CROSSFIRE IMMUNE
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Systemic (peripheral blood) immune monitoring data of Irreversible Electroporation and Stereotactic Ablative Body Radiotherapy treated patients with locally advanced Pancreatic cancer - flow cytometry - ElISpot Assay Data is provided for each figure presented in the manuscript
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FACS analysis was run on a BD LSRFortessa™ cell analyzer (BD Biosciences, Franklin Lakes, New Jersey) and included effector T cells, B cells, dendritic cells (DCs), natural killer (NK) cells, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs), all with appropriate FMO-controls (fluorescence minus one). Additionally, cell subsets were examined for expression of activation markers (HLA-DR, CD25, Ki67, CD40, CD80, CD86) and immune checkpoint markers (PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3). Tumor antigen-specific T cell responses were determined in 20 randomly selected participants following a previously described in vitro (re)-stimulation and expansion protocol. PBMCs were combined with one of the following peptide pools (JPT Peptide Technologies) at 1 µg/ml per peptide: 1) a pool of 110 15-mer overlapping peptides of Wilms tumor-1 (WT-1) protein; 2) a pool of 110 15-mer overlapping peptides covering Survivin protein, and; 3) CEFT positive control pool of 27 peptides selected from defined HLA Class I and II-restricted epitopes from CMV, EBV, Influenza and Tetanus Toxoid to ascertain recall antigen reactivity as a measure of immune competence. Survivin and WT-1 were selected as tumor antigens of choice based on their selective overexpression on PDAC cells observed in 80% and 75% of PDAC patients respectively, and not at all in healthy pancreatic tissue. PBMC were then cultured for 10 days in the presence of IL-2 (10 IU/ml; Novartis) and IL-15 (10 ng/ml; eBioscience). Cells were then harvested and seeded in a multiscreen 96-well plate (Millipore) coated with an IFNγ catching antibody (Mabtech). Cells were either rechallenged with the peptides overnight or cultured with a DMSO-vehicle control and plates were developed following manufacturer’s instructions (Mabtech) the next day. An automated Elispot reader system (AID) was used to count the spots. Tumor-antigen specific T cell frequencies were calculated by subtracting the mean number of spots in the Dimethyl Sulfoxide (DMSO) wells from the mean number of spots in the corresponding experimental wells (Survivin, WT-1, CEFT). Tumor-antigen specific T cell frequencies were considered a positive response when I) the absolute mean spot count in the experimental wells exceeded those in the DMSO control wells by at least 10, and IIa) the mean spot counts in the experimental wells exceeded those in the DMSO control wells ≥ 1.5-fold, or IIb) the mean spot counts in the experimental wells was significantly higher than the mean spot counts in the DMSO control wells as determined by an unpaired T-test. A CEFT-specific T cell response was considered a measure of immune competence. Participants with absolute post-treatment CEFT-specific mean spot counts < 200 per 100.000 T cells, unless exceeding the DMSO control wells > 3-fold, were considered generally immune compromised and were excluded for further correlation with survival and flowcytometry data.