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 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 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 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