Published August 14, 2015, doi:10.4049/jimmunol.1500325 The Journal of Immunology Priming of Human Resting NK Cells by Autologous M1 Macrophages via the Engagement of IL-1b, IFN-b, and IL-15 Pathways Irene Mattiola,*,†,‡,1 Matthieu Pesant,*,1 Paolo F. Tentorio,† Martina Molgora,*,‡ Emanuela Marcenaro,x Enrico Lugli,† Massimo Locati,*,‡,2 and Domenico Mavilio†,‡,2 The cross talk between NK cells and macrophages is emerging as a major line of defense against microbial infections and tumors. This study reveals a complex network of soluble mediators and cell-to-cell interactions allowing human classically activated (M1) macrophages, but not resting (M0) or alternatively activated (M2) macrophages, to prime resting autologous NK cells. In this article, we show that M1 increase NK cell cytotoxicity by IL-23 and IFN-b–dependent upregulation of NKG2D, IL-1b–dependent upreg- ulation of NKp44, and trans-presentation of IL-15. Moreover, both IFN-b–dependent cis-presentation of IL-15 on NK cells and engagement of the 2B4-CD48 pathway are used by M1 to trigger NK cell production of IFN-g. The disclosure of these synergic cellular mechanisms regulating the M1–NK cell cross talk provides novel insights to better understand the role of innate immune responses in the physiopathology of tumor biology and microbial infections. The Journal of Immunology, 2015, 195: 000–000. atural killer cells are important components of the in- the heterogeneous family of inhibitory NK cell receptors and nate immune system and play an active role in the clear- MHC class I molecules allow the NK cell–mediated killing via the N ance of tumor-transformed and viral-infected cells either engagement of a large group of activating NKRs (aNKRs) that bind lacking or down-modulating molecules of the MHC class I without specific ligands expressed on stressed (i.e., infected by pathogens, a prior sensitization (1). Impairments of the interactions between transplanted, inflamed) or tumor-transformed cells (2–4). Upon activation, NK cells also secrete a variety of proinflammatory cytokines and chemokines, which play important regulatory func- *Leukocyte Biology Unit, Humanitas Clinical and Research Center, I-20089 Roz- tions in the context of immune responses, hematopoiesis, and cel- † zano, Milan, Italy; Unit of Clinical and Experimental Immunology, Humanitas lular homeostasis (1, 5). Clinical and Research Center, I-20089 Rozzano, Milan, Italy; ‡Department of Med- ical Biotechnologies and Translational Medicine, University of Milan, I-20089 Roz- Macrophages are well-known for being important players in zano, Milan, Italy; and xDipartimento di Medicina Sperimentale and Centro di the physiopathology of microbial infections and cancer. In line with Eccellenza per le Ricerche Biomediche, Universita` degli Studi di Genova, I-16132 the Th1/Th2 T cell polarization, the stimulation of resting macro- Genoa, Italy phages (M0) with Th1 cytokines (i.e., IFN-g) and the TLR4 ligands 1I.M. and M.P. contributed equally to this work. 2 (i.e., LPS) induces the classical polarization toward M1 macro- M.L. and D.M. are joint senior authors on this work. phages (M1), which display strong microbicidal and tumoricidal ORCIDs: 0000-0003-3077-590X (M.L.); 0000-0001-6147-0952 (D.M.). activities and preferentially promote inflammatory responses. In Received for publication February 11, 2015. Accepted for publication July 19, 2015. contrast, the alternative polarization toward M2 macrophages (M2) This work was supported by Italian Ministry of Health (Bando Giovani Ricercatori) is induced by the Th2 cytokine IL-4 (6, 7). M2 are involved in the Grant GR-2008-1135082 (to D.M.), a Ministry of University and Research Fondo per gli Investimenti della Ricerca di Base “Futuro in Ricerca” project (to M.L.), Italian control of parasite infections and in tissue remodeling and fi- Association for Cancer Research Grants IG 9104 and 14687 (to D.M.) and IG 14685 brosis, and they are endowed with potent proangiogenic features (to M.L.), European Union Marie Curie International Reintegration Grant 322093 (to that favor tumor progression by virtue of their immunoregulatory E.L.) and Collaborative Project TIMER-HEALTH-F4-2011-281608 (to M.L.), the intramural research program of Humanitas Clinical and Research Center (to D.M.), functions (8, 9). The different biological properties of M1 and a European School of Molecular Medicine/Structured International Postdoctoral M2 are also mirrored by distinctive cytokine and chemokine rep- Marie Curie International Mobility Fellowship (to M.P.), and a “Mario e Valeria ertoires (10). Tumor-associated macrophages (TAMs), present in Rindi” Fellowship from the Italian Foundation for Cancer Research (to I.M.). solid tumors at high frequency, display both proangiogenic and I.M., M.P., M.L., and D.M. conceived and designed the experiments; I.M., M.P., P.F.T., M.M., and E.L. performed the experiments; I.M. and M.P. analyzed the data; immune-regulatory features resembling the M2 