Peptides and Cell Lines
The peptides gp100 209–2M (IMDQVPFSV), MART1 M26 (ELAGIGILTV), tyrosinase 370D (YMDGTMSQV), gp100 209 (ITDQVPFSV), MART1 27–35 (AAGIGILTV), CMV pp65 495–503 (NLVPMVATV), EBV BMLF1 280–288 (GLCTLVAML), and influenza MP 58–66 (GILGFVFTL) were produced at the Protein and Nucleic Acid Facility at Stanford University (Stanford, California, United States).
CD8+ T cell clones were derived and maintained as previously described [17
]. Briefly, clones were derived from melanoma patients or healthy donors expressing the human leukocyte antigen (HLA)-A2.1 MHC molecule. Clone 132.2 specifically binds gp100 209 or gp100 209–2M/HLA-A2.1. Clones 461.30 and 461.24 specifically bind MART 27–35 or M26/HLA-A2.1. Clone CMV94.3 specifically binds CMV pp65 495–503/HLA-A2.1 and was derived by fluorescence-activated cell sorter (FACS) separation of individual tetramer-positive cells from PBMCs from a healthy donor. T cell clones were cultured in CTL medium (Iscove's modified Dulbecco's medium, with 10% fetal calf serum, 2% human AB sera, and standard cell-culture concentrations of penicillin, streptomycin, and L-glutamine) supplemented with 50 U/ml IL-2. The clones were expanded by stimulation with phytohemagglutinin (Invitrogen, Carlsbad, California, United States) at a 1:100 dilution, followed by 14 d of culture in CTL medium with irradiated feeder cells and 50 U/ml of IL-2. Following expansion, clones were either cryopreserved or maintained in culture with CTL medium supplemented with 50 U/ml IL-2 or 2 ng/ml IL-15, and used within 2 wk. Cryopreserved cells were thawed at least 2 d prior to assays, and were suspended in CTL medium with 100 U/ml IL-2. At 1 d prior to experiments, the clones were transferred to fresh CTL medium without interleukins.
Preparation of pMHC Class I
The pMHC tetramers were developed in the Davis lab and were prepared as previously described [11
]. Alternatively, tetramers were purchased from Beckman Coulter (Allendale, New Jersey, United States). The pMHC dimers were purchased from BD Pharmingen (San Diego, California, United States) and prepared per manufacturer's protocol. All pMHC constructs were supplemented with glycerol prior to printing (2% final concentration).
A randomized phase II trial for patients with resected stages IIC/III and IV melanoma who were HLA A*0201-positive and expressed at least one of the following was conducted: HMB-45 (gp100), tyrosinase, or Melan-A (MART-1). Informed consent was obtained from all patients. A total of 60 patients were randomly allocated to receive three peptides at 1 mg each (gp100 209–2M, tyrosinase 370D, and MART-1 M26) emulsified with the adjuvant Montanide ISA 51 in a 1:1 ratio by volume with: (A) IL-12 at 30 ng per kilogram body weight, (B) IL-12 at 100 ng per kilogram, and (C) IL-12 at 30 ng per kilogram with GM-CSF at 83 μg per peptide emulsified. All patients had a CT scan of the chest/abdomen/pelvis and brain MRI required to show no evidence of disease within 28 d of initiation of vaccine treatment. Injections were given at weeks 0, 2, 4, 6, 10, 14, 18, and 26, then week 50 for a total of nine injections over 1 y. Leukopheresis was performed just prior to the first injection and within 2 wk after injection number 8 (week 28) for immune response assays. Smaller blood samples were obtained at 3, 9, and 12 mo. Clinical specimens were frozen and stored prior to use, as previously described [18
]. Tetramer flow cytometry on pretreatment blood samples showed no detectable melanoma antigen-specific T cells.
