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[Cancer Research 66, 4943-4951, May 1, 2006]
© 2006 American Association for Cancer Research

Clinical Research

Immunization of Stage IV Melanoma Patients with Melan-A/MART-1 and gp100 Peptides plus IFN-{alpha} Results in the Activation of Specific CD8+ T Cells and Monocyte/Dendritic Cell Precursors

Tiziana Di Pucchio1, Lorenzo Pilla5, Imerio Capone1, Maria Ferrantini1, Enrica Montefiore1, Francesca Urbani1, Roberto Patuzzo6, Elisabetta Pennacchioli6, Mario Santinami6, Agata Cova5, Gloria Sovena5, Flavio Arienti7, Claudia Lombardo7, Arianna Lombardi2, Patrizia Caporaso3, Stefania D'Atri3, Paolo Marchetti2, Enzo Bonmassar4, Giorgio Parmiani5, Filippo Belardelli1 and Licia Rivoltini5

1 Section of Experimental Immunotherapy, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità; 2 Division IV of Dermatology, 3 Laboratory of Molecular Oncology, Istituto Dermopatico dell'Immacolata; and 4 Department of Neurosciences, University of Rome "Tor Vergata," Rome, Italy; and Unit of 5 Immunotherapy of Human Tumor, 6 Melanoma and Sarcoma, and 7 Immunohematology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy

Requests for reprints: Filippo Belardelli, Section of Experimental Immunotherapy, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 39-06-4990-3290; Fax: 39-06-49902097; E-mail: belard{at}iss.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The use of IFN-{alpha} in clinical oncology has generally been based on the rationale of exploiting its antiproliferative and antiangiogenic activities. However, IFN-{alpha} also exhibits enhancing effects on T-cell and dendritic cell functions, which may suggest a novel use as a vaccine adjuvant. We have carried out a pilot phase I-II trial to determine the effects of IFN-{alpha}, administered as an adjuvant of Melan-A/MART-1:26-35(27L) and gp100:209-217(210M) peptides, on immune responses in stage IV melanoma patients. In five of the seven evaluable patients, a consistent enhancement of CD8+ T cells recognizing modified and native MART-1 and gp100 peptides and MART-1+gp100+ melanoma cells was observed. Moreover, vaccination induced an increase in CD8+ T-cell binding to HLA tetramers containing the relevant peptides and an increased frequency of CD45RA+CCR7– (terminally differentiated effectors) and CD45RA–CCR7– (effector memory) cells. In all patients, treatment augmented significantly the percentage of CD14+ monocytes and particularly of the CD14+CD16+ cell fraction. An increased expression of CD40 and CD86 costimulatory molecules in monocytes was also observed. Notably, postvaccination monocytes from two of the three patients showing stable disease or long disease-free survival showed an enhanced antigen-presenting cell function and capability to secrete IP10/CXCL10 when tested in mixed leukocyte reaction assays, associated to a boost of antigen and melanoma-specific CD8+ T cells. Although further clinical studies are needed to show the adjuvant activity of IFN-{alpha}, the present data represent an important starting point for considering a new clinical use of IFN-{alpha} and new immunologic end points, potentially predictive of clinical response. (Cancer Res 2006; 66(9): 4943-51)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IFN-{alpha} is a cytokine belonging to type I IFNs, which has been most frequently used in patients with certain types of cancer, including some hematologic malignancies and solid tumors, such as melanoma, renal carcinoma, and Kaposi's sarcoma. Despite many years of work in preclinical as well as in clinical settings, the mechanisms underlying the IFN-induced antitumor response are not well understood. For a long time, it was thought that the direct inhibitory effects on tumor cell growth/function were the major mechanisms of the IFN-mediated antitumor responses in patients. However, early experiments in mouse tumor models have shown that IFN-{alpha} plays an important role in the activation of a long-lasting antitumor response (1). Subsequent studies have also provided evidence for a role of type I IFNs in the differentiation of the Th1 subset, as well as in the generation of CTL and in the promotion of the in vivo proliferation and survival of T cells (reviewed in ref. 2). In mouse models, type I IFNs have been shown to be powerful adjuvants when administered with soluble proteins or with the human influenza vaccine (3, 4). Interestingly, in both these studies, the co-injection of type I IFNs with the antigen or the vaccine followed by additional IFN injections, both 1 and 2 days later, proved to be the optimal schedule for the induction of the antibody response (3, 4).

