Additional file 1: Table S1. of Anti-PD-1 increases the clonality and activity of tumor infiltrating antigen specific T cells induced by a potent immune therapy consisting of vaccine and metronomic cyclophosphamide

Primer sequences. Table S2. R9F-clone library. C3 tumor bearing mice (C57BL/6, n = 3) were treated with mCPA for 1 week and then vaccinated with DPX-R9F. Eight days later, mice were euthanized and spleens collected. R9F-specific CD8α + T cells were purified from spleens using FACS and TCRβ sequencing performed by Adaptive Biotechnologies. From the three mice, 26 different clones were identified at a frequency >1 % and considered R9F-specific. The table lists the amino acid sequence of each clone, the TCRbV and TCRbJ gene associated with each clone, and the frequency of each clone among the three samples. Highlighted in grey are the clones found in all samples. Highlighted in red are the most frequent clones of each sample. Figure S1. Upregulation of surface antigens on C3 tumor cells in vitro after culture with IFN-γ (50 U/mL, 48 hours). Surface expression determined by flow cytometry. Results representative of 3 separate experiments. Black: isotype control, blue: unstimulated, red: IFN-γ stimulated. Figure S2. (A, B) C57BL6 mice (n = 10) were implanted with B16-F10 tumors and treated with mCPA starting on day 3 for one week one, 1 week off. Mice were vaccinated with DPX containing the TRP2180-188 peptide (DPX-S9L) on days 3 and 17. Anti-PD-1 or isotype control administered on days 3, 6, 9, 17, 20, 23. (C) HLA-A2 transgenic mice (n = 5) were implanted subcutaneously with syngeneic ovarian tumors and treated using the same schedule as the B16-F10, except that mice were vaccinated with DPX-Survivac. Figure S3. Immunohistochemical staining of tumor sections for CD45 and CD8α expression. Samples were cryoprotected and snap frozen. Sections were fixed and blocked, then treated with anti-CD8α or anti-CD45 followed by biotinylated anti-rat IgG. Staining was visualized using Vectastain ABC kit (Vector Laboratories) and counterstained with Gill’s Hematoxylin. Microscopy was performed at a 10X magnification. (PPTX 478 kb)