Antibodies against human CD8 (HIT8a), HLA-A2 (BB7.2), IFNγ (NIB42, 4S.B3), TNFα (MAb1, MAb11), granzyme B (2CF/F5, GB11), GM-CSF (BVD2-23B6, BVD2-21C11), IL-2 (5344.111, B33–2), IL-4 (8D4–8, MP4-25D2), IL-5 (TRFK5, JES1-5A10), IL-10 (JES3-9D7, JES3-12G8), and IL-12p70 (20C2, C8.6) were purchased from BD Pharmingen (San Diego, California, United States); antibodies against IL-1a (4414.141, pAb BAF200), IL-1b (8516.311. pAb BAF201), IL-3 (4815.211, pAb BAF203), IL-6 (6708, pAb BAF206), IL-7 (7417, pAb BAF207), IL-13 (32116, pAb BAF213), IL-15 (34593, pAb BAF247), IL-17 (41809, pAb BAF317), lymphotactin (109001, pAb BAF695), IP-10/CXCL10 (33036.211, pAb BAF266), TGF-β1 (9005, 27240), TNFβ (5807, pAb BAF211), vascular endothelial growth factor (VEGF; pAb AF293NA, pAb BAF293), and VEGF-D (78902, 78923) were purchased from R&D Systems (Minneapolis, Minnesota, United States), and granzyme A (CLB-GA29, CLB-GA28) was purchased from Research Diagnostics Incorporated (Flanders, New Jersey, United States). Clone names are listed in parentheses, with biotinylated antibody clone names listed second, where appropriate. Polyclonal antibodies are noted with a pAb prefix.
Preparation of pMHC Functional Microarrays
Libraries of pMHC/antibody mixtures were prepared as follows: Each of the pMHC constructs was mixed with a panel of antibodies against potentially secreted factors (), such that each mixture contained a single pMHC construct (2.5 mg/ml final concentration) and a single antibody against a secreted factor (0.5–1mg/ml final concentration). Each of these pMHC-based mixtures was supplemented with 2% glycerol. A second library with 0.5 mg/ml of either anti-human CD8 or anti-human HLA-A2 (instead of pMHC) and an antibody against a secreted factor was prepared as a nonactivating control. A volume of 12 μl of each mixture was loaded into a 384-well plate (MJResearch, Waltham, Massachusetts, United States) and arrayed in triplicate onto three-dimensional substrates composed of microscope slides coated with a polyacrylamide gel (Perkin Elmer, Boston, Massachusetts, United States), preprocessed according to the manufacturer's instructions. Samples were dispensed using a non-contact piezoelectric arrayer (Perkin Elmer), such that each spot contained ten drops of approximately 0.45 nl each. Printed proteins were immobilized within the gel substrate by incubating the slides for 48 h at 4 °C in a humid chamber. Following the immobilization, the arrays were placed in a dry slide box, sealed with tape, and stored at 4 °C until use. Arrays were tested for specific capture of secreted factors using defined concentrations of recombinant human factors (Quantikines, R&D Systems) incubated on an unused array for 30 min at room temperature, followed by 12 h at 4 °C. Arrays were then washed, developed, and imaged, as described below.
Flow Cytometry Analysis
Patient PBMCs were analyzed for G209-2M-tetramer reactive cells by flow cytometry as described previously [13
]. Briefly, cells were reacted with G209-2M-tetramer-PE (Beckman Coulter Immunomics Operations, San Diego, CA, United States) at 1:200 dilution for 20 min at room temperature, followed by anti-CD19 FITC (Caltag Laboratories, Burlingame, California, United States) and anti-CD8 PerCP-Cy5.5 (BD Biosciences, San Jose, California, United States) antibodies at final staining dilution of 1:40 and 1:20, respectively, for an additional 20 min. Cells were then washed and analyzed using a FACSCalibur flow cytometer (Beckton Dickinson, San Jose, California, United States). Approximately 105
events were acquired from each sample and analyzed using FlowJo software (TreeStar, San Carlos, California, United States). Plotted CD19−, CD8+, tetramer+ lymphocytes were calculated as percent of total CD8+ lymphocytes for each sample.