Recently, several studies have shown that type I IFNs promote in vitro the differentiation of monocytes into dendritic cells and can markedly enhance dendritic cell activities (refs. 5–11 and reviewed in ref. 12). However, the pathways by which different subsets of monocytes can differentiate into distinct types of dendritic cells in response to IFNs in vivo remain poorly characterized. Human blood monocytes represent a heterogeneous cell population and several subsets can be distinguished on the basis of the expression of different membrane markers. Monocytes expressing the CD16 lymphocyte marker constitute the main monocyte subpopulation (13–15). Of note, CD14+CD16+ monocytes expressing low or high levels of CD14 seem to develop into dendritic cells or macrophages, respectively (16). In addition, several studies reported an increased number of circulating CD14+CD16+ in the course of infections, inflammation, and malignant diseases (17–20). Another subpopulation of circulating monocytes expresses the CD2 marker and these cells have been described as dendritic cells (21, 22). In particular, we have recently reported that peripheral blood monocytes may differentiate into highly active antigen-presenting cells and CD14+CD2+ monocytes can rapidly acquire the expression of the dendritic cell maturation marker CD83 after a short time (i.e., 4 hours) of incubation in the presence of IFN-{alpha} and granulocyte macrophage colony-stimulating factor (GM-CSF; ref. 23). As dendritic cells represent professional antigen-presenting cells for the generation of an immune response, these studies strongly suggest that IFN-{alpha} can play a pivotal role in linking innate and adaptive antitumor immunity and can be used as an adjuvant for cancer vaccines.

In spite of the evidence on the immune adjuvant activity of type I IFNs in viral and tumor models (1, 2), these cytokines have never been used as vaccine adjuvants in cancer patients. The main purpose of this pilot trial was to evaluate frequency, phenotype, and function of peptide-specific CD8+ T lymphocytes as well as phenotype and antigen-presenting cell activity of monocytes from stage IV melanoma patients vaccinated with MART-1 and gp100 peptides in combination with IFN-{alpha} as a vaccine adjuvant.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and treatment. This was an open-label, two-institution study to test the feasibility and the toxicity of Melan-A/MART-1 and gp100 peptides given in association with IFN-{alpha} as an adjuvant. Ten stage IV (American Joint Committee on Cancer) pretreated metastatic melanoma patients, molecularly typed as human leukocyte antigen HLA A*0201, were enrolled in the study. Patients were enrolled at the Istituto Nazionale per lo Studio e la Cura dei Tumori of Milan (n = 7) and the Istituto Dermopatico dell'Immacolata of Rome (n = 3). Of these patients, seven received at least one vaccine cycle and were considered assessable. Patients' characteristics are reported in Table 1 . Additional inclusion criteria were life expectancy of >6 months, Eastern Cooperative Oncology Group performance status of 0 to 1, and adequate hematopoietic, renal and hepatic functions. Patients were excluded if they had (a) brain metastases; (b) a concomitant malignant disease; (c) a severe cardiovascular disease; (d) chronically active infections; (e) a clinically significant autoimmune disease; (f) any illness requiring immunosuppressive therapy, previous chemotherapy, radiotherapy, or biological therapy received within 4 weeks before starting vaccination; (g) were pregnant or lactating; or (h) had a psychiatric illness that would interfere with patient compliance and informed consent. The study was approved by Ethics Committee of the Istituto Nazionale per lo Studio e la Cura dei Tumori and of the Istituto Dermopatico dell'Immacolata; approval and informed written consent were given by all patients. Patients included in the study were vaccinated according to a regimen consisting of two cycles of four vaccinations each (cycle 1: 250 µg of each peptide given i.d. every 2 weeks and 3 MU IFN-{alpha} given s.c. on days –1, 0, and +1 with respect to peptides, i.e., day 0; cycle 2: 250 µg of each peptide given monthly with a simultaneous single IFN-{alpha} dose of 3 MU). Both peptides and IFN-{alpha} were injected in close but separate sites next to local lymph nodes, changing the site of injection every two administrations. Toxicity was documented by evaluating the frequency and intensity of local adverse events as well as the clinically relevant changes in the laboratory variables categorized according to the WHO common toxicity criteria. Ophthalmologic examinations were carried out at 4, 8, and 12 weeks to assess possible autoimmune reactions caused by melanoma/retina cross-reacting differentiation antigens. Tumor evaluation was done at pretreatment visit, before the first (T73) and the fourth (T163) vaccine injection of the second cycle, and thereafter every 3 months and as clinically indicated. All time points of response were recorded from the time of the first peptide injection. This was done by the same sequential diagnostic imaging method. Clinical responses were defined according to Response Evaluation Criteria in Solid Tumors (24).


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  		Table 1. Characteristics of the assessable patients and their clinical response

 
Peptides and IFN-{alpha}. Two melanoma-associated peptides were used in this study: gp100:209-217(210M), IMDQVPFSV; Melan-A/MART-1:26-35(27L), ELAGIGILTV. The peptides were prepared under Good Manufacturing Practice conditions by Clinalfa (Laufelfingen, Switzerland) and were supplied as a water-soluble white powder in vials containing 250 µg of peptide. IFN-{alpha} (human leukocyte IFN{alpha}; Alfaferone) was supplied by Alfawassermann (Bologna, Italy) in commercial ampoules containing 3 MU of Alfaferone. The native Melan-A/MART-1:27-35 (AAGIGILTV) and gp100:209-217 (ITDQVPFSV) peptides, synthesized as previously described (25), together with the HLA-A*0201-binding peptides derived from HIV-NEF (VLEWRFDSRL), Flu A matrix M1 (GILGFVFTL), gp100 (280-288, YLEPGPVTA), and NY-ESO-1 (157-165V, SLLMWTTQV) proteins, were also used for in vitro studies.

