This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Walker, E. B. Articles by Urba, W. J. Search for Related Content PubMed PubMed Citation Articles by Walker, E. B. Articles by Urba, W. J.

gp100_209–2M Peptide Immunization of Human Lymphocyte Antigen-A2⁺ Stage I-III Melanoma Patients Induces Significant Increase in Antigen-Specific Effector and Long-Term Memory CD8⁺ T Cells

¹ Robert W. Franz Cancer Research Center, Earle A. Chiles Research Institute, Providence Portland Medical Center, Portland, Oregon; ² Department of Molecular and Microbiology and Immunology, Oregon Heath and Science University, Portland, Oregon; ³ Becton Dickinson Biosciences, San Jose, California; ⁴ Ludwig Institute for Cancer Research-Division of Oncology-Immunology, Lausanne, Switzerland; and ⁵ DMS–National Cancer Institute, Frederick, Maryland

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Thirty-five HLA-A2⁺ patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100_209–2M, emulsified in Montanide adjuvant. Direct ex vivo gp100_209–2M tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer⁺ CD8⁺ T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05–8.9%). Ex vivo IFN- cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100_209–2M in vitro activation showed that for all of the patients studied tetramer⁺ CD8⁺ T cells produced IFN-; however, some patients had significant numbers of tetramer⁺ IFN-^- CD8⁺T cells suggesting functional anergy. Additionally, 8 day gp100_209–2M in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer⁺ CD8⁺ T cells from postvaccine cells for 34 patients, and these IVS tetramer⁺ CD8⁺ T cells were functionally responsive by IFN- cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8⁺ T cells demonstrated the proliferation of functionally anergic gp100_209–2M- tetramer⁺ CD8⁺ T cells in several patients and also indicated interpatient variability of gp100_209–2M stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer⁺ CD8+ T cells (78.1 ± 4.2%) had either an "effector" (CD45 RA⁺/CCR7^-) or an "effector-memory" phenotype (CD45RA^-/CCR7^-). Notably, analysis of PBMCs collected 12–24 months after vaccine therapy demonstrated the durable presence of gp100_209–2M-specific memory CD8⁺ T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8⁺ T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In recent years the in vivo immunogenicity of melanoma peptides has been enhanced by altering the amino acid sequence in the HLA-binding region. This strategy has produced peptides with greater binding affinity for HLA-A2 molecules that have also been more effective when used for in vitro stimulation (IVS) of T-cell responses from melanoma patients (1 , 2) . The substitution of methionine for threonine at position 2 of the native gp100_209–217 peptide resulted in the modified peptide, gp100_209–2M (IMDQVPFSV), which was one of the first modified melanoma peptides tested in clinical trials (3, 4, 5, 6) . The initial gp100_209–2M peptide vaccine trials were conducted in patients with metastatic melanoma. Peptide-specific T-cell responses were usually detected only after extensive IVS of the postvaccine peripheral blood mononuclear cells (PBMCs) of patients, and were rarely detectable by direct ex vivo analysis after restimulation of cryopreserved cells with either the native or modified gp100 peptide (3, 4, 5) . Thus, immunomonitoring of the early clinical trials was influenced by the fact that the patients had advanced disease and potentially higher levels of immunological anergy to peptide vaccination. Immunomonitoring was also hampered by the fact that the IFN--specific ELISA used to monitor patient functional responses did not yield any information about either the phenotype(s) or the number of peptide-specific T cells.

Clinical studies have demonstrated the potential value of sensitive, quantitative functional assays such as cytokine flow cytometry (CFC) and ELISPOT analysis combined with antigen-specific, MHC-I-restricted tetramer analysis to measure peptide-specific CD8⁺ T-cell immune responses in vitro. The value of this strategy was demonstrated recently in gp100_209–2M vaccination studies of metastatic melanoma patients where the combined use of tetramer and CFC(IFN-) analysis demonstrated that only 50% of circulating gp100_209–2M tetramer⁺ CD8⁺ T cells in vaccinated patients produced IFN- after brief in vitro activation with native or modified gp100 peptide (7) . Thus, this type of ex vivo analysis can produce more complete information on the functional characteristics of circulating tumor antigen-specific CD8⁺ T cells. However, the presence of circulating functionally quiescent tumor antigen-specific T cells may not necessarily reflect a state of terminal functional anergy. Thus, recent work has also demonstrated that limited cognate-gp100_209–2M peptide-directed IVS of PBMCs from gp100_209–2M-vaccinated metastatic melanoma patients increased the mean frequency of CFC(IFN-)⁺ cells from 17% (by direct ex vivo analysis) to 84% of all of the gp100_209–2M tetramer⁺ CD8⁺ T cells, suggesting that circulating tetramer⁺ CD8⁺ T cells may be similarly responsive to peptide stimulation in vivo (8) . The utility of such combined phenotype and functional analysis of IVS-expanded CD8⁺ T cells is best realized only when pre-existing "baseline" ex vivo postvaccine phenotype and functional data are also available for individual patients. Comparison of ex vivo "baseline" tetramer and functional profiles to the IVS responses for each patient can reveal important interpatient differences in in vitro proliferation and functional potential, which may be indicative of differences in the strength of the immune response of individual patients to therapeutic melanoma peptide vaccination.

