Additional file 1: of Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

Figure S1. T cell stimulated with αCD3/αCD28 microbeads proliferate in the presence of dexamethasone.Healthy donor T cells were cultured for four days with the indicated ratio of αCD3/αCD28 microbeads:total T cells in the presence of vehicle or dexamethasone. A, Representative flow cytometry plots of CellTrace violet dilution. Plots were derived from gated CD4 (top row) or CD8 (bottom row) T cells. B-D, Proliferation analyses of CD4 T cells (top) and CD8 T cells (bottom) performed on the samples shown in (A). Precursor Frequency (B), Expansion Index (C), and Proliferation Index (D) are shown. Samples were plated in duplicate and analyzed with an unpaired students T test. Data are representative of three independent experiments. (PDF 3563 kb

Additional file 4: of Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

Figure S4. Increased co-stimulation ameliorates the inhibitory effects of dexamethasone. Negatively-selected healthy donor T cells were cultured with 5 μg/mL αCD3 and increasing concentrations of CD80 in the presence of vehicle or dexamethasone. A-B. CD8 T cells cultured with vehicle (A) or dexamethasone (B). Flow cytometry plots showing proliferation of cells cultured with the indicated concentration of CD80 (left) and total numbers of naïve (TN), central memory (TCM), effector memory (TEM), and terminal effector (TTE) T cells following four days of culture (right) are shown. Differentiation subsets were assessed by CD45RO and CCR7 staining. Each condition was plated in duplicate, and data are representative of three independent experiments. Data were analyzed with an unpaired, two-tailed T Test. (PDF 2573 kb

Additional file 3: of Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

Figure S3. T cell differentiation subsets formed during in vitro stimulation with ιCD3/CD80 stimulation. Negatively-selected healthy donor T cells were cultured with 5 Οg/mL ιCD3 and the indicated concentration of CD80. T cell differentiation subsets were quantified following four days of culture. A, Flow plot of gating strategy to identify the indicated T cell differentiation subsets. B, Flow plots of CD4 (top) and CD8 (bottom) T cells cultured under the indicated conditions. (PDF 3995 kb

Additional file 5: of Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

Figure S5 PD-1 blockade does not rescue dexamethasone-mediated proliferation defects. A, Flow cytometry analysis of PD-1 surface expression on CD4 (left) or CD8 (right) T cells stimulated with αCD3/αCD28 microbeads. Unstimulated (dashed line), stimulated in presence of vehicle (solid line), and stimulated in presence of dexamethasone (filled red line) are shown. B, Geometric median fluorescence intensity (gMFI) of PD-1 staining on CD4 or CD8 T cells. Cells cultured with vehicle (black bars) and dexamethasone (red bars) are shown. Data are an average of duplicate samples. C, Expression of PD-1 by qPCR of T cells stimulated in the presence of vehicle or dexamethasone. Data are representative of four independent experiments. D-E. Healthy donor T cells were stimulated for four days in the presence of vehicle or dexamethasone and nivolumab or ipilimumab F(ab’)2 antibody as indicated. Precursor frequency of CD4 and CD8 T cells was quantified by FlowJo. The ratio of dexamethasone to vehicle for CD4 (C) and CD8 (D) T cells is shown. All samples were plated in duplicate and the ratios were analyzed with a one-way ANOVA. Data are representative of n = 4 healthy donors. (PDF 2522 kb

Additional file 7: of Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

Figure S7. Quantification of Treg and checkpoint molecules in tumor-bearing mice. GL261 ffluc-mCherry tumor-bearing mice were randomized into the indicated cohorts based on bioluminescence values from tumor. Vehicle or dexamethasone treatment was initiated on day 7, and isotype or CTLA-4 blocking antibody were administered on days 13, 16, and 19 following tumor implantation. Mice were euthanized on day 23 and tissues were harvested for flow cytometry analysis. A, Treg cell number from tumor-bearing brain hemisphere (left; n = 8) or the cervical tumor-draining lymph nodes (right; n = 10). B, The percentage of CD4 (top two plots) or CD8 (bottom two plots) T cells expressing the indicated checkpoint molecules. Co-expression of molecules was quantified using a Boolean gating strategy. Data were analyzed using a unpaired students T test. (PDF 1891 kb

In-vivo investigation of X-PACT application to BALB/c mice with syngeneic 4T1-HER2 tumors.

<p>Error bars are 1 standard error of the mean. A generalized linear model that used an autoregressive correlation structure to account for within-mouse correlations showed the rate of tumor growth after initiation of treatment to differ significantly among the 4 groups (p<0.0001; interaction between time and group). The rate of tumor growth within the phosphor + X-ray and X-PACT groups was significantly slower than that observed in the saline (p<0.0001 and p<0.0001, respectively) and AMT + X-ray groups (p = 0.0073 and p = 0.0011, respectively).</p

Anti-tumor effects of X-PACT and its individual components on 4T1-HER2 cells.

<p><b>A:</b> cell viability after X-PACT (10μM 8-MOP equivalent dilution of UVADEX, 50μg/mL phosphor, 1Gy of 80kVp radiation) as determined by Guava flow cytometry. N is the number of independent measurements (different days), and error bars indicate one standard deviation. Radiation had a significantly different effect on cell viability when administered with X-PACT than with phosphor or UVADEX alone (p<0.0001 for interaction in two-way ANOVA). With X-PACT, radiation resulted in a significant decrease in cell viability relative to no x-ray dose. <b>B:</b> the Annexin V (+) fraction of viable cells shown in 3A. Radiation had a significantly different effect on Annexin V when administered with X-PACT, as opposed to individual components (p<0.0001). <b>C and D:</b> Cell viability illustrated by methyl blue staining for identical plates each receiving 1Gy of 80kVp x-rays. Each plate contained wells including no additives (control), three concentrations of phosphor only (25, 50, & 100μg/mL with DLC), UVADEX only (10uM 8-MOP equivalent dilution), and three combination X-PACT regimes.</p

Light output from X-PACT phosphors under x-ray stimulation.

<p>The new phosphors (red line) are much brighter than previous versions evaluated in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162078#pone.0162078.ref025" target="_blank">25</a>] (green line). The absorption spectrum of psoralen is shown for comparison (blue line).</p

X-Ray Psoralen Activated Cancer Therapy (X-PACT) - Fig 3

<p><b>A</b>: UV light activated psoralen was observed to reduce viable cells in 3 cell lines (data from Cell-Titer-Glo^® Luminescence Cell Viability Assay under UV light). N = 4 for each cell line at each UV light condition (0, 0.25, 0.5, 1.0 J/cm²). The psoralen concentration was 40 μM. Within each cell line, cell viability decreased as radiation dose increased (p<0.001; linear regression). <b>B:</b> in CT2A cells, X-PACT cytotoxicity increases with X-ray dose (0, 0.67 and 1.00 Gy respectively), concentration of 8-MOP psoralen (10, 20 and 40 μM respectively), and phosphor (50 and 100 μg/ml) respectively. Multiple linear regression showed significant reductions in cell viability with increasing radiation doses (p<0.001), increasing phosphor dose (p = 0.011), and increasing psoralen dose (p<0.001).</p

a) Representative tumor size for each arm: day 25 after tumor cell implantation.

<p>a) Representative tumor size for each arm: day 25 after tumor cell implantation.</p