Fig 2: Vorinostat effect on TNBC cells and PBMCs co-cultured together(A) MDA-MB231 cells untreated or treated with increasing doses of vorinostat were collected 24 and 48 hours after treatment and stained for flow cytometry analysis to quantify PD-L1 expression (expressed as fold change relative to the control). Statistical comparisons are relative to respective control at 24 or 48 hours. (B) MDA-MB231 cells were cultured alone or in presence of PBMCs obtained from healthy donors. Cells were untreated or treated with vorinostat (1.5µM) for 24, 48 and 72 hours and then collected and stained with a comprehensive multicolor flow cytometry panel. Representative flow cytometric plot for PD-L1 expression in MDA-MB231 with or without vorinostat treatment. (C) Flow cytometric quantification of PD-L1 expression (expressed as fold change relative to the control) in MDA-MB231 cells alone or co-cultured with PBMCs with or without vorinostat treatment. (D) Flow cytometric quantification of HLA-DR expression (expressed as fold change relative to the control) in MDA-MB231 cells treated or untreated with increasing doses of vorinostat for 24 and 48 hours. Statistical comparisons are relative to respective control. (E) Representative flow cytometric plot for HLA-DR expression in MDA-MB231 with or without vorinostat treatment. (F) Flow cytometric quantification of HLA-DR expression (expressed as fold change relative to the control) in MDA-MB231 cells alone or co-cultured with PBMCs with or without vorinostat treatment. (G) Representative flow cytometric plots for Foxp3+ CTLA-4high cells in PBMCs with or without vorinostat treatment. (H) Flow cytometric quantification (expressed as fold change relative to the control) of CD4+ Foxp3+ CTLA-4high T cells in PBMCs from healthy donors alone or in presence of MDA-MB231 after 24, 48 and 72 hours of vorinostat treatment. (I) Flow cytometric quantification of the number of CD4+ Foxp3+ CTLA-4high T cells in PBMCs with or without vorinostat treatment for 24, 48 and 72 hours. (J) Representative flow cytometric plots for CD25/CTLA-4 co-expression in live CD4+, CD127low, Foxp3+ PBMCs with or without vorinostat treatment. (K) Flow cytometric quantification of CD25high CTLA-4high Treg in PBMCs of healthy donors with or without vorinostat treatment for 24 and 48 hours. Plots in (J) and (K) are pre-gated on live CD4+ CD127low Foxp3+ cells. Data are presented as the mean ± SEM. Statistical significance is indicated by p-values as * P = 0.05; ** P = 0.01; *** P = 0.001; **** P = 0.0001.

Fig 3: Resident memory T (TRM) and non-TRM phenotypic profiles and susceptibility of cancer stem cell (CSC) to cytotoxic T lymphocyte (CTL)-mediated killing. (A) Flow cytometry analyses of CD8, LFA-1, CD103, CD49a, CD69, PD-1, CTLA-4, TGFBR2 and VEGFR2 on CD8+CD103+ TRM (Heu171) and CD8+CD103- non-TRM (H32-22) clones. Mean immunofluorescence intensity (MFI) are in parentheses.(B) Cytotoxic activity of the TRM clone (Heu171) towards autologous IGR-Heu, Heu-CSC, CSC-1 and CSC-2 target cells. Percent of specific lysis are shown at indicated effector to target (E:T) ratios. (C) Quantification of transforming growth factor (TGF)-ß in conditioned media from IGR-Heu, Heu-CSC, CSC-1 and CSC-2 by multi-analyte flow assay. Ratios of active/total TGF-ß normalized to IGR-Heu are included. Results are presented as mean±SEM of duplicates. (D) Cytotoxicity of the non-TRM clone (H32-22) towards autologous IGR-Heu, Heu-CSC, CSC-1 and CSC-2 target cells. (E) Inhibition of TRM-cell-mediated killing. CSC-1 cells are preincubated in the presence of isotype control, anti-major histocompatibility complex class I (MHC-I) or anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing monoclonal antibody (mAb) and then CTL were added at 5:1 E:T ratio. Symbols represent replicates and horizontal bars represent means±SEM (n=3). P value was determined by two-way analysis of variance test. *p<0.05, **p<0.01,***p<0.001,****p<0.0001. Data are from one representative experiment out of three.