We described previously the results of direct ex vivo gp100_209–2M tetramer analysis of pre- and postvaccine PBMCs from 29 stage I-III melanoma patients vaccinated with gp100_209–2M peptide emulsified in Montanide ISA 51 (9) . This preliminary report demonstrated that 28 of 29 patients showed a significant increase in tetramer ⁺ CD8⁺ T cells after vaccination (median, 0.34%). Herein we present a comprehensive summary of the ex vivo gp100_209–2M tetramer analysis of all 35 of the evaluable patients in the clinical trial. Additional experiments were performed to examine interpatient variability of cognate-peptide-specific proliferation and functional responses of CD8⁺ T cells after 8-day gp100_209–2M directed IVS of postvaccine PBMCs. Postvaccine gp100_209–2M tetramer ⁺ CD8⁺ T cells were also interrogated using multiparameter flow cytometry to characterize the phenotype distribution of effector (CD45RA⁺/CCR7^-), effector-memory (CD45RA^-/CCR7^-), and central-memory (CD45RA^-/CCR7⁺) T cells. In addition to the detailed phenotype and functional analysis of postvaccine cells, PBMCs from long-term (12–24 months after vaccine therapy) blood collections were examined for the durable presence of gp100_209–2M-specific CD8⁺ T cells. These studies were performed to determine whether immunization with a MHC class I restricted peptide in Montanide and in the apparent absence of MHC class II restricted help could result in an antigen-specific CD8⁺ memory T-cell response. This study confirms the importance of the integrated use of both T-cell receptor (TCR)-specificity assays (tetramer analysis) and antigen-specific functional assays such as CFC(IFN-) for accurate assessment of the immune response in vaccinated cancer patients. The data also support the combined use of direct ex vivo analysis of cryopreserved PBMCs followed by limited cognate-antigen IVS and reanalysis of patient cells to determine more accurately the proliferative and functional potential of circulating tumor antigen-specific T cells.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Clinical Protocol.
HLA-A2⁺ patients with completely resected stage I-III melanoma were vaccinated with the gp100_209–2M peptide as described elsewhere (9) . Patients were randomized to receive s.c. injections of gp100_209–2M peptide and a control "reporter" peptide, human papillomavirus (HPV) 16E7_12–20, every 2 weeks (13 vaccinations) or every 3 weeks (9 vaccinations) for 6 months. The HLA-A2-restricted gp100_209–2M (NSC #683472) and HPV16E7_12–20 (NSC #673925) peptides were provided by the Cancer Therapy Evaluation Program under a National Cancer Institute Investigational New Drug Application (BB6123). Peptides were administered s.c. after emulsification in the Montanide ISA 51 (NSC #675756), manufactured by Seppic, Inc. and provided by Cancer Therapy Evaluation Program. A pre- and postvaccine therapy leukapheresis was performed on each patient; PBMCs were harvested using standard Ficoll-Hypaque density gradient centrifuge procedures and frozen in liquid nitrogen at 50 x 10⁶-100 x 10⁶ per ampule in 90% fetal bovine serum (Life Technologies, Inc., Rockville, MD) with 10% DMSO (Sigma). The postvaccine therapy leukapheresis was collected 2–4 weeks after the last vaccination. Cryopreserved PBMCs were also available from 50-ml blood draws collected just before each booster vaccination and approximately every 3–4 months out to 24 months after vaccination. The clinical protocol was reviewed by the Cancer Therapy Evaluation Program, National Cancer Institute, and approved by the Providence Health System Institutional Review Board. All of the patients gave their written informed consent before screening for eligibility.

gp100 Peptides and HLA-A2-Restricted Dimers.
Both freshly thawed PBMCs and IVS-expanded cells from individual patients were stimulated directly with either native gp100_209–217 (ITDQVPFSV) or modified gp100_209–2M (IMDQVPFSV) peptide; all of the peptides were purchased from ResGen Invitrogen Corp. (Huntsville, AL). IVS and freshly thawed T cells were also stimulated with a gp100 peptide-loaded HLA-A2 dimer molecule produced at our institution (10) . The recombinant, "empty," dimer coexpresses the ₁, ₂, and ₃ domains of the HLA-A2 heavy chain combined with a human ß2 microglobulin-IgG1-Fc fusion protein expressed in Drosophila S2 cells. Either native or modified gp100 or a control peptide was loaded into the "empty" dimer molecule by combining soluble dimer plus peptide at a weight:weight ratio of 10:1 (dimer to peptide) and incubating at room temperature for 2 h. Thereafter the peptide-loaded dimer could be maintained at 4°C for up to 1 month.

Autologous Dendritic Cells (DCs).
DCs were prepared from prevaccine leukapheresis PBMCs from each patient. Fifty to 70 x 10⁶ cryopreserved PBMCs were cultured in X-VIVO 15 medium (BioWhittaker, Walkersville, MD) supplemented with 5% heat-inactivated human AB serum (Irvine Scientific, Irvine, CA) for 2 h at 37°C/5% CO₂ in T-75 tissue culture flasks to allow for attachment of adherent monocytes. After 2-h nonadherent cells were washed off with prewarmed medium and fresh medium supplemented with 1000 units/ml of granulocyte macrophage colony-stimulating factor and 500 units/ml of interleukin 4 (PeproTech, Rocky Hill, NJ) was added. DC cultures were maintained for 7 days at 37°C/5% CO₂ without feeding, whereupon cells were harvested using cold (4°C) PBS supplemented with 2% human AB serum and then maintained at 4°C on ice in X-VIVO 15 medium plus 5% human AB serum until they were used as antigen presenting cells in CFC assays. Typically DCs were >90% CD11c⁺/HLA-DR⁺/lineage negative.

gp100_209–2M IVS T-Cell Cultures.
Pre- and postvaccine therapy cryopreserved PBMCs were placed in a single 8-day cycle of gp100_209–2M-directed IVS before being tested in CFC functional assays. The IVS expansion culture consisted of PBMCs in a 96-well round-bottomed plate at 2.5 x 10⁵/well (200 µl) in X-VIVO 15 medium supplemented with 5% human AB serum, human interleukin 15 at 25 g/ml (PeproTech) and gp100_209–2M peptide (10 µg/ml) for 2 days. Subsequently 60 IU/ml of interleukin 2 (Chiron, Emeryville, CA) was added to each well, and cultures were maintained for an additional 6 days before effector cells were harvested and used in CFC assays.

