Mature neutrophils suppress T cell immunity in ovarian cancer microenvironment
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
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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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 2

Suppressed T cells are viable and responsive to secondary stimulation.

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Suppressed T cells are viable and responsive to secondary stimulation. T...
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) Results are consistent with soluble anti-CD3/CD28 Ab or anti-CD3/CD28 microbeads as T cell stimulus. (B) ASC (n = 31) were stratified into 3 categories based on the induction of a PMN suppressor phenotype, where X equals a reduction in proliferation as compared with anti-CD3/CD28–stimulated T cells alone: suppressors (SUPP, line 3; X ≥ 1 log10), intermediate suppressors (INTERMED, line 2; 0.5 log10 ≤ X < 1 log10), and nonsuppressors (NON-SUPP, line 1; X < 0.5 log10). SUPP-A and -B illustrate that a subset of ascites supernatants induced PMN suppressors X ≥ 2 log10. Bars are representative of the stratification of suppressor status defined in Table 2. (C–E) PMN suppressor phenotype fully suppressed anti-CD3/CD28–stimulated naive (CD3+CD45RA+RO–CD62L+) (C), central memory (CD3+CD45RA–RO+CD62L+) (D), and effector memory (CD3+CD45RA–RO+CD62L–) (E) T cell populations (n = 2). (F) T cells were annexin-V–negative (≥70%) after 72-hour coculture with ASC (n = 3) and/or PMN. Fas ligand was added to stimulated T cells as a positive control for apoptosis. (G and H) Stimulated T cell proliferation was suppressed after 72 hours with ASC and PMN (G), but was restored after ASC removal and anti-CD3/CD28 restimulation (n = 5) (H). (I) Addition of rIL-2 (100 IU) at 48 hours did not rescue T cell proliferation, as assessed at 72 hours. Symbols represent individual samples (n), and bars represent ± SEM. Statistical comparisons were by ANOVA with Tukey post hoc test (*P < 0.05; ***P < 0.001). Results were consistent between CD4+ and CD8+ T cells.