NK cells: receptors and functions Eric Vivier and Sophie Ugolini

Natural killer (NK) cells were identified in 1975 as lymphocytes of the innate many processes that were originally restricted to adaptive immunity, such immune system that can kill tumour cells. Since then, NK cells have been as priming, education and memory, are now known to occur in NK cells. shown to kill an array of ‘stressed’ cells and secrete cytokines that Indeed, NK cells undergo sophisticated processes of adaptation that allow participate in shaping adaptive immune responses. A key feature of NK cells them to be tuned to their environment. There is also a growing interest in resides in their capacity to distinguish stressed cells (such as tumour cells, manipulating NK cells in innovative therapeutic settings. For example, the infected cells and damaged cells) from normal cells. Although NK cells are understanding of NK cell inhibition by MHC class I-specific receptors has generally considered to be components of early innate immune defence, prompted the design of innovative anticancer therapies.

The NK cell detection system includes numerous receptors, Key receptors on human NK cells the engagement of which dictates the quality and intensity Inhibitory receptors Activating receptors, adhesion molecules or co-stimulatory molecules of the NK cell response. NK cells use inhibitory receptors to gauge the absence of constitutively expressed self molecules Target cell Nectin 2 SLAMF7 Sialic CLEC2D (CD112, PVRL2) NECL2 L-selectin NECL5 AICL NTB-A (CRACC, on susceptible target cells. As a consequence, NK cells can Receptors for MHC class I and class I-Iike molecules B7-H6 acid Cadherins (LLT1) CMV pp65 NECL5 (CADM1) (CD62L) (CD155, PVR) (CLEC2B) CEACAM1 (SLAMF6) CS1, CD319) recognize ‘missing self’ on haematopoietic cells. By interacting HLA HLA-A or MICA, MICB, Collagen ? ? ? Viral HA BAT3 Viral HA (CD155, PVR) CD70 with MHC class I molecules that are constitutively expressed by HLA-E class I HLA-C HLA-B HLA-CHLA-E ULBP1 (RAET1I) HLA-C IgG CD72 CD48 most healthy cells under steady-state conditions but that may be lost under conditions of stress, MHC class I-specific inhibitory receptors provide a way for NK cells to remain tolerant to healthy self cells while being toxic towards stressed cells. CD16 NKp46 NKp30 NKp44

Supplement to Nature Publishing Group Supplement to Nature By contrast, NK cell activating receptors detect self molecules KIR2DS1 NKG2D (FcaRIIIA) (NCR1) (NCR3) (NCR2) that are expressed under conditions of cell stress. Only human NK cell receptors are shown and the list is not exhaustive. ITIM DAP12 There are several differences in NK cell receptors between YxxM ITSM mice and humans. In mice, inhibitory MHC class I-specific ITAM DNAM1 LAIR1 SIGLEC-3 (CD33) KLRG1 NKR-P1A CD94– LIR1 KIR2DL1 KIR2DL5A KIR3DL1 KIR3DL3 CD160 FcRa FcRa FcRa (CD226) CRTAM CD27 PSGL1 CD96 CD100 NKp80 CEACAM1 NTB-A SLAMF7 receptors are lectin-like dimers of the Ly49 family. Although SIGLEC-7 (KLRB1) NKG2A (ILT2, KIR2DL2 KIR2DL5B KIR3DL2 KIR2DS2, KIR2DS3, DAP12 DAP12 DAP10 (BY55) DAP12 (Tactile) (SEMA4D) (KLRF1, (SLAMF6) (CRACC, CD244 (TYROBP) (HCST) several activating NK cell receptors are present in humans and SIGLEC-9 LILRB1) KIR2DL3 KIR2DS4, KIR2DS5, CD94– CD3c CD3c CD3c CLEC5C) CS1, CD319) (2B4, mice (such as CD16, NKp46, DNAM1 and NKG2D), commonly KIR3DS1 NKG2C/E SLAMF4) used mouse strains lack orthologues of NKp30 and NKp44. NK cell

Acquisition of NK cell function Biological function of NK cells and cellular crosstalk NK cell subsets and NK-like innate lymphoid cells