gp100_209–2M Tetramer Binding Assay.
Phycoerythrin-conjugated, HLA-A2-restricted, gp100_209–2M and HIV (pol; ILKEPVHGV) peptide-specific tetramers were produced at the Ludwig Institute (Lausanne, Switzerland). A pretitered optimal concentration of tetramer reagent was added to 10⁶ freshly thawed pre- and postvaccine PBMCs or to 10⁶ pre- and postvaccine IVS cells from each patient. Cells were incubated at room temperature in 100 µl of staining buffer (10% BSA and 0.1% NaN₃ in PBS) for 1 h in the dark with gentle mixing every 15 min; staining was performed in 12 x 75 mm polystyrene Falcon (#352054) tubes. Cells were washed twice in cold (4°C) staining buffer, resuspended in 100 µl of staining buffer, and stained simultaneously with optimal titered concentrations of antihuman CD3-PerCP, CD8-allophycocyanin (APC), and CD56-FITC (BD Biosciences, San Jose, CA). Cells were incubated at 4°C for 30 min, washed twice in cold staining buffer, and fixed in 500 µl of 1% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) made up in staining buffer. Fixed cells were stored in the dark at 4°C for at least 2 h before flow cytometry analysis, which was always performed within 24 h of staining. Cells were analyzed using a four-color acquisition format on a BD Calibur flow cytometry system. Data were collected on 5 x 10⁴ gated CD8⁺/CD3⁺ T cells and analyzed using CellQuest software (BD Biosciences). Negative controls consisted of fluorochrome-matched isotype controls for each test antibody and a phycoerythrin-conjugated HIV (pol), HLA-A2⁺ tetramer, and net tetramer⁺ frequencies were calculated by subtracting HIV (pol) tetramer frequency values from the percentage of gp100_209–2M tetramer⁺ cells.

CFC(IFN-) Assay Using Cryopreserved PBMCs.
Direct ex vivo CFC(IFN-) analysis was performed using freshly thawed pre- and postvaccine PBMCs. One x 10⁶ PBMCs/well were placed in a round-bottomed 96-well plate in 200 µl of X-VIVO 15 medium supplemented with 5% human AB serum. Cells were rested overnight (37°C/5% CO₂) before stimulation with native gp100_209–217 peptide or with gp100_209–2M peptide at 2.5 µg/well (10 µg/ml). HIV (pol) peptide was added at the same concentration to negative control cultures, and cells were incubated 1 h before 2.5 µg/well (10 µg/ml) of brefeldin A (Sigma) was added to each reaction well. Cells were incubated for an additional 4 h before the assay was collected. Twenty five µl of a 20 mM solution of EDTA (Sigma) was added to each well to stop the IFN- response and facilitate the efficient recovery of antigen-stimulated adherent CD8⁺ T cells, and plates were incubated for 10 min at room temperature before collection. Cell samples from replicate wells were pooled, washed twice with 3 ml of staining buffer, and 2 x 10⁶ cells were resuspended in 50 µL to 100 µl of staining buffer for fixation, permeabilization, and staining. Cells were fixed in 1–2 ml of 1x fluorescence-activated cell sorter (FACS) lysis buffer (BD Biosciences) for 10 min at room temperature. All of the samples were washed 2x in cold staining buffer and 500 µl of FACS permeabilization buffer (BD Biosciences) was added in each sample staining tube, gently mixed, and incubated at room temperature for 15 min. Permeabilized cells were washed twice and resuspended in 50–100 µl of staining buffer. Cells were stained with anti-IFN- (FITC)/CD69(PE)/CD8(peridinin chlorophyll protein-cyanine 5.5)/CD3(APC) monoclonal antibodies (BD Biosciences) and incubated at room temperature for 30 min. Matched isotype control stains consisting of CD3 (APC)/2a (FITC)/1(PE)/2a(peridinin chlorophyll protein-cyanine 5.5) were set up for each PBMC test group. After staining, cells were washed twice in staining buffer, fixed in 500 µl of 1% paraformaldehyde (Electron Microscopy Sciences), and stored at 4°C in the dark before analysis; analysis was performed within 24 h of staining. Cells were acquired through a standard lymphocyte (90° versus forward angle light scatter) light scatter gate, and 5 x 10⁴ to 1 x 10⁵ gated CD8⁺/CD3⁺ T cells were analyzed for IFN- staining using a FACS Calibur flow cytometry system and CellQuest software (BD Biosciences). Net frequencies of IFN-⁺ cells were calculated by subtracting background IFN- expression values due to HIV (pol) peptide stimulation from IFN- ⁺ CD8⁺ frequencies after gp100_209–2M stimulation.

CFC(IFN-) Assay Using gp100_209–2M IVS T Cells.
Autologous DCs were placed in a 96-well round-bottomed plate at 12 x 10³/well in 180 µl of X-VIVO 15 medium supplemented with 5% human AB serum. Dephosphorylated Lipid A (Avanti Polar Lipids, Alabaster, AL) was added to culture wells at 100 g/ml (20 g/well) overnight to mature the DCs (11) . Lipid A was made up in a 10% ethanol-in-water stock solution at 100 µg/ml before use. After overnight maturation 2.5 µg/well of gp100_209–2M peptide or native gp100_209–217 peptide or a negative control HIV (pol) peptide was added (10 µl aliquot/well) to appropriate wells and incubated for 2 h before the addition of IVS expanded T cells. Eight-day gp100_209–2M IVS pre- and postvaccine therapy PBMCs were added at 2.5 x 10⁵/well to replicate reaction wells containing peptide-pulsed DCs. Eight replicate wells were set up for each test condition (i.e., 2 x 10⁶ effector cells per test group), and cells were cultured for 1 h before the addition of brefeldin A at 5 µg/ml (1.25 µg/well) and additional overnight (12–14 h) incubation. Cells from replicate wells were pooled and interrogated for IFN- cytokine expression. Parallel stimulation cultures were also set up using IVS PBMCs at 2.5 x 10⁵/well except gp100_209–217, gp100_209–2M, or HIV (pol) peptide-loaded dimers were added to each reaction well at 0.25 µg/well. Similarly, dimer-stimulated cultures were incubated for 1 h before adding 5 µg/ml of brefeldin A and subsequently cultured overnight (12–14 h) before CFC(IFN-) analysis. Cells were harvested by adding 25 µl of a 20 mM solution of EDTA to each well for 10 min at room temperature before collection and pooling. CFC (IFN-) analysis was carried out exactly as described for cryopreserved cells.

