Assessment of variation in immunosuppressive pathway genes reveals TGFBR2 to be associated with risk of clear cell ovarian cancer.

BACKGROUND: Regulatory T (Treg) cells, a subset of CD4+ T lymphocytes, are mediators of immunosuppression in cancer, and, thus, variants in genes encoding Treg cell immune molecules could be associated with ovarian cancer. METHODS: In a population of 15,596 epithelial ovarian cancer (EOC) cases and 23,236 controls, we measured genetic associations of 1,351 SNPs in Treg cell pathway genes with odds of ovarian cancer and tested pathway and gene-level associations, overall and by histotype, for the 25 genes, using the admixture likelihood (AML) method. The most significant single SNP associations were tested for correlation with expression levels in 44 ovarian cancer patients. RESULTS: The most significant global associations for all genes in the pathway were seen in endometrioid ( p = 0.082) and clear cell ( p = 0.083), with the most significant gene level association seen with TGFBR2 ( p = 0.001) and clear cell EOC. Gene associations with histotypes at p < 0.05 included: IL12 ( p = 0.005 and p = 0.008, serous and high-grade serous, respectively), IL8RA ( p = 0.035, endometrioid and mucinous), LGALS1 ( p = 0.03, mucinous), STAT5B ( p = 0.022, clear cell), TGFBR1 ( p = 0.021 endometrioid) and TGFBR2 ( p = 0.017 and p = 0.025, endometrioid and mucinous, respectively). CONCLUSIONS: Common inherited gene variation in Treg cell pathways shows some evidence of germline genetic contribution to odds of EOC that varies by histologic subtype and may be associated with mRNA expression of immune-complex receptor in EOC patients

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

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

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

<p>A) Representative quantification of MDSCs. Splenocytes from WT and p47<i>^phox−/−</i> 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⁺Ly6C^Low) 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 p47<i>^phox−/−</i> 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.</p

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

<p>Cell-free supernatants collected from ascites from WT and p47<i>^phox−/−</i> 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.</p

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

<p>Kaplan-Meier plots of WT and NADPH oxidase-deficient (p47<i>^phox−/−</i>) mice (10 mice/group) showed similar survival after i.p. MOSEC challenge (log-rank, p = 0.25).</p

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

<p>Myeloid cells and granulocytic cells from PECs of MOSEC-bearing WT and p47<i>^phox−/−</i> 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 p47^phox−/− 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⁺Ly6C^low).</p

The p47<i>^phox</i> component is required for NADPH oxidase activity in granulocytic MDSCs.

<p>PECs from WT and p47<i>^phox−/−</i> 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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069631#s3" target="_blank">Results</a> are representative of two experiments.</p