Binding and Secretion Assays
Binding and secretion assays were performed with either patient CD8+ T cells or cultured human CD8+ T cell clones. CD8+ T cells were isolated from 5 × 107 PBMCs from patients on the above-described vaccine protocol, using a CD8− isolation column (Miltenyi Biotec). The CD8+ T cells were then resuspended in 200 μl of incubation medium (RPMI supplemented with 5% FCS, glutamine, and standard concentrations of penicillin and streptomycin). Alternatively, 1 × 106 CD8+ T cell clones, as described above, were resuspended in 200 μl of incubation medium. For pMHC binding analysis, the single cell suspension was incubated on the pMHC array for 10–30 min at 20 °C. At the end of the incubation period, the array was washed in calcium- and magnesium-free PBS (CMF) to remove unbound cells and imaged as detailed below. To analyze cellular secretion, the cells were incubated on the array in 400 μl of incubation medium at 37 °C for 2 h (CD8+ T cell lines) or for 24 h (patient samples). To determine the secretion of factors, the arrays were washed in CMF and incubated in 200 μl of pooled biotinylated antibodies in staining medium (10% FCS in CMF) for 20 min at 20 °C. The biotinylated antibodies were each matched to a single, printed antibody specific against different epitopes of the same secreted factor. The final concentration of each biotinylated antibody was based upon concentrations recommended for ELISA or ELISpot, and titrated as necessary. After incubation with biotinylated antibodies, the array was washed twice in CMF and stained with 3.3 μg/ml streptavidin-phycoerythrin (BD Pharmingen) in 200 μl of staining medium for 20 min at 20 °C in the dark. The array was dip-washed twice again in CMF and then imaged as detailed below.
Image Acquisition and Analysis
Imaging was performed using a Zeiss Axiovert-200 microscope (Oberkochen, Germany) fitted with a high-speed piezo electric z-motor stage (Applied Scientific Instrumentation, Eugene, Oregon, United States), a 10× Zeiss Fluor objective, a CCD camera (Roper Scientific, Trenton, New Jersey, United States), and dual excitation and emission filter wheels (Sutter Instruments, Novato, California, United States). DIC and Cy3 images were collected from each spot on the array. Image acquisition was controlled by Metamorph (Universal Imaging, Downingtown, Pennsylvania, United States). Image analysis, feature extraction, and data analysis were performed using Metamorph, ImageXpress (Molecular Devices, Union City, California, United States), and Matlab Software (The MathWorks, Natick, Massachusetts, United States).
Analysis of Patient Data
Scoring of patient samples was performed in a blinded fashion. Coded samples were scored without prior information regarding patient age, sex, therapy, clinical, or immunological outcome. Scoring was based on a five-point scale (i.e., 0–4), with 0 representing background signal. A cell count score for IFNγ and TNFα secretion was based upon the number of responding cells per spot: 0, no response; 1, 1–5; 2, 6–10; 3, 11–20; and 4, more than 21 responding cells. A second score, for intensity, was based on average integrated pixel fluorescence over all replicate spots (after subtraction of the average integrated pixel fluorescence of control spots containing only pMHC). Each of the averaged intensities was normalized by a value greater than the highest intensity for that particular secreted factor, across all patients and expressed as a percentage of that value. The intensity score was assigned as follows: 0, 0%–5%; 1, 6%–25%; 2, 26%–50%; 3, 51%–76%; and 4, 77%–100%. A combined score for IFNγ and TNFα was obtained by adjusting the cell count score up or down by 1 if the intensity score was higher or lower than the cell count score. Scores for secreted factors lacking clear and consistent focal secretion across all patients (including granzyme B, IL-2, TGFβ, IL-1b, IL-6, GM-CSF, IL-1a, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-17, lymphotactin, IP-10, TNFβ, VEGF, VEGF-D, and granzyme A) received only intensity scores according to the following scale: 0, 0%–5%; 1, 6%–19%; 2, 20%–50%; 3, 51%–80%; and 4, 81%–100%. Six clinical specimens were initially tested using the pMHC functional array. Five additional specimens were tested to assess consistency of findings and expand the number of analyzed specimens.