HLA typing and peripheral blood mononuclear cell samples. Molecular class I typing was done on genomic DNA by PCR with sequence-specific primers (PCR-SSP), following reported conditions and international guidelines (26, 27). For immunologic monitoring, 30 mL of heparinized blood were obtained from each patient before vaccination (pre-Tx and T0) and 28 and 73 days after the first vaccination. For some patients, samples from the T117 vaccination time point were also available for testing. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation, frozen in aliquots by a computer-assisted freezing system (Microgitcool, IMV Technologies Italia, Piacenza, Italy), and stored in liquid nitrogen. For immunologic assays, PBMCs obtained before and at different times during vaccination were simultaneously thawed and incubated overnight at 37°C in RPMI 1640 (BioWhittaker Europe, Verviers, Belgium) supplemented with 10% AB human serum (BioWhittaker Europe) before doing functional and phenotypic assays. PBMC recovery after thawing was ≥60% whereas viability after overnight incubation was ≥ 95% as assessed by trypan blue staining.

Delayed-type hypersensitivity reactions. Delayed type hypersensivity (DTH) reaction to the two vaccine peptides was done at the baseline (T0), at T42 (before the fourth vaccine injection), at T117 (between the sixth and the seventh vaccine injection), and then at 2 months after the end of the treatment. At least 5 mm of induration or erythema read 48 hours after intradermal injection was required to score a gp100 or Melan-A/MART-1 skin test as positive. However, no positive DTH reaction was observed.

ELISPOT and HLA/peptide tetramer staining. IFN-{gamma} ELISPOT was done at the Istituto Nazionale per lo Studio e la Cura dei Tumori for all patients as previously described (28) and according to the instructions manufacturer (MabTech, Nacka, Sweden). Data were evaluated by a computer-assisted ELISPOT reader (Bioline, AID, Turin, Italy). PBMCs (1.67 x 105 per well) were tested in triplicates against the TAP-deficient line T2 (1.67 x 104 per well) pulsed with Melan-A/MART-1 and gp100 native and modified peptides, or with an HIV-derived epitope (NEF), the Flu A matrix M1 peptide, gp100:280-288, and NY-ESO-1:157-165V, used as controls. The HLA-A*0201+ Melan-A/MART-1+gp100+ melanoma cell line 501mel (29) was also included in the assay for evaluating T-cell recognition of endogenously processed antigens. The HLA-A*0201+ colon carcinoma cell line Colo 206 (ref. 30; purchased from American Type Culture Collection, Manassas, VA) was used as negative control. To assess interassay variability, IFN-{gamma} production by the anti–Melan-A/MART-1(27-35) HLA-A*0201-restricted T-cell clone A42 (31), releasing 120 ± 9 spots/500 cells (n = 21) in response to T2 cells pulsed with Melan-A/MART-1(27-35) peptide, was included in each test. HLA blocking experiments were carried out by preincubating target cells with the anti–class I HLA (A, B, and C) immunoglobulin M antibody (clone A6-136; kindly provided by Dr. Daniela Pende, INT, Genoa, Italy) or with the anti-HLA-DR monoclonal antibody (mAb) L243, as negative control (32). For statistical evaluation, a t test for unpaired samples was used to compare prevaccine and postvaccine spots of the same patient or to evaluate statistical significance of HLA blocking experiments. P < 0.05 was considered statistically significant.

Staining with HLA/peptide tetramers. HLA-A*0201 tetramers containing Melan-A/MART-1:26-35(27L), Melan-A/MART-1:27-35, gp100:209-217 (210M), or gp100:209-217 were purchased from Beckman Coulter (San Diego, CA). Tetramer binding was evaluated after staining with the iMASC Gating Kit (Beckman Coulter), containing FITC-anti-CD8 mAb, together with PC5-anti-CD4, CD13, and CD19 mAbs. Data were reported as percentage of tetramer+CD8+, CD4/CD13/CD19– cells. As negative control, we used iTAgTM HLA class I human negative tetramers SA-PE (Beckman Coulter), developed for assessing the level of background PE fluorescence. They were loaded with a peptide which was shown to tightly bind to HLA-A2 and was proven not to be recognized by any T cells from HLA-A2+ individuals. Cells were analyzed using FACSCalibur and CellQuest software (Becton Dickinson, San José, CA).

Monocyte-derived cell cultures. Monocyte-derived cultures were prepared and tested at the Istituto Superiore di Sanità. PBMCs were isolated from 50 mL of heparinized blood by Ficoll gradient and frozen. To test the functional properties of monocytes in mixed leukocyte reaction assays, monocytes were isolated by negative selection using monocyte-isolation kits (Miltenyi Biotech, Bergisch Gladbach, Germany) and plated at a concentration of 2 x 106/mL in AIMV medium supplemented with 2.5% human serum for 24 hours.

