Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment
Kelly L. Singel, … , Emese Zsiros, Brahm H. Segal
Kelly L. Singel, … , Emese Zsiros, Brahm H. Segal
Published February 7, 2019
Citation Information: JCI Insight. 2019;4(5):e122311. https://doi.org/10.1172/jci.insight.122311.
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Research Article Immunology Oncology

Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment

Abstract

Epithelial ovarian cancer (EOC) often presents with metastases and ascites. Granulocytic myeloid–derived suppressor cells are an immature population that impairs antitumor immunity. Since suppressive granulocytes in the ascites of patients with newly diagnosed EOC were morphologically mature, we hypothesized that PMN were rendered suppressive in the tumor microenvironment (TME). Circulating PMN from patients were not suppressive but acquired a suppressor phenotype (defined as ≥1 log10 reduction of anti-CD3/CD28–stimulated T cell proliferation) after ascites supernatant exposure. Ascites supernatants (20 of 31 supernatants) recapitulated the suppressor phenotype in PMN from healthy donors. T cell proliferation was restored with ascites removal and restimulation. PMN suppressors also inhibited T cell activation and cytokine production. PMN suppressors completely suppressed proliferation in naive, central memory, and effector memory T cells and in engineered tumor antigen–specific cytotoxic T lymphocytes, while antigen-specific cell lysis was unaffected. Inhibition of complement C3 activation and PMN effector functions, including CR3 signaling, protein synthesis, and vesicular trafficking, abrogated the PMN suppressor phenotype. Moreover, malignant effusions from patients with various metastatic cancers also induced the C3-dependent PMN suppressor phenotype. These results point to PMN impairing T cell expansion and activation in the TME and the potential for complement inhibition to abrogate this barrier to antitumor immunity.

Authors

Kelly L. Singel, Tiffany R. Emmons, ANM Nazmul H. Khan, Paul C. Mayor, Shichen Shen, Jerry T. Wong, Kayla Morrell, Kevin H. Eng, Jaron Mark, Richard B. Bankert, Junko Matsuzaki, Richard C. Koya, Anna M. Blom, Kenneth R. McLeish, Jun Qu, Sanjay Ram, Kirsten B. Moysich, Scott I. Abrams, Kunle Odunsi, Emese Zsiros, Brahm H. Segal

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Figure 3

PMN suppressor phenotype requires contact between PMN and T cells, complement C3 activation, and complement receptor 3.

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PMN suppressor phenotype requires contact between PMN and T cells, compl...
T cells (CD3+) and PMN were used in autologous coculture at 1:1. PMN and/or ascites supernatants (ASC; 50% final well volume) were added to anti-CD3/CD28–stimulated T cells. After 72 hours of coculture, T cell proliferation was measured by [3H] thymidine incorporation (16–18 hours). (A) T cells were stimulated with anti-CD3/CD28 in the bottom chamber. PMN and ASC added to the transwell insert did not suppress T cell proliferation (n = 4). (B) T cells treated with anti–ICAM-1 Ab (1–10 μg) for 1 hour prior to coculture had no effect on proliferation. PMN treated with anti-CD11b Ab for 1 hour prior to coculture abrogated the suppressor phenotype. Treatment of T cells or PMN with IgG1 isotype (1–10 μg) had no effect on proliferation (n = 5). (C) PMN pretreated with C3b or iC3b (40–160 μg/ml) prior to coculture were unable to induce the suppressor phenotype. (D) ASC were heat inactivated (HI-ASC; 56°C, 1 hour) prior to coculture and abrogated the PMN suppressor phenotype (n = 5). (E–H) Two formulations of compstatin, CS and Cp40, were used to inhibit C3 activation. Addition of CS (250 μM) to ASC (CS-ASC) 2 hours prior to coculture with PMN and T cells abrogated the suppressor phenotype (n = 27) (E–G). Addition of Cp40 (20 μM) to ASC (Cp40-ASC) 2 hours prior to coculture also abrogated the suppressor phenotype, while scramble peptide (SCR-ASC, 20 μM) had no effect (n = 10) (H). (I) A titration study showed that 5 μM Cp40 was sufficient to fully abrogate the suppressor phenotype (n = 3). (J) ASC were pretreated with neutralizing Ab anti-C5 or -C7, or with OmCI, a peptide inhibitor of C5, prior to coculture. Anti-C5 and OmCI partially abrogated the suppressor phenotype, as compared with their respective controls, whereas anti-C7 did not affect the suppressor phenotype. (K) Malignant effusions (ME), including pleural fluid and ascites from patients with a number of metastatic cancers, induced the PMN suppressor phenotype, which was abrogated by Cp40 treatment in all of the tested samples (n = 7; Table 3). Symbols represent individual samples (n), and bars represent ± SEM. Statistical comparisons were by ANOVA with Tukey post hoc test or by Mann-Whitney (*P < 0.05; **P < 0.01; ***P < 0.001).