Four-Color Flow Cytometry Phenotype Analysis.
Freshly thawed PBMCs from the postvaccine leukapheresis were stained with several different combinations of monoclonal antibodies that have been used to delineate subsets of human memory/effector CD8⁺ T cells. Each sample (2 x 10⁶ PBMCs) was stained with phycoerythrin-conjugated gp100_209–2M tetramer and a custom-conjugated (clone 2ST8.5H7; Beckman Coulter, Brea, CA) antihuman CD8 ß chain-specific monoclonal antibody linked to the tandem dye, phycoerythrin-cyanine 7. The CD8ß heterodimer is expressed by TCR ß⁺ CD8⁺ T cells, whereas human TCR ⁺ CD8⁺ T cells and CD8⁺ natural killer cells express only the CD8 homodimer (12) . A series of different two-antibody staining combinations were added to this basic 2-color reagent set. Thus, CD45RA(FITC) and CD45RO(APC) were added simultaneously to define the CD45 isoform subset profile on gp100_209–2M tetramer⁺ CD8⁺ T cells. Additionally, CD45RA(APC) plus FITC-conjugated CD27, CD28, CD57, or CCR7, were combined to quantitate the number of tetramer⁺ CD8⁺CD45RA⁺ T cells expressing each of the four effector/memory T-cell markers. After the addition of the monoclonal antibodies, cells were incubated at 4°C for 30 min, washed twice in staining buffer, and fixed in 500 µl of 1% paraformaldehyde. Cells were stored in the dark at 4°C until analysis, which occurred within 24 h of staining. Flow cytometry data were collected on 10⁴ gated CD8ß⁺/gp100_209–2M tetramer⁺ T cells. All of the analysis was performed on a four-color FACS Calibur instrument using CellQuest software. Appropriate compensation and isotype controls were run for all of the samples.

Statistical Methods.
Data in this study were evaluated using standard descriptive techniques, graphical methods, correlation and regression analyses, independent and paired tests, and paired nonparametric tests. We performed various tests on distributions of immune parameter values to test assumptions underlying the procedures. For ease in interpretation of within-subject analyses (e.g., pre- versus postresults, tetramer versus CFC assay results, ratios, and so forth) we report two-sided probability values obtained from Wilcoxon signed ranks tests.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ex Vivo gp100_209–2M Tetramer Staining of Pre- and Postvaccine PBMCs.
Direct ex vivo gp100_209–2M tetramer staining and flow cytometry analysis were performed on cryopreserved pre- and postvaccine therapy PBMCs from 35 patients. The results for the first 29 patients were reported previously (9) . The results for 5 of the final 6 patients examined are shown in Fig. 1 ; patient EA33 had no gp100_209–2M-specific response detected by ex vivo tetramer analysis. Similar to the results reported previously, EA32 and EA34-EA37 demonstrated no gp100_209–2M-specific tetramer staining on prevaccine PBMCs, but did show bright, well-resolved tetramer staining of postvaccine cells (range, 0.36%–1.59%). Pre- and postvaccine PBMCs were comparably negative for the HIV (pol) negative control tetramer stain.

View larger version (56K): [in this window] [in a new window]   Fig. 1. Ex vivo detection of circulating gp100209–2M-specific CD8+ T cells. Cryopreserved pre- and postvaccine therapy peripheral blood mononuclear cells from 5 patients were thawed and stained with a phycoerythrin-conjugated HLA-A2+/gp100209–2M tetramer reagent in combination with antihuman CD8 (allophycocyanin) and CD3 (PerCP) monoclonal antibodies. Negative control staining was assessed using an HLA-A2+-restricted HIV (pol) peptide tetramer. Two-parameter dot plots of CD8 (Y-axis) versus gp100209–2M or HIV (pol) tetramer (X-axis) show the quantitation of HIV (pol) and gp100209–2M tetramer-binding cells expressed as a percentage of gated CD8+/CD3+ T cells.

Ex Vivo CFC(IFN-) Analysis of Pre- and Postvaccine PBMCs.
Fig. 2 shows the results of multiple direct ex vivo CFC(IFN-) assays of postvaccine cryopreserved cells from 12 representative patients and the matched ex vivo gp100_209–2M tetramer-staining results for the same patients. Tetramer staining and CFC(IFN-) analysis were performed 3–10 times on individual patients. The tetramer staining and CFC(IFN-) results were reproducible for all of the patients; all of the coefficients of variation were 22%. Statistical analysis demonstrated that mean tetramer percentages were significantly higher than mean CFC(IFN-) percentages (P < 0.05) for 9 of the 12 patients (i.e., EA02, EA08, EA11, EA12, EA14, EA16, EA24, EA28, and EA30). The overall difference between the tetramer analysis and the functional response was most noticeable for EA08 and EA12 where the mean CFC(IFN-) frequencies were 35% and 32% of the tetramer-binding frequencies, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Mean ex vivo gp100209–2M tetramer+ and cytokine flow cytometry (CFC; IFN-)+ frequencies of cryopreserved CD8+ T cells from 12 representative patients. CFC(IFN-) analysis and correlated gp100209–2M tetramer staining were performed on multiple samples (3–10/patient) of cryopreserved postvaccine peripheral blood mononuclear cells. CFC(IFN-) assays were performed using 10 µg/ml of gp100209–2M peptide or HIV (pol) peptide (negative control) to stimulate in vitro cultures of cryopreserved cells. Bar graphs represent the mean ± SE tetramer response () and the mean ± SE CFC (IFN-) response () for each patient. Data are expressed as the net mean frequency of gated CD8+/CD3+ T cells that bind tetramers or produce IFN-.

Comparison of Tetramer Staining to CFC(IFN-) Responses of Pre- and Postvaccine IVS CD8⁺ T Cells.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Summary of correlated gp100209–2M tetramer binding and cytokine flow cytometry (CFC; IFN-) responses by in vitro stimulation (IVS) T cells. Data represent a comparative summary of the frequency of gp100209–2M tetramer+ CD8+ cells after IVS expansion of postvaccine peripheral blood mononuclear cells () with corresponding dendritic cell (DC) -stimulated (), and/or dimer-stimulated CFC(IFN-; ) responses for each vaccinated patient. Data are expressed as a net percentage of CD8+/CD3+ T cells, and were obtained from a single set of in vitro assays for each patient. DC-stimulated CFC(IFN-) responses were not measured in every patient.

