rivoltini L, et al: Calcium ionophore treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells. J Immunol 159:3823–3837, 1997[Abstract]

10. Koski GK, Schwartz GN, Weng DE, et al: Calcium mobilization in human myeloid cells results in acquisition of individual dendritic cell-like characteristics through discrete signaling pathways. J Immunol 163:82–92, 1999[Abstract/Free Full Text]

11. Butterfield L, Ribas A, Dissette V, et al: Determinant spreading associated with clinical response in dendritic cell-based therapy for malignant melanoma. Clin Cancer Res 9:998–1008, 2003[Abstract/Free Full Text]

12. Lau R, Wang F, Jeffrey G, et al: Phase I trial of intravenous peptide-pulsed dendritic cells in patients with metastatic melanoma. J Immunother 24:66–78, 2001

13. Morse M, Deng Y, Coleman D, et al: A phase I study of active immunotherapy with carcinoembryonic antigen peptide (CAP-1)-pulsed, autologous human cultured dendritic cells in patients with metastatic malignancies expressing carcinoembryonic antigen. Clin Cancer Res 5:1331–1338, 1999[Abstract/Free Full Text]

14. Murphy G, Tjoa B, Simmons S, et al: Infusion of dendritic cells pulsed with HLA-A2 specific prostate-specific membrane antigen peptides: A phase II prostate cancer vaccine trial involving patients with hormone-refractory metastatic disease. Prostate 38:73–78, 1999[CrossRef][Medline]

15. Dhodapkar M, Steinman R, Krasovsky J, et al: Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med 193:233–238, 2001[Abstract/Free Full Text]

16. Jonuleit H, Giesecke-Tuettenberg A, Tutling T, et al: A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T cell responses in humans following intranodal injection. Int J Cancer 93:243–251, 2001[CrossRef][Medline]

17. Lambert L, Gibson G, Maloney M, et al: Intranodal immunization with tumor lysate-pulsed dendritic cells enhances protective antitumor immunity. Cancer Res 61:641–646, 2001[Abstract/Free Full Text]

18. Therasse P, Arbuck S, Eisenhauer E, et al: New guidelines to evaluate the response to treatment in solid tumors. J Natl Cancer Inst 92:205–217, 2000[Abstract/Free Full Text]

19. Faries M, Bedrosian I, Xu S, et al: Calcium signaling inhibits IL-12 production and activates CD83+ dendritic cells that induce Th2 development. Blood 98:2489–2497, 2001[Abstract/Free Full Text]

20. Barth R, Mule J, Asher A, et al: Interferon gamma and tumor necrosis factor have a role in tumor regressions mediated by murine CD8+ tumor-infiltrating lymphocytes. J Exp Med 173:647–658, 1991[Abstract/Free Full Text]

21. Mosca P, Hobeika A, Clay T, et al: A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment. Blood 96:3499–3504, 2000[Abstract/Free Full Text]

22. Hilkens CM, Kalinski P, de Boer M, et al: Human dendritic cells require exogenous interleukin-12-inducing factors to direct the development of naive T-helper cells toward the Th1 phenotype. Blood 90:1920–1926, 1997[Abstract/Free Full Text]

23. Snijders A, Kalinski P, Hilkens C, et al: High-level IL-12 production by human dendritic cells requires two signals. Int Immunol 10:1593–1598, 1998[Abstract/Free Full Text]

24. Xu S, Koski G, Faries M, et al: Rapid high efficiency sensitization of CD8+ T cells to tumor antigens by dendritic cells leads to enhanced functional avidity and direct tumor recognition through an IL-12 dependent mechanism. J Immunol 171:2251–2261, 2003[Abstract/Free Full Text]

Submitted April 4, 2003; accepted August 4, 2003.

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