Human NK Cells: Surface Receptors, Inhibitory Checkpoints, and Translational Applications
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www.nature.com/cmi Cellular & Molecular Immunology REVIEW ARTICLE Human NK cells: surface receptors, inhibitory checkpoints, and translational applications Simona Sivori 1, Paola Vacca2, Genny Del Zotto 3, Enrico Munari4,5, Maria Cristina Mingari1,6 and Lorenzo Moretta 2 NK cells play important roles in innate defenses against viruses and in the control of tumor growth and metastasis. The regulation/ induction of NK cell function is mediated by an array of activating or inhibitory surface receptors. In humans, major activating receptors involved in target cell killing are the natural cytotoxicity receptors (NCRs) and NKG2D. Activating receptors recognize ligands that are overexpressed or expressed de novo upon cell stress, viral infection, or tumor transformation. The HLA-class I- specific inhibitory receptors, including KIRs recognizing HLA-class I allotypic determinants and CD94/NKG2A recognizing the class- Ib HLA-E, constitute a fail-safe mechanism to avoid unwanted NK-mediated damage to healthy cells. Other receptors such as PD-1, primarily expressed by activated T lymphocytes, are important inhibitory checkpoints of immune responses that ensure T-cell tolerance. PD-1 also may be expressed by NK cells in cancer patients. Since PD-1 ligand (PD-L1) may be expressed by different tumors, PD-1/PD-L1 interactions inactivate both T and NK cells. Thus, the reliable evaluation of PD-L1 expression in tumors has become a major issue to select patients who may benefit from therapy with mAbs disrupting PD-1/PD-L1 interactions. Recently, NKG2A was revealed to be an important checkpoint controlling both NK and T-cell activation. Since most tumors express HLA-E, mAbs targeting NKG2A has been used alone or in combination with other therapeutic mAbs targeting PD-1 or tumor antigens (e.g., EGFR), with encouraging results. The translational value of NK cells and their receptors is evidenced by the extraordinary therapeutic success of haploidentical HSCT to cure otherwise fatal high-risk leukemias. Keywords: Human NK cells, NK receptors, Inhibitory checkpoints, Immunotherapy Cellular & Molecular Immunology (2019) 16:430–441; https://doi.org/10.1038/s41423-019-0206-4 INTRODUCTION AND GENERAL OVERVIEW OF HUMAN NK molecules in both humans and in mice. Remarkably, while the CELLS first identified human receptors specific for human leukocyte Natural killer (NK) cells play major roles in first-line innate defenses antigen (HLA)-cl I molecules belong to the immunoglobulin (Ig) against viral infections, tumor growth, and metastatic spread. Both superfamily (and were subsequently named killer-cell immuno- the constitutive expression of efficient, ready-to-function lytic globulin-like receptors, KIRs), the murine receptors (Ly49) are machinery, and the rapid release of interferon-gamma (IFN-γ) and members of the lectin family. Thus, humans and mice developed tumor necrosis factor-alfa (TNF-α) following cell activation allow two molecularly different receptors fulfilling the same function, prompt intervention by NK cells, resulting in target cell killing and i.e., the recognition of MHC-cl I molecules and the delivery of the initiation of an inflammatory response. inhibitory signals to NK cells, resulting in NK cell inactivation.5 The NK cells were discovered in the mid-70s;1,2 however, the discovery of KIRs was made possible by the availability of two mechanism by which they discriminate between tumor or virus- important technologies, namely, monoclonal antibody technology infected cells and healthy cells remained a mystery for many years. and a high-efficiency lymphocyte cloning system. The latter The missing self-hypothesis, proposed in the late 80s by Karre and technology allowed the clonal expansion of virtually 100% of Ljunggren,3 represented a true milestone, inspiring subsequent human T lymphocytes.6 Thus, it has been possible to analyze the groundbreaking discoveries in the early 90s. This hypothesis was frequency of cells endowed with a given function (e.g., cytolytic based on the finding that murine NK cells could kill a lymphoma activity) and to establish correlations between the surface cell line that had lost major histocompatibility complex-class I phenotype and function of any T-cell population.7 After suitable (MHC-cl I) molecules, while parental MHC-cl I+ cells were resistant adaptation of this cloning technique, NK cells were also revealed to lysis. Thus, NK cells appear to sense the absence of MHC-cl I on to be clonogenic and suitable for precise functional studies. NK cells (i.e., the missing self).4 Research aimed to identify the cell clones were used for mouse immunization and the production molecular mechanism(s) involved in this phenomenon, leading to