Fig. 4 shows the mean (±SE) for multiple (3, 4, 5, 6, 7, 8, 9, 10) CFC(IFN-) and correlated tetramer-binding assays performed on IVS-expanded CD8⁺ T cells from 6 patients. CFC(IFN-) analysis was performed after stimulation with gp100_209–2M-loaded HLA-A2⁺ dimers. The data demonstrate that similar to ex vivo results (Fig. 2) both assays were also reproducible using IVS T cells (coefficients of variation 25%). Four patients, EA07, EA14, EA16, and EA19, had mean CFC(IFN-) responses comparable with mean tetramer-binding responses (P < 0.05). By contrast, patients EA12 and EA24 showed statistically higher (P < 0.05) mean tetramer-binding values compared with their mean CFC(IFN-) responses, with CFC(IFN-):tetramer ratios of 0.14 and 0.69, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Mean gp100209–2M tetramer staining and cytokine flow cytometry (CFC; IFN-) frequencies for postvaccine in vitro stimulation (IVS) CD8+ T cells. CFC(IFN-) analysis and correlated gp100209–2M tetramer staining were performed on multiple samples (3–10/patient) of IVS-expanded postvaccine peripheral blood mononuclear cells from six representative patients. IVS cells were stimulated in vitro with gp100209–2M-loaded dimers. Bar graphs represent the mean ± SE tetramer response () and the mean ± SE CFC(IFN-) response () for each patient. Data are presented as the net mean frequency of gated IVS CD8+/CD3+ T cells that were positive for tetramer binding or for IFN- expression.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Postvaccine gp100209–2M in vitro stimulation (IVS) CD8+ T cells recognize native gp100209–217 peptide. A, data show the cytokine flow cytometry (CFC;IFN-) response of postvaccine IVS CD8+ T cells from 3 patients after stimulation over a five-log titration (5 µg/ml to 0.0005 µg/ml) of native gp100209–217 or modified gp100209–2M peptide. Nonlinear curve fitting analysis and EC50 calculations were performed using Prism Graphics software; "r" values for all curves shown were >0.95. B, also shown are the comparative CFC(IFN-) responses of IVS CD8+ T cells from 15 patients stimulated with the native and modified peptide at 5 µg/ml.

Vaccinated Patients Have Long-Term Memory (LTM) CD8⁺ T Cells.
Cryopreserved PBMCs collected 12–24 months after the last vaccine were directly stained ex vivo with gp100_209–2M tetramers and interrogated by flow cytometry (Fig. 6A) . The frequencies of circulating LTM gp100_209–2M-tetramer⁺ CD8⁺ T cells for 2 representative patients were significantly diminished at 1.1% (EA08) and 0.10% (EA13) compared with the matched frequencies of tetramer⁺ T cells in PBMCs obtained 2–4 weeks postvaccine therapy, 8.86% (EA08) and 2.42% (EA13). To determine the proliferative potential of LTM cells, they were exposed to 8-day IVS expansion with gp100_209–2m peptide. The bottom two rows of Fig. 6A show gp100_209–2M tetramer staining of IVS-expanded prevaccine, postvaccine, and LTM T cells. EA08 shows a 4.8-fold increase (8.86% to 42.9%) of gp100_209–2M-specific CD8⁺ T cells after IVS expansion of the postvaccine PBMCs as compared with a 16.3-fold increase (1.1% to 17.9%) of LTM PBMCs. A similar comparison for patient EA13 indicates the number of gp100_209–2M tetramer⁺ CD8⁺ T cells increased from 0.10% (ex vivo) to 2.59% after IVS (25.9-fold increase). By contrast, the IVS expansion of PBMCs collected only 2 weeks after vaccine therapy increased from 2.42% to 21% (8.7-fold increase). The observed difference in the expanded percentage of antigen-specific T cells after IVS suggests the PBMCs at 12 months contain circulating gp100_209–2M-specific memory T cells, and these circulating long-term memory T cells are capable of a heightened proliferative response. Fig. 6B shows the matched CFC(IFN-) functional response of CD8⁺ T cells from the same IVS cultures of LTM PBMCs from patients EA08 and EA13. Both patients demonstrated elevated levels of functionally active cells after restimulation with gp100_209–2M peptide-loaded dimers. Both CFC(IFN-) responses were greater than the matched tetramer values for the same IVS effector/memory cells for each patient. This observation may be attributable to the down-regulation of gp100 TCR expression to below levels detectable by flow cytometry (i.e., 1000 copies/cell) after gp100_209–2M IVS. Although cells may have diminished gp100 peptide TCR expression the remaining TCR complexes were sufficient to allow for activation of detectable IFN- production by cognate gp100_209–2M peptide. Table 2 shows the antigen-specific CD8⁺ T cell fold-increase values for 2–4 week postvaccine therapy PBMCs and for the paired LTM PBMCs after gp100_209–2M directed IVS. Ten of 11 patients showed amplified gp100_209–2M-specific proliferation by the LTM PBMCs compared with PBMCs collected shortly after vaccine therapy.

View larger version (34K): [in this window] [in a new window]   Fig. 6. Flow cytometry analysis of gp100209–2M tetramer binding and cytokine flow cytometry (CFC;IFN-) expression by pre- and postvaccine, and long-term memory CD8+ T cells. A, data are presented for 2 patients as the percentage of gated CD8+/CD3+ T cells that were gp100209–2M tetramer+ for freshly thawed prevaccine and postvaccine (2–4 weeks postvaccine therapy) peripheral blood mononuclear cells (PBMCs), and for long-term memory (15 months postvaccine therapy) PBMCs. The bottom two rows show the results of the same analysis using 8-day gp100209–2M IVS CD8+ T cells for both patients. B, also shown are the CFC(IFN-) responses of 8-day IVS long-term memory CD8+ T cells after in vitro restimulation with HIV (pol)-loaded dimer or gp100209–2M-loaded dimer for both patients.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

At present there is general agreement that antigen-directed IVS of PBMCs may alter the phenotype and function of circulating antigen-specific T cells in vaccinated patients, and thereby yield an inaccurate characterization of the in vivo immune response (24) . This conclusion seems intuitively correct in the context of any immunomonitoring situation where the immune response of patients is so weak that direct ex vivo analysis cannot detect recall-antigen-specific functional responses, or even demonstrate the presence of antigen-specific T cells. However, if initial ex vivo assays readily detect both the frequency and function of antigen-specific T cells, as in this study, the follow-up analysis of IVS-expanded cells could then be compared to the ex vivo results for each patient, and thus be used to assess antigen-specific immune response differences between patients. Once baseline postvaccine ex vivo numbers of antigen-specific T cells and their functional status are determined, recall-antigen IVS may then reveal potentially important interpatient differences in the proliferation and functional responsiveness of antigen-specific T cells, as well as the functional avidity for cognate antigen, which may empirically prove to be valuable immune status index parameters that could potentially correlate with clinical outcome. More extensive data from larger clinical trials will be required to confirm this type of clinical correlation.