Immunophenotypic analysis. PBMCs were thawed and resuspended in PBS containing 1% human serum and incubated with fluorochrome-conjugated mAbs for 30 minutes at 4°C. The following mAbs were used for immunofluorescence staining: anti-CD14 (Becton Dickinson), CD16, CD40, CD86 (PharMingen, San Diego, CA), and anti-CD2 (clone SFCI3Pt2H9; Beckman Coulter). Samples were collected and analyzed by using a FACSort and data analysis was done with CellQuest software (Becton Dickinson).

Mixed leukocyte reaction. Allogeneic CD3+ T lymphocytes were sorted using anti-CD3 conjugated magnetic microbeads (Miltenyi Biotech) and seeded into 96-wells plates at 105 per well. Patient monocyte-derived cells were added to each well in triplicate at different stimulator-to-responder ratios. After 5 days, 1 µCi of [3H]thymidine (Amersham Pharmacia, Uppsala, Sweden) was added to each well and incubation was continued for additional 18 hours. Cells were finally collected by a Mach II Mcell harvester and thymidine uptake was quantitated by liquid scintillation counting on 1205 Betaplate.

Chemokine detection. Chemokine production was evaluated by commercial ELISAs (R&D Systems, Minneapolis, MN).

Statistical analysis. ANOVA was used to calculate the significance of the differences obtained with prevaccine (T0) and postvaccine PBMCs from the same patient. Statistical analysis for ELISPOT data was done by t test for paired samples, comparing results obtained with prevaccine (T0) and postvaccine (T28, T73, and T117) PBMCs from the same patient. P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical results. A total of 10 HLA-A*0201 positive patients with stage IV metastatic melanoma were vaccinated with gp100:209-217(210M) and Melan-A/MART-1:26-35(27L) peptides, administered in association with IFN-{alpha}. Seven of these patients completed at least the first vaccination cycle (four IFN-{alpha}/peptide injections) and were considered assessable for immune and clinical responses. Patients' characteristics are listed in Table 1. Three patients received fewer than four vaccine injections because of disease progression and were not included in the analysis. No severe side effects (grade 3/4, WHO criteria) were observed whereas grade 1/2 toxicities were detected in all but two patients (patients 4 and 7). Symptoms observed were fatigue, fever, and anorexia. No signs of autoimmunity were observed as assessed by physical examination for skin depigmentation (vitiligo) and ophthalmic examination (uveitis). Clinical response was not a primary end point of the current study. However, no major responses were observed whereas two patients experienced long-lasting disease stabilization (+24 and +13 months for patients 3 and 7, respectively) and prolonged disease-free interval (+11 months) was observed in a third patient vaccinated without evidence of disease (Table 1). It is worth mentioning that both patients with stable disease after vaccination had been previously treated with multiple cycles of chemotherapy and biological therapy.

Evaluation of peptide-specific T-cell responses. The immunologic monitoring was initially focused on assessing whether the administration of IFN-{alpha}, in conjunction with Melan-A/MART-1 and gp100-modified peptides, could result in a significant activation of specific CD8+ T cells. To this aim, IFN-{gamma} production in response to both modified and native peptides was assessed by ELISPOT in PBMCs obtained before and at different times during vaccination. As depicted in Fig. 1A , five of the seven patients tested showed a progressive increase in the frequency of T cells producing IFN-{gamma} in response to both modified and native Melan-A/MART-1 and, to a lesser but still significant extent, gp100 peptides. Enhanced cytokine release could be detected at T28 (i.e., before the third vaccination) and was still evident at T73 (i.e., before the fifth vaccination). Interestingly, no significant change in the recognition of an HLA-A2-restricted Flu-derived peptide was instead observed throughout the vaccination period. In addition, an increase in the recognition of T2 cells loaded with gp100:280-288 peptide, a melanoma-associated epitope not included in the vaccine, was observed in patients 4 (in T28) and 7 (T73), possibly due to in vivo epitope spreading or reawakening of spontaneous antitumor T-cell responses by IFN{alpha}/peptide vaccine (Fig. 1B). On the contrary, no significant modification of T cells recognizing NY-ESO-1:157-165V peptide, which was clearly detectable in uncultured PBMCs, was observed (data not shown). The increased recognition of modified and native peptides was paralleled by an improved ability of PBMCs to recognize Melan-A/MART-1+gp100+HLA-A2+ melanoma cells (Fig. 1C), thus indicating that a boost of T cells recognizing the same peptides when endogenously presented by tumor cells has likely occurred in vivo during vaccination. On the contrary, no change in the IFN-{gamma} release in response to Melan-A/MART-1–gp100–HLA-A2+ colon carcinoma cells was observed in the same patients (Fig. 1D). Both the peptide- and the melanoma cell–specific recognition was mediated by CD8+ T cells, as IFN-{gamma} release could be significantly blocked by the addition of an anti–HLA class I (Fig. 1D) but not an anti–HLA class II (data not shown) mAb (Fig. 1E). In three of these five patients, the increase of Melan-A/MART-1 and gp100-specific CD8+ T cells after vaccination was confirmed by HLA tetramer staining (Fig. 2A-C ). Indeed, the percentage of CD8+ T cells binding HLA-A2 tetramers carrying either Melan-A/MART-1 or gp100 peptides (either in their native or modified sequence) increased during vaccination. It is worth noting that the frequency of these peptide-specific T cells, as well as the increase induced by the vaccine, varied among patients, ranging from 0.01% to 1.8% in prevaccine PBMCs and from 0.15% to 7.4% in postvaccine samples. However, no significant change in the CD8+ T-cell staining with negative HLA tetramers was observed during vaccination.


