Search

Figure S12. Numbers of NK cells are not affected by combination therapy. Peritoneal cells harvested from mice treated with vehicle (Veh); chemotherapy (Chemo); anti-IL-10, 2′3’-cGAMP, and anti-PD-L1 immunotherapy (IT); or both Chemo and IT (Combo) were assessed by flow cytometry 13 days after initiation of treatment. Bar graph shows quantification of NK cells (NK1.1+CD3−) and their expression of activation makers CD44 and PD-1. Experiment was performed twice with n = 4 biological replicates. Statistics were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SEM * p ≤ 0.05, ** p ≤ 0.01. (PDF 73 kb)

Figure S7. Combination therapy increases the proportion of differentiated CD8+ T cells. Peritoneal cells harvested from mice treated with vehicle (Veh); chemotherapy (Chemo); anti-IL-10, 2′3’-cGAMP, and anti-PD-L1 immunotherapy (IT); or both Chemo and IT (Combo) were assessed by flow cytometry 4 days after initiation of treatment. Flow cytometry gating of subsets of CD69 and CD107a expressing CD8+ T cells are shown as scatter plots and quantified at right. Experiment was performed twice with n = 4 biological replicates. Statistics were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SEM * p ≤ 0.05. (PDF 83 kb)

Figure S5. Galectin3 is upregulated after chemotherapy treatment. Mice were treated with vehicle (Veh) or chemotherapy (Chemo) 8 days after ID8-Vegf-Defb29 tumor inoculation and peritoneal cells were assessed by flow cytometry 4 days after initiation of treatment. Histograms of Gal3+ expression on CD11b+ myeloid cells are shown and the mean fluorescence intensity was quantified on the right. Experiment was performed twice with n = 5 biological replicates. Statistics were calculated using a two-sided unpaired t-test. Data are presented as mean ± SEM ** p ≤ 0.01 (PDF 97 kb)

Figure S6. Proportion of CD4+ and CD8+ T cells is not affected shortly after treatment. Peritoneal cells harvested from mice treated with vehicle (Veh); chemotherapy (Chemo); anti-IL-10, 2′3’-cGAMP, and anti-PD-L1 immunotherapy (IT); or both Chem and IT (Combo) were assessed by flow cytometry 4 days after initiation of treatment. Flow cytometry gating of subsets of CD4+ and CD8+ expressing CD3+ T cells are shown as scatter plots and quantified at right. Experiment was performed twice with n = 4 biological replicates. Statistics were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SEM. (PDF 76 kb)

Figure S8. Expression of IFNγ or PD-1 on T cells is not affected shortly after treatment. Peritoneal cells harvested from mice treated with vehicle (Veh); chemotherapy (Chemo); anti-IL-10, 2′3’-cGAMP, and anti-PD-L1 immunotherapy (IT); or both Chemo and IT (Combo) were assessed by flow cytometry 4 days after initiation of treatment. Flow cytometry gating of subsets of PD-1+ and IFNγ+ g CD4+ and CD8+ T cells are shown as scatter plots and quantified at right. Experiment was performed twice with n = 4 biological replicates. Statistics were calculated using one-way ANOVA with Tukey’s multiple comparisons test. Data are presented as mean ± SEM. (PDF 84 kb)

Figure S3. Gating strategy used in flow cytometric analysis of immune cells harvested from the peritoneal cavity after treatment. Flow cytometric data were analyzed using FlowJo software. (PDF 423 kb)

Figure S4. Chemotherapy induces an increase in granulocytic but not monocytic MDSCs. Mice were inoculated orthotopically with ID8-Vegf-Defb29 ovarian cancer cells. Eight days later, the mice were injected with vehicle (Veh) or chemotherapy (Chemo). Two days later, peritoneal cells were harvested and assessed by flow cytometry. (a) Increased numbers of granulocytic MDSCs (Ly6G+Ly6C+) are observed following chemotherapy. (b) Numbers of monocytic MDSCs (Ly6G−Ly6C+) and their expression of immunosuppressive ARG1 and IL-10 are quantified. The experiment was performed twice with n = 4 biological replicates. Statistics were calculated using a two-sided unpaired t-test. Data are presented as mean ± SEM **** p ≤ 0.0001. (PDF 78 kb)

Figure S2. Functional enrichment analysis of unsorted cells highlights the importance of the innate immune system. Mice were inoculated with ID8-Vegf-Defb29 cancer cells. Eight days later, mice were injected with either vehicle or paclitaxel and carboplatin (Chemo). Two days thereafter, peritoneal cells were recovered and assessed by gene expression analysis. (a) The top 84 significantly upregulated genes after chemotherapy treatment with a fold change â Ľ1 were queried for their pathway interactions using the ToppGene Suite gene list enrichment analysis tool. (b) The genes induced by chemotherapy had a significant correlation with myeloid cell populations. (PDF 1020 kb)

Figure S9. Flow cytometry analysis confirms that CD11b+ myeloid cells, CD8a+ T cells, and CD4+ T cells are depleted following administration of appropriate antibodies. (a) Plots are shown for leukocytes isolated from blood after initiation of treatment. (b) Quantification of depletion is representative of n = 6 mice, and the experiment was performed twice. Statistics were calculated using a two-sided unpaired t-test. Data are presented as mean ± SEM **** p ≤ 0.0001. (PDF 114 kb)