To study the effect of limited gp100_209–2M IVS on the rate of proliferation and functional activation of antigen-specific CD8⁺ T cells in each patient, we performed 8-day gp100_209–2M-directed IVS for all 35 of the patients. A single cycle of IVS induced significant increases in the percentage of tetramer⁺ CD8⁺ T cells above baseline ex vivo levels for 34 of 35 patients (Fig. 3) . The combined use of the CFC(IFN-) assay and tetramer-binding analysis on IVS cells showed that many patients, e.g. EA13 and EA16 (Fig. 3) , had comparable responses in both assays, indicating that the IVS protocol used resulted in the expansion of functionally competent, antigen-specific cells. Other patients, exemplified by EA12 and EA31 (Fig. 3) , showed significantly lower percentages of functionally responsive cells compared with the net percentage of tetramer⁺ CD8⁺ T cells after IVS suggesting the presence of a subpopulation of anergic CD8⁺ T cells, even after IVS. Theoretically, IVS could result in the down-modulation of functional activity of proliferating, antigen-specific cells. Our observations to date suggest that this is not the case, because IVS usually results in either the retention of observed ex vivo patterns of functional anergy or in an increase in the frequency of functionally responsive tetramer⁺ CD8⁺ T cells (Figs. 2 and 4 ). Thus, if IVS pertubates the ex vivo functional response profile it appears to do so by favoring proliferation of functionally responsive antigen-specific CD8⁺ T cells or by inducing functional activity in cells that were quiescent by ex vivo interrogation. Either response could indicate interpatient differences predictive of clinical response. The data also suggest these anergic, antigen-specific CD8⁺ T cells had a high proliferative response to cognate-antigen stimulation. Comparison of IVS T-cell tetramer-binding data to the matched ex vivo tetramer-binding values for each patient also revealed important interpatient differences in the overall proliferation potential of gp100_209–2M-specific CD8⁺ T cells. This was most apparent in patients with essentially equivalent baseline ex vivo tetramer⁺CD8⁺ frequencies, such as EA02, EA12, EA16, and EA20, who subsequently demonstrated significantly different tetramer⁺ T-cell frequencies after IVS (Fig. 5) . This observation of interpatient heterogeneity with regard to proliferation potential after cognate-antigen stimulation would not have been made if our immunomonitoring strategy had relied only on direct ex vivo interrogation; and it may reflect potentially important in vivo differences in patient responses to a melanoma tumor antigen vaccine.

There was heterogeneity among patient in vitro response to native gp100_209–217 versus gp100_209–2M stimulation. Rosenberg et al. (3) documented differences in IFN- response by IVS-expanded cells from immunized patients after restimulation in vitro with T2 cells pulsed with native gp100_209–217 peptide or with gp100_209–2M. Herein we also demonstrated patient-to-patient variation in the in vitro response of IVS cells to native gp100_209–217 peptide versus gp100_209–2M. Fig. 5B shows the comparative CFC(IFN-) response of IVS cells from 15 patients to both peptides at an optimal antigen concentration of 5 µg/ml. The mean ratio (± SE) of native peptide over modified peptide CFC(IFN-) response for all 15 of the patients was 69.8 ± 4.4% (range, 42–94%). This mean ratio value for IVS cells is comparable with the corresponding ex vivo ratio value of 71.5% we reported previously for 10 of the same patients analyzed in Fig. 5B (9) , and suggests that gp100_209–2M IVS expansion of postvaccine PBMCs did not alter the functional avidity of CD8⁺ T cells for the native peptide compared with the modified peptide antigen. It is axiomatic that the levels of melanoma-associated native gp100 peptide "seen" by memory/effector CTLs in melanoma patients in vivo may be substantially lower than the levels on dimers or stimulator cells used in vitro. In this regard, it is noteworthy that of 19 patients selected from this clinical trial, gp100_209–2M IVS T cells produced IFN- after restimulation by HLA-A2⁺/gp100⁺ melanoma tumor cell lines in 13 patients (68%),⁶ suggesting the functional avidity of IVS-expanded CD8⁺ T cells from immunized patients was strong enough to recognize the levels of native peptide expressed on melanoma cells. This observation was also reinforced by the correlated data from Meijer et al.,⁶ which demonstrated that cryopreserved postvaccine PBMCs from 6 of 9 patients studied (67%) also produced IFN- after direct ex vivo stimulation by HLA-A2⁺/gp100⁺ melanoma cell lines.