Figure 1
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  		Figure 1. Increased IFN-{gamma} release by peptide- and melanoma-specific CD8+ T cells in PBMC of vaccinated patients. PBMCs obtained before and at different times during vaccination with IFN and peptides were tested by ELISPOT for the ability to release IFN{gamma} in response to T2 cells pulsed with Melan A/MART-1 and gp100 modified (M) and native peptides, or with NEF and Flu peptides, as negative and positive controls, respectively. Background spots obtained with T2 cells alone were subtracted (A); IFN{gamma} ELISPOT in response to T2 cells loaded with gp100:280-288 peptide, tested as melanoma-associated epitope not included in the vaccine (B); HLA-A2+ Melan A/MART-1+gp100+ melanoma cell line (501mel; C), or a HLA-A2+ Melan A/MART-1–gp100– colon carcinoma line (Colo 206), used as negative control (D). E, an example of blocking experiments that were done by pretreating peptide-pulsed T2 cells or 501mel cells with an anti–HLA class I mAb (A6-136) before adding PBMCs for IFN{gamma} ELISPOT. *, P < 0.05, statistical significance with respect to untreated target cells. No blocking was observed when an anti–HLA class II (L243) was used (data not shown). Representative of two or more independent experiments.

 

Figure 2
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  		Figure 2. Increase in CD8+ HLA/peptide tetramer+ T cells in PBMC of vaccinated patients. PBMCs obtained before and at different times during vaccination with IFN and peptides were tested for binding to HLA-A*0201 tetramers containing either the modified or the native MART-1 and gp100 peptides. PBMCs were stained with FITC-CD8 (positive gate), PC5-CD4, -CD13, -CD19 (negative gate) as well as with PE-HLA-A*0201/peptide tetramers, and analyzed by four-color flow cytometry (FACScalibur and CellQuest). Numbers represent the percentage of CD8+, tetramer+ cells (A, patient 1; B, patient 5; C, patient 7). As negative control, iTAgTM HLA class I human negative tetramers SA-PE (Beckman Coulter), developed for assessing the level of background PE fluorescence, were used (Neg). Representative of two independent experiments.

 
To further characterize the phenotype of peptide-specific CD8+ T cells induced during vaccination, the expression of CD45RA and CCR7, markers defining memory versus effector T cells, was evaluated in HLA/peptide tetramer+ CD8+ T lymphocytes obtained before and after vaccination. An increase in the CD45RA+CCR7– peptide-specific T cells, identified as terminally differentiated effector cells (33), was observed in all tested patients, although to different extent (Fig. 3 ). However, only three patients (patients 3, 5, and 7) displayed a boost in the CD45RA–CCR7– cells (representing the effector memory population; ref. 33), which instead decreased in the remaining two cases (patients 1 and 4). It is worth noting that the patients showing increase in tetramer-binding CD8+ T cells with effector-memory phenotype were clinical nonprogressors (i.e., undergoing either stable disease or long-lasting disease-free interval on vaccination) whereas patients with enhancement confined to terminally differentiated effector cells underwent disease progression.


Figure 3
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  		Figure 3. Changes of CD45RA and CCR7 expression in CD8+ HLA/peptide tetramer+ T cells in PBMC of vaccinated patients. HLA-A*0201/MART-1 tetramer+ CD8+ T cells, present in PBMCs obtained before or after (T28 or T73) vaccination, were analyzed for CD45RA and CCR7 expression by a four-color flow cytometry (FACScalibur and CellQuest). Representative of at least two independent experiments.

