CROSS-TALK BETWEEN HUMAN NK CELLS AND MACROPHAGES: INFLUENCE OF THE TUMOR MICRO-ENVIRONMENT

Natural killer (NK) cells are important effectors of innate immune responses providing cellular immunity against tumor-transformed and virally-infected cells. The existence of cross-talks between NK cells and myeloid cells, in particular dendritic cells, is well established, but information on the cross-talk between NK cells and macrophages is scanty. These interactions have been analyzed using an in vitro reconstituted tumoral micro-environment, as a simplified model to define soluble factors involved and/or cell contact dependency. Autologous human NK cells and monocyte-derived macrophages were obtained from buffy coats of healthy donors after magnetic beads cell purification. Macrophages were polarized into M0, M1 and M2, using well described stimuli. First, the influence of human polarized macrophages on NK cell anti-tumoral activities was studied. The co-cultures between NK cells and macrophages were performed in direct contact or by treating NK cells with macrophage-conditioned media. Activating receptors expression and degranulation ability (CD107a assay) of NK cells were evaluated by flow cytometry. IFN-\u3b3 production by NK cells was quantified by RT-PCR and ELISA. Then, the effect of NK cell-derived IFN-\u3b3 on macrophage polarization was assessed. Gene expression of markers, cytokines and chemokines well described to characterized M1 or M2 polarization were evaluated by RT-PCR. In parallel, cytokine and chemokine secretion were detected by ELISA. M1 polarization was required to enhance IFN-\u3b3 production and degranulation by resting NK cells. M1 ability to activate NK cells was further confirmed by the upregulation of CD69 activation marker. Importantly, either soluble mediators and direct contact interactions were involved in this process. However, the level of expression of NKp44 and NKG2D resulted increased only when NK cells were treated with M1-conditioned medium (M1-primed NK cells). Higher NKp44 and NKG2D expression correlated with enhanced NK cell degranulation towards altered cells. Although both NK cell subsets upregulated both receptors, M1-secreted IL-1\u3b2 was responsible for NKp44 induction on CD56dim population, whereas IFN-\u3b2 released by M1 favored increased expression of NKG2D by the CD56bright counterpart. Importantly, M1 secretion of IFN-\u3b2 triggered NK cell expression of IL-15 and IL-15R\u3b1, inducing a mechanism of IL-15 cis-presentation. IL-15 cis-presentation strongly enhanced IFN-\u3b3 secretion, that was further sustained by 2B4-CD48 interactions during direct co-cultures. On the contrary, NKG2D upregulation was responsible for increased degranulation by M1-primed NK cells. In parallel, IL-15 trans-presentation mediated by M1, together with NKG2D and NKp30 engagement, were needed to trigger NK cell degranulation during direct contact interactions. On the other hand, IFN-\u3b3 secreted by M1-primed NK cells was sufficient not only to downmodulate CD206 and ALOX15 expression by alternatively-activated macrophages, but also to induce pro-inflammatory cytokine (IL-1\u3b2 and IL-15) and chemokine (CCL-5, CXCL-9 and CXCL-10) production. Importantly, also CD80 and IL-15R\u3b1, which expression is strictly associated to M1 phenotype, were upregulated. In conclusion, we demonstrate for the first time in a human model that IL-15/IL-15R\u3b1 complex plays a key role in the crosstalk between NK cells and M1 polarized macrophages. Both, cis and trans-presented IL-15 favors NK cell secretion of high amount of IFN-\u3b3 and enhances NK cell cytotoxic activity towards tumor cells. Furthermore, having determined a functional correlation between M1-derived IL-1\u3b2 and NKp44 expression, we propose new effects of IL-1\u3b2 on NK cell biology. Finally, we demonstrate that IFN-\u3b3 provided by activated NK cells is sufficient to partially revert the anti-inflammatory phenotype typical of alternatively-activated macrophages into a pro-inflammatory one. This confers to NK cells a potential involvement in TAMs re-education

The macrophage tetraspan MS4A4A enhances dectin-1-dependent NK cell-mediated resistance to metastasis

Fondazione Cariplo (grant no. 2015–0564 to A.M.)Cluster Alisei (grant no. MEDINTECH CTN01_00177_962865 to A.M.)European Research Council (grant no. 669415-PHII to A.M.)Italian Association for Cancer Research (AIRC IG-2016 grant no. 19014 to A.M.; AIRC 5 × 1000 grant no. 21147 to A.M.; AIRC IG-2016 grant no. 19213 to M.L.)Medical Research Council (Pathobiology of Early Arthritis Cohort grant no. 36661 to C.P.)Arthritis Research UK Experimental Treatment Centre (grant no. 20022 to C.P.

The macrophage tetraspan MS4A4A enhances dectin-1-dependent NK cell–mediated resistance to metastasis

The plasma membrane tetraspan molecule MS4A4A is selectively expressed by macrophage-lineage cells, but its function is unknown. Here we report that MS4A4A was restricted to murine and human mononuclear phagocytes and was induced during monocyte-to-macrophage differentiation in the presence of interleukin 4 or dexamethasone. Human MS4A4A was co-expressed with M2/M2-like molecules in subsets of normal tissue-resident macrophages, infiltrating macrophages from inflamed synovium and tumor-associated macrophages. MS4A4A interacted and colocalized with the β-glucan receptor dectin-1 in lipid rafts. In response to dectin-1 ligands, Ms4a4a-deficient macrophages showed defective signaling and defective production of effector molecules. In experimental models of tumor progression and metastasis, Ms4a4a deficiency in macrophages had no impact on primary tumor growth, but was essential for dectin-1-mediated activation of macrophages and natural killer (NK) cell–mediated metastasis control. Thus, MS4A4A is a tetraspan molecule selectively expressed in macrophages during differentiation and polarization, essential for dectin-1-dependent activation of NK cell–mediated resistance to metastasis

Granzyme A and CD160 expression delineates ILC1 with graded functions in the mouse liver

Type 1 innate lymphoid cells (ILC1) are tissue-resident lymphocytes that provide early protection against bacterial and viral infections. Discrete transcriptional states of ILC1 have been identified in homeostatic and pathological contexts. However, whether these states delineate ILC1 with different functional properties is not completely understood. Here, we show that liver ILC1 are heterogeneous for the expression of distinct effector molecules and surface receptors, including granzyme A (GzmA) and CD160, in mice. ILC1 expressing high levels of GzmA are enriched in the liver of adult mice, and represent the main hepatic ILC1 population at birth. However, the heterogeneity of GzmA and CD160 expression in hepatic ILC1 begins perinatally and increases with age. GzmA+ ILC1 differ from NK cells for the limited homeostatic requirements of JAK/STAT signals and the transcription factor Nfil3. Moreover, by employing Rorc(γt)-fate map (fm) reporter mice, we established that ILC3-ILC1 plasticity contributes to delineate the heterogeneity of liver ILC1, with RORγt-fm+ cells skewed toward a GzmA–CD160+ phenotype. Finally, we showed that ILC1 defined by the expression of GzmA and CD160 are characterized by graded cytotoxic potential and ability to produce IFN-γ. In conclusion, our findings help deconvoluting ILC1 heterogeneity and provide evidence for functional diversification of liver ILC1