Myeloid-derived suppressor cells modulate immune responses independently of NADPH oxidase in the ovarian tumor microenvironment in mice

Abstract

The phagocyte NADPH oxidase generates superoxide anion and downstream reactive oxidant intermediates in response to infectious threat, and is a critical mediator of antimicrobial host defense and inflammatory responses. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are recruited by cancer cells, accumulate locally and systemically in advanced cancer, and can abrogate anti-tumor immunity. Prior studies have implicated the phagocyte NADPH oxidase as being an important component promoting MDSC accumulation and immunosuppression in cancer. We therefore used engineered NADPH oxidase-deficient (p47 (phox-/-)) mice to delineate the role of this enzyme complex in MDSC accumulation and function in a syngeneic mouse model of epithelial ovarian cancer. We found that the presence of NADPH oxidase did not affect tumor progression. The accumulation of MDSCs locally and systemically was similar in tumor-bearing wild-type (WT) and p47 (phox-/-) mice. Although MDSCs from tumor-bearing WT mice had functional NADPH oxidase, the suppressive effect of MDSCs on ex vivo stimulated T cell proliferation was NADPH oxidase-independent. In contrast to other tumor-bearing mouse models, our results show that MDSC accumulation and immunosuppression in syngeneic epithelial ovarian cancer is NADPH oxidase-independent. We speculate that factors inherent to the tumor, tumor microenvironment, or both determine the specific requirement for NADPH oxidase in MDSC accumulation and function.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1.Time. to tumor progression requiring euthanasia is not altered by NADPH oxidase.

Kaplan-Meier plots of WT and NADPH oxidase-deficient (p47phox−/−) mice (10 mice/group) showed similar survival after i.p. MOSEC challenge (log-rank, p = 0.25).

Figure 2. Effect of NADPH oxidase in local and systemic accumulation of MDSCs in tumor-bearing mice.

A) Representative quantification of MDSCs. Splenocytes from WT and p47phox−/− mice at day 42 and 90 after MOSEC administration were analyzed for MDSC accumulation. Gating on myeloid (CD11b+) cells, the proportion of monocytic MDSCs (R1; Ly6C+Ly6G−) and granulocytic MDSCs (R2; Ly6G+Ly6CLow) significantly increased at day 90 versus day 42. All gates were set based on isotypes. This approach was used to quantify MDSCs in PECs, lymph nodes, and spleens. B) Proportion of MDSCs in myeloid PECs on day 42 and 90. The proportion with granulocytic and monocytic MDSC markers was greater in advanced (day 90) versus early (day 42) stage tumor burden in both genotypes. C) In draining lymph nodes, there was a trend toward increased monocytic MDSC accumulation in p47phox−/− versus WT mice at day 42 but not at day 90. There was no effect of NADPH oxidase on granulocytic MDSC accumulation at either time point. D) In spleens, there was an increased accumulation of MDSCs, particularly granulocytic MDSCs, in mice with advanced versus early disease, but no effect of mouse genotype. Data (± SEM) are from at least 3 mice per genotype per time point, and are representative of 3 separate experiments. Comparison between genotypes: p = NS.

Figure 3. Role of NADPH oxidase in cytokine production in the ovarian tumor microenvironment.

Cell-free supernatants collected from ascites from WT and p47phox−/− mice at day 90 after MOSEC administration were analyzed by ELISA for pro-inflammatory cytokines, G-CSF, and VEGF. N = 8 mice per genotype. Comparison between genotypes: p = NS.

Figure 4. The p47phox component is required for NADPH oxidase activity in granulocytic MDSCs.

PECs from WT and p47phox−/− mice harvested at day 90 after MOSEC challenge were stimulated with PMA, and intracellular ROI production in CD11b+Ly6G+ cells was assessed by H2DCFDA fluorescence. A) Gating on all non-aggregated cells (Gate 1), CD11b+Ly6G+ cells (Gate 2) were defined using respective isotype controls. B and C) Stimulated ROI production was detectable in WT (B), but not in NADPH oxidase-deficient (C), granulocytic MDSCs. White plot = PMA stimulation; shaded plot = no-stimulation. Results are representative of two experiments.