MHC class I-specific MHC class I-specific MHC NK cells can recognize various stressed cells that Various NK cell subsets can be found in *WOCP /QWUG Lymphoid inhibitory receptor MHC inhibitory receptor class I have or have not been opsonized by antibodies. many lymphoid and non-lymphoid NKp46 NK1.1 NKp46 tissues class I NK cell activation triggered by this recognition can Healthy DNA damage, tissues, including the liver, lungs, thymus, (NKR-P1C) cell tumour transformation, lead to target cell lysis, as well as to the production pancreas and uterus. CD56hi intracytoplasmic + CD27– Educating – Healthy of various cytokines and chemokines, depending In humans, NK cells can be divided CD62L microbial infection hi CD11b– NK cell + IFNa CD94 cell cell on the nature of the stimulation. NK cells also according to the density of CD56 – NK cell hi CD57 Education engage in crosstalk with DCs in many different expression. There is evidence that CD56 ways, including NK cell killing of 1RUQPK\CVKQP NK cells give rise to mature cytotoxic NK1.1 NKp46 No activation Activating Activating CD56low NK cells, which can be further receptor ligand immature DCs (iDCs) and the Antibody divided based on expression of PEN5, a promotion of DC differentiation CD27+ IL-15Rα IL-12 Tolerance by NK cell-derived IFNγ and Stressed Stressed carbohydrate epitope present on PSGL1, CD16 CD11b– cell cell NKp46 IL-15 IL-18 TNF. Through these KLRG1, CD57, CD94 and CD62L. Uterine γ IL-12R hi –/low c biological activities, Elimination NK cells are CD56 CD16 and secrete CD56low Cytotoxicity + NK cells participate a specific set of chemokines, including CD62L NK1.1 NKp46 IL-2/15Rβ IL-18R low in shaping the CXCL8, CXCL10, CXCL12 and VEGF. CD94 Stressed Lysis Activation Lysis CD57– NK cell + subsequent In mice, four subsets of circulating Cytotoxicity cell IFNa + Priming NK cell immune NK cells can be distinguished: immature CD27 – – CD11b+ IFNAR response. CD27 CD11b NK cells, intermediate Missing self CD27+CD11b– and CD27+CD11b+ IFNa IL-21R Activation IL-21 Type I IFNs IFNa NK cell subsets, and mature CD16 KLRG1 KLRG1 IFNa NK cell Regulation CD27–CD11b+ NK cells. PSGL1 IL-10 NKp46 NK1.1 NKp46 To acquire the capacity to recognize target cells with low TNF Although NKp46 is a conserved marker MHC class I expression (‘missing self’ recognition), NK cells that best defines NK cells in mammals, CD56low Activation Cytotoxicity Cytotoxicity have to be educated by the detection of host MHC class I – Macrophage CD62L– – Stressed cells that express NCRs (NKp46 in mice, CD27 molecules by their cognate inhibitory receptors (a process also + Uptake of target IL-12 CD94– + cell Activation, IFNa NKp46, NKp30 and NKp44 in humans) CD11b cell debris IL-15 CD57+ referred to as tuning or licensing). NK cells also adapt to their NK cell proliferation IL-10 Lysis IL-18 and produce IL-22 are found in mucosal IFNa environment through the process of ‘priming’ by cytokines, such and survival IFNa Type I IFNs tissues. These cells are not cytotoxic, Blood Activation as IL-15 (trans-presented by DCs), IL-18 and IL-12. The integration do not secrete IFNγ and express the of various pathways (inhibitory and activating) following interaction Stress-induced self transcription factor RORγt instead of NKp46 NK1.1 s NKp46 with neighbouring cells governs the dynamic equilibrium that regulates E4BP4 (also known as NFIL3), which is NK cell activation and determines whether NK cells are activated to kill target IL-12 crucial for bona fide NK cells. IL-22 IL-22 iDC mDC T cell cells and produce cytokines. The innate immune system is generally thought to lack These IL-22-producing NCR+ innate CD56+ Maturation Activation the capacity for immunological memory. However, recent findings show that some lymphoid cells are therefore distinct Mucosal NK cells can be long-lived and mount a robust recall response to haptens or viruses. from conventional NK cell subsets. tissues