 
Immunophenotype of blood monocytes and monocyte/dendritic cell subsets. We then evaluated whether the IFN/peptide vaccination induced in vivo phenotypic changes in the monocyte/dendritic cell compartment. Figure 4 shows the percentage of monocytes and monocyte subpopulations in PBMCs of all patients analyzed before and 2 days after the first and fourth IFN/peptide treatment, as detailed in Materials and Methods. In all patients, a significant increase in the percentage of total CD14+ monocytes was observed after the first IFN/peptide treatment (T2) with respect to the percentage detected before treatment (T0; Fig. 4A). Before the fourth IFN/peptide vaccination (T42), the percentage of monocytes had returned to the pretreatment level in four patients while maintaining the same augmented level observed at T2 in two patients (Fig. 4A). On average, a significant increase in the percentage of CD14+ monocytes was again observed after the fourth treatment (T44; Fig. 4A). Interestingly, in two analyzed nonprogressing patients (patients 3 and 5), an increase in the percentage of CD14+ monocytes was detected throughout the entire vaccination period, with values at T73 (i.e., before the fifth IFN/peptide treatment) higher (14% and 42%, respectively) than those observed before vaccination (T0; 5% and 27%) and before the fourth treatment (T42; 7% and 32%). A significant increase was also observed in the percentage of CD14+CD16+ monocytes after both the first and fourth IFN/peptide treatment with respect to the pretreatment values (Fig. 4B). Additionally, a trend toward an increase of the percentage of CD14+CD2+ monocytes was observed although the differences between the median values in prevaccine versus postvaccine PBMCs did not reach statistical significance (Fig. 4C). In the three patients who experienced stable disease or prolonged disease-free survival on vaccination (patients 3, 5 and 7), the ex vivo analysis of the CD14+ monocyte subsets was characterized in details (Fig. 5 ). In all the three patients, an increase of the CD14dimCD16+ monocyte population was observed during the course of IFN/peptide vaccination although at different time points and extent for each patient (Fig. 5A). Interestingly, in patient 7, a 2- to 3-fold increase in the percentage of CD14dimCD16+ monocytes was observed after the first and fourth IFN/peptide treatment with respect to the corresponding pretreatment level (Fig. 5A). In the same three patients, higher percentages of CD14+CD2+ monocytes were detected after the fourth (patient 7) or the fifth (patients 3 and 5) IFN/peptide vaccination as compared with the prevaccination levels (Fig. 5B).


Figure 4
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  		Figure 4. Analysis of monocytes and monocyte subpopulations in patients' PBMC. PBMC obtained before (T0) and at the indicated times of IFN/peptide vaccination were analyzed for the expression of CD14 (A), CD14 and CD16 (B), and CD14 and CD2 (C) differentiation markers. For each patient, identified by the same symbol, the reported values indicate the percentage of positive cells over total PBMC. Columns, median values at each time point. Patients: 1 ({blacklozenge}), 2 (+), 3 ({blacksquare}), 4 ({blacktriangleup}), 5 (x), 6 (bullet), 7 (*).

 

Figure 5
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  		Figure 5. Immunophenotypic analysis of monocyte subpopulations in patients with stable disease. Dot plots showing the immunophenotypic analysis of CD14+CD16+ (A) and CD14+CD2+ (B) monocytes before (T0) and at the indicated times of IFN/peptide vaccination in patients 3, 5, and 7. A, the numbers in the dot plots indicate the percentage of CD14dimCD16+ cells over total PBMC.

 
We also did the ex vivo analysis of monocytes expressing CD40, CD80, and CD86 costimulatory molecules before and after the first and fourth IFN/peptide vaccination. A strong increase in the percentage of CD14+ monocytes expressing CD40 was observed in the majority of patients 2 days after both IFN/peptide treatments with respect to the prevaccination level (Fig. 6A ). Figure 6B illustrates the CD40 expression on CD14+ monocytes of patient 3 who experienced stable disease. In this patient, CD40 expression was found to be up-regulated on CD14+ monocytes recovered right after the first IFN/peptide treatment (T2), with respect to the pretreatment counterparts (T0), as well as at time points distant from the IFN/peptide treatment [i.e., 14 (T117) and 30 (T163) days after the fifth and the seventh vaccination, respectively].


Figure 6
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  		Figure 6. Analysis of costimulatory molecules expressed on monocytes of vaccinated patients. A, expression of CD40 on monocytes derived from patients before (T0) and at the indicated times of IFN/peptide vaccination. For each patient, identified by the same symbol as in Fig. 4, the reported values indicate the percentage of CD14+CD40+ cells over total PBMC population. Columns, median values at each time point. B, dot plots showing the expression of CD40 on monocytes obtained from patient 3 before (T0) and at the indicated times of IFN/peptide vaccination. C, expression of CD86 on monocytes derived from patients before (T0) and at the indicated times of IFN/peptide vaccination. For each patient, identified by the same symbol as in Fig. 4, the reported values indicate the mean fluorescence intensity (MFI) of CD86 expression on CD14+ monocytes. Columns, median values at each time point.

 
Concerning the CD86 expression, whereas no significant changes were observed in the percentage of CD14+CD86+ monocytes before and after vaccination, a trend towards an enhancement of CD86 expression was observed in terms of fluorescence intensity after each IFN/peptide treatment with respect to the corresponding pretreatment levels (Fig. 6C). In contrast, CD80 expression was not detected in circulating monocytes either before or after vaccination (data not shown).

