5 Dead 6304p Protoc ol 118 04/06/2016 Appendix B Laboratory Analysi s A. Antigens used in the immunologic assays Antigen Designation Assay Concentration No antigen Background control All NA CMV lysate Positive control CFC* 2.5 μg/ml CMV HLA -A2 pp65 peptide Positive control CFC and MHC tetramer 10 μg/ml tt protein Positive control CFC 1 μg/ml HER2 ICD overlapping peptide pools Experimental CFC 0.7 μg/ml per peptide HER2 ECD overlapping peptide pools Experimental CFC 0.7 μ g/ml per peptide Individual HER2 class II peptides Experimental CFC 10 μg/ml Individual HER2 class I peptides Experimental CFC and MHC tetramer 10 μg/ml CEA overlapping peptide pool Experimental (for epitope spreading) CFC 0.7 μg/ml per peptide CEA CAP -1 class I peptide Experimental (for epitope spreading) CFC and MHC tetramer 10 μg/ml PHA Mitogen CFC 2.5 μg/ml * All CFC antigen would be operative for ELIspot analysis B. Immunologic analysis of antigen specific T cells 1. Detection of HER2 specifi c T-cell responses by cytokine flow cytometry (CFC) . CFC is a highly quantitative method that will enumerate both CD4+ and CD8+ antigen specific T cells. Either 1 ml of whole blood or 1x106 PBMC in 1 ml culture media (EHAA/RPMI, 10% AB serum, penicillin, streptomycin, L -glutamine) is stimulated with no antigen (PBS only), 1 μg/ml HER2 peptide mix in overlapping peptide pools, 10 μg individual HER2 CD4+ or CD8+ peptides, CMV HLA -A2 pp65 peptide, tt protein, or PHA, concentrations listed above. 2 μg/ml costimulatory molecules (CD28 and CD49d cocktail) and 10 mg/ml Brefeldin A are added to the reaction mixture and incubated for 6 hours at 37°C+5%CO 2. To remove the adherent cells, 2 mM EDTA is added and incubated at room temperature for 15 minutes. 9 mls 1X FACS lysing solution (BD Biosciences, San Jose, CA) is added and incuba ted at room temperature for 10 minutes to lyse the red cells and fix the PBMC. The reaction mixture is stored at - 80°C freezer overnight. Cells are quickly thawed in a 37°C water bath, centrifuged and 2 mls of FACS permeabilizing solution 2 (BD Bioscienc es) is added. After a 10 minute incubation in the dark, the cells are washed with 10 mls wash buffer (1X PBS/0.5% BSA). Cells are thoroughly resuspended in 650 μ l wash buffe r and 100 μl aliquoted to 6 microcentrifuge tube s. Antibodie s (examples) are add ed as follows: Tube #1: CD3 cychrome/CD8 FITC/IFN -gamma PE Tube #2: CD3 cychrome/CD4 FITC/ IFN -gamma PE Tube #3: CD3 cychrome/CD69 FITC/ IFN -gamma PE Tube #4: CD3 cychrome/CD8 FITC/IL -4 PE Tube #5: CD3 cychrome/CD4 FITC/IL -4 PE Tube #6: CD3 cychrome/CD69 FITC/IL -4 PE 6304p Protocol 118 04/06/2016 After a 30 minute incubation at room temperature in the dark, the cells are washed twice with wash buffer. The cells are resuspended in 300 μl 1% paraformaldehyde and then analyzed using three color flow cytometry. % responding cells are calculated counting a minimum of 50,000 events (CD8+ or CD4+ T cells). Antigen specific CD8+ or CD4+ IFN γ or IL -4 secreting cells will be reported as % resp onding cells for a particular population. A precursor frequency based on the total number of CD3+ cells can be calculated from this value. 2. Detection of HER2 specific T -cell responses by ELIspot . Briefly, on day 1, PBMC are plated into 96 -well plates in 6 -well replicates in 200 μl of RPMI -1640 containing L -glutamine, penicillin, streptomycin and 10% AB s e r um ( T c e l l m e d i um ) i n t h e p r e se n c e o r a b s e n c e o f 1 0 μ g / m l pe p ti d e a n t i g e n o r 1 μ g /m l TT . Th e c e l l s a r e incubated at 370C+5% CO 2. On day 2 (48 hours), 2.5 x 105/well irradiated autologous PBMC and 10 μg/ml antigens are added. Also on day 2, nitrocellulose -backed 96- well plates (NC -plate) are coated with 10 μg/ml anti-IFN-gamma antibody in PBS at 50 μl/well. On day 3 the NC -plate is washed 3X with PBS and blocked for 2 hours wit h PBS containing 2% bovine serum albumin followed by 3 washes with PBS. On day 3, the cells are gently re -suspended, pooled, centrifuged, and the media is replaced. The cells are transferred into the NC -plate i n a v o l u m e o f 1 0 0 μ l / w e l l i n T -cell medium. The NC -plate is incubated at 37oC for a further 20 -24 hours followed by washing 3X using PBS containing 0.05% Tween- 20. The plate is then incubated for 2.5 hours at room temperature in 50 μl/well PBS containing 5μg/ml biotinylated anti -IFN-gamma antibody, washed 3X with PBS, and further incubated with 100 μl/well streptavidin -alkaline phosphatase at a dilution of 1:1000 in PBS for 2 hours at room temperature. After washing 3X in PBS, the plate is incubated with 100 μl/well AP -colorimetric substrate for 20- 30 minutes, rinsed with cool tap water, and allowed to completely dry. Resultant spots are then enumerated using a dissecting microscope. Precursor frequencies are calculated by subtracting the mean number of spots obtained from the no antigen control wells from the mean number obtained in the experimental wells. Statistical analysis is performed using the Student’s t test (Microsoft Excel 97). A positive response is defined as a precursor frequency that was both significantly (p<0.05) greater than th e mean of control no antigen wells and detectable (i.e. >1:100,000).