of monoclonal antibodies (mAbs) that were selected for their the discovery of inhibitory receptors specific for MHC-cl I ability to either inhibit or induce NK cell cytolytic activity or 1Department of Experimental Medicine (DIMES) and Centre of Excellence for Biomedical Research (CEBR), University of Genoa, Genoa, Italy; 2Department of Immunology, IRCCS Bambino Gesù Children’s Hospital, Rome, Italy; 3Department of Research and Diagnostics, Istituto G. Gaslini, Genoa, Italy; 4Department of Diagnostics and Public Health, University of Verona, Verona, Italy; 5Department of Pathology, Sacro Cuore Don Calabria, Negrar, VR, Italy and 6UOC Immunologia, IRCCS Ospedale Policlinico San Martino Genova, Genoa, Italy Correspondence: Lorenzo Moretta ([email protected]) Received: 14 January 2019 Accepted: 22 January 2019 Published online: 18 February 2019 © CSI and USTC 2019 Human NK cells: surface receptors, inhibitory checkpoints, and. S Sivori et al. 431 cytokine production. Two selected mAbs, originally defined as sensing different HLA-E-bound CMV peptides and undergo p58.1 and p58.28–10, recognized two highly homologous mole- selective proliferation only in response to given HLA-E-peptide cules expressed by partially overlapping NK cell subsets. These combinations.31 Such specific expansion, together with a number mAbs represented prototypes of HLA-cl I-specific inhibitory of adaptive characteristics, is compatible with the memory-like receptors, and were found to recognize allotypic determinants function of these cells, which can allow for prompt control of CMV shared by the two main groups of HLA-C alleles.10–13 reactivation.32 Other HLA-cl I-specific receptors were identified (see below). Of While NK cells have been known for over 45 years, other cells relevance, the HLA-E-specific NKG2A,14 expressed earlier than KIR that are developmentally related to NK cells were identified only during NK cell maturation, was recently found to be of great 10 years ago and collectively termed innate lymphoid cells potential interest as a target of checkpoint inhibitors in tumor (ILCs).33,34 Similar to NK cells, ILCs derive from CD34+ hemato- immunotherapy15,16(see below). poietic precursors and share with NK cells a common ID2+ Remarkably, the pool of mature NK cells is equipped with at lymphoid precursor.35 Absent or infrequent in PB from healthy least one inhibitory receptor for self HLA-cl I antigens (whether KIR individuals, ILCs reside in mucosal tissues, skin, and lymphoid or NKG2A). Only very few peripheral blood (PB) NK cells lack such organs. Unlike NK cells, the other ILCs (termed “helper ILCs”) are receptors and are anergic. The repertoire of inhibitory NK noncytolytic and secrete typical sets of cytokines that differ for receptors is shaped during NK cell maturation and is the result each subset, including IFN-γ (ILC1), IL-5, IL-13, and small amounts of a process of selection termed NK cell “licensing” or “educa- of IL-4 (ILC2), IL-22 (NCR+ ILC3), IL-17, and TNF-α (NCRneg ILC3 or tion”.17–19 Accordingly, only NK cells expressing receptors for self LTi-like cells).36 Interestingly, helper ILCs mimic corresponding HLA-cl I molecules acquire full functional potential, while the CD4+ T-cell subsets in terms of the type of cytokines produced. remaining are either deleted or anergic. Therefore, these cytokines can shape not only adaptive but also “Off” signals are necessary to prevent NK-mediated autoreac- innate immune responses, although with different timing. In tivity, implying the existence of “on” signals responsible for NK cell general, ILCs contribute to innate defenses against various activation. Indeed, in the absence of inhibitory interactions, NK pathogens and are involved in tissue repair and homeostasis cells kill target cells and produce cytokines. Again, the use of NK and in lymphoid organogenesis, particularly during fetal life. While cell clones and the selection of mAbs were fundamental for the they will not be discussed further in this review, which is devoted discovery of prototypes that activate NK receptors, namely, to NK cells, it is worth noting that ILCs may share some markers NKp46,20–22 NKp4423 and NKp30,24 collectively termed “natural and the NCR activating receptors with NK cells.37 25 1234567890();,: cytotoxicity receptors” (NCRs). mAbs directed to these surface NK cell traffic in the circulation and towards tissues and molecules were selected based on their ability to modulate NK lymphoid organs is regulated by chemokines and chemokine clone-mediated cytotoxicity against selected tumor targets. It receptors, directing the given NK cell subsets to specific sites.38 became evident that NK cell activation is not due to a master Thus, while the expression of CD62L, CXCR3, and CCR7 will direct receptor (such as TCR and BCR in T and B cells, respectively) but, in CD56bright NK