Allostimulatory activity of prevaccination versus postvaccination patients' monocytes. As IFN/peptide vaccination induced changes in the percentage of circulating monocytes displaying an activated phenotype, we evaluated by mixed leukocyte reaction assays the functional activity of monocytes obtained before and after IFN/peptide vaccination in six patients (patients 1, 3, 4, 5, 6, and 7), of which all but one (patient 6) experienced increased frequency of CD8+ peptide-specific T cells. Purified monocytes were cultured in serum-free medium for 24 hours and then cocultured with allogeneic CD3+ T lymphocytes. As depicted in Supplementary Fig. S1, in three of the six cases analyzed (i.e., patients 1, 4, and 6), monocytes derived from PBMCs did not change their ability to stimulate the proliferation of allogeneic T cells during the course of vaccination. In contrast, a trend of increased proliferation was observed with monocytes derived from patients 3, 5, and 7 (right, solid lines) at postvaccination times with respect to prevaccination (T0). It is worth mentioning that these latter patients were clinical nonprogressors (i.e., undergoing either stable disease or long-lasting disease-free interval on vaccination). We also analyzed the levels of interleukin (IL)-5, IL-10, and IP-10/CXCL10 in the cocultures of allogeneic T lymphocytes with monocytes isolated from the same six patients. No detectable IL-5 and IL-10 secretion was found in any of the cocultures and time points analyzed. Interestingly, higher levels of IP-10/CXCL10 were found in the cultures of allogeneic T lymphocytes with monocytes isolated from patients 3, 5, and 7 at postvaccination times with respect to the amounts secreted in the cocultures with prevaccination monocytes (T0; Supplementary Fig. S1). In contrast, constant low levels of IP-10/CXCL10 were detected in the cultures of allogeneic T lymphocytes with either prevaccination or postvaccination monocytes derived from patients 1, 4, and 6 (Supplementary Fig. S1).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One of the critical issues for the development of more effective cancer vaccines is how to combine them with safe and powerful immune adjuvants capable of breaking tolerance against self antigens and promoting the expansion and survival of effector memory CD8+ T cells. In view of its well-known effects on the mobilization and activation of dendritic cells, GM-CSF is the cytokine most frequently used as adjuvant of cancer vaccines in clinical trials, but its effectiveness is still a matter of debate. In spite of the existence of much information on the clinical use of IFN-{alpha} in cancer patients, this cytokine had never been used as vaccine adjuvant in humans. Although early studies in mice had already suggested the possible advantages of using IFN-{alpha} as an immune adjuvant in cancer treatment (for reviews, see refs. 1, 2), only recent reports showing the promoting effects of these cytokines on the differentiation and activations of human dendritic cells (5–12) have led to a new interest in testing the possible effectiveness of IFN-{alpha} as a vaccine adjuvant in cancer patients.

This work represents the first report on the testing of IFN-{alpha} as an adjuvant in vaccination of cancer patients. In our pilot clinical trial, HLA-A2 stage IV melanoma patients were injected with MART-1 and gp100 peptides and IFN-{alpha} was administered in close spatial and temporal proximity to the peptide vaccine, a modality conceptually more suitable for a vaccine adjuvant. Although major clinical responses were not achieved, three of the seven evaluable patients showed durable disease stabilization after vaccination. A significant increase of peptide- and melanoma-specific CD8+ T lymphocytes, detected as increment of HLA class I–restricted IFN-{gamma} release in response to vaccine peptides or antigen-expressing melanoma cells, and as an increase in tetramer-positive cells, was observed in five patients, including those experiencing stable disease on vaccination. In addition, an increase in the recognition of T2 cells loaded with gp100:280-288 peptide, a melanoma-associated epitope not included in the vaccine, was observed in two patients, suggesting a potential role of the vaccine in epitope spreading (34, 35) or in vivo reawakening of spontaneous antitumor T-cell responses, as recently shown to occur in vaccinated melanoma patients (36).

Furthermore, vaccine administration promoted the increase of antigen-specific effector-memory (CD45RA–CCR7–) cells (33) only in patients with stable disease after vaccination and not in progressing patients. This evidence suggests that IFN-{alpha} may play a role in promoting the expansion of an effector memory cell pool that could eventually differentiate into terminal effector cells, as recently shown for CpG by Speiser et al. (37). In this study (37), the coadministration of CpG with the Melan-A/MART-1:26-35(27L) peptide formulated in incomplete Freund's adjuvant resulted in a significant increase of peptide-specific CD8+ T-cell frequencies with respect to the prevaccination values. Interestingly, the authors suggest that the magnitude and durability of the peptide-specific CD8+ T-cell response can be dependent on the amount of IFN-{alpha} induced by CpG oligodeoxynucleotides (37). The results of our study are consistent with this interpretation, as the increase in the peptide-specific CD8+ T-cell responses observed in the vaccinated patients is likely the result of IFN-{alpha} administration. In fact, the injection of peptides in the absence of adjuvant does not induce any detectable CD8+ T-cell response (38, 39). Now that the importance of IFN-{alpha} in the promotion of a protective immune response is fully recognized, it is tempting to speculate that the therapeutic role of IFN-{alpha} therapy in melanoma patients can at least, in part, rely on the ability of the cytokine to enhance CD8+ T-cell responses against tumor-associated antigens, as shown for patients with chronic myelogenous leukemia responding to IFN-{alpha} therapy (40). However, studies specifically addressing this issue are still missing, in spite of some evidence of a positive correlation between the presence of T lymphocytes in the infiltrates of melanoma lesions and the response to IFN-{alpha} (reviewed in ref. 2).