Figure 5. Analysis of peritoneal exudate cells from MOSEC-bearing mice after enrichment for myeloid cells and granulocytic cells.

Myeloid cells and granulocytic cells from PECs of MOSEC-bearing WT and p47phox−/− mice were column-purified using anti-CD11b and anti-Ly6G, respectively. Analysis of post-enriched fractions showed concordance between cytology and flow cytometry. Representative analysis of PECs from p47phox−/− mice collected on 90 after MOSEC administration is shown. A) The CD11b-negative fraction contained a preponderance of tumor cells (based on cytology), while myeloid cells were rare. B) The CD11b-enriched fraction contained a mixed myeloid cell population, with a preponderance of macrophages based on cytology and surface markers (CD11b+F4/80+). C) The Ly6G-enriched fraction contained a preponderance of granulocytic cells (arrows), with the majority of myeloid cells expressing granulocytic MDSC markers (CD11b+Ly6G+Ly6Clow).

Figure 6. Myeloid peritoneal cells in MOSEC-bearing mice suppress T cell proliferation independently of NADPH oxidase.

Purified myeloid (CD11b+) PECs from MOSEC-bearing WT and p47phox−/− mice were co-cultured with CFSE-labeled splenocytes from naïve mice (E∶T ratio: 1∶1) in anti-CD3/B7.1-coated plates. After 72 h of culture, CD4+ and CD8+ T cell proliferation was assessed based on CFSE dilution as described in methods. A) Representation histograms showing that CD11b+, but not CD11b−, PECs from MOSEC-bearing WT and p47phox−/− mice (day 90) completely suppress anti-CD3/B7.1-stimulated CD4+ T cells proliferation. Anti-CD3/B7.1 stimulated and unstimulated CD4+ T cells are used as positive and negative controls, respectively. B) CD11b-enriched PECs from MOSEC-bearing WT and p47phox−/− mice at day 42 equally suppress both CD4+ and CD8+ T cell proliferation. In contrast, CD11b− PECs from the same mice incompletely suppressed anti-CD3/B7.1-stimulated T cell proliferation. C) CD11b-enriched PECs from MOSEC-bearing WT and p47phox−/− mice at day 90 completely suppressed T cell proliferation while the CD11b-negative fraction had no significant effect on T cell proliferation. Myeloid PECs collected on day 42 were pooled because of limited cell number, while non-pooled PECs from individual mice were analyzed from day 90 harvests. Individual experiments were performed using PECs from 3 mice per genotype per time point, and each experiment was repeated at least once using PECs from different mice, with similar results. Comparison between genotypes: p = NS.

Figure 7. Peritoneal and splenic granulocytic MDSCs from tumor-bearing mice suppress T cell proliferation independently of NADPH oxidase.

Ly6G-enriched PECs and splenocytes from MOSEC-bearing WT and p47phox−/− mice (day 90) were co-cultured with splenocytes from non-tumor-bearing WT mice (E∶T ratio: 1∶1). A) Ly6G-enriched PECs completely suppressed anti-CD3/B7.1-stimulated CD4+ and CD8+ T cell proliferation. PECs from 3 mice per genotype were evaluated. B) In Ly6G-enriched splenocytes, the majority of cells had a granulocytic morphology (arrows), and 86% of CD11b+ cells expressed granulocytic MDSC markers (Ly6G+Ly6Clow). C) Ly6G-enriched splenocytes from WT and p47phox−/− mice modestly suppressed anti-CD3/B7.1-stimulated CD4+ and CD8+ T cell proliferation. N = 3 mice per genotype were used in this experiment, and results are representative of 3 experiments. Comparison between genotypes: p = NS.