phenotype (11). It E.M. provided reagents; and I.M., M.P., M.L., and D.M. wrote the manuscript. is now widely accepted that TAMs sustain cancer growth by en- Address correspondence and reprint requests to Prof. Massimo Locati or Prof. Do- gaging vicious cellular cross talks with malignant cells and other menico Mavilio, Leukocyte Biology Unit, Department of Medical Biotechnologies immune effectors within the tumor mass, thus worsening clinical and Translational Medicine, University of Milan School of Medicine, Humanitas Clinical and Research Center, Via Alessandro Manzoni 113, I-20089 Rozzano, Mi- prognosis and favoring disease progression (12–14). lan, Italy (M.L.) or Clinical and Experimental Immunology Unit, Department of Over the past decade, several lines of evidence clearly demon- Medical Biotechnologies and Translational Medicine, University of Milan School strated that NK cells are able to engage bidirectional interactions of Medicine, Humanitas Clinical and Research Center, Via Alessandro Manzoni 113, I-20089 Rozzano, Milan, Italy (D.M.). E-mail addresses: massimo.locati@ with other members of innate immunity, such as autologous den- unimi.it (M.L.) or [email protected] (D.M.) dritic cells (DCs), macrophages, and neutrophils (2, 15–20). The The online version of this article contains supplemental material. final outcome of these synergic interactions mediated by both cell- Abbreviations used in this article: aNKR, activating NKR; DC, dendritic cell; ILC, to-cell contacts and soluble mediators is the coordination and innate lymphoid cell; IL-15Ra, IL-15R a-chain; rh, recombinant human; RT, room optimization of both innate and adaptive immune responses. In temperature; TAM, tumor-associated macrophage. particular, the NK cell–macrophage cross talk is highly relevant Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 in the context of host–pathogen interactions (15, 16, 19) and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500325 2 PRIMING OF NK CELLS BY AUTOLOGOUS M1 MACROPHAGES during the course of viral infections (20). However, the mechanism(s) Flow cytometry and cell sorting adopted by activated human macrophages to regulate autologous For direct multicolor flow-cytometry analysis (FACSCanto II; BD Bio- resting NK cell functions are largely unknown. A proper activation sciences) or cell sorting (FACSAria II; BD Biosciences), NK cells, mono- of NK cells can be achieved with different proinflammatory cyto- cytes, and polarized macrophages were washed with HBSS (Lonza) kines, such as IL-2, IL-12, IL-15, IL-18, and type I IFNs (1, 21, 22). supplemented with 2% FBS and incubated for 20 min at 4˚C with the In particular, IL-15 has been shown to play an important role in the following directly conjugated Abs labeled with indicated fluorochromes: CD3/CD56-FITC/PC5 (Beckman Coulter), CD3-FITC (BD Pharmingen), context of NK development, homeostasis, and functions. After its CD3-Brilliant Violet 650 (Biolegend), CD14-FITC (BD Pharmingen), CD14- binding to the high-affinity IL-15R a-chain (IL-15Ra) expressed Brilliant Violet 570 (Biolegend), CD16-PE-Cy7 (BD Pharmingen), CD16- by DCs and monocytes/macrophages, IL-15 is trans-presented to PerCP-Cy5.5 (BD Pharmingen), CD19-allophycocyanin-Cy7 (BD Pharmingen), surrounding cells bearing the lower-affinity IL-15Rbg-chains (22, CD48-PE (BD Pharmingen), CD56-Brilliant Violet 421 (Biolegend), CD69- Pe-Cy7 (BD Pharmingen), CD80-PE (BD Pharmingen), CD117-PE-Cy7 23). The membrane-associated IL-15–IL-15Ra complex can be (Biolegend), CD127-PE-Cy5 (eBioscience), CD206-FITC (BD Pharmingen), also cleaved from cell surface and released in biologic fluids, such CD209-PE (BD Pharmingen), M-DC8-allophycocyanin (Miltenyi), NKp46- as plasma (24, 25). More recently, it emerged that IL-15 can also PE (Beckman Coulter), NKp44-PE (Beckman Coulter), NKp30-PE (Beckman be presented by IL-15Ra to IL-15Rbg-chains expressed on the Coulter), NKG2D-PE (Beckman Coulter), DNAM-1–PE (Beckman Coulter), same cell, thus allowing a mechanism of IL-15 cis-presentation that 2B4-PE (R&D Systems), IL-15Ra–PE (Biolegend), IL-1RII–PE (R&D Sys- tems), and their appropriate isotype controls. For IL-1RI indirect staining, triggers NK cell effector functions (26). Another potent proin- NK cells were incubated for 45 min at room temperature (RT) with purified flammatory cytokine produced by M1 is IL-1b, which has been goat anti-human IL-1RI (R&D Systems) or its polyclonal IgG control and reported to modulate homeostasis and activation of innate lym- for 30 min at RT with mouse anti-goat Alexa Fluor 647–labeled sec- phoid cells (ILCs) in secondary lymphoid tissues (27–29). ILCs ondary Ab (Invitrogen). For sorting experiments, thawed NK cells were stained for 15 min at RTwith LIVE/DEAD Aqua (Invitrogen), followed have been recently identified as an additional lymphocytic pop- by incubation for 20 min at RT with fluorochrome-conjugated Abs and ulation that could be divided in different subsets (ILC1, ILC2, then sorted.