Ovarian cancer ascites-activated neutrophils suppress T cell proliferation in a contact-dependent mechanism

Ovarian cancer (OC) is often diagnosed at advanced stages. Necrosis is a hallmark of advanced cancer and releases DAMPs that activate innate immune responses. It is well known that CD8 + T cells mediate antitumor immunity and immunosuppressive myeloid cells can impair T cell responses. In contrast, there is limited knowledge on the role of activated neutrophils in the tumor microenvironment. In patients with newly diagnosed OC, ~90% of ascites cells were inflammatory (CD45 + ) with varying proportions of neutrophils, monocytes and lymphocytes. Although patient variability existed, neutrophils comprised ~15% of CD45 + cells and the neutrophil:CD8 + T cell ratio was 1:1. In ex vivo studies, cell-free ascites (CFA) attracted normal donor neutrophils (NDN). Neutrophil extracellular traps (NETs) are a distinct mode of death, and characterized by extracellular release of DNA, histones, and granular products. We found that CFA induced NDN to generate NETs ex vivo . In co-culture studies, CD3/CD28-stimulated normal donor T cells were incubated with media; purified NDN 1:1; CFA; or NDN + CFA. After 72h, T cell proliferation was measured by [ 3 H] thymidine incorporation. While CFA or NDN alone did not suppress T cell proliferation, CFA-activated NDN completely suppressed proliferation. Suppression did not affect T cell viability or induce apoptosis, and was not reversed by the addition of arginine, N-acetyl-L-cysteine or IL-2. Suppression required T cell contact with NDN. These results support a model in which ascites-activated neutrophils suppress T cell responses, thereby abrogating antitumor immunity. Further studies will identify ascites constituents that activate neutrophils and mechanisms for neutrophil-mediated T cell suppression.