In our detailed four-color flow cytometry analysis of postvaccine cryopreserved PBMCs from 6 patients the majority of tetramer⁺ CD8⁺ T cells (65%–78%) were in effector and effector-memory subpopulations as delineated by the differential expression of the CD45RA, CD27, CD28, CD57, and CCR7 molecules (Table 1) . Similar subpopulations of CD8⁺ T cells have been described in melanoma patients vaccinated with Melan-A/MART-1, tyrosinase, and gp100 peptides, as well as in unvaccinated melanoma patients. Pittet et al. (25) showed that the frequency of circulating Melan-A/MART-1_26–35-specific CD8⁺ T cells in a single patient increased >20-fold after multiple immunizations. Multiparameter flow cytometry analysis indicated that the increase in functionally responsive Melan-A tetramer⁺ CD8⁺ T cells was associated with a concomitant increase in the percentage of tetramer⁺ CD8⁺ T cells that expressed an "activated/memory" phenotype characterized by simultaneous CD45RA^low/CD27^-/CD28^-/CD57⁺ staining. Notably, the percent of Melan-A tetramer⁺ cells with this phenotype increased over time with repetitive immunization (25) . In a related study, Valmori et al. (15) characterized ex vivo the functional properties and cell surface phenotype of constitutively expressed tyrosinase_368–376 tetramer⁺ CD8⁺ T cells in a single unimmunized melanoma patient. The tyrosinase tetramer⁺ cells comprised >5% of the circulating CD8⁺ T cells in this patient and were detectable over a 3-year period. The tyrosinase-specific CD8⁺ T cells were directly lytic ex vivo for tyrosinase⁺ tumor lines, and were predominantly CD45RA⁺/CD27^-CD28^-/CD57⁺ expressing the so-called terminal effector phenotype (15) . Fourteen-day IVS expansion down-regulated CD45RA expression completely, but longer (21-day) IVS-cultured CD8⁺ T cells re-expressed CD45RA concomitantly with low cell surface expression of CD45RO; the fidelity of the CD27^-/CD28^-/CD57⁺ staining was maintained even over long-term IVS. Importantly, after sorting, these "terminally differentiated" effector CD8⁺ T cells could proliferate after mitogen stimulation. The in vitro ability to proliferate and modulate CD45 isoform expression suggested that they might have the same differentiation/proliferation properties in vivo (15) . In another report Monsurro et al. (8) demonstrated that 13 melanoma patients vaccinated multiple times with the gp100_209–2M peptide had an average increase in circulating gp100_209–2M tetramer⁺ cells equal to 1.2% of CD8⁺ T cells. These antigen-specific CD8⁺ T cells were predominantly effector, CD45RA⁺/CD27^- (43% ± 6%), or effector-memory, CD45RA^-/CD27^- (30 ± 4.1%), phenotype. However, ex vivo functional analysis using a CFC (perforin) assay indicated that the tetramer⁺ cells were poorly responsive after stimulation with gp100_209–2M-pulsed melanoma cell lines (17% perforin positive). Although gp100_209–2M-specific IVS followed by 10-day expansion in interleukin 2 dramatically increased the frequency of perforin⁺ cells to 84 ± 3.6%, these functionally active cells now unexpectedly displayed increased expression of CD27⁺; i.e., 67% of all of the tetramer⁺ CD8⁺ T cells were CD27⁺ regardless of CD45RA expression. The predominate phenotypes after IVS were 23% CD45RA⁺/CD27⁺ (naïve) and 44% CD45RA^-/CD27⁺ (memory; Ref. 8 ). The plasticity of these predominantly CD27^- effector and effector-memory T cells after cognate-antigen IVS suggested that the cells were not terminally differentiated CD8⁺ T lymphocytes and that CD27 expression may be a marker of their activation status rather than end-stage differentiation as suggested by Valmori et al. (15) . Similarly the subsequent increased expression of the CD45RA⁺/CD27⁺ naive phenotype on 23% of all of the tetramer⁺ CD8⁺ T cells after IVS also suggested that this so-called naïve staining profile was not uniquely characteristic of antigen-inexperienced cells. Thus, our description of the predominant effector and effector-memory phenotypes of the gp100_209–2M-specific CD8⁺ T cells in this report agrees with the phenotype descriptions of melanoma peptide-specific T cells in previous studies. However, a comparative analysis of the data from previous studies also suggests that the description of the natural history of the CD8⁺ T cell effector-memory differentiation pathway remains unresolved due to the proliferation potential and phenotype plasticity after activation of presumably end-stage memory/effector CD8⁺ T-cell subpopulations (8 , 15, 16, 17, 18) . Additional work involving the detailed ex vivo six to eight-color flow cytometry analysis of antigen-specific CD8⁺ T cells to assess the overlapping expression of markers like CD27, CD28, CCR7, and CD57 on the CD45RA or CD45RO background is required, as well as similar detailed phenotype and functional interrogation of sorted subsets of tetramer⁺ CD8⁺ T cells after IVS expansion with cognate-recall antigen to determine the phenotype and functional stability of CD8⁺ T-cell subpopulations.

To date, there have been few descriptions of the detailed functional and/or phenotype characterization of long-term memory/effector CD8⁺ T cells (i.e., 12 months postvaccine therapy) from vaccinated nonmetastatic melanoma patients. In this study ex vivo tetramer analysis demonstrated the sustained presence of low frequencies of tetramer⁺ CD8⁺ T cells (range, 0.08–1.1%) in peptide-vaccinated patients at time points out to 1 and 2 years after vaccination. We also demonstrated that these circulating long-term tetramer⁺ CD8⁺ T cells had elevated proliferation responses after IVS, a trait characteristic of memory T cells (26) . Thus, gp100_209–2M immunization resulted in the maintenance of long-term memory T cells that were capable of accelerated proliferation response and were functionally active by CFC(IFN-) analysis after IVS expansion. Antigen-specific effector and memory CD8⁺ T-cell proliferation and activation is governed by many factors, including the amount of antigen available in vivo (27 , 28) and the presence of CD4 helper T-cell function at the time of CD8 T-cell priming (29 , 30) . Presently we have no mechanistic explanation for the durable CD8 memory T-cell response observed in the absence of an exogenous source of CD4 MHC class II-restricted antigen. However, the chemical structure of the adjuvant, Montanide ISA 51, may provide some insight into the immune stimulation of a memory CD8⁺ T-cell response by the gp100_209–2M peptide. The principal surfactant in Montanide is a Mannide mono oleate lipid complex (31) . Montanide adjuvant formulations and related lipid-complexed adjuvants made up of palmitic acid groups admixed with or conjugated to class I-restricted peptides have been shown to stimulate weak memory CD8 T-cell responses (32 , 33) . Recent data suggests that DCs copresenting class I restricted peptides and the CD1d-binding glycolipid -galactosyl-ceramide can prime CTL responses in a class II independent fashion by causing the activation of natural killer T cells. Natural killer T cells, in turn, provide help to facilitate antigen-specific CD8 T-cell priming by as-yet poorly understood mechanisms (34 , 35) , which may involve the reciprocal maturation of DCs (36) . It is unknown whether a similar CD1d-mediated DC-natural killer T nexus is facilitated by the Mannide mono oleate lipid surfactant found in Montanide. However, the absence of any known CD4 antigenic stimulus in Montanide suggests that some component of the lipid chemistry of the adjuvant is supporting long-term CD8⁺ memory to the MHC class I-restricted peptide antigen perhaps by a mechanism similar to that of glycolipid -galactosyl-ceramide.

