Fig. 4

Quantitative and qualitative characterization of antigen specific T cell response post-vaccination. The durability of the antigen specific T cell response after peptide pulsed DC vaccination was determined by enumerating the difference in antigen specific T cells using the PBMCs prepared from peripheral blood drawn prior to vaccination and d56 post-vaccination. a The tetramer staining for the Cap1, Tert, and SRV shows the T cells reactive to these epitopes in the PBMCs for various patients at different time points. Thin red lines indicate patients with stable disease; thin black lines patients with progression. The line obtained from the data of long survivor patient has patient ID #13 marked next to it. Thick black line indicates fitted regression model. The data was acquired using BD FACS Aria and analyzed using FlowJo software. b ELISPOT assay was performed as detailed in the methods. The antigen specific re-stimulation with the tumor peptide epitope leading to secretion of the effector cytokine IFNγ was determined by quantifying the differences in ELISPOT’s between PBMC from pre vaccination (d0) vs. post-vaccination (d56) samples. Overnight stimulation of the pre-vaccination and post-vaccination PBMCs with the three tumor epitope peptides and CEF peptide pool was done. The CEF peptide stimulation served as positive control for the assay. Subtracting the spots seen in the unstimulated well normalized the data, and IFNγ spots were plotted at baseline vs. at day 56. Patient data is indicated by unique symbols on the plot, with red for stable disease and black for progression. Patient with long survival is indicated with black circle around his/her symbol in all of the plots. An x = y line is included to demonstrate changes from baseline (points above the line indicate increases; points below the line indicate decreases). c The supernatant collected after overnight re-stimulation of the pre-vaccination and post-vaccination PBMCs with the tumor epitope peptides was used to determine the IFNγ levels (pg/ml) using ELISA. The differences in pre-vaccination and post-vaccination levels were plotted. The PBMCs were also stimulated in parallel with the CEF peptide pool that served as positive control for the assay. Patient data is indicated by unique symbols on the plot, with red for stable disease and black for progression. Patient with long survival is indicated with black circle around his/her symbol in all of the plots. An x = y line is included to demonstrate changes from baseline (points above the line indicate increases; points below the line indicate decreases)