Isolation of NK cells with STEMCELL Technologies Species Selection Starting Sample Product Catalog # Abbreviations KIR, killer cell immunoglobulin-like receptor; KLRG, killer cell lectin-like Affiliations The isolation of highly purified, functional NK cells is critical for successful NK cell NK cell isolation kits from STEMCELL Technologies Human Negative PBMC EasySep™ Human NK Cell 17955 Eric Vivier and Sophie Ugolini are at the Centre d’Immunologie de Marseille-Luminy γc, common cytokine receptor γ-chain; AICL, activation-induced C-type receptor subfamily G; LAIR1, leukocyte-associated immunoglobulin-like research. STEMCELL Technologies Inc. offers fast and easy isolation products for consistently provide high purities (up to 99%), and high Isolation Kit Whole Blood EasySep™ Direct Human 19665 lectin; BAT3, HLA-B associated transcript 3; CADM1, cell adhesion receptor 1; LIR1, leukocyte immunoglobulin-like receptor 1; LLT1, (CIML), INSERM-CNRS-Université de la Méditerranée, Marseille, France. E.V. is also at NK cells using the unique immunomagnetic and immunodensity cell separation recoveries. Purified NK cells can be used immediately in NK Cell Isolation Kit molecule 1; CD62L, CD62 ligand; CEACAM1, carcinoembryonic lectin-like transcript 1; mDC, mature DC; MIC, MHC class I polypeptide- the Assistance Publique des Hôpitaux de Marseille, Hôpital de la Conception, systems, EasySep™ and RosetteSep™. NK cell isolation kits from STEMCELL any downstream assay. RosetteSep™ Human 15025 antigen-related cell adhesion molecule 1; CLEC, C-type lectin domain related sequence; NCR, natural cytotoxicity receptor; NECL, nectin-like; Marseille, France. e-mails: [email protected]; [email protected] Technologies have supported cutting-edge research, including analysis on NK cell NK Cell Enrichment Cocktail family; CMV, cytomegalovirus; CRACC, CD2-like-receptor activating NFIL3, nuclear factor IL-3-regulated protein; NKG2, NK group 2; function1,2, investigations into the anti-leukemic activity of NK cells3 and the role To learn more about our specialized products for Positive PBMC EasySep™ Release Human 17755 Acknowledgements CD56 Positive Selection Kit cytotoxic cells; CRTAM, class I MHC-restricted T cell-associated NKR-P1A, NK cell receptor protein 1A; NTB-A, natural killer, T and B cell of NK cells in HIV pathogenesis and immunology.4,5 Untouched human NK cells NK cell research or to request a sample, visit E.V. and S.U. are supported by grants from the European Research Council, Agence www.stemcell.com/nkcells. EasySep™ Human CD56 17855 molecule; CS1, CD2 subset 1; CXCL, CXC-chemokine ligand; antigen; PSGL1, P-selectin glycoprotein ligand 1; PVR, poliovirus can be isolated from PBMC in as little as 8 minutes using column-free EasySep™ Positive Selection Kit II DC, dendritic cell; DNAM1, DNAX accessory molecule 1; E4BP4, E4 receptor; R, receptor; RAET1I, retinoic acid early transcript 1I; RORγt, Nationale de la Recherche, Ligue Nationale contre le Cancer (Equipe labellisée ‘La Ligue’) technology, or directly from whole blood during a standard density gradient 1. Zhu S et al. (2014) Blood 124(3): 403–411. Buffy Coat EasySep™ Human Buffy Coat 18085 promoter binding-protein 4; FcR, Fc receptor; HA, haemagglutinin; retinoic acid receptor-related orphan receptor-γt; SEMA4D, semaphorin and institutional grants from INSERM, CNRS and Université de la Méditerranée to the CIML. centrifugation with RosetteSep™. For full automation of EasySep™ NK cell 2. Briercheck EL et al. (2015) J Immunol 194(4): 1832–1840. CD56 Positive Selection Kit HCST, haematopoietic cell signal transducer; IFN, interferon; IL, 4D; SIGLEC, sialic acid-binding immunoglobulin-like lectin; SLAM, isolations, RoboSep™ instruments can process up to sixteen samples Mouse Negative Spleen or other EasySep™ Mouse NK Cell 19855 The authors declare competing financial interests: see Web version for details. 3. Vasu S et al. (2016) Blood. tissues Isolation Kit interleukin; ILT, immunoglobulin-like transcript; ITAM, immunoreceptor signalling lymphocytic activation molecule family; TNF, tumour necrosis simultaneously with minimal sample handling and no cross-contamination. 4. Norman JM et al. (2011) Nat Immunol 12(10): 975–983. Positive EasySep™ Mouse CD49b 18755 tyrosine-based activation motif; ITIM, immunoreceptor tyrosine-based factor; TYROBP, TYRO protein tyrosine kinase-binding protein; ULBP1, Edited by Lucy Bird; copyedited by Isabel Woodman; designed by Simon Bradbrook. DOCUMENT # 28771 | VERSION 1.1.0 5. Huang Q-Q et al. (2015) Nat Commun 6: 7086. Positive Selection Kit inhibitory motif; ITSM, immunoreceptor tyrosine-based switch motif; UL16-binding protein 1; VEGF, vascular endothelial growth factor. © 2010 Nature Publishing Group. http://www.nature.com/nri/posters/nkcells