Key objectives of this clinical study were to determine whether the gp100_209–2M vaccine regimen used resulted in significant immune stimulation in this high-risk patient population, and to characterize interpatient differences in immune response using ex vivo analysis and correlated analysis of IVS T cells. Additionally we were interested in determining whether the MHC class I-restricted peptide antigen, gp100_209–2M, generated a long-term memory response. Overall, the experimental results demonstrate that resected nonmetastatic melanoma patients can mount a strong antigen-specific immune response when immunized multiple times with the gp100_209–2M peptide over a 6-month period, and this response appears to include the induction of long-lived antigen-specific memory CD8⁺ T cells. The resulting data suggest the most accurate method to perform such immunomonitoring analysis in the future should include the combined use of functional and (where possible) assays of TCR specificity, such as tetramer binding, to quantitate antigen-specific immune responses. Additionally, the data also support the general concept of combining direct ex vivo functional and tetramer analysis with follow-up duplicate assays of cognate-antigen IVS CD8⁺ T cells, because there may be patient variation in antigen-specific proliferative response by both functionally active and functionally anergic T cells after antigen reactivation. Thus, patient differences in T-cell functional and phenotype changes after IVS compared with baseline ex vivo quantitation of antigen-specific, functionally responsive T cells may yet empirically prove to be important index parameters predictive of clinical response. The utility of this approach will only be validated after more extensive clinical studies involving combined ex vivo and IVS analysis of antigen-specific T cells have been completed.

	ACKNOWLEDGMENTS

 
We thank Dr. Andrew Weinberg for reviewing the paper, and Melinda Palmer and Jessie Herron for excellent secretarial assistance in preparation of the manuscript. We are also indebted to Dr. Jeffrey Weber for very helpful critique of the manuscript.

	FOOTNOTES

 
Grant support: Supported by NIH 1R21-CS82614-01 (W. J. U.), the M. J. Murdock Charitable Trust, and The Chiles Foundation.

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.

Requests for reprints: Edwin B. Walker, Earle A. Chiles Research Institute, Robert W. Franz Cancer Research Center, Providence Portland Medical Center, 4805 NE Glisan, 5F40, Portland, OR 97213. Phone: (503) 215-2620; Fax: (503) 215-6841; Email: edwin.walker{at}providence.org

⁶ S. L. Meijer, A. Dols, S. Jensen, H. M. Hu, W. Miller, E. B. Walker, P. Romero, B. A. Fox, and W. J. Urba. Antimelanoma activity of circulating CD8⁺ T cells following adjuvant vaccination of melanoma patients with gp100_209–2M peptide, submitted for publication.

Received 8/26/03; revised 11/18/03; accepted 11/20/03.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Parkhurst M. R., Salgaller M. L., Southwood S., Robbins P. F., Sette A., Rosenberg S. A., Kawakami Y. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-a*0201-binding residues. J. Immunol., 157: 2539-2548, 1996.[Abstract]
2. Salgaller M. L., Afshar A., Marincola F. M., Rivoltini L., Kawakami Y., Rosenberg S. A. Recognition of multiple epitopes in the human melanoma antigen gp100 by peripheral blood lymphocytes stimulated in vitro with synthetic peptides. Cancer Res., 55: 4972-4979, 1995.[Abstract/Free Full Text]
3. Rosenberg S. A., Yang J. C., Schwartzentruber D. J., Hwu P., Marincola F. M., Topalian S. L., Restifo N. P., Dudley M. E., Schwarz S. L., Spiess P. J., Wunderlich J. R., Parkhurst M. R., Kawakami Y., Seipp C. A., Einhorn J. H., White D. E. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat. Med., 4: 321-327, 1998.[CrossRef][Medline]
4. Rosenberg S. A., Yang J. C., Schwartzentruber D. J., Hwu P., Marincola F. M., Topalian S. L., Restifo N. P., Sznol M., Schwarz S. L., Spiess P. J., Wunderlich J. R., Seipp C. A., Einhorn J. H., Rogers-Freezer L., White D. E. Impact of cytokine administration on the generation of antitumor reactivity in patients with metastatic melanoma receiving a peptide vaccine. J Immunol., 163: 1690-1695, 1999.[Abstract/Free Full Text]
5. Lee P., Wang F., Kuniyoshi J., Rubio V., Stuges T., Groshen S., Gee C., Lau R., Jeffery G., Margolin K., Marty V., Weber J. Effects of interleukin-12 on the immune response to a multipeptide vaccine for resected metastatic melanoma. J. Clin. Oncol., 19: 3836-3847, 2001.[Abstract/Free Full Text]
6. Lau R., Wang F., Jeffery G., Marty V., Kuniyoshi J., Bade E., Ryback M. E., Weber J. Phase I trial of intravenous peptide-pulsed dendritic cells in patients with metastatic melanoma. J. Immunother., 24: 66-78, 2001.
7. Nielsen M. B., Monsurro V., Migueles S. A., Wang E., Perez-Diez A., Lee K. H., Kammula U., Rosenberg S. A., Marincola F. M. Status of activation of circulating vaccine-elicited CD8+ T cells. J. Immunol., 165: 2287-2296, 2000.[Abstract/Free Full Text]
8. Monsurro V., Nagorsen D., Wang E., Provenzano M., Dudley M. E., Rosenberg S. A., Marincola F. M. Functional heterogeneity of vaccine-induced CD8(+) T cells. J. Immunol., 168: 5933-5942, 2002.[Abstract/Free Full Text]
9. Smith J. W., Walker E. B., Fox B. A., Haley D., Wisner K. P., Doran T., Fisher B., Justice L., Wood W., Vetto J., Maecker H., Dols A., Meijer S., Hu H. M., Romero P., Alvord W. G., Urba W. J. Adjuvant immunization of HLA-A2-positive melanoma patients with a modified gp100 peptide induces peptide-specific CD8+ T-cell responses. J. Clin. Oncol., 21: 1562-1573, 2003.[Abstract/Free Full Text]
10. Hu H. M., Dols A., Meijer S. L., Floyd K., Walker E. B., Urba W. J., Fox B. A. Immunological monitoring of melanoma patients following peptide vaccination using soluble peptide/HLA-A2 dimer complexes. J. Immunother., 27: 48-59 2003.
11. Ismaili J., Rennesson J., Aksoy E., Vekemans J., Vincart B., Amraoui Z., Van Laethem F., Goldman M., Dubois P. M. Monophosphoryl lipid A activates both human dendritic cells and T cells. J. Immunol., 168: