12367 Version Date: 10/24/2017 Version: 09 I FN mAb (clone B6-1, MabTech). After incubation plates are further washed with P BST and then incubated biotin conjugated with alkaline peroxidase for a further hour. Afterwards plates are then washed with PBST and then incubated with a substrate, BCIP (5-Bromo-4-chloro-3-indolyl phosphate) to visualize IFN  secreting cells. ELISPOT data are presented as the median of triplicates with background (PBMCs incubated with media ) IFN spots subtracted. 9.2.2 Cellular immune response assays – Proliferation Antigen-specific T cell proliferation to PA2024, PAP and PHA are assayed by means a standa rd tritiated thymidine (3H -thymidine) incorporation assay using round bottom 96 well tissue culture plates . Cryopreserved PBMC, thawed and rested overnight in medium a re then plated in triplicate at 1x105 PBMC/well in a total volume of200 L/well with either medium alone or with medium containing antigen (PA2024: PAP-GMCSF fusion protein, PAP or PHA). Plates are incubated for 5 days, after which the wells are pulsed with 0.5Ci of 3H-thymidine overnight, and the amount of 3H-thymidine incorporation into the nucleus Is quantified by means of a - radiation counter with the degree of proliferation expressed as a stimulation index (SI), defined as 3H-thymidine incorporation in the presence of antigen divided by 3H-thymidine incorporation with media alone. 9.2.3 Humoral response assays Antibody responses against PA2024 and PAP are determined by means of standard antibody ELISA (enzyme-linked immunosorbent assay). 96 well flat bottomed plates are coated with either PA2024, PAP or Tetanus (an assay control, and measure of immunocompetence) overnight, after which time plates are blocked with PBS/casein and then washed with PBST. Serially diluted serum is then added in duplicate to each set of plates and incubated at room temperature for 2 hours, after which time plates are washed with PBST and then incubated with a mixture of anti-IgM and anti-IgG (Jackson Immunoresearch) for an hour. After this time plates are washed again with PBST and then incubated for an hour with horse radish phosphatase conjugated anti IgG+amti-IgM. Plates were further washed with PBST and O-phenylenediamine dihydrochloride substrate (Sigma) was then added for 15 minutes after which the developing reaction was stopped by the addition of 50 µl/well of 2N HCL (Sigma). Plates were read on a Synergy HT spectrophotometer (BioTEK) at 492 nm and the endpoint titer was determined as being the last dilution of serum that yielded an O.D. reading equivalent to assay background. 9.3 Analysis of PBMCs 9.3.1 Analysis of pSTAT3 in PBMCs Analysis of pSTAT3 will be conducted through previously reported techniques (Kortylewski et al. Nature Med 2007). PBMCs will be immersed in a mixture of PBS, 2% FCS and 0.1% (wt/vol) sodium azide with Fc III/IIR-specific antibody to block nonspecific binding and stained the cells with different combinations of fluorochrome- coupled antibodies to CD4, CD14, CD15, CD25, CD33, FoxP3 or HLA-DR as well as 7AAD for dead cell exclusion (BD Biosciences). We collected fluorescence data on Page 32 of 50 IRB Protocol No.