Highlights d Tumor-secreted CXCR1 and CXCR2 ligands induce extrusion of NETs d NETs protect tumor cells from CTL and NK cytotoxicity in 3D cultures d Inhibition of NETosis sensitizes tumors to PD-1+CTLA-4 dual checkpoint blockade d NETs impair contact of immune cytotoxic cells with tumor cells in living mice Authors

Weak and ineffective antitumor cytotoxic T lymphocyte (CTL) responses can be rescued by immunomodulatory monoclonal antibodies (mAbs) targeting PD-1 or CD137. Using Batf3-/- mice, which are defective for cross-presentation of cell-associated antigens, we show that Batf3-dependent dendritic cells (DCs) are essential for the response to therapy with anti-CD137 or anti-PD-1 mAbs. Batf3-/- mice failed to prime an endogenous CTL-mediated immune response toward tumor-associated antigens, including neoantigens. As a result, the immunomodulatory mAbs could not amplify any therapeutically functional immune response in these mice. Moreover, administration of systemic sFlt3L and local poly-ICLC enhanced DC-mediated cross-priming and synergized with anti-CD137- and anti-PD-1-mediated immunostimulation in tumor therapy against B16-OVA-derived melanomas, whereas this function was lost in Batf3-/- mice. These experiments show that cross-priming of tumor antigens by Flt3L- and Batf3-dependent DCs is crucial to the efficacy of immunostimulatory mAbs and represents a very attractive point of intervention to enhance their clinical antitumor effects.

Purpose: Myeloid-derived suppressor cells (MDSC) are considered an important T-cell immunosuppressive component in cancer-bearing hosts. The factors that attract these cells to the tumor microenvironment are poorly understood. IL8 (CXCL8) is a potent chemotactic factor for neutrophils and monocytes.Experimental Design: MDSC were characterized and sorted by multicolor flow cytometry on ficoll-gradient isolated blood leucokytes from healthy volunteers (n ¼ 10) and advanced cancer patients (n ¼ 28). In chemotaxis assays, sorted granulocytic and monocytic MDSC were tested in response to recombinant IL8, IL8 derived from cancer cell lines, and patient sera. Neutrophil extracellular traps (NETs) formation was assessed by confocal microscopy, fluorimetry, and time-lapse fluorescence confocal microscopy on short-term MDSC cultures.Results: IL8 chemoattracts both granulocytic (GrMDSC) and monocytic (MoMDSC) human MDSC. Monocytic but not granulocytic MDSC exerted a suppressor activity on the proliferation of autologous T cells isolated from the circulation of cancer patients. IL8 did not modify the T-cell suppressor activity of human MDSC. However, IL8 induced the formation of NETs in the GrMDSC subset.Conclusions: IL8 derived from tumors contributes to the chemotactic recruitment of MDSC and to their functional control.