Fitc conjugated anti mouse cd8a

All of the procedures were performed as previously described in^{109 (link)}. Briefly, splenocytes of RT-immunized or vector-immunized mice were stimulated with RT-derived peptides RT202–210 wt, RT202–210 dr, RT207–215 wt, RT207–215 dr, RT205–220 wt, and RT205–220 dr (Table 2) and stained for viability with the Fixable Viability Stain 660 (FSV660; BD Horizon #564405). Thereafter, cell surface staining was performed with a mixture of antibodies including FITC-conjugated anti-mouse CD8a (BD Pharmingen #553031), APC-H7-conjugated anti-mouse CD4 (BD Pharmingen #560181), and PerCP-conjugated anti-mouse CD3 (BD Pharmingen #553067). The cells were then fixed, permeabilized, washed, and stained with PE-Cy7-conjugated anti-mouse IFN-γ antibodies (BD Pharmingen #557649). Stained samples were analysed on a FACSVerse cytometer (BD Biosciences, USA). Data were exported as FCS3.0 files with the use of FACSuite software and read using BioConductor’s^{127 (link)} package flowCore^{128 (link)} in the R software package. Final normalization of the data was performed with the flowStats package^{129 (link)} and the data were gated. First, a general lymphocyte area was defined and viable cells were identified by the lack of FSV660 staining. From the viable population, single cells were defined by the expression of surface markers and IFN-γ production.

Groups of C57BL/6 mice (5 per group) were challenged with TC-1 tumor cells and treated with bortezomib and/or SAHA as described above. To detect HPV16 E7-specific CD8⁺ T cells in peripheral blood, peripheral blood mononuclear cells (PBMCs) were harvested from the tail vein one week after the last treatment. The cells were stained with FITC-conjugated anti-mouse CD8a (BD Pharmingen, San Diego, CA, USA) and PE-conjugated HPV16 E7 aa49-57 peptide loaded H-2D^b tetramer and acquired with FACSCalibur.
To detect HPV16 E7-specific CD8⁺ T cells in the tumor, single cell suspensions were stimulated with HPV16 E7 aa49-57 peptide (1 μg/ml) in the presence of GolgiPlug (BD Pharmingen, San Diego, CA, USA) overnight at 37°C. The cells were then stained with PE-conjugated anti-mouse CD8a. After permeabilization and fixation, the cells were stained with FITC-conjugated anti-mouse IFN-γ followed by flow cytometry analysis. The data were analyzed with FlowJo or CellQuest Pro software.