As for the study of phenotype/function of circulating monocytes/dendritic cell precursors, in all the seven patients analyzed for monocyte/dendritic cell phenotype and function, the percentage of CD14+ monocytes was significantly augmented after IFN/peptide vaccination and, within this cell population, the percentage of CD14+CD16+ cells was specifically increased soon after the IFN/peptide treatment. Recently, CD14+CD16+ monocytes have been indicated as cells in transitional stage of development of monocytes to either macrophages or dendritic cells. The CD14highCD16+ subsets can differentiate into macrophages whereas dendritic cells arise from CD14dimCD16+ cells (16). Of interest, the CD14+CD16+ subset of human monocytes has been shown to exhibit special capabilities to migrate (41) and to stimulate CD4+ T cells (42). The physiologic function of these populations, however, is still a matter of debate. Notably, alterations in the number and phenotype/function of CD14+CD16+ monocytes have been reported in patients with cancer, infectious diseases, or inflammatory disorders (17–20). In this study, we have also evaluated the percentage of blood CD14+CD2+ cells, as these cells have been described as circulating dendritic cell precursors (21–23) as well as potential precursors of the IFN-derived dendritic cells (23). The ex vivo fluorescence-activated cell sorting analysis showed a slight augment in the percentage of CD14+CD2+ monocyte/dendritic cell subset, which did not reach statistical significance, and it was not consistently observed after IFN/peptide treatment but rather progressively increased during vaccination in two patients with stable disease. In addition, the expression of CD40 and CD86 costimulatory molecules on CD14+ monocytes was generally enhanced after IFN/peptide treatment.

In six patients, we have also determined the allostimulatory activity of monocytes cultured for 24 hours in vitro. In two of the three nonprogressing patients, a clear-cut higher allostimulatory ability was exhibited by postvaccination monocytes as compared with the prevaccination counterparts. In addition, high levels of IP-10/CXCL10 were secreted after coculture of allogeneic lymphocytes with postvaccination monocytes derived from patients with stable disease. In contrast, no increase in the allostimulatory activity and IP-10/CXCL10 production was exhibited from monocytes obtained from patients undergoing a rapid disease progression. Chemokines play an important role in the regulation of the host immune reactivity against both pathogens and neoplastic cells and the balance of their production can result in a Th1 or a Th2 type of immune response (43). Among the many chemokines secreted by reactive host cells, the IP-10/CXCL10 lymphoid chemokine is produced by mature dendritic cells in lymphoid tissues and mediates the migration of plasmacytoid dendritic cells directly from blood and the preferential recruitment of Th1 lymphocyte cells (44). Of note, IP-10/CXCL10 is also produced by dendritic cells generated after a short-term exposure of monocytes to IFN-{alpha} and GM-CSF (6) and its expression seems to correlate with the clinical response to immunotherapy in melanoma patients.8 Thus, we speculate that the high production of IP-10/CXCL10 in the mixed leukocyte reaction cultures from patients with stable disease showing enhanced antigen-presenting cell activity (Supplementary Fig. S1), which is consistent with the detection of high levels of secretion of this chemokine in the supernatants of the same patients' monocytes cultured alone for 24 in fresh medium (data not shown), can represent a marker of IFN-{alpha} adjuvant activity.

In conclusion, our study indicates that the IFN/peptide vaccination can affect the phenotypic and functional properties of blood circulating monocytes inducing the generation of defined subsets of monocytes/dendritic cells endowed with different functions, possibly associated with the clinical effect of vaccination. Of note, the IFN/peptide vaccination induced a general increase of the percentage of blood circulating dendritic cell precursors/CD14+ monocytes with activated phenotype, suggesting that some changes can occur in all the treated patients. The phenotypic and functional changes mediated by IFN in monocyte/dendritic cell precursors could be responsible for the observed in vivo priming of antigen-specific effector-memory CD8+ T cells with enhanced antitumor activity. Although further clinical studies are needed to assess vaccine adjuvant activity of IFN-{alpha} in cancer patients, the present data represent an important starting point for considering a new clinical use of this cytokine aimed at promoting the expansion of effector memory CD8+ T cells and mobilizing highly active monocyte-derived antigen-presenting cells. Lastly, we propose that the characterization of blood monocyte/dendritic cell phenotype and function should be considered as an additional variable in the immunomonitoring of patients enrolled in protocols based on the use of immunopotentiators, including IFN-{alpha}.


   Acknowledgments
 
Grant support: Italian Ministry of Health (Special project no. 0AP/F entitled "New antitumor therapies with Interferon: from the basic research to a clinical trial with cancer vaccines"), Italian Association for Cancer Research, and Fondo per gli investimenti della ricerca di base-Ministero dell'Istruzione, dell'Università e della Ricerca (projects RBNE017B4C and RBNE01N9EE).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Alessandra Di Pucchio for her help in the statistical analysis, Cinzia Gasparrini for secretarial assistance, and Dr. Giuseppe Viscomi (Alfawassermann, Bologna, Italy) for providing human leukocyte IFN{alpha} (Alfaferone).


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

T. Di Pucchio and L. Pilla contributed equally to this work.

8 F. Marincola and M. Panelli, unpublished results. Back

Received 9/21/05. Revised 1/27/06. Accepted 3/ 1/06.


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